Serotype b of Aggregatibacter actinomycetemcomitans triggers pro-inflammatory responses and amyloid beta secretion in hippocampal cells: a novel link between periodontitis and Alzheimer´s disease?

Introduction: Previous reports have proposed that Periodontal disease (PDis) predisposes to Alzheimer's disease (AD), both highly prevalent pathologies among the elderly. The bacteria Aggregatibacter actinomycetemcomitans (Aa), associated with the most aggressive forms of PDis, are classified in different serotypes with distinct virulence according to the antigenicity of their lipopolysaccharide (LPS). Methods: Here, we determined the effects of purified LPS, from serotypes a, b or c of Aa, on primary cultures of microglia or mixed hippocampal cells. Results: We found that both culture types exhibited higher levels of inflammatory cytokines (IL-1ß, IL-6 and TNFα) when treated with serotype b-LPS, compared with controls, as quantified by qPCR and/or ELISA. Also, cultures treated with serotype a-LPS displayed increased mRNA levels of the modulatory cytokines IL-4 and IL-10. Mixed hippocampal cultures treated with serotype b-LPS exhibited severe neuronal morphological changes and displayed increased levels of secreted Aß1-42 peptide. These results indicate that LPS from different Aa serotypes triggers discriminatory immune responses, which differentially affect primary hippocampal cells. Conclusion: Altogether, our results show that treatment with serotype b-LPS triggers the secretion of proinflammatory cytokines by microglia, induces neurite shrinking, and increases the extracellular Aß1-42 levels, all features strongly associated with the etiology of AD.

Six-month Follow-up of the Effect of Nonvital Bleaching on IL-1ß and RANK-L: A Randomized Clinical Trial.

OBJECTIVES: It has been reported that bleaching generates an increase in the activity of osteoclasts in vitro. We quantified the RANK-L and IL-1ß biomarkers in a double-blind, randomized clinical trial evaluating the in vivo effect of hydrogen peroxide (35%) and peroxide carbamide (37%) six months after whitening. METHODS AND MATERIALS: Fifty volunteers participated, each with color change in a nonvital tooth. Fifty teeth were randomly divided into two groups (n=25), and the teeth were bleached using either 35% hydrogen peroxide (G1) or 37% carbamide peroxide (G2). Intracoronal bleaching was carried out by a technical "walking bleach" over four sessions. Gingival crevicular fluid samples were collected and used to quantify the IL-1ß and RANK-L secreted levels. Samples of six periodontal sites (three vestibular and three palatal) were collected for up to six months (at the beginning of the study [baseline] and at one week, one month, and six months posttreatment). The color change was visually monitored using the Vita Bleached Guide (ΔSGU). RESULTS: Comparing each time to baseline assessment, a significant increase in the levels of IL-1ß and RANK-L across time points was detected (p<0.05). The color change was 4 in G1 and G2, and a statistically significant difference (p<0.05) was found at the month time point between the groups. Using the Spearman test, a strong correlation (>0.8) between the IL-1ß and RANK-L levels in both groups at all time points was detected. CONCLUSIONS: Nonvital bleaching using a technical walking bleach induces an increase in the IL-1ß and RANKL production in periodontal tissues, which persists for six months after treatment. Both biomarkers were highly correlated in both groups and at all time points.

Th1/Th17/Th22 immune response and their association with joint pain, imagenological bone loss, RANKL expression and osteoclast activity in temporomandibular joint osteoarthritis: A preliminary report.

