CCL3 and CXCL12 production in vitro by dental pulp fibroblasts from permanent and deciduous teeth stimulated by Porphyromonas gingivalis LPS.

OBJECTIVE: The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS). MATERIAL AND METHODS: Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0-10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results. RESULTS: Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF. CONCLUSION: These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation.

CCL3 and CXCL12 production in vitro by dental pulp fibroblasts from permanent and deciduous teeth stimulated by Porphyromonas gingivalis LPS

ABSTRACT Objective: The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS). Material and Methods: Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0 – 10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results. Results: Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF. Conclusion: These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation. .

Avaliação do papel dos receptores TLR2 e TLR4 na produção de citocinas por fibroblastos humanos periodontais deficientes desses receptores / The role of TLR2 and TLR4 in cytokines production by human periodontal fibroblasts knocked down for these receptors

Os fibroblastos são atualmente considerados componentes ativos da resposta imune porque estas células expressam receptores do tipo Toll (TLRs), são capazes de reconhecer padrões moleculares associados a patógenos e mediar a produção de citocinas e quimiocinas durante a inflamação. A resposta imune inata do hospedeiro a lipopolissacarídeos (LPS) de Porphyromonas gingivalis é incomum, já que diferentes estudos relataram que este LPS pode ser um agonista para TLR2 e um antagonista ou agonista para TLR4. A sinalização por TLRs envolve proteínas adaptadoras, como MyD88 e TRAM, que são necessárias para a transdução do sinal até o núcleo para que ocorra a transcrição de RNAm para os mediadores da inflamação. O objetivo deste estudo foi investigar e comparar se a sinalização por meio de TLR2 ou TLR4 poderia afetar a produção de Interleucina (IL)-6, IL-8 e CXCL12 em fibroblastos humanos gengivais (HGF) e fibroblastos humanos de ligamento periodontal (HPLF). Objetivamos também comparar a participação das moléculas adaptadoras MyD88 e TRAM na expressão do RNAm dos mesmos alvos. Material e Métodos: Após silenciamento mediado por RNA de interferência de TLR2, TLR4, MyD88 ou TRAM, confirmado por RT-qPCR, HGF e HPLF, provenientes de três dadores voluntários, foram estimulados com LPS de Porphyromonas gingivalis ou com dois agonistas sintéticos de TLR2, Pam2CSK4 e Pam3CSK4, por 6 horas. A expressão do RNAm e das proteínas IL-6, IL-8, e CXCL12 foram avaliados por qRT-PCR e ELISA, respectivamente. Resultados: A expressão do RNAm de TLR2 foi regulada em HGF, mas não em HPLF por todos os estímulos. O silenciamento de TLR2 diminuiu IL-6 e IL-8 em resposta ao LPS de P. gingivalis, Pam2CSK4 e Pam3CSK4 de maneira semelhante, em ambas as subpopulações de fibroblastos (p<0,05). Por outro lado, a produção de CXCL12 permaneceu inalterada pelo silenciamento de TLR2 ou TLR4. No caso do silenciamento de MyD88 e TRAM, em ambos os subtipos de fibroblastos, o RNAm para os mesmos alvos também...

Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen associated molecular patterns and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual in that different studies have reported that it can be an agonist for TLR2 and an antagonist or agonist for TLR4. TLRs signaling pathway involves adaptor proteins, like MyD88 and TRAM, which are crucial for signal transduction to the nucleus and mRNA expression of inflammatory mediators. This study investigated and compared whether signaling through TLR2 or TLR4 could affect the production of IL-6, IL-8 and CXCL12 in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). The role of MyD88 and TRAM on the mRNA expression of the same targets were also evaluated. Methods: After small interfering RNA-mediated silencing of TLR2, TLR4, MyD88 or TRAM, confirmed by RT-qPCR, HGF and HPLF from three volunteer donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein production were evaluated by RT-qPCR and ELISA, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, Pam2CSK4 and Pam3CSK4 in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. For MyD88 or TRAM silencing, IL-6 and IL-8 mRNA were also decreased, in both fibroblasts subtypes. However CXCL12 mRNA constitutive expression was increased by siMyD88 or siTRAM. Conclusion: These results suggest that signaling through TLR2 by fibroblasts, the most numerous resident cells in gingiva and periodontal...

