Frequency of Porphyromonas gingivalis fimA in smokers and nonsmokers after periodontal therapy

Porphyromonas gingivalis is one of the most important Gram-negative anaerobe bacteria involved in the pathogenesis of periodontitis. P. gingivalis has an arsenal of specialized virulence factors that contribute to its pathogenicity. Among them, fimbriae play a role in the initial attachment and organization of biofilms. Different genotypes of fimA have been related to length of fimbriae and pathogenicity of the bacterium. Objectives: The aim of this study was to identify 5 types of fimA genotype strains in smokers and nonsmokers with periodontitis, before and after periodontal therapy. Material and Methods: Thirty-one patients with periodontitis harboring P. gingivalis were selected: 16 nonsmokers (NS) and 15 smokers (SM). Clinical and microbiological parameters were evaluated at baseline and 3 months after periodontal treatment, namely: plaque index, bleeding on probe, probing depth, gingival recession and clinical attachment level. The frequency of P. gingivalis and fimA genotype strains were determined by polymerase chain reaction. Results: Type I fimA was detected in the majority of SM and NS at baseline, and the frequency did not diminish after 3 months of treatment. The frequency of type II genotype was higher in SM than NS at baseline. After 3 months, statistical reduction was observed only for types II and V fimA genotypes in SM. The highest association was found between types I and II at baseline for NS (37.5%) and SM (53.3%). Conclusion: The most prevalent P. gingivalis fimA genotypes detected in periodontal and smoker patients were genotypes I and II. However, the presence of fimA genotype II was higher in SM. Periodontal treatment was effective in controlling periodontal disease and reducing type II and V P. gingivalis fimA

Aggregatibacter actinomycetemcomitans cytolethal distending toxin effect in osteoclast activity.

Aggregatibacter actinomycetemcomitans está associado à periodontite agressiva, caracterizada pela intensa reabsorção do osso alveolar. Esta espécie produz a toxina distensora citoletal (AaCDT) que possui atividade de DNAse, e promove o bloqueio das células alvo na fase G2 ou G1/ G2. Por outro lado, CDT ativa a cascata apoptótica pela atividade de PIP3, regulando a proliferação e sobrevivência de linfócitos, pelo bloqueio de Akt. Em monócitos, AaCDT induz aumento da produção de citocinas pró-inflamatórias e inibe a produção de óxido nítrico e fagocitose. Células precursoras de osteoclastos têm origem hematopoiética e sofrem diferenciação em osteoclastos, mediada pelo RANKL, mas outros fatores co-estimulatórios estão envolvidos. A AaCDT induz a produção de RANKL por fibroblastos. Assim, formulamos a hipótese se CDT influenciaria a homeostase óssea por afetar a diferenciação de células precursoras de osteoclastos. O estudo visou determinar o efeito de AaCDT sobre a sobrevivência, diferenciação e atividade em RAW264.7 e BMC. Os dados sugerem que a CDT interfere na homeostase óssea, favorecendo a indução da diferenciação de células precursoras de osteoclastos e alterando o perfil de citocinas produzidas.Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis, characterized by severe alveolar bone resorption. This species produces a distending toxin cytolethal (AaCDT) which has DNase activity, and promotes the blocking of target cells in G2 or G1 / G2 phase. On the other hand, CDT activates the apoptotic cascade by PIP3 activity, regulating lymphocyte proliferation and survival by blocking Akt. In monocytes, AaCDT enhances the production of proinflammatory cytokines and inhibits nitric oxide production and phagocytosis. Osteoclast precursor cells are of hematopoietic origin and must undergo differentiation into osteoclasts mediated by RANKL although other co-stimulatory factors are involved. AaCDT induces the production of RANKL by fibroblasts. Thus, CDT is hypothesized to influence bone homeostasis by affecting the differentiation of precursor cells into osteoclasts. This study aimed to determine the effect of AaCDT on survival, differentiation and activity of osteoclasts precursor cells. The data suggested that CDT interfere in bone homeostasis, favoring the differentiation of osteoclasts precursors cells and by altering their cytokines profile