It is well accepted that the presence of cytokines belonging to the Th1/Th17/Th22 axis of immuno-inflammatory response in the joint environment, such as IL-1ß, IL-17 and IL-22, respectively, are associated with pathogenesis of several synovial joint degenerative disorders. During temporomandibular joint osteoarthritis (TMJ-OA), IL-1ß and IL-17 have been implicated in the inflammation and resorption of sub-chondral bone; however, the role of Th22 response in the TMJ-OA pathophysiology has not been established. This study aimed to compare the expression of Th1/Th17/Th22-type cytokines, chemokines and chemokine receptors in synovial fluid samples obtained from TMJ-OA or disk displacement with reduction (DDWR) patients. In addition, it aimed to associate these levels with joint pain, imagenological signs of bone degeneration, RANKL production, osteoclastogenesis and osteoclast-induced bone resorption. Higher levels of IL-1ß, IL-17 and IL-22 were expressed in TMJ-OA compared with DDWR subjects, and these increased levels significantly correlated with RANKL expression, joint pain and articular bone degeneration. Higher levels of CCR5, CCR6 and CCR7, as well as their respective ligands CCL5 and CCL20, responsible for recruitment of IL-1ß, IL-17 and IL-22-producing cells, were over-expressed in TMJ-OA compared with DDWR subjects. Osteoclastogenesis and osteoclast-induced bone resorption were significantly greater in presence of synovial fluid from TMJ-OA compared with DDWR subjects. These data demonstrate that cytokines, CCLs and CCRs associated with the Th1/Th17/Th22 axis of immuno-inflammatory response are involved in TMJ-OA pathogenesis. These findings suggest that IL-22 is involved in the RANKL expression in TMJ-OA, which in turn induces differentiation of osteoclasts and subsequent resorption of sub-chondral bone.

Does the Use of a "Walking Bleaching" Technique Increase Bone Resorption Markers?

OBJECTIVE: This randomized clinical trial evaluated the effect of 35% hydrogen peroxide in comparison with 37% carbamide peroxide in a nonvital bleaching technique of "walking bleaching" (four sessions of treatment) on periodontal markers: nuclear factor kappa B-ligand (RANK-L-process of root resorption marker) and interleukin 1ß (IL-1ß-inflammatory response marker). METHODS AND MATERIALS: Fifty volunteers presenting with discoloration of nonvital teeth and endodontic treatment in good condition participated. Fifty teeth were randomly divided into two study groups according to bleaching gel: HP = 35% hydrogen peroxide (n=25) and 37% carbamide peroxide (n=25). Nonvital bleaching was performed with a walking bleaching technique consisting of four sessions of bleach application. Gingival crevicular fluid samples were taken in order to quantify the RANK-L and IL-1ß levels by enzyme-linked immunosorbent assay. Samples were obtained from six periodontal sites for each bleached tooth: three vestibular and three palatine (mesial, middle, and distal) at seven time periods: baseline, after each of the four sessions of nonvital bleaching, at one week, and at one month after nonvital bleaching. Tooth color variations were analyzed in each session by VITA Bleachedguide 3D-MASTER (ΔSGU). RESULTS: Significant increments in the RANK-L and IL-1ß levels were detected in each evaluated time compared with baseline ( p<0.05); however, no differences were detected between hydrogen peroxide and carbamide peroxide on increments of the biomarkers studied. The change of color was effective for both nonvital bleaching therapies ( p<0.05). CONCLUSIONS: Nonvital bleaching induced a significant increment in the RANK-L and IL-1ß levels in periodontal tissues around bleached, nonvital teeth.

Human periodontal ligament fibroblasts synthesize C-reactive protein and Th-related cytokines in response to interleukin (IL)-6 trans-signalling.

