Diferencia en la expresión del ligando de receptor activador para el factor nuclear K B, en tejido tumoral frente a la infección por diferentes patógenos periodontales / Difference in receptor activator of nuclear factor K B expression in tumoral tissue by different periodontal pathogens

La enfermedad periodontal es una de las principales causas de pérdida dentaria. Clínicamente, esta patología, mediada por la desregulación del sistema inmune producto de una disbiosis ocurrida en el surco gingival, inicia con la inflamación de la encía y evoluciona con el daño irreversible de los tejidos que rodean el diente. El hueso alveolar es uno de los tejidos afectados esta patología, esto debido a la activación de osteoclastos por la sobreexpresión de la proteína RANKL en el huésped. El propósito de este trabajo es determinar el nivel de sobreexpresión de RANKL, en un modelo de células tumorales U2OS, frente a la infección con Porphyromonas gingivalis y Prevotella intermedia. Para identificar el nivel de RANKL, se definieron cuatro grupos: Un grupo control, no tratado; Grupo PG, tratado con P. gingivalis; Grupo PI, tratado con P. Intermedia; y un grupo PG+PI, tratado con ambas bacterias. El nivel relativo de la proteína RANKL fue determinado en el sobrenadante y en los extractos celulares de manera independiente, mediante la técnica Western blot. En sobrenadantes, el grupo PG mostró mayores niveles de RANKL comparados con PI (p < 0,05). En extractos celulares los niveles fueron mayores en el grupo PG+PI (p < 0,05). El grupo PI mostró los niveles más bajos de RANKL. La infección polimicrobiana resulta en una mayor expresión de RANKL en células tumorales U2OS, mientras que frente a la infección P. gingivalis, se observó mayor cantidad de RANKL soluble.

SUMMARY: Periodontal disease is one of the main causes of tooth loss. Clinically, this pathology, mediated by the deregulation of the immune system due to a dysbiosis occurred in the gingival sulcus, begins with the inflammation of the gum and evolves with the irreversible damage of the tissues that surround the tooth. Alveolar bone is one of the most affected tissues by this disease, due to the activation of osteoclasts by the upregulation of RANKL in the host. The aim of this study is to determine the increase of RANKL, in a U2OS tumor cells model, inoculated with Porphyromonas gingivalis and Prevotella intermedia. To identify the level of RANKL, four groups were defined: A control group, not treated; PG group, treated with P.gingivalis; PI group, treated with P. intermedia; and a PG+PI group, treated with both bacteria. The relative level of RANKL was determined in the supernatant and cell extracts independently, using the Western blot technique. In supernatants, the PG group showed higher RANKL levels compared to PI (p < 0.05). In cell extracts the levels were higher in the PG+PI group (p < 0.05.). The PI group showed the lowest levels of RANKL.Polymicrobial infection results in a greater expression of of soluble RANKL was observed.

Composite-derived monomers affect cell viability and cytokine expression in human leukocytes stimulated with Porphyromonas gingivalis

Abstract Objectives: Dental composites release unreacted resin monomers into the oral environment, even after polymerization. Periodontal cells are, therefore, exposed to substances that potentially elicit the immune inflammatory response. The underlying molecular mechanisms associated with the interaction between resin monomers and human immune cells found in the gingival crevicular fluid are not fully understood yet. This study investigated the ability of bisphenol A-glycidyl methacrylate (BISGMA), urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) to induce apoptosis and cytokine release by human leukocytes stimulated with a periodontal pathogen. Methodology: Peripheral blood mononuclear cells (PBMC) from 16 healthy individuals were included in this study. To determine the toxicity, the PBMC were incubated for 20 hours, with monomers, for the analysis of cell viability using MTT assay. To evaluate cell death in the populations of monocytes and lymphocytes, they were exposed to sub-lethal doses of each monomer and of heat-inactivated Porphyromonas gingivalis (P. gingivalis) for 5 hours. Secretions of IL-1β, IL-6, IL-10 and TNF-α were determined by ELISA after 20 hours. Results: UDMA and TEGDMA induced apoptosis after a short-time exposure. Bacterial challenge induced significant production of IL-1β and TNF-α (p<0.05). TEGDMA reduced the bacterial-induced release of IL-1β and TNF-α, whereas UDMA reduced IL-1β release (p<0.05). These monomers did not affect IL-10 and IL-6 secretion. BISGMA did not significantly interfere in cytokine release. Conclusions: These results show that resin monomers are toxic to PBMC in a dose-dependent manner, and may influence the local immune inflammatory response and tissue damage mechanisms via regulation of bacterial-induced IL-1β and TNF-α secretion by PBMC.

