Flávia Sammartino Mariano

The effects of nicotine and cotinine on Porphyromonas gingivalis colonisation of epithelial cells.

Smoking is a risk factor for development of periodontitis. Porphyromonas gingivalis is an important colonizer of the subgingival crevice and is a major pathogenic agent in the initiation and progression of severe forms of periodontal disease. However, the effect of major cigarettes derivatives on P. gingivalis is poorly understood. The purpose of this study was to determine the influence of nicotine and cotinine on bacterial colonisation to epithelial cells. KB cells monolayers and P. gingivalis ATCC 33277 were exposed to 0.1, 10 and 100 microg/mL of nicotine and cotinine concentrations. The epithelial cells were incubated for 24 h while P. gingivalis was exposed to these substances until reach early logarithmic phase. After the incubation period, P. gingivalis ability to colonize KB cells was assayed. The number of cell-associated/invasive bacteria was assessed by counting the colony-forming units. 100 microg/mL cotinine significantly increased P. gingivalis association and invasion of epithelial cells, when the bacteria was exposed to this substance (p<0.05; ANOVA-Tukey test). No other condition or drug altered the bacteria colonisation ability (p>0.05). These data indicated that cotinine may interfere with P. gingivalis ability to associate and invade the epithelial cells. Further studies are needed to investigate whether oral cells might be more susceptible to be colonized by P. gingivalis in smokers.

Adhesion and invasion of Candida albicans from periodontal pockets of patients with chronic periodontitis and diabetes to gingival human fibroblasts

The objectives of this study were to evaluate clinical isolates of Candida albicans, particularly their adhesion to and invasion of gingival human fibroblasts in culture and to measure nitric oxide concentration (NO) produced by fibroblasts in the presence of these yeasts. Sixteen strains of C. albicans isolated from patients with chronic periodontitis and diabetes mellitus type II were divided on the basis of phenotypic tests into two groups, i.e., highly or weakly hydrophobic. Primary cultures of human fibroblasts were isolated from gingival biopsies and after subsequent subcultures, the cells were seeded into culture plates and incubated for 24 h. C. albicans strains were inoculated into these plates and maintained for 2 and 4 h to assess their adhesion and invasion, respectively. The number of adherent or invasive yeasts was evaluated by assessing colony-forming units (CFU). The production of NO by fibroblasts was also quantified. The results showed that strains with high hydrophobicity had a greater ability to adhere and invade fibroblasts (p < 0.05, ANOVA and Tukey). The production of NO was higher for the most hydrophobic strains, but did not reach statistical difference with the weakly hydrophobic isolates. These data indicated that the hydrophobicity may play a role in the adhesion and invasion of C. albicans in fibroblast cultures.

Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects

Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.

CovR Regulates Streptococcus mutans Susceptibility To Complement Immunity and Survival in Blood

ABSTRACT Streptococcus mutans, a major pathogen of dental caries, may promote systemic infections after accessing the bloodstream from oral niches. In this study, we investigate pathways of complement immunity against S. mutans and show that the orphan regulator CovR (CovR Sm ) modulates susceptibility to complement opsonization and survival in blood. S. mutans blood isolates showed reduced susceptibility to C3b deposition compared to oral isolates. Reduced expression of covRSm in blood strains was associated with increased transcription of CovR Sm -repressed genes required for S. mutans interactions with glucans (gbpC, gbpB, and epsC), sucrose-derived exopolysaccharides (EPS). Consistently, blood strains showed an increased capacity to bind glucan in vitro. Deletion of covRSm in strain UA159 (UAcov) impaired C3b deposition and binding to serum IgG and C-reactive protein (CRP) as well as phagocytosis through C3b/iC3b receptors and killing by neutrophils. Opposite effects were observed in mutants of gbpC, epsC, or gtfBCD (required for glucan synthesis). C3b deposition on UA159 was abolished in C1q-depleted serum, implying that the classical pathway is essential for complement activation on S. mutans. Growth in sucrose-containing medium impaired the binding of C3b and IgG to UA159, UAcov, and blood isolates but had absent or reduced effects on C3b deposition in gtfBCD, gbpC, and epsC mutants. UAcov further showed increased ex vivo survival in human blood in an EPS-dependent way. Consistently, reduced survival was observed for the gbpC and epsC mutants. Finally, UAcov showed an increased ability to cause bacteremia in a rat model. These results reveal that CovR Sm modulates systemic virulence by regulating functions affecting S. mutans susceptibility to complement opsonization.

