RESUMO
BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis infection induces apoptosis inhibition in gingival epithelial cells; however, it is not fully understood which bacterial effectors are involved in this process. The aim of this study is to evaluate whether the P. gingivalis lipopolysaccharide (LPS), specifically the O-antigen region, affects adherence, invasion, viability and apoptosis of gingival epithelial cells. MATERIAL AND METHODS: Gingival epithelial cells (OKF6/TERT2 line) were infected by different freshly prepared P. gingivalis clinical isolates, obtained from subjects with chronic periodontitis (CP3 and CP4) and healthy individuals (H1 and H3). Periodontitis and healthy isolates show differences in O-antigen production, as healthy isolates lack the O-antigen region. In addition, cells were infected by a site-specific mutant lacking the O-antigen portion. After 24 h postinfection, cell proliferation, viability and apoptosis were evaluated by Trypan blue, MTS and annexin V assays, respectively. Bacterial invasion, adhesion and proliferation were measured by gentamicin/metronidazole protection assays. Finally, toll-like receptor (TLR)2 and TLR4 mRNA expression was evaluated by quantitative reverse transcription-polymerase chain reaction. Statistical analysis was performed using ANOVA, Tukey's or Dunnett's tests (p < 0.05). RESULTS: At 24 h postinfection, strains lacking the O-antigen region (healthy isolates and O-antigen ligase-deficient strain) were unable to increase proliferation and viability, or decrease apoptosis as compared with strains producing intact LPS (periodontitis isolates and reference strain). However, the presence of the O-antigen neither contributed to changes in the ability of the bacteria to adhere to or invade cells, nor to intracellular survival. The presence of O-antigen also increased the expression of TLR4 (nearly sixfold), which correlated with inhibition of apoptosis. CONCLUSION: The O-antigen region of P. gingivalis LPS is required to increase gingival epithelial cell viability upon infection by bacteria and this increase is attributable to a reduction in apoptosis. Moreover, although bacterial internalization is required, the effects observed are not due to alterations in P. gingivalis adherence, invasion or intracellular survival. Interestingly, inhibition of apoptosis correlates with increased TLR4 expression, suggesting a role for TLR4 in this process.
Assuntos
Apoptose/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Antígenos O/farmacologia , Porphyromonas gingivalis/fisiologia , Infecções Bacterianas , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica , Gengiva/citologia , Gengiva/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Periodontite , Porphyromonas gingivalis/isolamento & purificação , RNA Mensageiro/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
BACKGROUND: The mechanisms involved in reactive oxygen species and matrix metalloproteinase (MMP)-mediated periodontal tissue breakdown are unknown. OBJECTIVE: To determine the effect of H2 O2 in MMP-2 and MMP-9 activity, and the involvement of nuclear factor kappa B (NFκB) and Ca(2+) -mediated signals in human periodontal ligament fibroblasts. MATERIAL AND METHODS: Primary cultures were characterized for their phenotype and exposed for 24 h to sublethal doses (2.5-10 µm) of H2 O2 or control media. NFκB involvement was evaluated through immunofluorescence of p65 subunit, using the NFκB blocking peptide SN50 and catalase. Ca(2+) signals were analyzed by loading the cells with Fluo4-AM and recording the fluorescence changes in a confocal microscope before and after the addition of H2 O2 . 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl was used to chelate intracellular Ca(2+) . The activity and levels of MMP-2 and MMP-9 were analyzed by gelatin zymogram and densitometric scanning, and enzyme-linked immunosorbent assay, respectively. Statistical analysis was performed with stata V11.1 software using the ANOVA test. RESULTS: H2 O2 at concentrations of 2.5-5 µm induced Ca(2+) signaling and NFκB subunit p65 nuclear translocation, whereas catalase, SN50 and BAPTA-AM prevented p65 nuclear translocation. H2 O2 at 2.5-5 µm significantly increased MMP-9 and MMP-2 activity, while SN50 resulted in lower MMP-2 and MMP-9 activity rates compared with controls. CONCLUSION: Sublethal H2 O2 induces Ca(2+) -dependent NFκB signaling with an increase in MMP gelatinolytic activity in human periodontal ligament.