Toll-like receptor agonists Porphyromonas gingivalis LPS and CpG differentially regulate IL-10 competency and frequencies of mouse B10 cells

Abstract IL-10 expressing regulatory B cells (B10) play a key role in immune system balance by limiting excessive inflammatory responses. Effects of toll-like receptor signaling and co-stimulatory molecules on B10 activity during innate and adaptive immune responses are not fully understood. Objective This study is to determine the effects of P. gingivalis LPS and CpG on B10 cell expansion and IL-10 competency in vitro. Material and Methods Spleen B cells were isolated from C57BL/6J mice with or without formalin-fixed P. gingivalis immunization. B cells were cultured for 48 hours under the following conditions: CD40L, CD40L+LPS, CD40L+CpG, and CD40L+LPS+CpG in the presence or absence of fixed P. gingivalis. Percentages of CD1dhiCD5+ B cells were measured by flow cytometry. IL-10 mRNA expression and secreted IL-10 were measured by real-time quantitative PCR and by ELISA respectively. Results P. gingivalis LPS plus CD40L significantly increased CD1dhiCD5+ B cell percentages and secreted IL-10 levels in both immunized and non-immunized mice B cells in the presence or absence of P. gingivalis, compared with control group. Secreted IL-10 levels were significantly increased in CD40L+LPS treated group compared with CD40L treatment group in the absence of P. gingivalis. CpG plus CD40L significantly decreased CD1dhiCD5+ B cell percentages, but greatly elevated secreted IL-10 levels in immunized and non-immunized mice B cells in the absence of P. gingivalis, compared with CD40L treatment group. Conclusions P. gingivalis LPS and CpG differentially enhance IL-10 secretion and expansion of mouse B10 cells during innate and adaptive immune responses.

Efeito da severidade da doença periodontal sobre a expressão do Receptor Ativado por Protease do Tipo 2 (PAR-2) / Effect of the severity of periodontal disease on the expression of Protease Activated Receptor Type 2 (PAR-2)

Pesquisas recentes investigando o receptor ativado por protease do tipo 2 (PAR-2) sugerem uma associação entre este receptor e a inflamação periodontal. Além disso, é sabido que a gingipaína, protease bacteriana secretada por um importante periodontopatógeno, Porphyromonas gingivalis (Pg), tem a capacidade de ativar o PAR-2. Ademais, um estudo anterior do grupo, verificou que quanto mais profunda a bolsa periodontal, maior era a expressão do receptor PAR-2. No entanto, não é sabido se a expressão de PAR-2 é proporcional a severidade de doença periodontal e a quantidade de gingipaína expressa na bolsa periodontal. Desta forma, o presente estudo, verificou no fluido gengival a correlação entre a expressão gênica de PAR-2 (Real Time-PCR) com os parâmetros clínicos periodontais, e a expressão gênica da protease gingipaína em pacientes com periodontite crônica severa e moderada, antes e após o tratamento periodontal não-cirúrgico. A expressão de PAR-2 e da protease gingipaína foi estatisticamente maior nos pacientes do grupo periodontite crônica severa (PS) em comparação com os pacientes do grupo periodontite crônica modera (PM) e controle (C). Além disso, o tratamento periodontal levou à redução significativa (p<0.05) da expressão de PAR-2 nos pacientes com periodontite crônica moderada. Em conclusão, dentro dos limites do presente estudo, nós demonstramos que a severidade da doença periodontal e a expressão de gingipaína influenciaram a expressão de PAR-2 no fluido gengival de pacientes com periodontite crônica.