Avaliação do papel dos receptores TLR2 e TLR4 na produção de citocinas por fibroblastos humanos periodontais deficientes desses receptores / The role of TLR2 and TLR4 in cytokines production by human periodontal fibroblasts knocked down for these receptors

Os fibroblastos são atualmente considerados componentes ativos da resposta imune porque estas células expressam receptores do tipo Toll (TLRs), são capazes de reconhecer padrões moleculares associados a patógenos e mediar a produção de citocinas e quimiocinas durante a inflamação. A resposta imune inata do hospedeiro a lipopolissacarídeos (LPS) de Porphyromonas gingivalis é incomum, já que diferentes estudos relataram que este LPS pode ser um agonista para TLR2 e um antagonista ou agonista para TLR4. A sinalização por TLRs envolve proteínas adaptadoras, como MyD88 e TRAM, que são necessárias para a transdução do sinal até o núcleo para que ocorra a transcrição de RNAm para os mediadores da inflamação. O objetivo deste estudo foi investigar e comparar se a sinalização por meio de TLR2 ou TLR4 poderia afetar a produção de Interleucina (IL)-6, IL-8 e CXCL12 em fibroblastos humanos gengivais (HGF) e fibroblastos humanos de ligamento periodontal (HPLF). Objetivamos também comparar a participação das moléculas adaptadoras MyD88 e TRAM na expressão do RNAm dos mesmos alvos. Material e Métodos: Após silenciamento mediado por RNA de interferência de TLR2, TLR4, MyD88 ou TRAM, confirmado por RT-qPCR, HGF e HPLF, provenientes de três dadores voluntários, foram estimulados com LPS de Porphyromonas gingivalis ou com dois agonistas sintéticos de TLR2, Pam2CSK4 e Pam3CSK4, por 6 horas. A expressão do RNAm e das proteínas IL-6, IL-8, e CXCL12 foram avaliados por qRT-PCR e ELISA, respectivamente. Resultados: A expressão do RNAm de TLR2 foi regulada em HGF, mas não em HPLF por todos os estímulos. O silenciamento de TLR2 diminuiu IL-6 e IL-8 em resposta ao LPS de P. gingivalis, Pam2CSK4 e Pam3CSK4 de maneira semelhante, em ambas as subpopulações de fibroblastos (p<0,05). Por outro lado, a produção de CXCL12 permaneceu inalterada pelo silenciamento de TLR2 ou TLR4. No caso do silenciamento de MyD88 e TRAM, em ambos os subtipos de fibroblastos, o RNAm para os mesmos alvos também...

Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen associated molecular patterns and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual in that different studies have reported that it can be an agonist for TLR2 and an antagonist or agonist for TLR4. TLRs signaling pathway involves adaptor proteins, like MyD88 and TRAM, which are crucial for signal transduction to the nucleus and mRNA expression of inflammatory mediators. This study investigated and compared whether signaling through TLR2 or TLR4 could affect the production of IL-6, IL-8 and CXCL12 in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). The role of MyD88 and TRAM on the mRNA expression of the same targets were also evaluated. Methods: After small interfering RNA-mediated silencing of TLR2, TLR4, MyD88 or TRAM, confirmed by RT-qPCR, HGF and HPLF from three volunteer donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein production were evaluated by RT-qPCR and ELISA, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, Pam2CSK4 and Pam3CSK4 in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. For MyD88 or TRAM silencing, IL-6 and IL-8 mRNA were also decreased, in both fibroblasts subtypes. However CXCL12 mRNA constitutive expression was increased by siMyD88 or siTRAM. Conclusion: These results suggest that signaling through TLR2 by fibroblasts, the most numerous resident cells in gingiva and periodontal...

Periodontal ligament and gingival fibroblasts participate in the production of TGF-ß, interleukin (IL)-8 and IL-10.

