RESUMO
Studies have been conducted on the pathogenicity of periodontopathogens in cultures of endothelial cells on two-dimensional (2D) polystyrene surfaces, where the monolayer formed is not exposed to proteins of the subendothelial matrix. In this work, we developed a culture system by seeding human coronary artery endothelial cells (HCAECs) onto three-dimensional (3D) scaffolds of collagen type I, a subendothelial protein. The inflammatory responses of the HCAEC monolayers, formed either on 3D scaffolds or directly on a 2D polystyrene plate, to lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) were evaluated. The transcription of 3 genes, the secretion of 40 cytokines and 2 prostanoids, and the adhesion of monocytes to 2D and 3D cultures with or without exposure to lipopolysaccharides (control) were assessed. HCAECs exhibited differences in transcriptional and secretory profiles between the 3D and 2D models. In addition, the inflammatory responses of HCAEC to Aa-LPS and Pg-LPS differed between the two models. In 3D cultures treated with Aa-LPS, the levels of IL-8, RANTES, G-CSF, ICAM-1, IL-6, and TXA2 were significantly higher than those in the controls. In 2D cultures treated with Aa-LPS, IL-8, RANTES, G-CSF, ICAM-1, TNF-RI, PGI2, and TXA2 levels were significantly higher than those in their controls. In the presence of Aa-LPS, monocyte adhesion did not differ between treated and control 3D cultures but was significantly higher in treated 2D cultures than in the controls. In response to Pg-LPS, cytokine-prostaglandin secretion and monocyte adhesion did not differ between 3D and 2D cultures. These data indicate that HCAECs respond differently to these two types of LPS.
Assuntos
Adesão Celular , Técnicas de Cultura de Células , Células Endoteliais , Lipopolissacarídeos , Alicerces Teciduais , Aggregatibacter actinomycetemcomitans/química , Humanos , Monócitos/fisiologia , Porphyromonas gingivalis/químicaRESUMO
Chemical hosts bind their guests by the same physical mechanisms as biomolecules and often display similarly subtle structure activity relationships. The cyclodextrins have found increasing application as inert, nontoxic carriers of active compounds in drug formulations. The present study was conducted to prepare inclusion complexes of chlorhexidine:ß-cyclodextrin (Cx:ß-cd), and evaluate their interactions with bacterial membrane through: scanning electron microscopy (SEM) and transmission electron microscopy (TEM); and measuring morphology alterations, roughness values, and cell weights by atomic force microscopy (AFM). It was found that the antimicrobial activity was significantly enhanced by cyclodextrin encapsulation. SEM analysis images demonstrated recognizable cell membrane structural changes and ultrastructural membrane swelling. By TEM, cellular alterations such as vacuolization, cellular leakage, and membrane defects were observed; these effects were enhanced at 1:3 and 1:4 Cx:ß-cd. In addition, AFM analysis at these ratios showed substantially more membrane disruption and large aggregates mixing with microorganism remains. In conclusion, nanoaggregates formed by cyclodextrin inclusion compounds create cluster-like structures with the cell membrane, possibly due to a hydrogen rich bonding interaction system with increasing surface roughness and possibly increasing the electrostatic interaction between cationic chlorhexidine with the lipopolysaccharides of Gram negative bacteria.
Assuntos
Membrana Celular/ultraestrutura , Bactérias Gram-Negativas/ultraestrutura , Microscopia de Força Atômica/métodos , Microscopia Eletrônica de Transmissão/métodos , Nanopartículas/química , beta-Ciclodextrinas/síntese química , Aggregatibacter actinomycetemcomitans/química , Aggregatibacter actinomycetemcomitans/ultraestrutura , Membrana Celular/efeitos dos fármacos , Clorexidina/administração & dosagem , Clorexidina/síntese química , Avaliação Pré-Clínica de Medicamentos/métodos , Bactérias Gram-Negativas/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Microscopia Eletrônica de Varredura/métodos , Nanopartículas/administração & dosagem , Tamanho da Partícula , beta-Ciclodextrinas/administração & dosagemRESUMO
OBJECTIVES: Previous studies suggest differences between geographically and racially distinct populations in the prevalence of periodontopathic bacteria as well as greater periodontal destruction associated with infection by highly leucotoxic Actinobacillus actinomycetemcomitans. The present study examined these hypotheses in Brazilians with aggressive or chronic periodontitis. MATERIALS AND METHODS: Clinical, radiographical, and microbiological assessments were performed on 25 aggressive periodontitis and 178 chronic periodontitis patients including 71 males and 132 females, 15-69 years of age. RESULTS: The prevalence of Porphyromonas gingivalis was similar to that of other South American populations. The prevalence of A. actinomycetemcomitans and its highly leucotoxic subgroup was higher in Brazilians. Highly leucotoxic A. actinomycetemcomitans was more prevalent in aggressive periodontitis (chi2=27.83) and positively associated with deep pockets (>6 mm, chi2=18.26) and young age (<29 years, chi2=18.68). Greater mean attachment loss was found in subjects with highly leucotoxic A. actinomycetemcomitans than in subjects with minimally leucotoxic (p=0.0029) or subjects not infected (p=0.0001). CONCLUSION: These data support the hypothesis of differences between populations in the prevalence of periodontopathic bacteria and of greater attachment loss in sites infected with highly leucotoxic A. actinomycetemcomitans. Detection of highly leucotoxic A. actinomycetemcomitans in children and adolescents may be a useful marker for aggressive periodontitis.
