Actinobacillus actinomycetemcomitans lipopolysaccharide induces interleukin-6 expression through multiple mitogen-activated protein kinase pathways in periodontal ligament fibroblasts

Actinobacillus actinomycetemcomitans plays a major role in the pathogenesis of aggressive periodontitis. Lipopolysaccharide (LPS) derived from A. actinomycetemcomitans is a key factor in inflammatory cytokine generation within periodontal tissues. In this study, we identify major mitogen-activated protein kinase (MAPK) signaling pathways induced by A. actinomycetemcomitans LPS, Escherichia coli LPS and interleukin-1 beta (IL-1 beta) in a murine periodontal ligament (mPDL) fibroblast cell line. Immunoblot analysis was used to assess the phosphorylated forms of p38, extracellular-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) MAPK following stimulation with A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta. IL-6 mRNA induction was detected via reverse transcription-polymerase chain reaction, while protein levels were quantified via enzyme-linked immunosorbent assays (ELISA). We utilized biochemical inhibitors of p38, ERK and JNK MAPK to identify the MAPK signaling pathways needed for IL-6 expression. Additional use of stable mPDL cell lines containing dominant negative mutant constructs of MAPK kinase-3 and -6 (MKK-3/6) and p38 null mutant mouse embryonic fibroblast (MEF) cells were used to substantiate the biochemical inhibitor data. Blocking p38 MAPK with SB203580 reduced the induction of IL-6 mRNA by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta by > 70%, > 95% and similar to 60%, respectively. IL-6 ELISA indicated that blocking p38 MAPK reduced the IL-6 protein levels induced by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta by similar to 60%, similar to 50% and similar to 70%, respectively. All MAPK inhibitors significantly reduced the IL-6 protein levels induced by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta whereas only p38 inhibitors consistently reduced the A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta induction of IL-6 mRNA steady-state levels. The contribution of p38 MAPK LPS-induced IL-6 expression was confirmed using MKK-3/6 dominant negative stable mPDL cell lines. Wild-type and p38 alpha(-/-) MEF cells provided additional evidence to support the role of p38 alpha MAPK in A. actinomycetemcomitans LPS-stimulated IL-6. Our results indicate that induction of IL-6 by E. coli LPS, IL-1 beta and A. actinomycetemcomitans LPS requires signaling through MKK-3-p38 alpha ERK, JNK and p38 MAPK in mPDL cells.

Leukotoxic activity of Actinobacillus actinomycetemcomitans isolated from Brazilian periodontal patients

The leukotoxic activity of 31 Actinobacillus actinomycetemcomitans isolates from Brazilian periodontal patients [nine from Localized Juvenile Periodontitis (LJP) patients, 22 from patients with AIDS-associated Necrotizing Ulcerative Periodontitis (AIDS/NUP)], and from the reference strain A. actinomycetemcomitans ATCC43718, were analyses for their cytotoxicity on human monocytes. A cytotoxicity inhibitory assay of the isolate P35 and the reference strain ATCC 43718 with sera from ten LJP patients and ten healthy subjects was also performed and leukotoxin reactivity was evaluated with serum from rabbits immune to leukotoxin from A. actinomycetemcomitans ATCC 43718. The cytotoxicity results were not statistically different among groups of A. actinomycetemcomitans isolates from LJP and AIDS/NUP patients, but the individual analysis of each isolate showed two isolates (P24 and P35) from LJP patients with high leukotoxic activity (P<0.05). Also, a high leucotoxic inhibitory effect with LJP patients' sera compared with healthy subjects with sonic extract from isolate P35 (P<0.05) and the reactivity of rabbit antiserum to leukotoxin were observed. Both leukotoxic and non-leukotoxic activity is more frequent in PJL than AIDS/NUP patients. Even though A. actinomycetemcomitans exhibits leukotoxic activity, there is an immune response to the leucotoxin in LJP patients. (C) 2000 Academic Press.

Actinobacillus actinomycetemcomitans lipopolysaccharide-mediated experimental bone loss model for aggressive periodontitis

Background: Bacterial constituents, such as Gram-negative derived lipopolysaccharide (LPS), can initiate inflammatory bone loss through induction of host-derived inflammatory cytokines. The aim of this study was to establish a model of aggressive inflammatory alveolar bone loss in rats using LPS derived from the periodontal pathogen Actinobacillus actinomycetemcomitans.Methods: Eighteen female Sprague-Dawley rats were divided into LPS test (N = 12) and saline control (N = 6) groups. All artimals received injections to the palatal molar gingiva three times per week for 8 weeks. At 8 weeks, linear and volumetric alveolar bone loss was measured by micro-computed tomography (mu CT). The prevalence of inflammatory infiltrate, proinflammatory cytokines, and osteoclasts was assessed from hematoxylin and eosin, immunohistochemical, or tartrate-resistant acid phosphatase (TRAP)-stained sections. Statistical analysis was performed.Results: A. actinomycetemcomitans LPS induced severe bone loss over 8 weeks, whereas control groups were unchanged. Linear and volumetric analysis of maxillae by mu CT indicated significant loss of bone with LPS, administration. Histologic examination revealed increased inflammatory infiltrate, significantly increased immunostaining for interleukin IL-6 and -1 beta and tumor necrosis factor-alpha, and more TRAP-positive osteoclasts in the LPS group compared to controls.Conclusion: Oral injections of LPS derived from the periodontal pathogen A. actinomycetemcomitans can induce severe alveolar bone loss and proinflammatory cytokine production in rats by 8 weeks.

