Interleukin-6 and Interferon-α Levels in Gingival Crevicular Fluid in HIV-1 Patients with Chronic Periodontitis / Niveles de Interleukin-6 e Interferon-α de Fluido Crevicular Gingival en Pacientes VIH-1 con Periodontitis Crónica

ABSTRACT: The influence of cytokine on the progression of chronic periodontitis in human immunodeficiency virus (HIV) patients is still controversial and poorly investigated. This study aimed to analyze and compare IL-6 and IFN-α levels in the gingival crevicular fluid of HIV-1-positive and HIV-1-negative patients with chronic periodontitis and different grades of tissue destruction and inflammation. Samples from the gingival crevicular sulcus were obtained from 35 HIV-1-positive individuals with chronic periodontitis and 35 seronegative patients with chronic periodontitis. Probing depth and clinical attachment level, as well as the results of the Enzyme-Linked Immunosorbent Assay for confirmation of patient diagnostics, were evaluated. Statistical analyses were performed using Student t, Mann-Whitney and Spearman tests. IL-6 levels were significantly lower, while IFN-α levels were significantly higher in HIV-1 patients. Clinical attachment level was directly associated with IFN-α levels in HIV-1 carriers, connected to probing depth in these patients. Clinical data in association with gingival crevicular fluid cytokine levels may reveal a localized immunological response pattern, which may contribute to the understanding of periodontitis pathogenesis in HIV-1 carriers.

RESUMEN: La influencia de la citocina en la progresión de la periodontitis crónica en pacientes con el virus de la inmunodeficiencia humana (VIH) sigue siendo controvertida y poco investigada. Este estudio tuvo como objetivo analizar y comparar los niveles de interleuquina-6 (IL6) e interferón-α (IFN-α) en el líquido crevicular gingival de pacientes VIH-1-positivos y VIH-1-negativos con periodontitis crónica y diferentes grados de destrucción e inflamación tisular. Se obtuvieron muestras del surco crevicular gingival de 35 individuos VIH-1 positivos con periodontitis crónica y 35 pacientes seronegativos con periodontitis crónica. Se evaluaron la profundidad de sondeo y el nivel de inserción clínica, así como los resultados del Ensayo Inmunoabsorbente Ligado a Enzimas para la confirmación del diagnóstico del paciente. Los análisis estadísticos se realizaron utilizando pruebas t de Student, Mann-Whitney y Spearman. Los niveles de IL-6 fueron significativamente más bajos, mientras que los niveles de IFN-a fueron significativamente más altos en los pacientes con VIH-1. El nivel de inserción clínica se asoció directamente con los niveles de IFN-α en los portadores del VIH1, conectados a la profundidad del sondaje en estos pacientes. Los datos clínicos en asociación con los niveles de citoquinas de los fluidos creviculares gingivales pueden revelar un patrón de respuesta inmunológica localizado, que puede contribuir a la comprensión de la patogénesis de la periodontitis en los portadores del VIH-1.

Cytokines and MMPs levels in gingival crevicular fluid from patients with chronic periodontitis before and after non-surgical periodontal therapy

The study of host response in periodontal disease may provide a mechanism to monitor disease progression. the purpose of the present research was to determine the levels of IL-1alfa, IL-1beta, TNF-alfa, IL-6, IL-6sR, IL-8, IL-10, MMP-3 and MMP-8 in gingival crevicular fluid (GCF), before and after non-surgical periodontal treatment (NSPT) in order to evaluate therapy response. methodology: eleven patients diagnosed with chronic periodontitis and eleven healthy subjects were selected for this study. clinical measurements, including probing depth (PD) and clinical attachment loss (CAL) were carried out in patients diagnosed with chronic periodontitis and periodontal healthy controls. the clinical indexes evaluated were: gingival index (GI) and plaque index (PI). samples of GCF were taken from one tooth per quadrant before and 45 days after NSPT. the levels of inflammatory mediators were measured by ELISA. results: the values of all clinical parameters decressed significsntly after treatment. the concentration levels of all cytokines and MMP-3 and MMP-8 in the GCF sample were higher in patients diagnosed with chronic periodontitis compared to the healthy group. all inflammatory mediators decreased after therapy, but did not reach control values; IL-6, Il-6sR, IL-10 and TNF-alfa, attained the highest reduction (70 percent -54 percent); the vales of MMP3, IL-1alfa, IL-1beta and IL-8 were reduced between 50 percent ­ 34 percent; and MMP-8 showed the lowest decrease (28 percent). conclusion: all clinical parameters and cytokines levels decreased after NSPT. the mediators TNF-alfa IL-6, IL-6sR, and IL-10 showed the largest variation between before and after NSPT and could thus be used to evaluate therapy response.