Assuntos
Sinalização do Cálcio , Fibroblastos/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Estresse Fisiológico , Adulto , Células Cultivadas , Feminino , Fibroblastos/enzimologia , Fibroblastos/fisiologia , Humanos , Masculino , Ligamento Periodontal/citologiaRESUMO
La periodontitis crónica es una patología infecciosa, causada por un complejo de especies bacterianas, que afecta principalmente los tejidos de inserción de los dientes. La respuesta inmune-inflamatoria producida se caracteriza por la presencia de un infiltrado inflamatorio, en el cual los macrófagos representan entre 5 al 30 por ciento. Es sabido que los macrófagos se activan mediante dos vías: Clásica y Alterna, caracterizadas por la presencia de marcadores indirectos: IFN-y e IL-6 para la vía clásica e IL-4 para la vía alterna, ampliamente abordados. Recientemente, se ha descrito a la subunidad A del factor XIII de la coagulación (FXIII-A) como un buen marcador de la vía alterna. El objetivo de este estudio consiste en determinar la presencia de IFN-y, IL-6, FXIII-A e IL-4 como marcadores de las vías de activación de los macrófagos, en pacientes con periodontitis crónica. Para tal efecto, se realizó inmunohistoquímica y Western-Blot para los cuatro marcadores junto a CD-68, marcador de macrófagos, en 18 biopsias de tejido periodontal sano y 18 con periodontitis crónica. Se detectó la presencia de IFN-y, IL-6, IL-4 y FXIII-A junto a CD68+, en todas las muestras de pacientes sanos y con periodontitis. Los resultados obtenidos sugieren que al estar presente IFN-y, IL-6, IL-4 y FXIII-A, los macrófagos se activarían a través de ambas vías, lo cual, produciría una respuesta tanto proinflamatoria (Th1) como antinflamatoria (Th2). Son necesarios más estudios para determinar si existe una vía preferencial de activación.
Periodontitis is a chronic infectious disease caused by a bacterial species complex, which affects mainly the insertion tissues of the teeth. The immune-inflammatory response produced is characterized by an inflammatory infiltrate in which macrophages represent between 5 to 30 percent. It is known and has been widely discussed that macrophages are activated in two ways: Classical and Alterna, characterized by the presence of indirect markers: IFN-y and IL-6 for the classical pathway and IL-4 for the alternative pathway. Recently the subunit A of the clotting factor XIII (FXIII-A) has been described as a good marker of the alternative pathway. The objective of this study is to determine the presence of IFN-y, IL-6, IL-4 and FXIII-A as markers of the macrophage activation pathways in patients with chronic periodontitis. To this end, we performed immunohistochemistry and Western blot for the four markers with CD68 macrophage marker, in 18 healthy periodontal tissue biopsies and 18 with chronic periodontitis. We detected the presence of IFN-y, IL-6, IL-4 and FXIII-A with CD68 +, in all samples of healthy patients and periodontitis. The results suggest that when present, IFN-y, IL-6, IL-4 and FXIII-A, activate macrophages through both routes, which would produce a proinflammatory response (Th1) as antiinflammatory (Th2). Further studies are necessary to determine whether there is a preferential pathway activation.
Assuntos
Humanos , Adulto , Ativação de Macrófagos , Macrófagos/imunologia , Biomarcadores/análise , Periodontite Crônica/patologia , Fator XIIIa/análise , Imuno-Histoquímica , Interferon gama/análise , /análise , Periodontite Crônica/imunologiaRESUMO
Porphyromonas gingivalis es un microorganismo fuertemente asociado con la etiología de la periodontitis. Esta bacteria posee varios factores de virulencia, dentro de los que destacan las gingipaínas, debido a sus múltiples acciones relacionadas con la destrucción de la matriz extracelular del tejido conectivo periodontal, la modulación del sistema inmune del hospedero y la estimulación de la expresión de citoquinas pro-inflamatorias. Estas proteinasas tienen afinidades específicas siendo Arg-gingipaínas (RgpA y RgpB, codificadas por los genes rgpA y rgpB, respectivamente) y Lys-gingipaínas (Kgp, codificada por el gen kgp). Se ha descrito que existen polimorfismos en los genes que codifican para esta proteinasas. El objetivo del presente estudio fue describir la frecuencia de los genotipos identificados para los genes rgpA y kgp en aislados clínicos de P. gingivalis, obtenidos desde pacientes con periodontitis. Para ello se utilizó amplificación por PCR de los genes rgpA y kgp, seguido de análisis de restricción. De un total de 47 aislados provenientes de 4 individuos con periodontitis crónica y 2 con periodontitis agresiva, se genotipificaron 38 aislados para el gen rgpA, exhibiendo la totalidad de éstos el patrón electroforético A (100 por ciento). Para el gen kgp se genotipificaron 43 aislados, presentando 28 de ellos (65.2 por ciento) el perfil electroforético kgp-I y 15 aislados (34.8 por ciento) el perfil kgp-II. En los aislados provenientes de un individuo fue posible apreciar ambos genotipos descritos para el gen kgp. Los resultados indican un predominio del patrón electroforético A (rgpA) y que el genotipo kgp-I fue el más frecuentemente encontrado de los genotipos kgp.