Alteration of Homeostasis in Pre-osteoclasts Induced by Aggregatibacter actinomycetemcomitans CDT

The dysbiotic microbiota associated with aggressive periodontitis includes Aggregatibacter actinomycetemcomitans, the only oral species known to produce a cytolethal distending toxin (AaCDT). Given that CDT alters the cytokine profile in monocytic cells, we aimed to test the hypothesis that CDT plays a role in bone homeostasis by affecting the differentiation of precursor cells into osteoclasts. Recombinant AaCDT was added to murine bone marrow monocytes (BMMC) in the presence or absence of RANKL and the cell viability and cytokine profile of osteoclast precursor cells were determined. Multinucleated TRAP+ cell numbers, and relative transcription of genes related to osteoclastogenesis were also evaluated. The addition of AaCDT did not lead to loss in cell viability but promoted an increase in the average number of TRAP+ cells with 1-2 nuclei in the absence or presence of RANKL (Tukey, p < 0.05). This increase was also observed for TRAP+ cells with ≥ 3nuclei, although this difference was not significant. Levels of TGF-β, TNF-α and IL-6, in the supernatant fraction of cells, were higher when in AaCDT exposed cells, whereas levels of IL-1β and IL-10 were lower than controls under the same conditions. After interaction with AaCDT, transcription of the rank (encoding the receptor RANK), nfatc1 (transcription factor) and ctpK (encoding cathepsin K) genes was downregulated in pre-osteoclastic cells. The data indicated that despite the presence of RANKL and M-CSF, AaCDT may inhibit osteoclast differentiation by altering cytokine profiles and repressing transcription of genes involved in osteoclastogenesis. Therefore, the CDT may impair host defense mechanisms in periodontitis

The cytolethal distending toxin of Aggregatibacter actinomycetemcomitans inhibits macrophage phagocytosis and subverts cytokine production

Aggregatibacter actinomycetemcomitans is an important periodontal pathogen that can participate in periodontitis and other non-oral infections. The cytolethal distending toxin (Cdt) is among the virulence factors produced by this bacterium. The Cdt is also secreted by several mucosa-associated Gram-negative pathogens and may play a role in perpetuating the infection by modulating the immune response. Although the toxin targets a wide range of eukaryotic cell types little is known about its activity on macrophages which play a key part in alerting the rest of the immune system to the presence of pathogens and their virulence factors. In view of this, we tested the hypothesis that the A. actinomycetemcomitans Cdt (AaCdt) disrupts macrophage function by inhibiting phagocytic activity as well as affecting the production of cytokines. Murine macrophages were co-cultured with either wild-type A. actinomycetemcomitans or a Cdt(-) mutant. Viable counts and qPCR showed that phagocytosis of the wild-type strain was significantly reduced relative to that of the Cdt(-) mutant. Addition of recombinant Aa(r)Cdt to co-cultures along with the Cdt(-) mutant diminished the phagocytic activity similar to that observed with the wild type strain. High concentrations of Aa(r)Cdt resulted in decreased phagocytosis of fluorescent bioparticles. Nitric oxide production was modulated by the presence of Cdt and the levels of IL-1β, IL-12 and IL-10 were increased. Production of TNF-α did not differ in the co-culture assays but was increased by the presence of Aa(r)Cdt. These data suggest that the Cdt may modulate macrophage function in A. actinomycetemcomitans infected sites by impairing phagocytosis and modifying the pro-inflammatory/anti-inflammatory cytokine balance.FAPESP, 09/54178-0FAPESP, 09/54849-1NIDCR, R01 DE12212USPHS, DE012593National Institutes of Healt

RESEARCH ARTICLE Brazilian Red Propolis Attenuates Inflammatory Signaling Cascade in LPS- Activated Macrophages