Occurrence of Actinobacillus actinomycetemcomitans in patients with chronic periodontitis, aggressive periodontitis, healthy subjects and children with gingivitis in two cities of the state of São Paulo, Brazil

The aim of this study was to determine the frequency of isolation of Actinobacillus actinomycetemcomitans (Aa) in 100 patients with chronic periodontitis, 14 patients with aggressive periodontitis, 142 pre-school children with gingivitis and 134 periodontally healthy subjects. Samples of subgingival plaque were taken using sterilized paper points introduced into periodontal pockets or gingival crevice for 60 seconds and inoculated on TSBV agar, which was incubated under anaerobiosis at 37°C, for 4 days. Microbial identification was performed through biochemical methods and morphocellular and morphocolonial analysis. Aa was detected in 40.3% of healthy subjects, 68% of patients with chronic periodontitis, 92.86% of patients with aggressive periodontitis and 40.14% of children with gingivitis. The rate of recovery of Aa in the tested human groups proved to be higher than previously reported and in agreement with participation of this facultative anaerobe as a member of native microbiota of the periodontium and its relation with aggressive and chronic periodontitis in Brazil.

Transmission of Aggregatibacter actinomycetemcomitans between Brazilian women with severe chronic periodontitis and their children

This study evaluated the transmission of Aggregatibacter actinomycetemcomitans (Aa) in women with severe chronic periodontitis and their children. Thirty women (mean age = 36.1±6.0 years) who were mothers of at least one child aged 7 to 16 years were enrolled. In order to investigate mother-child transmission of Aa, the children were also evaluated when their mothers were colonized by the bacterium. Subgingival plaque samples of each woman were collected from 3 sites (mean probing depth of 7.3±1.2 mm and mean clinical attachment level of 7.9±1.5 mm) and pooled in reduced transport fluid (RTF). These samples were processed, inoculated onto TSBVagar selective medium and incubated at 37°C in microaerophilic atmosphere for 5 days. Aa was identified on the basis of colony morphology, Gram staining, catalase and oxidase reactions. Aa was found in 8 out of 30 women. Therefore, 8 children from these women (mean age= 12 ± 3.7 years) were evaluated, but Aa was found only in 2 of them. Aa strains of the two mother-child pairs were evaluated by arbitrarily-primed polymerase chain reaction (AP-PCR), although it was not found similarity between the amplitypes of each pair. No Aa transmission was found between Brazilian women with severe chronic periodontitis and their children.

Local application of tetracycline solution with a microbrush: An alternative treatment for persistent periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

O tabagismo como fator de risco para as doenças periodontais: aspectos microbiológicos

O fumo é considerado importante fator predisponente para muitas doenças, incluindo-se as periodontopatias. Desde que as doenças periodontais representam a inter-relação entre os fatores de virulência da microbiota subgengival sobre um hospedeiro susceptível, foi objetivo avaliar a freqüência de isolamento de três periodontopatógenos em indivíduos sadios e pacientes com doença periodontal, fumantes ou não, com níveis variados de higiene bucal; verificar a relação entre o número de microrganismos produtores de sulfeto de hidrogênio na placa subgengival de fumantes e não fumantes e sua condição clínica. Foram examinados 189 pacientes e indivíduos sadios, dos quais 60 foram selecionados para análise microbiológica. O índice de placa foi registrado de acordo com o índice de O'Leary e os espécimes de placa subgengival coletados e processados de acordo com SLOTS35 (1982). A identificação dos isolados foi obtida pelas suas características morfocelulares, morfocoloniais e bioquímico-fisiológicas. Verificou-se que a freqüência de isolamento dos bastonetes anaeróbios produtores de pigmento negro, Fusobacterium nucleatum e bactérias produtoras de sulfeto de hidrogênio foi similar entre fumantes e não fumantes, sendo mais elevada nos pacientes com doença periodontal. Já Actinobacillus actinomycetemcomitans foi isolado mais freqüentemente em sadios fumantes do que sadios não fumantes.