Porphyromonas gingivalis is a microorganism strongly associated with the etiology of periodontitis. This periodontal bacterium possesses an array of virulence factors, among which gingipains have a key importance, being involved with extracellular matrix destruction of periodontal tissues, modulation of host immune response and stimulation in the production of pro-inflammatory cytokines by different types of cells. These proteinases have specific affinities, being Arg-gingipains (RgpA and RgpB, encoded by rgpA and rgpB genes, respectively) and Lys-gingipains (Kgp, encoded by the kgp gene). It has been described that there are polymorphisms in the genes encoding for gingipains. Therefore, the aim of the present study was to describe the frequency of rgpA and kgp genotypes in clinical isolates of P. gingivalis obtained from periodontitis patients. For determining the rgpA and kgp genotypes, we used PCR amplification and restriction analysis. From 47 isolates obtained from 4 individuals with chronic periodontitis and 2 subjects with aggressive periodontitis, 38 were typified for rgpA gene and all exhibited the electrophoretic pattern A (100 percent). For kgp gene, we characterized 43 isolates, 28 of them (65.2 percent) with the kgp-I electrophoretic profile and 15 isolates (34.8 percent) with the kgp-II profile. In the isolates belonging to one individual, we found both genotypes of kgp gene. The results indicate a clear predominance of the electrophoretic pattern A (for rgpA gene) and kgp-I genotype was the most frequently found of the kgp genotypes.
Assuntos
Humanos , Adesinas Bacterianas/genética , Cisteína Endopeptidases/genética , Porphyromonas gingivalis/isolamento & purificação , Porphyromonas gingivalis/genética , Amplificação de Genes , Genótipo , Reação em Cadeia da Polimerase , Periodontite/genética , Periodontite/microbiologiaRESUMO
The purpose of this study was to assess the prevalence of dental caries, tooth loss, and risk factors among adult population of Chile. Furthermore, age, gender, and behavioural specific differences in caries prevalence and tooth loss were examined. A national stratified multistage probabilistic sample design in two-age cohorts was applied to the Chilean population. A sample of 1553 adults, comprising 1088 individuals aged 35-44 and 465 senior individuals aged 65-74, were examined. The DMFT was evaluated following WHO recommendations using diagnostic criteria of caries lesions into dentin. The data were analyzed by univariate and multivariate models using logistic regression analyses. Results showed a mean DMFT of 15.06 in the 35-44-year-old group and of 21.57 in the 65-74 group. Factors related to tooth loss in the 35-44 group through univariate logistic regression were depression (OR 1.9 CI 95% 1.26-2.85), education level <12 years (OR 2.24 CI 95% 1.31-3.73), personal income (OR 1.51 CI 95% 1.04-2.19), and familiar income (OR 2.05 CI 95% 1.34-3.13), and through multivariate logistic regression in the same age group were depression (OR 1.93 CI 95% 1.24-3.0), education level <12 years (OR 1.94 CI 95% 1.2-3.14), and familiar income (OR 1.71 CI 95% 1.09-2.68). Factors related to tooth loss in the 65-74-year-old group through univariate logistic regression were education level <12 years (OR 2.54 CI 95% 1.3-4.96) and personal income (OR 1.66 CI 95% 1.05-2.63), and for multivariate logistic regression in the same age group, it was education level <12 years (OR 2.51 CI 95% 1.21-5.18). In conclusion, adult population in Chile showed a high prevalence of dental caries and tooth loss, as age, education level, personal and familiar incomes, and depression are being the main risk factors.
RESUMO
Periodontitis is an infection characterized by the occurrence of supporting tissue destruction with an episodic nature. Disease progression is often determined by the loss of attachment level or alveolar bone, and sequential probing of periodontal attachment remains the most commonly utilized method to diagnose progressive destruction of the periodontium. The tolerance method has been the most extensive clinical method used in recent years to determine site-specific attachment level changes. There is abundant evidence that major tissue destruction in periodontal lesions results from the recruitment of immune cells. Considerable effort has been made to study the host cell and mediator profiles involved in the pathogenesis of chronic periodontitis, but the definition of active sites, where current periodontal breakdown occurs, and consecutive characterization of the mediators involved are still among the main concerns. In the present review, we summarize periodontopathic bacteria and host factors, including infiltrating cell populations, cytokines, and host matrix metalloproteinases, associated with under-going episodic attachment loss that could partly explain the mechanisms involved in destruction of the supporting tissues of the tooth.