Influence of VicRK and CovR on the interactions of Streptococcus mutans with phagocytes

OBJECTIVE Streptococcus mutans are members of the oral microbiota that are implicated in dental caries and infective endocarditis. To adapt to environmental stresses encountered during host colonization, these bacteria employ two-component regulatory systems, which modulate global changes in gene expression. These include the systems VicRK and CovR. In this study, we investigate the influence of VicRK and CovR in S. mutans interactions with mononuclear and polymorphonuclear (PMN) phagocytes. METHODS Patterns of S. mutans uptake by murine macrophages were determined in strains, which differ in the production of proteins regulated by VicRK and CovR. Bacterial uptake by murine macrophages and by PMN in human blood was analyzed in vicK and covR knockout mutants obtained in strains UA159 and LT11. RESULTS Inactivation of covR did not affect uptake by macrophages, while vicK inactivation transiently reduced uptake only in LT11 (P < 0.05). In the two strains, inactivation of vicK and covR impaired uptake by PMN for a period of 1 h or more (P < 0.01-0.05). Mutant complementation with vicK or covR restored the PMN uptake phenotypes. CONCLUSION This study indicates that VicRK and CovR regulate functions that influence bacterial susceptibility to phagocytosis, suggesting a novel role for these systems in the virulence of S. mutans.

HaCaT anchorage blockade leads to oxidative stress, DNA damage and DNA methylation changes

Cell adhesion plays an important role in neoplastic transformation. Thus, anchorage-independent growth and epithelial-mesenchymal transition, which are features associated to anoikis-resistance, are vital steps in cancer progression and metastatic colonization. Cell attachment loss may induce intracellular oxidative stress, which triggers DNA damage as methylation changes. HaCaT lineage cells were submitted to periods of 1, 3, 5 and 24 h of anchorage blockage with the purpose of study of oxidative stress effect on changes in the DNA methylation pattern, derived from attachment blockade. Through this study, HaCaT anchorage blockage-induced oxidative stress was reported to mediate alterations in global DNA methylation changes and into TP53 gene promoter pattern during anoikis-resistance acquisition. Furthermore, at the first experimental time-periods (1, 3 and 5 h), genome hypermethylation was found; however, genome hypomethylation was observed in later time-periods (24 h) of attachment impediment. The TP 53 methylation analyses were performed after 24 h of replated anoikis-resistance cells and same methylation pattern was observed, occurring an early (1 and 3 h) hypermethylation that was followed by late (5 and 24 h) hypomethylation. However, LINE-1, a marker of genomic instability, was perceived in time-dependent hypomethylation. The mRNA levels of the DNMTs enzymes were influenced by cell attachment blockage, but non-conclusive results were obtained in order to match DNMTs transcription to pattern methylation results. In conclusion, DNA damage was found, leaded by oxidative stress that has come up from HaCaT anchorage blockade, which rises a global genome hypomethylation tendency as consequence, which might denote genomic instability.

The role of immune system in the development of periodontal disease: a brief review

Periodontitis is a highly complex and multi-factorial disease. This review summarizes some immunological factors involved in the development and control of this oral disease, such as: the participation of inflammatory cells in local inflammation, the synthesis of chemotaxis proteins with activation of the complement system and a range of antimicrobial peptides, such as defensins, cathelicidin and saposins. The integration of pathogen-associated molecular patterns (PAMPs) from microorganisms with their surface receptors in the immune cells, induces the production of several cytokines and chemokines that presents either a pro- and/or anti-inflammatory role by stimulating the secretion of a great variety of antibody subtypes and the activation of mechanisms of controlling the disease, such as the regulatory T cells. Although several studies have tried to clarify some of the immune mechanisms involved in periodontal disease, more studies must be conducted to understand its development and progression and consequently to discover new alternatives for the prevention and treatment of this severe inflammatory disease.

Microbiological, lipid and immunological profiles in children with gingivitis and type 1 diabetes mellitus

Abstract Objective The aim of this study was to compare the prevalence of periodontal pathogens, systemic inflammatory mediators and lipid profiles in type 1 diabetes children (DM) with those observed in children without diabetes (NDM), both with gingivitis. Material and methods Twenty-four DM children and twenty-seven NDM controls were evaluated. The periodontal status, glycemic and lipid profiles were determined for both groups. Subgingival samples of periodontal sites were collected to determine the prevalence of periodontal microorganisms by PCR. Blood samples were collected for IL-1-β, TNF-α and IL-6 analysis using ELISA kits. Results Periodontal conditions of DM and NDM patients were similar, without statistical differences in periodontal indices. When considering patients with gingivitis, all lipid parameters evaluated were highest in the DM group; Capnocytophaga sputigena and Capnocytophaga ochracea were more prevalent in the periodontal sites of DM children. “Red complex” bacteria were detected in few sites of DM and NDM groups. Fusobacterium nucleatum and Campylobacter rectus were frequently found in both groups. Similar levels of IL-1-β, TNF-α and IL-6 were detected in DM and NDM children. Conclusion Clinical and immunological profiles are similar between DM and NDM children. The presence of Capnocytophaga sputigena and Capnocytophaga ochracea were associated with gingivitis in DM children.