O efeito adjunto de uma suplementação alimentar à base de ômega-3 no tratamento da periodontite: um estudo clínico e imunológico / The adjunctive effects of Omega-3 dietary supplementation in the treatment of periodontitis: a clinical and immunological study

O objetivo foi avaliar se a suplementação com ômega (ω)-3 no tratamento não cirúrgico da periodontite melhora os resultados clínicos e imunológicos. Avaliar os níveis séricos dos ω-3 e ω-6 na doença periodontal, investigar se o tratamento periodontal associado ou não a suplementação com ω-3 afeta os níveis séricos destes. Assim como avaliar a expressão de um painel de citocinas relacionadas a osteoclatogênese no fluido gengival (FG) de sítios dos pacientes com gengivite e periodontite, adicionalmente relacionar estas citocinas a densidade óptica do osso alveolar destes sítios. Por fim, investigar se as terapias propostas neste estudo afetam os parâmetros clínicos periodontais, a densidade óptica do osso alveolar e a expressão das citocinas no FG. Para isso foi realizado um ensaio clínico piloto, randomizado, duplo-cego, controlado-placebo, com vinte e um pacientes com periodontite e dezesseis com gengivite. Estes foram investigados para os níveis séricos dos ácidos graxos poli-insaturados de cadeia longa (AGPI-CL), ácido eicosapentaenóico (EPA), ácido docosahexaenóico (DHA), ácidos docosapentaenóico (DPA), o ácido araquidônico (AA) usando cromatografia gasosa. O FG dos sítios controle-gengivite, controle-periodontite e destruído de pacientes com periodontite foi avaliado para Receptor ativador do fator nuclear kappa-B ligante (RANK-L), osteoprotegerina (OPG), a osteocalcina (OC), fator de necrose tumoral (TNF)-α, interferon (IFN)-γ, interleucina (IL) -1ß, IL-4, IL-6 e IL-10, utilizando imunoensaio multiplex. Pacientes com periodontite foram subagrupados em grupos com e sem Síndrome Metabólica para avaliação dos AGPI-CL. Sítios dos pacientes com periodontite foram subagrupados em sítios sem e com considerável desmineralização ópticas baseado na avaliação da densidade óptica alveolar representada pelos valores de pixels das radiografias intraorais digitais para avaliação das citocinas no FG. Pacientes com periodontite foram randomizados em raspagem e alisamento radicular mais suplementação com ω-3 (RAR+ω-3) ou placebo (RAR+placebo), e foram reavaliados depois de quatro meses para AGPI-CL no soro, citocinas no FG e densidade alveolar óptica. Os níveis significativamente maiores dos AGPI-CLs foram observados em pacientes com periodontite em comparação com gengivite. A profundidade de bolsa mostrou uma correlação positiva significativa com DHA, DPA e AA. Após RAR+ω-3, DPA, AA, AA/EPA e AA/DHA reduziram significativamente, e depois da RAR+placebo todos os níveis dos AGPI-CLs reduziram significativamente. Os níveis das citocinas, IFN-γ, IL-4, IL-10, RANK-L e OC, no FG de sítios controle-gengivite foram menores comparados aos sítios controle-periodontite e destruído. O mesmo resultado foi observado para os níveis da IL-1ß, IL-6 e OPG em comparação com sítios destruídos. Pacientes com periodontite apresentaram níveis mais baixos de IL-1ß e OPG em sítios controle-periodontite comparado aos destruídos. Após RAR+ω-3, o nível da IL-4 aumentou e os níveis de OC, IFN-γ e IL-10 diminuíram. Não houve diferenças significativas entre os grupos experimentais para a densidade alveolar óptica e parâmetros. Em conclusão, os níveis séricos dos AGPI-CLs são afetados pela gravidade de doença periodontal e diferem com a presença da síndrome metabólica. Citocinas relacionadas a osteoclatogênese no FG refletem a gravidade periodontal e diferem com densidade óptica alveolar alterada. A suplementação com ω-3 não melhora clinica ou imunologicamente o resultado do tratamento periodontal.