AIM: To characterize the potential of human periodontal ligament fibroblasts (HPLF) to synthesize CRP and Th-related cytokines in response to IL-6 in periodontal health and apical inflammation. METHODOLOGY: Primary HPLF stimulated with IL-6, soluble(s) IL-6 receptor (R) and controls were assayed for CRP, Th1, Th2, Th17 and Treg-related cytokines by quantitative real-time PCR and ELISA, respectively. IL-6R mRNA expression and its soluble protein levels were screened in HPLF cultures, and ex vivo samples of healthy periodontal ligaments (n = 5) and apical lesions (n = 13). Data were analysed with ANOVA or unpaired t-test. RESULTS: 0.5 ng mL-1 IL-6 plus 1 ng mL-1 of its soluble receptor (sIL-6R) for 24 h was effective in inducing CRP production. IL-6 alone had a mild dose-dependent effect; co-stimulation with sIL-6R significantly enhanced this effect, whereas it was completely abolished by the addition of IL-6R blocking antibody (P < 0.05). Similarly, higher mRNA expression and protein levels of Th1, Th17 and partially Treg-related cytokines were found for IL-6 combined with its soluble receptor versus the nonstimulated group and IL-6R antibody (P < 0.05). IL-6R mRNA expression was slightly induced by IL-6 compared to THP-1 cells, but sILR-6 protein could not be detected in HPLF. High sIL-6R levels were detected in apical lesions and were immunolocalized to mononuclear inflammatory cells and proliferating epithelium. CONCLUSION: IL-6 trans-signalling induced Th1 and Th17-related cytokines and represents an extra-hepatic mechanism for PCR synthesis in human periodontal ligament fibroblasts, contributing to explain the bone-destructive phenotype of apical lesions and eventually its systemic complications.

Effectiveness and Impact of the Walking Bleach Technique on Esthetic Self-perception and Psychosocial Factors: A Randomized Double-blind Clinical Trial.

OBJECTIVE: This trial evaluates the impact of psychosocial and esthetic self-perceptions of patients undergoing nonvital tooth bleaching using the walking bleach technique. We also assessed the clinical effectiveness of bleaching tooth discoloration. METHODS: Fifty volunteers with nonvital tooth discoloration were enrolled. Teeth were randomized into two groups: 35% hydrogen peroxide (n=25) and 37% carbamide peroxide (n=25). Intracoronal bleaching was performed over four sessions using the walking bleach technique. Tooth color was evaluated at each session to measure total color variation. The shade guide was arranged from highest (B1) to lowest (C4) values to assess the color and calculate the color change in the number of shade guide units. Subjective and objective assessments were compared with the tooth counterpart. Esthetic self-perception and psychosocial factors were assessed before and after treatment. RESULTS: Color change was 15.48<5.17 for hydrogen peroxide and 14.02<4.85 for carbamide peroxide. There was no significant difference at any time point (p>0.05) except at sessions 3 and 4 (p<0.05). Overall, whitened teeth values were similar to those of counterpart teeth (p>0.05). There was a decrease in Oral Health Impact Profile and Psychosocial Impact of Dental Esthetics questionnaire scores after treatment compared with baseline (p<0.05). CONCLUSION: The walking bleach technique was highly effective on nonvital teeth and had a positive effect on self-esthetic perception and psychological impact for the patients.

Increased levels of the T-helper 22-associated cytokine (interleukin-22) and transcription factor (aryl hydrocarbon receptor) in patients with periodontitis are associated with osteoclast resorptive activity and severity of the disease.

BACKGROUND AND OBJECTIVE: Two new T-helper (Th) phenotypes have been recently described and named Th9 and Th22 lymphocytes; however, their role in the pathogenesis of periodontitis remains unclear. This study was aimed to assess whether Th9 and Th22 lymphocytes, through interleukin (IL)-9 and IL-22 production, respectively, are associated with the severity of periodontitis and bone resorption. MATERIAL AND METHODS: Gingival crevicular fluid samples and biopsies were obtained from patients with moderate-to-advanced chronic periodontitis and gingivitis, and healthy controls. The levels for the Th9 and Th22-associated cytokines and master-switch transcription factors Spi-B and aryl hydrocarbon receptor (AhR) were quantified by enzyme-linked immunosorbent assay, real-time reverse-transcription quantitative polymerase chain reaction and flow cytometry. In addition, the osteoclast activity in response to tissue homogenates from periodontitis and healthy samples was analyzed quantifying the number of TRAP-positive cells and areas of bone resorption pits produced, in the presence or absence of recombinant human IL-22 and anti-IL-22 neutralization antibody. RESULTS: Higher levels of IL-22 and AhR were detected in patients with periodontitis compared with gingivitis and healthy individuals. In addition, higher levels of IL-9 and Spi-B were detected in gingivitis patients compared with periodontitis and healthy individuals. In patients with periodontitis, a significant positive correlation was detected between secreted levels of IL-22 and clinical attachment level of the sampled periodontal pockets. When osteoclasts were exposed to tissue homogenates obtained from patients with periodontitis, higher levels of resorptive activity were observed as compared with the same cells exposed to tissue homogenates obtained from healthy individuals, and this increment was dependent on the presence and neutralization of IL-22. CONCLUSION: Increased levels of IL-22 produced by Th22 lymphocytes are associated with the pathogenesis of periodontitis, in particular, with osteoclast resorptive activity and severity of disease.