The Effect of a Supragingival Plaque-Control Regimen on the Subgingival Microbiota in Smokers and Never-Smokers: Evaluation by Real-Time Polymerase Chain Reaction

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Periodontal status in smokers and never-smokers: Clinical findings and real-time polymerase chain reaction quantification of putative periodontal pathogens

Background: Smoking is a well-known risk factor for destructive periodontal disease, but its relationship with periodontal status and subgingival microbiota remains unclear. Inherent limitations of microbiological methods previously used may partly explain these mixed results, and real-time polymerase chain reaction (PCR) has been presented as a valid alternative. The aim of the present study was to investigate the clinical condition and microbiological profile of patients with chronic periodontitis as related to the habit of smoking.Methods: Fifty patients (33 to 59 years old), 25 smokers and 25 never-smokers, constituted the sample. The visible plaque index (VPI), gingival bleeding index (GBI), bleeding on probing (BOP), periodontal probing depth (PD), clinical attachment loss (CAL), and gingival crevicular fluid (GCF) volume were recorded. Real-time PCR quantified Porphyromonas gingivalis, Micromonas micros, Dialister pneumosintes, Actinobacillus actinomycetemcomitans and total bacteria in subgingival samples.Results: Smokers and never-smokers showed similar values for VPI, GBI, and BOP. Smokers had deeper PD in buccal/lingual sites and higher CAL independently of the tooth surface. The GCF volume was smaller in smokers, independent of the PD. Similar amounts of total bacteria and P. gingivalis were observed for both groups. Significantly higher numbers of D. pneumosintes and M. micros were present in smokers and associated with moderate and deep pockets. When heavy smokers were considered, higher counts of total bacteria, M. micros, and D. pneumosintes were observed.Conclusions: Smoking seems to have a detrimental impact on the periodontal status and microbiological profile of patients with periodontitis. Compared to never-smokers, smokers had deeper pockets, greater periodontal destruction, and higher counts of some putative periodontal pathogens.

Transcriptional activation of MMP-13 by periodontal pathogenic LPS requires p38 MAP kinase

Matrix metal loprotease-13 (MMP-13) is induced by pro-inflammatory cytokines and increased expression is associated with a number of pathological conditions such as tumor metastasis, osteoarthritis, rheumatoid arthritis and periodontal diseases. MMP-13 gene regulation and the signal transduction pathways activated in response to bacterial LPS are largely unknown. In these studies, the role of the mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-13 induced by lipopolysaccharide was investigated. Lipopolysaccharide from Escherichia coli and Actinobacillus actinomycetemcomitans significantly (P < 0.05) increased MMP-13 steady-state mRNA (average of 27% and 46% increase, respectively) in murine periodontal ligament fibroblasts. MMP-13 mRNA induction was significantly reduced by inhibition of p38 MAP kinase. Immunoblot analysis indicated that p38 signaling was required for LPS-induced MMP-13 expression. Lipopolysaccharide induced proximal promoter reporter (-660/+32 mMMP-13) gene activity required p38 signaling. Collectively, these results indicate that lipopolysaccharide-induced murine MMP-13 is regulated by p38 signaling through a transcriptional mechanism.

Microbiologic and radiographic analysis of ligature-induced peri-implantitis with different dental implant surfaces

Purpose: The goal of this study was to evaluate microbiota and radiographic peri-implant bone loss associated with ligature-induced peri-implantitis. Materials and Methods: Thirty-six dental implants with 4 different surfaces (9 commercially pure titanium, 9 titanium plasma-sprayed, 9 hydroxyapatite, and 9 acid-etched) were placed in the edentulous mandibles of 6 dogs. After 3 months with optimal plaque control, abutment connection was performed. On days 0, 20, 40, and 60 after placement of cotton ligatures, both microbiologic samples and periapical radiographs were obtained. The presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia/nigrescens, Campylobacter spp, Capnocytophaga spp, Fusobacterium spp, beta-hemolytic Streptococcus, and Candida spp were evaluated culturally. Results: P intermedia/nigrescens was detected in 13.89% of implants at baseline and 100% of implants at other periods. P gingivalis was not detected at baseline, but after 20 and 40 days it was detected in 33.34% of implants and at 60 days it was detected in 29.03% of dental implants. Fusobacterium spp was detected in all periods. Streptococci were detected in 16.67% of implants at baseline and in 83.34%, 72.22%, and 77.42% of implants at 20, 40, and 60 days, respectively. Campylobacter spp and Candida spp were detected in low proportions. The total viable count analysis showed no significant differences among surfaces (P = .831), although a significant difference was observed after ligature placement (P < .0014). However, there was no significant qualitative difference, in spite of the difference among the periods. The peri-implant bone loss was not significantly different between all the dental implant surfaces (P = .908). Discussion and Conclusions: These data suggest that with ligature-induced peri-implantitis, both time and periodontal pathogens affect all surfaces equally after 60 days.

Estudo das condições de saúde bucal e fatores sócioeconômico-culturais, comportamentais e microbiológicos de pacientes autistas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo longitudinal da terapia antibiótica local de bolsas periodontais residuais. Análise clínica e microbiológica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)