The aim of this study was to quantify and compare the production of transforming growth factor beta (TGF-ß), interleukin (IL)-8 and IL-10 by human cultured periodontal ligament and gingival fibroblasts both obtained from the same donors challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Fibroblasts were exposed to 0.1-10 µg/mL of LPS from P. gingivalis and after 24 h the supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA). TGF-ß protein production was upregulated in a concentration-dependent manner, mainly in gingival fibroblasts, which was statistically significant when challenged by 10 µg/mL LPS. Additionally, at this concentration, gingival fibroblasts had almost a two-fold increase in the amount of TGF-ß when compared to periodontal ligament fibroblasts. Both periodontal ligament and gingival fibroblasts showed an increase in IL-8 production when challenged with 1 µg/mL and 10 µg/mL LPS. IL-10 production remained unaffected when challenged by any of the LPS concentrations tested in either periodontal ligament or gingival fibroblasts. Our results demonstrate that periodontal ligament and gingival fibroblasts when challenged by LPS from P. gingivalis with 24 h may play a critical role in producing TGF-ß and IL-8 but not IL-10.

Periodontal ligament and gingival fibroblasts participate in the production of TGF-β, interleukin (IL)-8 and IL-10

The aim of this study was to quantify and compare the production of transforming growth factor beta (TGF-β), interleukin (IL)-8 and IL-10 by human cultured periodontal ligament and gingival fibroblasts both obtained from the same donors challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Fibroblasts were exposed to 0.1-10 µg/mL of LPS from P. gingivalis and after 24 h the supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA). TGF-β protein production was upregulated in a concentration-dependent manner, mainly in gingival fibroblasts, which was statistically significant when challenged by 10 µg/mL LPS. Additionally, at this concentration, gingival fibroblasts had almost a two-fold increase in the amount of TGF-β when compared to periodontal ligament fibroblasts. Both periodontal ligament and gingival fibroblasts showed an increase in IL-8 production when challenged with 1 µg/mL and 10 µg/mL LPS. IL-10 production remained unaffected when challenged by any of the LPS concentrations tested in either periodontal ligament or gingival fibroblasts. Our results demonstrate that periodontal ligament and gingival fibroblasts when challenged by LPS from P. gingivalis with 24 h may play a critical role in producing TGF-β and IL-8 but not IL-10.

Produção diferencial de pró-colágeno tipo I e citocinas por fibroblastos humanos de ligamento periodontal e de gengiva estimulados por lipopolissacarídeo de Porphyromonas gingivalis / Differential production of pro-collagen type I and cytokines by cultured human periodontal ligament and gingival fibroblasts challenged with lipopolyssacharide from Porphyromonas gingivalis

O ligamento periodontal e o tecido gengival são formados por tecido conjuntivo frouxo, sendo constituídos por diversas células, dentre as quais os fibroblastos são as mais numerosas. Ao serem submetidas a agressões diversas, estas células respondem com a liberação de substâncias, tais como citocinas e quimiocinas, que participam de maneira ativa no processo inflamatório, e com a produção de componentes da matriz extracelular fundamentais para o reparo, como por exemplo, o colágeno. Assim sendo, este trabalho teve como objetivo: Avaliar e comparar a expressão e a produção de pró-colágeno tipo I, IL-6, MIP-1a e SDF-1 por fibroblastos humanos, cultivados de ligamento periodontal e de tecido gengival, estimulados por lipopolissacarídeo (LPS) de Porphyromonas gingivalis. Foram coletados ligamentos periodontais de terceiros molares não irrompidos e biópsias de gengiva de um mesmo indivíduo (n=3). Estes tecidos foram picotados e mantidos em meio de cultura adequado para fibroblastos, que foram utilizados na quarta passagem. Após adesão dos fibroblastos a placas de 24 poços, o meio de cultura contendo 0,1 - 10 µg/mL de LPS de P. gingivalis foi adicionado às placas em duplicata. O sobrenadante e as células foram coletados após 1, 6 e 24 horas e analisados por ELISA e PCR em tempo real, respectivamente. A análise estatística foi realizada por meio do programa GraphPad Prism, aplicando-se o teste ANOVA a 1 critério com nível de significância de 5%. A expressão de pró-colágeno tipo I mostrou-se ligeiramente diferente entre fibroblastos de ligamento periodontal e de gengiva. A produção de IL-6, MIP-1a e SDF-1 foi significativamente maior em fibroblastos gengivais.