Assuntos
Perda do Osso Alveolar/imunologia , Periodontite Crônica/imunologia , Periodontite Crônica/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Perda do Osso Alveolar/microbiologia , Citocinas/metabolismo , Progressão da Doença , Fatores de Transcrição Forkhead/metabolismo , Humanos , Tolerância Imunológica , Metaloproteinases da Matriz/metabolismo , Ligante RANK/metabolismo , Explosão Respiratória , Linfócitos T/imunologiaRESUMO
BACKGROUND AND OBJECTIVE: Matrix metalloproteinase-8 (MMP-8) is a central mediator in chronic periodontitis. Recently developed MMP-8-deficient mice show an impaired polymorphonuclear neutrophil response and more severe alveolar bone loss in Porphyromonas gingivalis-induced experimental periodontitis. The main mediators involved in neutrophil and monocyte/macrophage recruitment and in bone loss include lipopolysaccharide-induced CXC chemokine (LIX/CXCL5), stromal-derived factor-1/CXC chemokine ligand 12 (SDF1/CXCL12) and RANKL. Therefore, the aim of this study was to characterize the expression of LIX/CXCL5, SDF1/CXCL12 and RANKL in Porphyromonas gingivalis-induced experimental periodontitis in MMP-8â»/â» (knockout) and wild-type mice. MATERIAL AND METHODS: MMP-8 null and WT P. gingivalis-infected and uninfected mice were included. Histopathological changes were assessed and LIX/CXCL5, SDF1/CXCL12 and RANKL were immunodetected and quantified. RESULTS: Typical histopathological features of chronic periodontitis were seen in P. gingivalis-infected groups. LIX/CXCL5 expression was restricted to the gingival papilla in all four groups. Significantly lower expression of LIX/CXCL5 was seen in the knockout group compared with the wild-type infected group (p < 0.05). SDF1/CXCL12 and RANKL expression was mainly localized to the alveolar crest, including inflammatory leukocytes, vascular endothelium, osteoblasts and osteoclasts. Significant increases of SDF1/CXCL12 and RANKL were seen in both knockout and wild-type P. gingivalis-infected groups compared with uninfected groups (p < 0.05). CONCLUSION: RANKL and SDF1/CXCL12 are up-regulated in P. gingivalis-induced periodontitis and they appear to be associated with the pathogenesis of the disease. MMP-8 is associated with a reduced expression of LIX/CXCL5 in the P. gingivalis-induced experimental periodontitis model.
Assuntos
Perda do Osso Alveolar/metabolismo , Quimiocina CXCL5/biossíntese , Periodontite Crônica/metabolismo , Metaloproteinase 8 da Matriz/metabolismo , Perda do Osso Alveolar/microbiologia , Animais , Quimiocina CXCL12/biossíntese , Quimiocina CXCL12/genética , Quimiocina CXCL5/genética , Periodontite Crônica/microbiologia , Regulação da Expressão Gênica , Lipopolissacarídeos/farmacologia , Masculino , Metaloproteinase 8 da Matriz/deficiência , Metaloproteinase 8 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Porphyromonas gingivalis , Ligante RANK/biossíntese , Ligante RANK/genéticaRESUMO
La enfermedad periodontal requiere de un hospedero susceptible para su desarrollo y progresión. Dentro de las características del hospedero se encuentra la respuesta T reguladora, que otorga tolerancia frente a antígenos propios, participa durante las enfermedades infecciosas limitando el daño tisular, sin disminuir la respuesta antibacteriana. El presente estudio tiene por objetivo determinar la presencia, reclutamiento y función de Tregs en pacientes con periodontitis crónica. En 10 biopsias de tejido periodontal sano y con periodontits crónica se realizó inmunohistoquímica para marcadores (CD4, CD25, Foxp3), quimioquinas (CCL17, CCL22) y citoquinas (TGF-B, IL-10) de Tregs. Además de Western-Blot para detectar las citoquinas. Los resultados obtenidos sugieren una posible asociación entre células Tregs y la infección periodontal, ya que se confirma su reclutamiento y presencia. Sin embargo, son necesarios más estudios del posible desbalance con su contraparte pro-inflamatoria Th17, que expliquen en parte la compleja etiopatogenia de la enfermedad periodontal.
Periodontal disease requires a susceptible host to initiation, development and progression. T regulatory response is one of these inmunoregulatory characteristics of the susceptible host, which provide tolerance, tissular protection during infection without impairing the control of periodontopathogens. The aim of this study is to determinate the presence, homing and function of T regulatory cells (Tregs) in patients with chronic periodontitis. Ten biopsies were taken from pockets, the presence of Tregs markers (CD4, CD25, Foxp3), chemokines (CCL17, CCL22) and cytokines (TGF-p, IL-10) were determinate by immunohistochemistry. Cytokines also were detected with Western-Blot. Our results suggest a possible association between Tregs and periodontal infection, confirming homing and presence of Tregs. However, further studies are required to determine the possible imbalance with pro-inflammatory part Th17, that might explain the complex etiopathogenesis of periodontal disease.