The aim was to test whether omega (ω)-3 supplementation associated to non-surgical treatment of periodontitis improves clinical and immunological results. Evaluate the serum levels of ω-3 and ω-6 in periodontal disease, hence, to investigate whether periodontal treatment with or without ω-3 supplementation affects serum levels of these. Therefore, to evaluate the expression of a panel of cytokines related to osteoclastogenesis in the gingival crevicular fluid (GCF) sites of patients with gingivitis and periodontitis, additionally relate these cytokines with optical alveolar bone density in these sites. Finally, to investigate whether the therapies proposed in this study affect the clinical periodontal parameter, the optical density of the alveolar bone and the expression of cytokines in GCF. Then was conducted a pilot clinical trial, randomized, double-blind, placebo-controlled, with twenty-one patients with periodontitis and sixteen with gingivitis. These were investigated for blood levels of long chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), arachidonic acid (AA) using gas chromatography. The GCF of the sites of control-gingivitis, control-periodontitis and destroyed from patients periodontitis were evaluated for receptor activator of kappa-B ligand nuclear factor (RANK-L), osteoprotegerin (OPG), osteocalcin (OC), tumor necrosis factor (TNF) -α, interferon (IFN) -γ, interleukin (IL) -1ß, IL-4, IL-6 and IL-10 using multiplex immunoassay. Patients with periodontitis were subgrouped in groups with and without metabolic syndrome to evaluate the LC-PUFA. Sites of patients with periodontitis were subgrouped at sites with and without considerable optical demineralization based on the evaluation of alveolar optical density represented by the pixel values of the digital intraoral radiographs for evaluation of cytokines in GCF. Patients were randomized into the periodontal scaling and root planing more ω-3 supplementation (ω-3 SRP +) or placebo (SRP + placebo), and were assessed after four months to LC-PUFA in serum, cytokines in GCF and alveolar optical density. The significantly higher levels of LC-PUFA were observed in patients with periodontitis compared to gingivitis. The pocket depth showed a significant positive correlation with DHA, DPA and AA. After RAR + ω-3, APD, AA, AA / AA and EPA / DHA reduced significantly, and, after SRP + placebo, all levels of LC-PUFA reduced significantly. The levels of cytokines, IFN-γ, IL-4, IL-10, and OC RANK-L, from controlgingivitis sites GCF were lower compared to control-periodontitis and destroyed sites. The same result was observed for levels of IL-1ß, IL-6 and OPG compared to destroyed sites. Periodontitis patients had lower levels of IL-1ß and OPG in control-periodontitis compared to destroyed sites. After SRP + ω-3, the level of IL-4 and OC increased and levels of IFN-γ and IL-10 decreased. There were no significant differences between the experimental groups to optical density and clinical parameters. In conclusion, serum levels of LC-PUFA are affected by periodontal disease severity and differ in the presence of the metabolic syndrome. Cytokines related to osteoclastogenesis in GCF reflect the periodontal severity and differ with impaired alveolar optical density. Ω-3 supplementation does not improve clinical or immune result of non-surgical periodontal treatment.

Effectiveness of non-surgical treatment to reduce il-18 levels in the gingival crevicular fluid of patients with periodontal disease

The aim of this study was to evaluate the effectiveness of the non-surgical periodontal treatment in reducing the gingival crevicular fluid (GCF) levels of IL-18 from inflamed periodontal sites. Fourteen patients with periodontal disease were included, being 9 patients with chronic periodontitis (mean age: 48.8 SD ± 7.4 years) and 5 patients with gingivitis (mean age: 43.6 SD ± 11.8). The patients were divided in the following groups: gingivitis sites from periodontitis patients (sites GP), periodontitis sites from periodontitis patients (sites PP), and gingivitis sites from gingivitis patients (sites GG). Probing pocket depth (PPD), probing attachment level (AL), plaque index (PI) and gingival index (GI) were recorded, and gingival fluid samples were collected. The subjects received non-surgical treatment and were re-evaluated 30 days after treatment (day 30 AT). There was a significant reduction in PI in GG (1.0 ± 0.4 to 0.5 ± 0.2), GP (1.2 ± 0.3 to 0.5 ± 0.3), and in PP (1.3 ± 0.4 to 0.7 ± 0.3) 30 AT. There was also a significant reduction in the GI in GG (1.3 ± 0.3 to 0.7 ± 0.4). PPD reduced significantly in GG (2.4 ± 0.6 to 1.9 ± 0.1), and PP (6.7 ± 1.1 to 5.2 ± 0.9) 30 AT. When all the samples were analyzed together, there was a significant reduction in IL-18 (12.9 ± 7.2 to 10.0 ± 3.1). This study showed that non-surgical treatment was effective in reducing GCF levels of IL-18 from inflamed periodontal sites.