Recent studies investigating the protease-activated receptor type 2 (PAR-2) suggest an association between the receptor and periodontal inflammation. In addition, it is known that gingipain, a bacterial protease secreted by an important periodontopathogen, Porphyromonas gingivalis (Pg), has the ability to activate PAR- 2. Furthermore, a previous study from our group found that the deeper the periodontal pocket, the higher the expression of the PAR-2 receptor. However, it is not known whether the expression of PAR-2 is associated to the severity of periodontal disease and the amount of gingipain expressed in the periodontal pocket. Thus, the present study verified, in the gingival fluid, the correlation between the PAR-2 gene expression (Real Time-PCR) with the clinical periodontal parameters, and the gene expression of the gingipain protease in patients with moderate and severe chronic periodontitis before and after non-surgical periodontal treatment. PAR-2 expression and gingipain protease were statistically more expressed in patients of the severe chronic periodontitis group (PS) compared with those in the moderate chronic periodontitis group (PM) and control group (C). Furthermore, periodontal treatment led to a significant reduction (p <0.05) in the expression of PAR-2 in patients with moderate chronic periodontitis. In conclusion, within the limits of the present study, we demonstrated that the severity of periodontal disease and the expression of gingipain influenced the PAR-2 expression in the gingival fluid of patients with chronic periodontitis.

Periodontitis, Porphyromonas gingivalis y su relación con la expresión de quorum sensing / Periodontitis, Porphyromonas gingivalis and its relation to quorum sensing expression

Los mecanismos de señalización bacteriana desempeñan un papel fundamental en el establecimiento y progresión de la enfermedad periodontal. Dadas estas circunstancias es crucial profundizar en el entendimiento de estos mecanismos para intentar proveer estrategias terapéuticas novedosas. El presente artículo de revisión, de carácter narrativo, tiene como objetivo conducir un análisis crítico de la evidencia disponible sobre la influencia de Porphyromonas gingivalis (Pg) y expresión de quorum sensing (Qs) en enfermedad periodontal. Se realizó una búsqueda a través de bases de datos como Ovid (MEDLINE), ScienceDirect, Hinari. El conocimiento actual de estos mecanismos ofrece la posibilidad de desarrollar nuevos y profundos estudios (teóricos y experimentales) sobre la expresión del QS en pacientes con enfermedad periodontal y permitirá un novedoso campo de investigación con el que no se cuenta en la actualidad. Desde su descubrimiento, el QS se vislumbra como un espacio de investigación valioso en el cual se debe insistir de manera permanente. La anterior evidencia permite concluir que a través de la regulación de la expresión de determinados genes en bacterias como la PG, se puede efectuar la inhibición de la formación de las biopelículas que tiene efectos directos e indirectos sobre el desarrollo de la enfermedad periodontal(AU)

The bacterial signaling mechanisms play a key role in the establishment and progression of periodontal disease. Due to these circumstances it is crucial to deepen in the understanding of these mechanisms to try to provide novel therapeutic strategies. The objective of present narrative literature review was to make a critical analyze of the available evidence on the influence of Porphyromonas gingivalis (PG) and the quorum sensing expression in periodontal disease. Using the Ovid (MEDLINE) ScienceDirect, Hinari database we made a search. The current knowledge of these mechanisms offers the possibility of developing new and deep studies (theoretical and experimental) on the QS expression in patients presenting with periodontal disease allowing a novel research field not currently available. From its discovery the QS is discerned as a valuable research space in which we must to insist in a permanent way. The above mentioned evidence allows concluding that by the regulation of the expression of determined genes in bacteria like PG, it is possible to carry out the inhibition in the formation of the biofilms with direct and indirect effects on the periodontal disease development(AU)

Nanotechnology: Role in dental biofilms.

Biofilms are surface- adherent populations of microorganisms consisting of cells, water and extracellular matrix material Nanotechnology is promising field of science which can guide our understanding of the role of interspecies interaction in the development of biofilm. Streptococcus mutans with other species of bacteria has been known to form dental biofilm. The correlation between genetically modified bacteria Streptococcus mutans and nanoscale morphology has been assessed using AFMi.e atomic force microscopy. Nanotechnology application includes 16 O/ 18 O reverse proteolytic labeling,use of quantum dots for labeling of bacterial cells, selective removal of cariogenic bacteria while preserving the normal oral flora and silver antimicrobial nanotechnology against pathogens associated with biofilms. The future comprises a mouthwash full of smart nanomachines which can allow the harmless flora of mouth to flourish in a healthy ecosystem