Assuntos
Humanos , Masculino , Feminino , Adulto , Linfócitos T Reguladores/imunologia , Periodontite Crônica/imunologia , Western Blotting , Quimiocinas , Citocinas , Fatores de Transcrição Forkhead , Imuno-HistoquímicaRESUMO
BACKGROUND: Matrix metalloproteinase (MMP)-8 is a central mediator in chronic periodontitis. MMP-8 can be activated by the cooperative action of other MMPs such as MMP-14, reactive oxygen species, and microbial proteases. The aim of this study is to associate the levels, molecular forms, isoenzyme distribution, and degree of activation of MMP-8 and -14, myeloperoxidase (MPO), and tissue inhibitor of MMP (TIMP)-1 in gingival crevicular fluid (GCF) from patients with progressive periodontitis at baseline and after periodontal therapy. METHODS: In this longitudinal study, GCF samples from active (n = 25) and inactive (n = 25) sites of subjects with periodontitis were screened at baseline for GCF levels of MMP-8 by immunofluorometric assay, of MMP-14 by specific activity assay, and of MPO and TIMP-1 by enzyme-linked immunosorbent assay. MMP-8 and MPO were also measured after periodontal treatment. Molecular forms were determined by immuno-Western blot analyses and subjected to densitometric scanning and statistical analyses. RESULTS: High MMP-8 and MPO levels and a strong MPO/MMP-8 positive correlation were found in active and inactive sites at baseline. After treatment, decreases in MPO and MMP-8 were seen, except for active sites in which MMP-8 differences were not significant (P <0.05). CONCLUSIONS: We present initial data that show that GCF levels and associations between MPO and MMP-8 are related to progression episodes and treatment responses in patients with chronic periodontitis. Our results suggest an interaction between the MPO oxidative pathway and MMP-8 activation, and this cascade might be useful as a potential biomarker for treatment outcomes.
Assuntos
Periodontite Crônica/enzimologia , Líquido do Sulco Gengival/enzimologia , Metaloproteinase 14 da Matriz/análise , Metaloproteinase 8 da Matriz/análise , Peroxidase/análise , Adulto , Perda do Osso Alveolar/enzimologia , Periodontite Crônica/terapia , Raspagem Dentária , Progressão da Doença , Ativação Enzimática , Feminino , Seguimentos , Humanos , Isoenzimas/análise , Estudos Longitudinais , Masculino , Mesoderma/enzimologia , Pessoa de Meia-Idade , Neutrófilos/enzimologia , Higiene Bucal , Perda da Inserção Periodontal/enzimologia , Bolsa Periodontal/enzimologia , Aplainamento Radicular , Inibidor Tecidual de Metaloproteinase-1/análiseRESUMO
AIM: To study the expression of monocyte chemotactic protein-3 (MCP-3, also known as chemokine CCL-7) in tissue from apical lesions (AL) and to associate MCP-3 expression with symptomatic or asymptomatic apical periodontitis. METHODOLOGY: To determine the expression of MCP-3 in AL, biopsies obtained during tooth extraction procedures were fixed, subjected to routine processing and diagnosed as apical granuloma (AG) (n = 7) or radicular cyst (RC) (n = 5). As controls, apical periodontal ligament (PDL) specimens from healthy premolars extracted for orthodontics reasons were included (n = 7). All specimens were immunostained for MCP-3 and examined under a light microscope. In addition, homogenates from AL (n = 14) and healthy PDL samples (n = 7) were studied through immunowestern blot. Finally, periapical exudates samples were collected from root canals of teeth having diagnosis of symptomatic (n = 14) and asymptomatic apical periodontitis (n = 14) during routine endodontic treatments and analysed by immunowestern blot and densitometry. RESULTS: MCP-3 was detected in AG and RC and localized mainly to inflammatory leucocytes, whereas no expression was observed in healthy PDLs. MCP-3 was also detected in periapical exudate, and its levels were significantly higher in symptomatic than in asymptomatic apical periodontitis. CONCLUSIONS: MCP-3 was expressed in AL and its levels associated with clinical symptoms. MCP-3 might play a role in disease pathogenesis, possibly by stimulating mononuclear chemotaxis.