O objetivo desse estudo foi avaliar o efeito do tratamento periodontal não-cirúrgico sobre os níveis da IL-18 em sítios inflamados de pacientes com doença periodontal. Foram avaliados 14 pacientes com doença periodontal, sendo 9 pacientes com periodontite crônica generalizada (idade média: 48,8 DP ± 7,4 anos) e 5 pacientes com gengivite (idade média: 43,6 DP ± 11,8 anos). Os pacientes foram divididos nos seguintes grupos: sítios sem perda de inserção nos pacientes com periodontite (GP), sítios com perda de inserção nos pacientes com periodontite (PP) e sítios sem perda de inserção nos pacientes com gengivite (GG). Profundidade de bolsa (PB), nível de inserção (NI), índice de placa (IP) e índice gengival (IG) foram avaliados e amostras do fluido gengival foram coletadas. Os pacientes receberam terapia não-cirúrgica e foram reavaliados 30 dias após tratamento (AT). Houve uma redução significante no IP dos GG (1,0 ± 0,4 para 0,5 ± 0,2), GP (1,2 ± 0,3 para 0,5 ± 0,3) e PP (1,3 ± 0,4 para 0,7 ± 0,3) 30 AT. Também houve uma redução significante no IG do GG (1,3 ± 0,3 para 0,7 ± 0,4). A profundidade de bolsa reduziu no GG (2,4 ± 0,6 para 1,9 ± 0,1) e no PP (6,7 ± 1,1 para 5,2 ± 0,9) 30 AT. Quando todas as amostras foram analisadas juntas, houve uma redução significante do IL-18 (12,9 ± 7,2 para 10,0 ± 3,1). Esse estudo mostrou que a terapia periodontal não-cirúrgica é eficaz em reduzir os níveis de IL-18 no fluido gengival de sítios inflamados.

Asociación entre niveles de TNF-a en fluido crevicular gingival de dientes con periodontitis apical asintomática / Levels of TNF-a increase in gingival crevicular fluid of teeth with asymptomatic apical periodontitis

Introducción: Como consecuencia de necrosis pulpar séptica, el ingreso de productos bacterianos en el periápice induce la producción de citoquinas pro-inflamatorias como el Factor de Necrosis Tumoral (TNF)- a, que entre otras funciones participa en la diferenciación y activación de los osteoclastos para inducir reabsorción ósea, fenómeno característico de la periodontitis apical asintomática (PAA). El fluido crevicular gingival (FCG) ofrece un gran potencial como fuente de factores asociados con la actividad osteoclástica. El objetivo del presente estudio fue determinar los niveles de TNF- a en FCG de dientes con PAA y controles sanos contralaterales. Métodos: Se incluyeron 14 pacientes en la Clínica de Endodoncia de la Facultad de Odontología de la Universidad de Chile con diagnóstico clínico de PAA y se obtuvieron muestras de FCG con tiras de papel a partir de los dientes afectados y sanos contralaterales por un periodo estandarizado de 30 segundos. Se determinaron las concentraciones de proteínas totales mediante el método del ácido bisciconitico y los niveles de TNF- a, mediante ensayo ELISA. Los datos se analizaron con Test-t pareado utilizando el programa StataV11. Resultados: Se detectaron niveles de TNF- a significativamente mayores en el FCG de dientes con PAA estandarizados, tanto por 30 segundos de toma de muestra como por mg de proteínas totales. Conclusiones: Este estudio provee evidencia preliminar de que los niveles de TNF- a en el FCG reflejan la presencia de PAA y podría ser de utilidad como complemento al diagnóstico clínico y monitoreo del estado de salud o enfermedad de los tejidos perirradiculares.