Bone resorptive activity in symptomatic and asymptomatic apical lesions of endodontic origin.

OBJECTIVES: The aim of this study is to assess the levels and diagnostic accuracy of a set of bone resorption biomarkers, including TRAP-5, RANKL, and OPG in symptomatic and asymptomatic apical lesions and controls. MATERIALS AND METHODS: Apical tissues from symptomatic and asymptomatic apical periodontitis patients and periodontal ligaments from healthy teeth extracted for orthodontic reasons were processed for tissue homogenization and the levels of TRAP-5, RANKL, and OPG were determined by multiplex assay. Marker levels were analyzed by Kruskal Wallis test, and diagnostic accuracy was analyzed with ROC curves. RESULTS: Higher levels of RANKL, OPG, and RANKL/OPG ratio were determined in both types of apical lesions compared to healthy periodontal ligament, whereas higher TRAP-5 levels were found only in symptomatic apical lesions (p < 0.05). OPG, RANKL, and RANKL/OPG ratio showed diagnostic potential to identify apical lesions versus healthy controls (AUC = 0.69, p < 0.05); while TRAP-5 showed a potential to discriminate symptomatic versus asymptomatic apical periodontitis (AUC = 0.71, p < 0.05) and healthy controls (AUC = 0.83, p < 0.05). CONCLUSIONS: Apical lesions showed higher RANKL and OPG levels than healthy tissues. TRAP-5 levels were the highest in symptomatic apical lesions, suggesting that these represent a progressive state, and showed diagnostic potential. CLINICAL RELEVANCE: Clinically symptomatic apical periodontitis might represent biologically progressive apical lesions based on TRAP5 levels. TRAP5 has diagnostic potential to identify these lesions, representing a candidate prognostic biomarker.

The Porphyromonas gingivalis O antigen is required for inhibition of apoptosis in gingival epithelial cells following bacterial infection.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis infection induces apoptosis inhibition in gingival epithelial cells; however, it is not fully understood which bacterial effectors are involved in this process. The aim of this study is to evaluate whether the P. gingivalis lipopolysaccharide (LPS), specifically the O-antigen region, affects adherence, invasion, viability and apoptosis of gingival epithelial cells. MATERIAL AND METHODS: Gingival epithelial cells (OKF6/TERT2 line) were infected by different freshly prepared P. gingivalis clinical isolates, obtained from subjects with chronic periodontitis (CP3 and CP4) and healthy individuals (H1 and H3). Periodontitis and healthy isolates show differences in O-antigen production, as healthy isolates lack the O-antigen region. In addition, cells were infected by a site-specific mutant lacking the O-antigen portion. After 24 h postinfection, cell proliferation, viability and apoptosis were evaluated by Trypan blue, MTS and annexin V assays, respectively. Bacterial invasion, adhesion and proliferation were measured by gentamicin/metronidazole protection assays. Finally, toll-like receptor (TLR)2 and TLR4 mRNA expression was evaluated by quantitative reverse transcription-polymerase chain reaction. Statistical analysis was performed using ANOVA, Tukey's or Dunnett's tests (p < 0.05). RESULTS: At 24 h postinfection, strains lacking the O-antigen region (healthy isolates and O-antigen ligase-deficient strain) were unable to increase proliferation and viability, or decrease apoptosis as compared with strains producing intact LPS (periodontitis isolates and reference strain). However, the presence of the O-antigen neither contributed to changes in the ability of the bacteria to adhere to or invade cells, nor to intracellular survival. The presence of O-antigen also increased the expression of TLR4 (nearly sixfold), which correlated with inhibition of apoptosis. CONCLUSION: The O-antigen region of P. gingivalis LPS is required to increase gingival epithelial cell viability upon infection by bacteria and this increase is attributable to a reduction in apoptosis. Moreover, although bacterial internalization is required, the effects observed are not due to alterations in P. gingivalis adherence, invasion or intracellular survival. Interestingly, inhibition of apoptosis correlates with increased TLR4 expression, suggesting a role for TLR4 in this process.