Although previous studies suggested an anti-inflammatory property of Brazilian red propolis (BRP), the mechanisms involved in the anti-inflammatory effects of BRP and its activity on macrophages were still not elucidated. This study aimed to evaluate whether BRP attenu-ates the inflammatory effect of LPS on macrophages and to investigate its underlying mech-anisms. BRP was added to RAW 264.7 murine macrophages after activation with LPS. NO production, cell viability, cytokines profile were evaluated. Activation of inflammatory signal-ing pathways and macrophage polarization were determined by RT-qPCR andWestern blot. BRP at 50 μg/ml inhibited NO production by 78 % without affecting cell viability. Cd80 and Cd86 were upregulated whereasmrc1 was down regulated by BRP indicating macro-phage polarization at M1. BRP attenuated the production of pro-inflammatory mediators IL-12, GM-CSF, IFN-γ, IL-1β in cell supernatants although levels of TNF- α and IL-6 were slightly increased after BRP treatment. Levels of IL-4, IL-10 and TGF-β were also reduced by BRP. BRP significantly reduced the up-regulation promoted by LPS of transcription of genes in inflammatory signaling (Pdk1, Pak1, Nfkb1,Mtcp1,Gsk3b, Fos and Elk1) and of Il1β and Il1f9 (fold-change rate&gt; 5), which were further confirmed by the inhibition of NF-κB and MAPK signaling pathways. Furthermore, the upstream adaptor MyD88 adaptor-like (Mal), also known as TIRAP, involved in TLR2 and TLR4 signaling, was down- regulated in BRP treated LPS-activated macrophages. Given that BRP inhibited multiple signaling path-ways in macrophages involved in the inflammatory process activated by LPS, our data indi-cated that BRP is a noteworthy food-source for the discovery of new bioactive compounds and a potential candidate to attenuate exhacerbated inflammatory diseases

Bacteremia after chewing in a patient with severe chronic periodontitis and diabetes mellitus type 2: A brief report

This study evaluated systemic bacteremia caused by chewing in a 42-year-old male with chronic severe periodontitis and uncontrolled type 2 diabetes mellitus. Aerobic and anaerobic Gram-positive bacilli were detected by culture, and Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis by real time polymerase chain reaction in blood samples. Keywords: Bacteremia, Periodontitis, Diabetes type

Anti-Inflammatory Effects of (3<i>S</i>)-Vestitol on Peritoneal Macrophages

The isoflavone (3S)-vestitol, obtained from red propolis, has exhibited anti-inflammatory, antimicrobial, and anti-caries activity; however, few manuscripts deal with its anti-inflammatory mechanisms in macrophages. The objective is to elucidate the anti-inflammatory mechanisms of (3S)-vestitol on those cells. Peritoneal macrophages of C57BL6 mice, stimulated with lipopolysaccharide, were treated with 0.37 to 0.59 µM of (3S)-vestitol for 48 h. Then, nitric oxide (NO) quantities, macrophages viability, the release of 20 cytokines and the transcription of several genes related to cytokine production and inflammatory response were evaluated. The Tukey–Kramer variance analysis test statistically analyzed the data. (3S)-vestitol 0.55 µM (V55) lowered NO release by 60% without altering cell viability and diminished IL-1β, IL-1α, G-CSF, IL-10 and GM-CSF levels. V55 reduced expression of Icam-1, Wnt5a and Mmp7 (associated to inflammation and tissue destruction in periodontitis) and Scd1, Scd2, Egf1 (correlated to atherosclerosis). V55 increased expression of Socs3 and Dab2 genes (inhibitors of cytokine signaling and NF-κB pathway), Apoe (associated to atherosclerosis control), Igf1 (encoder a protein with analogous effects to insulin) and Fgf10 (fibroblasts growth factor). (3S)-vestitol anti-inflammatory mechanisms involve cytokines and NF-κB pathway inhibition. Moreover, (3S)-vestitol may be a candidate for future in vivo investigations about the treatment/prevention of persistent inflammatory diseases such as atherosclerosis and periodontitis