Assuntos
Quimiocina CCL7/análise , Quimiotaxia de Leucócito/imunologia , Periodontite Periapical/imunologia , Adulto , Doenças Assintomáticas , Biópsia , Western Blotting , Cavidade Pulpar/imunologia , Células Endoteliais/imunologia , Endotélio Vascular/imunologia , Exsudatos e Transudatos/imunologia , Humanos , Linfócitos/imunologia , Granuloma Periapical/imunologia , Tecido Periapical/imunologia , Ligamento Periodontal/imunologia , Plasmócitos/imunologia , Cisto Radicular/imunologiaRESUMO
OBJECTIVE: Neutrophils play a crucial role in the defense of invading bacteria by releasing biologically active molecules. The response of peripheral blood neutrophils was studied in periodontitis-affected patients and in healthy controls towards stimulation to Porphyromonas gingivalis (Pg) and Actinobacillus actinomycetemcomitans (Aa) extracts. MATERIALS AND METHODS: Peripheral venous blood was drawn from 23 adult patients with moderate to advanced chronic periodontitis (probing depth >or=5 mm, attachment loss >or=3 mm), and 30 healthy volunteers. Neutrophil response followed by metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8) secretion was assayed by zymography and enzyme-linked immunosorbent assay, respectively, on both whole blood and purified neutrophils. In addition to periodontal pathogen extracts, known stimulating agents were tested, such as Escherichia coli-lipopolysaccharide (LPS), phytohemagglutinin, and zymosan A. RESULTS: Neutrophil response, expressed as a secretion ratio under stimulated and non-stimulated conditions, measured in whole blood, showed no differences between periodontitis and healthy controls. Instead, in purified neutrophils from patients, MMP-9 exhibited a significantly higher secretion ratio with LPS and Pg (1.5- to 2-fold), whereas IL-8 showed a larger increase in secretion ratio (3- to 7-fold) in the presence of Pg, Aa, LPS, and zymosan A. CONCLUSION: Peripheral neutrophils of periodontitis-affected patients are more reactive as suggested by their significantly higher response toward periodontal pathogen extracts and other stimulating agents.
Assuntos
Interleucina-8/análise , Metaloproteinase 9 da Matriz/análise , Neutrófilos/metabolismo , Periodontite/microbiologia , Adulto , Aggregatibacter actinomycetemcomitans , Estudos de Casos e Controles , Índice de Placa Dentária , Feminino , Humanos , Masculino , Índice Periodontal , Periodontite/sangue , Porphyromonas gingivalisRESUMO
OBJECTIVES: The cytokine receptor activator of nuclear factor kappaB-ligand (RANKL) has been involved in both the physiological and pathological regulation of osteoclast life span and bone metabolism. Periapical granuloma is a periradicular lesion characterized by periapical bone destruction. The aims of this study were to associate the RANKL mRNA levels to periapical granulomas using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) technique and to determine the specific cell involved in RANKL synthesis. METHODS: In eight periapical granuloma and eight periodontal ligament samples from periodontally healthy volunteers, RANKL mRNA was detected by real-time RT-PCR. Expression of RANKL on infiltrate leukocytes was further investigated by flow cytometry in six periapical granulomas. RESULTS: Receptor activator of nuclear factor kappaB-ligand mRNA levels were higher in periapical granulomas than in healthy periodontal ligament as its RANKL mRNA cycle threshold (Ct) and DeltaCt were significantly lower than that of controls (33.07 +/- 1.24 vs 36.96 +/- 1.69 and 11.58 +/- 3.02 vs 15.60 +/- 3.31, respectively). A 16.2-fold (2.0-131.6) higher RANKL gene expression was detected in the granulomas compared with the control tissues. We determined by flow cytometry that lymphocytes were the predominant leukocyte cells (53.31%), and monocytes and dendritic cells were the main RANKL synthesizers in granuloma lesions. CONCLUSIONS: These data indicate that monocytes synthesized RANKL in periapical granulomas and suggest that RANKL is involved in bone loss associated with periapical lesions.
Assuntos
Perda do Osso Alveolar/metabolismo , Proteínas de Transporte/biossíntese , Glicoproteínas de Membrana/biossíntese , Granuloma Periapical/metabolismo , Ligamento Periodontal/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Humanos , Análise dos Mínimos Quadrados , Masculino , Monócitos/metabolismo , Ligante RANK , RNA Mensageiro/análise , Receptor Ativador de Fator Nuclear kappa-B , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: The aim of this work was to improve the assessment of the periodontal disease status through measurements of extracellular matrix metalloproteinases (MMPs) and their tissular inhibitors (TIMPs) in the gingival crevicular fluid from patients diagnosed with chronic periodontitis. METHODS: Gingival crevicular fluid samples from patients (n = 13) were taken from 60 sites initially, and from 51 and 41 sites, respectively, 3 and 6 months after scaling and root planing. Gingival crevicular fluid samples were also taken from healthy subjects (n = 11, 24 sites). The presence of MMP-9 and MMP-8 was assessed by zymography and immunowestern blotting, respectively. The actual MMP activity (gelatinase and collagenase) was measured using the fluorogenic substrate assay. TIMP-1 and -2 levels were measured by immunodot blot. RESULTS: The fluorogenic substrate assay determinations showed higher MMP activity in sites with probing depth > or = 4 mm, with significant reduction post-treatment. Gelatinase activity followed by zymography consisted mainly of MMP-9. A different pattern of MMP-8 in control and patient sites was found. Controls only showed species of a partially active form (69 kDa), whereas patient sites showed a high frequency of the active form (56 kDa), and in some cases the latent form (85 kDa) was also observed. The active form reduced its frequency in sites with probing depth > or = 4 mm. TIMP-1 and -2 levels in patients were significantly lower than in controls, and after treatment the recovery of TIMP-1 level similar to control was observed. CONCLUSION: Significant correlations between the severity of the periodontal disease and the actual MMP activity, the active form of MMP-8 and the low level of both TIMP-1 and TIMP-2 were found.