Serotype b of Aggregatibacter actinomycetemcomitans increases osteoclast and memory T-lymphocyte activation.

During periodontitis, alveolar bone resorption is associated with activation of T helper type 17 (Th17) lymphocytes and receptor activator of nuclear factor-κB ligand (RANKL) -induced osteoclasts. We previously reported that serotype b of Aggregatibacter actinomycetemcomitans has a higher capacity to trigger Th17-type differentiation and function in activated T lymphocytes and its lipopolysaccharide is a more potent immunogen compared with the other serotypes. This study aimed to investigate whether serotype b of A. actinomycetemcomitans induces higher Th17-associated RANKL production, RANKL-induced osteoclast activation, and antigen-specific memory T lymphocyte proliferation. On naive CD4(+) T lymphocytes stimulated with autologous dendritic cells primed with different A. actinomycetemcomitans serotypes, RANKL production, T-bet, GATA-3, RORC2 and Foxp3 expression, RORC2/RANKL intracellular double-expression, TRAP(+) osteoclast activation, and bone resorption were quantified. The frequency of proliferating memory T lymphocytes in response to A. actinomycetemcomitans serotypes was determined in periodontitis and healthy subjects. Naive CD4(+) T lymphocytes stimulated by serotype b-primed dendritic cells elicited higher levels of RANKL, RORC2, TRAP(+) osteoclasts, and bone resorption than the same cells stimulated with the other serotypes. RANKL positively correlated and co-expressed with RORC2. Memory T lymphocytes responding to serotype b were more frequently detected in periodontitis patients than healthy subjects. These results indicate that serotype b of A. actinomycetemcomitans is associated with higher production of RANKL and these increased levels are associated with Th17 lymphocyte induction, osteoclast activation, and bone resorption.

H2 O2 activates matrix metalloproteinases through the nuclear factor kappa B pathway and Ca(2+) signals in human periodontal fibroblasts.

BACKGROUND: The mechanisms involved in reactive oxygen species and matrix metalloproteinase (MMP)-mediated periodontal tissue breakdown are unknown. OBJECTIVE: To determine the effect of H2 O2 in MMP-2 and MMP-9 activity, and the involvement of nuclear factor kappa B (NFκB) and Ca(2+) -mediated signals in human periodontal ligament fibroblasts. MATERIAL AND METHODS: Primary cultures were characterized for their phenotype and exposed for 24 h to sublethal doses (2.5-10 µm) of H2 O2 or control media. NFκB involvement was evaluated through immunofluorescence of p65 subunit, using the NFκB blocking peptide SN50 and catalase. Ca(2+) signals were analyzed by loading the cells with Fluo4-AM and recording the fluorescence changes in a confocal microscope before and after the addition of H2 O2 . 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl was used to chelate intracellular Ca(2+) . The activity and levels of MMP-2 and MMP-9 were analyzed by gelatin zymogram and densitometric scanning, and enzyme-linked immunosorbent assay, respectively. Statistical analysis was performed with stata V11.1 software using the ANOVA test. RESULTS: H2 O2 at concentrations of 2.5-5 µm induced Ca(2+) signaling and NFκB subunit p65 nuclear translocation, whereas catalase, SN50 and BAPTA-AM prevented p65 nuclear translocation. H2 O2 at 2.5-5 µm significantly increased MMP-9 and MMP-2 activity, while SN50 resulted in lower MMP-2 and MMP-9 activity rates compared with controls. CONCLUSION: Sublethal H2 O2 induces Ca(2+) -dependent NFκB signaling with an increase in MMP gelatinolytic activity in human periodontal ligament.