Introduction: As a consequence of septic pulp necrosis, the entry of bacterial products into periapical tissues induces the release of pro-inflammatory cytokines, such as the tumor necrosis factor (TNF)- a. This pleiotropic cytokine is involved in the differentiation and activation of osteoclasts to induce bone resorption, a hallmark of asymptomatic apical periodontitis (AAP). Gingival crevicular fluid (GCF) has a great potential as a source of factors associated with osteoclastic activity. The aim of this study was to determine the levels of TNF- a in GCF of teeth with PAA and contralateral healthy controls. Methods: A total of 14 patients with clinical diagnosis of AAP were enrolled from the Clinic of Endodontics, Faculty of Dentistry, Universidad de Chile during 2009 and 30-second GCF samples were obtained with paper strips from AAP teeth and contralateral healthy controls. Total protein concentration and TNF- a levels were determined through bisciconitic acid method and ELISA assay, respectively. Paired t test and StataV11 software were used for statistical analysis. Results: Levels of TNF- a were significantly higher in GCF from teeth with AAP than controls when standardized by either 30s of sampling and total protein content. Conclusions: The present study provides preliminary data supporting that TNF- a levels in GCF reflect periapical status. Screening of TNF- a levels in GCF might represent a useful side-diagnostic tool to the monitoring of the apical status.

Efeito do tratamento periodontal não cirúrgico em pacientes com periodontite crônica e agressiva: achados microbiológicos e imunológicos / Effect of non surgical periodontal treatment in chronic and aggressive subjects: microbiological and immunological findings

O objetivo do presente estudo foi comparar a expressão de IL-1β, IL-4, IL-8, interferon-γ, atividade de elastase e a composição do perfil microbiano subgengival antes e depois do tratamento periodontal não cirúrgico em pacientes com doença peridontal crônica generalizada (PC) e agressiva generalizada (PA). Vinte pacientes com PC e quatorze com PA foram avaliados. Dados clínicos, fluido gengival e biofilme subgengival foram analisados na visita inicial (VI) e 3 meses (3M) após o tratamento periodontal não cirúrgico. Amostras de fluido gengival (FG) foram coletadas com tiras de papel e os níveis de: IL-1β, IL-4, IL-8 e INF-γ foram medidos, utilizando um tipo de imunoensaio multiplexado (Luminex). Atividade da elastase foi avaliada por um ensaio enzimático. Amostras de placa subgengival foram analisadas através do checkerboard DNA-DNA hybridization. Na avaliação de 3 meses após terapia periodontal foi encontrado melhora significativa para todos os parâmetros clínicos em ambos os grupos. Foram encontradas reduções significativas na atividade de elastase nos sítios rasos e profundos dos pacientes do grupo PA e nos sítios profundos do grupo PC, também foi achado um aumento significativo de INF-γ nos sítios rasos do grupo PA. Os dados microbiológicos, mostraram reduções significativas para os níveis dos membros do complexo vermelho (P. gingivalis, T. forsythia, T.denticola), e para as espécies E.nodatum e P.micra no grupo PC. No grupo PA, ocorreram reduções significativas no níveis de P. gingivalis, T. forsythia, Fusobacterium nucleatum ss polymorphum e Fusobacterium periodonticum. Quando as respostas clínica e imunológica 3M após terapia foram comparadas entre os grupos, apenas diferenças sutis foram observadas. Nenhuma diferença microbiológica foi encontrada entre os grupos após a terapia. Em conclusão, os achados suportam nossa hipótese de que as periodontites cronica e agressiva respondem de forma semelhante ao tratamento periodontal nao cirúrgico.

Our aim was to compare the expression of IL1β, IL-4, IL-8, INF-γ, elastase activity and the composition of the subgingival microbiota profile before and after non-surgical periodontal treatment in patients with untreated generalized chronic (GCP) and aggressive periodontitis (GAgP). Twenty patients with GCP and 14 with GAgP were evaluated. Clinical data, GCF and plaque were analyzed at baseline and 3 months after non-surgical periodontal treatment. IL-1β, IL-4, IL-8 and INFγ were analyzed in a luminex assay. Elastase activity was assessed by an enzymatic assay. Subgingival plaque samples were analyzed using checkerboard DNA-DNA hybridization. Three months after periodontal therapy significant improvement for all clinical parameters in both groups were found. We have found significant reductions in the elastase activity in shallow and deep sites from GAgP and in deep sites from GCP group. A significant increase in INF-γ in the shallow sites from GAgP group were also found. Microbiological data showed significant reductions in the levels of members of the red complex (P. gingivalis, T. forsythia, T. denticola), and for the species E.nodatum and P.micra on GCP group. In the GAgP group, there were significant reductions in levels of P. gingivalis, T. forsythia, Fusobacterium nucleatum and Fusobacterium polymorphum ss periodonticum. When the clinical and immunological responses after therapy were compared between groups, only slight differences were found. No microbiological difference was found between groups after therapy. In conclusion, the findings support our hypothesis that the chronic and aggressive periodontitis respond equally well to non-surgical periodontal treatment.