Assuntos
Líquido do Sulco Gengival/metabolismo , Metaloproteinases da Matriz/metabolismo , Periodontite/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Adulto , Análise de Variância , Western Blotting/métodos , Feminino , Fluorometria/métodos , Líquido do Sulco Gengival/enzimologia , Humanos , Estudos Longitudinais , Masculino , Metaloproteinases da Matriz/análise , Neutrófilos/enzimologia , Periodontite/enzimologia , Periodontite/terapia , Estatísticas não Paramétricas , Inibidores Teciduais de Metaloproteinases/análiseRESUMO
BACKGROUND: Oral prevalence studies are important to know the state of health and the needs of treatment. Our aim was to determine the prevalence of oral mucosal lesions and associated factors among aging Chileans. METHODS: A random sample by age, gender, and socioeconomic status was obtained, comprising 889 individuals older than 65 years. Individuals were interviewed and examined in Santiago, the capital of Chile, according to the World Health Organization guidelines. RESULTS: The prevalence of one or more oral mucosal lesions in the sample was 53%. Logistic regression model revealed that denture use increased the probability of one or more oral mucosal lesions by threefold, while age, gender, smoking, medication use, xerostomia, and social or cultural factors had no effect. The most common lesion was denture stomatitis (22.3%), followed by irritative hyperplasia (9.4%), oral mucosal varicosities (9%), solitary pigmented lesions (4%), traumatic ulcer (3.5%), angular cheilitis (2.9%), multiple pigmented lesions (2.8%), hemangioma (2.3%), lichen planus (2.1%), leukoplakia (1.7%), recurrent aphthous stomatitis (1.4%), nicotine stomatitis (1.3%), median rhomboid glossitis (0.9%), actinic cheilitis (0.9%), pyogenic granuloma (0.7%), oral squamous papiloma (0.6%), and mucocele (0.2%). One case of oral cancer was observed. Different factors increased the probability of specific oral mucosal pathologies. CONCLUSIONS: We can conclude that oral mucosal lesions are common in elderly people in Santiago, suggesting the necessity for improved standards of prevention, and diagnostic and opportune treatment of these lesions.
Assuntos
Doenças da Boca/epidemiologia , Fatores Etários , Idoso , Queilite/epidemiologia , Chile/epidemiologia , Tratamento Farmacológico/estatística & dados numéricos , Feminino , Humanos , Hiperplasia , Modelos Logísticos , Masculino , Úlceras Orais/epidemiologia , Transtornos da Pigmentação/epidemiologia , Prevalência , Fatores Sexuais , Fumar/epidemiologia , Classe Social , Estomatite sob Prótese/epidemiologia , Varizes/epidemiologia , Xerostomia/epidemiologiaRESUMO
BACKGROUND, AIMS: Neutrophil cells constitute the first defense barrier against the oral bacterial challenge in the periodontium. Reduction of neutrophils could impair this response against periopathogenic bacteria such as Porphyromonas gingivalis. Our previous work implicates the apoptosis of neutrophils in the pathogenesis of periodontitis. We now demonstrate that granulocyte monocyte-colony stimulating factor (GM-CSF) present in the gingival crevicular fluid (GCF) and secreted during the immune response reduces the apoptosis of neutrophils. METHOD: In this study, the presence of GM-CSF and tumor necrosis factor-alpha (TNF-alpha) in GCF was determined in samples obtained from adult patients with periodontitis and from control subjects with clinically healthy gingiva. GCF was collected for 30 s using Periopaper(R) strips, and cytokines were quantified by ELISA. We used ex vivo culture of gingival tissue biopsies for 2 and 4 days in the presence of GM-CSF. Apoptosis was determined using the terminal TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique, and expression of Bax by immunohistochemistry. RESULTS: The presence of GM-CSF and TNF-alpha was detected in the majority of sites from periodontal patients (83.3% and 63.3%, respectively), presenting a total amount of 27.65 and 42.38 pg, respectively. GM-CSF reduces the neutrophil apoptosis determined by double staining with TUNEL and myeloperoxidase and by a reduction of Bax expression. CONCLUSIONS: These findings suggest a novel mechanism by which neutrophils specifically accumulate in adult patients with periodontitis.