T-lymphocyte phenotype and function triggered by Aggregatibacter actinomycetemcomitans is serotype-dependent.

BACKGROUND AND OBJECTIVE: Based on lipopolysaccharide (LPS) antigenicity, different Aggregatibacter actinomycetemcomitans serotypes have been described. Serotype b strains have demonstrated a stronger capacity to trigger cytokine production on dendritic cells (DCs). As DCs regulate the development of T-lymphocyte lineages, the objective of this investigation was to study the response of T lymphocytes after being stimulated with autologous DCs primed with different bacterial strains belonging to the most prevalent serotypes of A. actinomycetemcomitans in humans: a-c. MATERIAL AND METHODS: Human DCs were primed with increasing multiplicity of infection (10(-1) -10(2) ) or the purified LPS (10-50 ng/mL) of A. actinomycetemcomitans serotypes a-c and then used to stimulate autologous naïve CD4(+) T lymphocytes. The T-helper (Th) type 1, Th2, Th17 and T-regulatory transcription factors T-bet, GATA-3, RORC2 and Foxp3, which are the master-switch genes implied in their specific differentiation, as well as T-cell phenotype-specific cytokine patterns were quantified by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, the intracellular expression of T-bet/interferon-γ, GATA-3/interleukin-4, RORC2/interleukin-17A and Foxp3/transforming growth factor-ß1 was analysed by double staining and flow cytometry. RESULTS: All the A. actinomycetemcomitans serotypes led to T-lymphocyte activation; however, when T lymphocytes were stimulated with DCs primed with the A. actinomycetemcomitans serotype b strain or their purified LPS, higher levels of Th1- and Th17-associated transcription factors and cytokines were detected compared with similar experiments with the other serotypes. CONCLUSION: These results demonstrate that serotype b of A. actinomycetemcomitans has a higher capacity of trigger Th1 and Th17 phenotype and function and it was demonstrated that their LPS is a more potent immunogen compared with the other serotypes.

Variabilidad de la síntesis de citoquinas por células dendríticas humanas estimuladas con los distintos serotipos de Aggregatibacter actinomycetecomitans / Variability in the cytokine synthesis by human dendritic cells in response to different Aggregatibacter actinomycetemcomitans serotypes

Objetivo: Sobre la base de la antigenicidad del polisacárido O del LPS, en A. actinomycetemcomitans se describen distintos serotipos bacterianos y entre ellos se ha especulado una patogenicidad e inmunogenicidad diferente. El objetivo de este trabajo es analizar las diferencias en la síntesis de citoquinas producidas por células dendríticas cuando son estimuladas con los distintos serotipos de A. actinomycetemcomitans. Metodología: Células dendríticas diferenciadas a partir de monocitos circulantes periféricos humanos fueron estimuladas a MOIs=10-1-10-2 con los serotipos a, b y c de A. Actinomycetemcomitans. Mediante PCR y ELISA se evaluaron los niveles de expresión y secreción de citoquinas. Resultados: En las células dendríticas, la producción de citoquinas fue diferente ante los distintos serotipos de A. actinomycetemcomitans, con mayores niveles de secreción de IL-1beta, IL-6, IL-12, IL-23, IFN-gamma y TNF-alfa cuando el microorganismo estimulante fue la cepa ATCC® 43718™ (serotipo b). Conclusión: El serotipo b de A. actinomycetemcomitans posee un mayor potencial inmuno-estimulador de células dendríticas comparado con los otros serotipos bacterianos y potencialmente contribuiría a inducir un patrón de respuesta inmune tipo Th1 y/o Th17 durante las periodontitis.