Serum leveis of inflammatory markers in type 2 diabetes patients with chronic periodontitis

Diabetes has been associated with periodontitis, but the mechanisms through which periodontal diseases affect the metabolic control remain unclear.Objective:This study aimed to evaluate serum leveis of inflammatory markers, IL-8, IL-6 and monocyte chemoattractant protein 1 (MCP-1), in type 2 diabetic patients in the presence of chronic periodontitis.Material and Methods:Forty two individuals were enrolled in this study and assigned to one of five groups: diabetes mellitus with inadequate glycemic control and periodontitis (DMI+P, n = 10), diabetes mellitus with adequate glycemic control and periodontitis (DMA+P, n = 10), diabetes mellitus without periodontitis (DM, n = 10), periodontitis without diabetes (P, n=6), and neither diabetes nor periodontitis (H, n = 6). Periodontal clinical examination included visible plaque index (PL), gingival bleeding index (GB), probing depth (PD), attachment level (AL) and bleeding on probing (BP). Glycemic control was evaluated by serum concentration of glycated hemoglobin (HbAlc). Inflammatory serum markers IL-8, IL-6 and (MCP-1) were measured by ELISA. Results: DMI+P and DMA+P groups presented higher PD (p=0.025) and AL (p=0.003) values when compared to the P group. There were no significant differences among groups for IL-6, IL-8 and MCP-1 serum levels. Conclusions:Although periodontitis was more severe in diabetic patients, the serum levels of the investigated inflammatory markers did not differ among the groups.University of São Paulo Institute of Biomedical Sciences Department of MicrobiologyUniversity of São Paulo School of Dentistry Department of StomatologyFederal University of São Paulo School of Medicine Department of EndocrinologyUNIFESP, EPM, Department of EndocrinologySciEL

Oral and fecal microbiome in molar-Incisor pattern periodontitis

In order to improve our understanding on the microbial complexity associated with Grade C/molar-incisor pattern periodontitis (GC/MIP), we surveyed the oral and fecal microbiomes of GC/MIP and compared to non-affected individuals (Control). Seven Afro-descendants with GC/MIP and seven age/race/gender-matched controls were evaluated. Biofilms from supra/subgingival sites (OB) and feces were collected and submitted to 16S rRNA sequencing. Aggregatibacter actinomycetemcomitans (Aa) JP2 clone genotyping and salivary nitrite levels were determined. Supragingival biofilm of GC/MIP presented greater abundance of opportunistic bacteria. Selenomonas was increased in subgingival healthy sites of GC/MIP compared to Control. Synergistetes and Spirochaetae were more abundant whereas Actinobacteria was reduced in OB of GC/MIP compared to controls. Aa abundance was 50 times higher in periodontal sites with PD≥ 4 mm of GC/MIP than in controls. GC/MIP oral microbiome was characterized by a reduction in commensals such as Kingella, Granulicatella, Haemophilus, Bergeyella, and Streptococcus and enrichment in periodontopathogens, especially Aa and sulfate reducing Deltaproteobacteria. The oral microbiome of the Aa JP2-like+ patient was phylogenetically distant from other GC/MIP individuals. GC/MIP presented a higher abundance of sulfidogenic bacteria in the feces, such as Desulfovibrio fairfieldensis, Erysipelothrix tonsillarum, and Peptostreptococcus anaerobius than controls. These preliminary data show that the dysbiosis of the microbiome in Afro-descendants with GC/MIP was not restricted to affected sites, but was also observed in supragingival and subgingival healthy sites, as well as in the feces. The understanding on differences of the microbiome between healthy and GC/MIP patients will help in developing strategies to improve and monitor periodontal treatment