Assuntos
Apoptose , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Neutrófilos/fisiologia , Periodontite/imunologia , Proteínas Proto-Oncogênicas c-bcl-2 , Fator de Necrose Tumoral alfa/fisiologia , Adulto , Estudos de Casos e Controles , Doença Crônica , Feminino , Expressão Gênica , Líquido do Sulco Gengival/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/análise , Humanos , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Masculino , Pessoa de Meia-Idade , Periodontite/fisiopatologia , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/biossíntese , Fator de Necrose Tumoral alfa/análise , Proteína X Associada a bcl-2RESUMO
BACKGROUND: Apoptosis is an evolutionary form of physiological cell death. Previous studies suggest that apoptosis is involved in the pathogenesis of periodontal diseases. Therefore, we studied the apoptotic events in the gingival tissue of chronic adult periodontitis patients. METHODS: Gingival tissue biopsies from 22 patients with chronic adult periodontitis and from 11 healthy controls were obtained. Criteria for patient inclusion in the periodontitis group were a minimum of 14 natural teeth, excluding third molars, with at least 10 posterior teeth; 5 to 6 sites with probing depth > or = 5 mm; attachment loss > or = 3 mm; and extensive radiographic bone loss. The control group included healthy subjects with no prior history of periodontal disease. Apoptosis was determined using the terminal TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique; electron microscopic analysis; and expression of Caspase-3, Fas, FasL, Bcl-2, and p53 by immunohistochemistry. RESULTS: TUNEL-positive cells and cells exhibiting chromatin condensation by electron microscopy were observed in the inflammatory infiltrate of biopsies obtained from periodontitis patients. Most of the TUNEL-positive cells belonged to neutrophil cell populations as they were stained with anti-myeloperoxidase. Positive staining for active-caspase 3, Fas, FasL, and p53 was only observed in the inflammatory infiltrate from periodontitis biopsies, whereas Bcl-2 cells were present in both periodontitis patients and healthy controls. CONCLUSIONS: Our findings establish that apoptosis is induced in the periodontal tissue by host and microbial factors and support the hypothesis that apoptotic mechanisms could be implicated in the inflammatory process associated with gingival tissue destruction observed in adult periodontitis patients.
Assuntos
Apoptose/fisiologia , Periodontite/patologia , Adulto , Perda do Osso Alveolar/patologia , Antígenos de Superfície/análise , Antígenos de Superfície/genética , Apoptose/genética , Biópsia , Caspase 3 , Caspases/análise , Caspases/genética , Doença Crônica , Dano ao DNA , Precursores Enzimáticos/análise , Precursores Enzimáticos/genética , Proteína Ligante Fas , Feminino , Gengiva/patologia , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Ligantes , Masculino , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Microscopia Eletrônica , Pessoa de Meia-Idade , Neutrófilos/patologia , Perda da Inserção Periodontal/patologia , Bolsa Periodontal/patologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína Supressora de Tumor p53/análise , Proteína Supressora de Tumor p53/genética , Receptor fas/análise , Receptor fas/genéticaRESUMO
A CPITN survey involving Chileans aged 35-44 and 65-74 was conducted. A random, stratified sample by age, gender, socio-economic status and educational level was obtained, comprising 1150 individuals. Prevalence of chronic inflammatory periodontal disease (Codes 3 + 4) was 90.89 per cent in subjects aged 35-44, and 100 per cent in subjects aged 65-74. The total prevalence for both age cohorts was 92.19 per cent. Prevalence of periodontal disease was slightly lower in females but severity was significantly higher in males. A significant association between socio-economic status and periodontal health was found. Prevalence (Code 3 + 4) was 56.44 per cent in subjects of high, 98 per cent in subjects of middle, and 100 per cent in subjects of low socio-economic status. Also, the mean number of sextants with pockets > 6 mm (1.12) and mean number of excluded sextants (1.43) were significantly higher in subjects of low socio-economic status. An association between educational level and periodontal health was apparent. The only subjects who were periodontally healthy were in the group with university education. Prevalence of CITN (Code 3 + 4) was also significantly lower in subjects with university education. There was also a significant association between educational level and loss of teeth. Concerning missing teeth, 22 per cent were lost due to periodontal disease and 77 per cent due to caries. The prevalence of periodontal disease found in this adult representative Chilean population indicates that the entire population needs oral hygiene instruction and scaling, and that 45.70 per cent need complex periodontal treatment.