Objective: A. actinomycetemcomitans expresses a number of virulence factors that contribute to direct tissue damage and, based on the antigenicity of LPS O-polysaccharide, distinct serotypes have been described. The aim of this study was to determine the pattern of cytokine expression and secretion on dendritic cells stimulated with A. actinomycetemcomitans serotypes a, b and c. Methods: Using different multiplicity of infections of the serotypes a, b, and c of A. actinomycetemcomitans, the mRNA expression and secretion levels for cytokines IL-1beta, IL-5, IL-6, IL-10, IL-12, IL-23, TNF-alpha, and IFN-gamma were determined in stimulated dendritic cells using PCR and ELISA. Results: A dose-dependent increase in the secretion levels for IL-1beta, IL-5, IL-6, IL-10, IL-12, IL-23, TNF-alpha, and IFN-gamma was elicited on dendritic cells following stimulation with each of the serotypes of A. actinomycetemcomitans. In addition, A. actinomycetemcomitans serotype b (ATCC® 43718™) induced higher levels of IL-1beta, IL-6, IL-12, IL-23, IFN-gamma y TNF-alpha compared with the other strains. Conclusion: These data demonstrate that the distinct A. actinomycetemcomitans LPS O-polysaccharide serotypes induce both quantitative and qualitative differences in the dendritic cell response. Furthermore, the observed dendritic cell response to A. actinomycetemcomitans b serotype was characteristic of a Th1 and Th17 pattern of cytokine expression.

Host-pathogen interactions in progressive chronic periodontitis.

Periodontitis is an infection characterized by the occurrence of supporting tissue destruction with an episodic nature. Disease progression is often determined by the loss of attachment level or alveolar bone, and sequential probing of periodontal attachment remains the most commonly utilized method to diagnose progressive destruction of the periodontium. The tolerance method has been the most extensive clinical method used in recent years to determine site-specific attachment level changes. There is abundant evidence that major tissue destruction in periodontal lesions results from the recruitment of immune cells. Considerable effort has been made to study the host cell and mediator profiles involved in the pathogenesis of chronic periodontitis, but the definition of active sites, where current periodontal breakdown occurs, and consecutive characterization of the mediators involved are still among the main concerns. In the present review, we summarize periodontopathic bacteria and host factors, including infiltrating cell populations, cytokines, and host matrix metalloproteinases, associated with under-going episodic attachment loss that could partly explain the mechanisms involved in destruction of the supporting tissues of the tooth.

Variabilidad de la síntesis de RANKL por linfocitos T frente a distintos serotipos capsulares de Porphyromonas gingivalis / Variability in the RANKL synthesis by T Lymphocytes in response to different Porphyromonas gingivalis capsular serotypes

Propósito: Las periodontitis representan un grupo heterogéneo de infecciones periodontales cuya etiología son las bacterias residentes en el biofilm subgingival. Aunque este biofilm está constituido por una amplia variedad de especies bacterianas, sólo un número limitado de especies, como Porphyromonas gingivalis, se ha asociado a la etiología de la enfermedad. P. gingivalis expresa diversos factores de virulencia que pueden causar daño directo a los tejidos del hospedero; sin embargo, su mayor patogenicidad involucra la inducción de una respuesta inmuno-inflamatoria, durante la cual se secretan una amplia variedad de citoquinas, quimioquinas y mediadores inflamatorios que pueden inducir la destrucción de los tejidos de soporte de los dientes y la pérdida de ellos. Método: En esta investigación, se evaluó si los distintos serotipos capsulares (K) de P. gingivalis pueden determinar los niveles de síntesis de RANKL, citoquina clave en la destrucción del hueso alveolar durante la periodontitis. Para ello, se cuantificaron los niveles de expresión de RANKL mediante PCR cuantitativa y los niveles de secreción mediante ELISA en linfocitos T activados en presencia de los serotipos capsulares K1-K6 de P. gingivalis, y estos se correlacionaron a los niveles de expresión de los factores de transcripción asociados a cada uno de los fenotipos de linfocitos efectores: Th1 (T-bet), Th2 (GATA-3), Th17 (RORC2) y Treg (Foxp3). Resultados: Mayores niveles de expresión y secreción de RANKL fueron detectados en linfocitos T activados en presencia de los serotipos K1 y K2 de P. gingivalis, en comparación a los detectados ante los otros serotipos. Además, estos mayores niveles de RANKL se correlacionaron positivamente con los niveles de expresión de RORC2. Conclusión: Estos datos demuestran que la síntesis de RANKL por linfocitos T se restringe a ciertos serotipos capsulares de P. gingivalis (K1 y K2) y permiten sugerir que los serotipos K1 y K2 de P. gingivalis podrían asociarse...