Assuntos
Periodontite/epidemiologia , Periodontite/microbiologia , Doença Aguda , Adolescente , Adulto , Idoso , Aggregatibacter actinomycetemcomitans/química , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Bacteroides/isolamento & purificação , Brasil/epidemiologia , Campylobacter rectus/isolamento & purificação , Doença Crônica , Contagem de Colônia Microbiana , Estudos Transversais , DNA Bacteriano/análise , Placa Dentária/microbiologia , Exotoxinas/análise , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Porphyromonas gingivalis/isolamento & purificação , Prevalência , Prevotella intermedia/isolamento & purificaçãoRESUMO
BACKGROUND: The use of whole saliva has shown to be promising in detecting Actinobacillus actinomycetemcomitans out of the subgingival environment. The objective of the present study was to evaluate the use of unstimulated saliva in detecting A. actinomycetemcomitans and to compare the subgingival and extracrevicular occurrence of this pathogen in Brazilian subjects with chronic periodontitis. METHODS: Sixty-six patients (mean age 38.01 9.28 years) with advanced generalized chronic periodontitis were sampled. Subgingival plaque samples were collected from eight sites per patient representing the two deepest sites of each quadrant. Samples of the mucous surfaces, including dorsal surface of the tongue and cheek, were collected with a sterile swab and placed in a microtube containing a reduced solution. Samples of unstimulated saliva were also collected in sterile tubes and 0.1 ml of whole saliva was diluted in 1 ml of reduced solution. The presence of A. actionomycetemcomitans was established using bacterial culture in trypticase soy bacitracin vancomycin selective media. Polymerase chain reaction (PCR) was used to differentiate highly from minimally leukotoxic strains in patients who presented A. actinomycetemcomitans in at least two sampled sites. RESULTS: A. actinomycetemcomitans was isolated from 63.63% of subgingival samples, 56.06% of saliva samples, and 45.45% of samples from mucous surfaces. No statistical difference was observed between subgingival and salivary occurrence of the microorganism. Linear regression showed an association between subgingival plaque and saliva (r(2) = 0.897; P = 0.015) and mucous membrane and saliva (r(2) = 0.152; P = 0.024). The same A. actinomycetemcomitans leukotoxic profile was observed in all sampled sites for a given patient. CONCLUSION: These results suggest that in advanced periodontitis, unstimulated saliva is representative of pooled subgingival plaque samples and its use is appropriate in the oral detection of A. actinomycetemcomitans.
Assuntos
Aggregatibacter actinomycetemcomitans/isolamento & purificação , Periodontite/microbiologia , Saliva/microbiologia , Adulto , Aggregatibacter actinomycetemcomitans/química , Doença Crônica , Placa Dentária/microbiologia , Exotoxinas/isolamento & purificação , Feminino , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Mucosa Bucal/microbiologia , Reação em Cadeia da PolimeraseRESUMO
An immune mechanism has been suggested in the pathogenesis of periodontal disease. Actinobacillus actinomycetemcomitants (Aa) has been implicated as one of the etiological agents that induces the major immune response together with a dense infiltrate of inflammatory cells. But the exact role of these immune cells in periodontal disease has not yet been clarified. In this study the T lymphocyte (TL) proliferative response was evaluated after having being exposed to free cell supernatant (SN) from Aa. Aa SN increased TL proliferation. This mitogenic effect of Aa SN was attenuated by pretreating TL with indomethacin (INDO) or acetylsalicylic acid (ASA) but not by polymyxin B. The inhibitory effect of INDO on cell proliferation was reversed by the addition of prostaglandin E2 (PGE2) to the culture assay. Moreover, when immune cells were exposed to Aa SN they were able to generate PGE2 at the same time as intracellular levels of cAMP decreased. Both, PGE2 release and decrease accumulation of cAMP in TL were blunted by treated lymphocytes with INDO. In this paper we demonstrate that cell free SN from Aa induces a mitogenic effect on murine lymphocytes. The mechanism involves the host's immunecompetent cells and the release of PGE2 and appears not to be induced by capsular-like polysaccharide antigen. Results show a paradoxical mitogenic effect of Aa SN accompanied by increased generation of PGE2 and decreased production of cAMP by lymphocytes.