A citocina IL-6 foi produzida de maneira tempo-dependente com LPS de P gingivalis, principalmente por fibroblastos gengivais. Para MIP-1a, os fibroblastos gengivais mostraram maior produção com a menor concentração de estímulo (0,1µg/ml). Para SDF-1, foi foi detectada produção constitutiva que foi inibida com o aumento da concentração de LPS ao longo do tempo nestas mesmas células. Já para fibroblastos de ligamento periodontal, não foi observado um padrão homogêneo e linear, apesar de a produção basal de SDF-1 também existir, porém em níveis bem mais discretos, como aquele observado para a produção de MIP-1a. A capacidade dos fibroblastos modificarem o padrão de produção dessas citocinas frente ao estímulo com LPS de P. gingivalis reforça a importância dessas células no contexto da resposta imune do indivíduo frente à doença periodontal.

The fibroblast is considered an important cell in periodontitis because it is the predominant cell type in the periodontal connective tissue. When challenged by different agents, fibroblasts respond through the release of substances, such as cytokines and chemokines that participate in an active way in the inflammatory process as well as the production of basic components of the extracellular matrix for repair, like collagen. The aim of this study was to: to evaluate and to compare the expression and production of type I pro-collagen, IL-6, MIP-1a and SDF-1 by cultured human periodontal ligament and gingival fibroblasts challenged with lipopolyssacharide from Porphyromonas gingivalis. Human periodontal ligament and gingival fibroblasts were cultured from biopsies of the same donor and were used on the fourth passage. After confluence in 24-well plates, the culture medium alone (control) or with 0,1- 10 ug/mL of LPS from P. gingivalis were added and after 1, 6 and 24 hours, the supernatant and the cells were collected and analysed by ELISA and Real time PCR, respectively. Data were analysed by GraphPad Prism Program (1 way- ANOVA test) and a significance level of 5% was adopted. Pro-collagen type I expression by Real Time PCR differ between periodontal ligament and gingival fibroblasts.

In vitro experiments revealed that IL-6, MIP-1a and SDF-1 production were significantly greater in gingival fibroblasts when compared to periodontal ligament. In addition, IL-6 was upregulated in a time-dependent manner, mainly by the gingival fibroblasts. On one hand, MIP-1a was stimulated with a low concentration (0,1ugml) of LPS by gingival fibroblasts. On the other hand, SDF-1 was constitutively secreted by the same cells but its production was inhibited when challenged by a higher concentration of LPS from P gingivalis. In general, periodontal ligament fibroblasts did not show a pattern of production of these cytokines under the challenge with LPS, despite of the basal production of SDF-1 in lower levels than gingival cells and the low production of MIP-1a over time. The differential ability of the gingival and periodontal ligament fibroblasts to secrete these cytokines emphasizes their crucial role in the inflammatory microenvironment and in the host immune response to periodontal disease.

Absenteísmo: uma revisão da literatura sobre a ausência ao trabalho relacionada à odontologia / Absenteeism: a revision of the literature about the absnece to the work related to the dentistry

Os fatores odontológicos que acarretam ausências ao trabalho têm sido de interesse crescente ao setor público e privado, razão esta que tem levado alguns pesquisadores a estudar os principais fatores que estão envolvidos com este tipo de absenteísmo. Diante disso, objetivou-se revisar a literatura em busca de resultados e conclusões que demonstrem a importância do assunto aos responsáveis pela saúde dos trabalhadores, abordando aspectos como: gênero, faixas etárias e principais problemas (odontológicos) que levam ao absenteísmo. A partir dos trabalhos revisados pode-se concluir que o gênero feminino e as faixas etárias mais jovens aparecem como as variáveis com maior freqüência de absenteísmo por causas odontológicas; e a carência por publicações e estudos mais aprofundados, a divergência de resultados e a construção e uso restrito de dados quantitativos limitados ao controle administrativo dos recursos humanos, dificultam o melhor entendimento dessa importante questão