Aim: Periodontitis represents a heterogenic group of periodontal infections elicited by bacteria residing at the subgingival biofilm. Although this biofilm is constituted by a broad variety of bacterial species, only a limited number has been associated with the periodontitis aetiology, among them Porphyromonas gingivalis. P. gingivalis express a number of virulence factors that contribute to direct tissue damage; however, their pathogenicity relies mainly on the induction of a host immuno-inflammatory response. This leads to the release of a broad array of cytokines, chemokines and inflammatory mediators, which cause destruction of the tooth-supporting alveolar bone and ultimately tooth loss. Method: In the present investigation, in order to determine whether different P. gingivalis serotypes might lead to a differential RANKL synthesis, a key cytokine involved in alveolar bone resorption, the mRNA expression and secretion of RANKL and the expression of transcription factors T-bet, GATA-3, RORC2 and Foxp3, the master-switch genes controlling the Th1, Th2, Th17, and Treg cell differentiation, respectively, were analyzed on human T cells activated with different P. gingivalis capsular (K) serotypes. Results: T lymphocytes responding to P. gingivalis serotypes K1 or K2, but not to the other serotypes, led to an increased expression and secretion of RANKL. In addition, these higher RANKL levels correlate with RORC2 expression upon activation with K1 or K2 serotypes. Conclusion: These data demonstrated that RANKL expression and secretion by T lymphocytes was restricted to particular P. gingivalis serotypes (namely K1 and K2), and allowed to suggest a link between these serotypes with alveolar bone destruction and teeth loosening during the periodontitis.

An orally active calcium-sensing receptor antagonist that transiently increases plasma concentrations of PTH and stimulates bone formation.

Daily subcutaneous administration of exogenous parathyroid hormone (PTH) promotes bone formation in patients with osteoporosis. Here we describe two novel, short-acting calcium-sensing receptor antagonists (SB-423562 and its orally bioavailable precursor, SB-423557) that elicit transient PTH release from the parathyroid gland in several preclinical species and in humans. In an ovariectomized rat model of bone loss, daily oral administration of SB-423557 promoted bone formation and improved parameters of bone strength at lumbar spine, proximal tibia and midshaft femur. Chronic administration of SB-423557 did not increase parathyroid cell proliferation in rats. In healthy human volunteers, single doses of intravenous SB-423562 and oral SB-423557 elicited transient elevations of endogenous PTH concentrations in a profile similar to that observed with subcutaneously administered PTH. Both agents were well tolerated in humans. Transient increases in serum calcium, an expected effect of increased parathyroid hormone concentrations, were observed post-dose at the higher doses of SB-423557 studied. These data constitute an early proof of principle in humans and provide the basis for further development of this class of compound as a novel, orally administered bone-forming treatment for osteoporosis.

Variability in the response of human dendritic cells stimulated with Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Dendritic cells are able to prime and polarize naïve T cells towards either a T helper 1 or a T helper 2 response, depending on the antigen type and concentration, the costimulatory signals and the local cytokine millieu. In this investigation, we analyzed the response of human dendritic cells to stimulation with different concentrations of Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans. MATERIAL AND METHODS: Using different concentrations of P. gingivalis ATCC33277 and A. actinomycetemcomitans ATCC 33384, we determined the expression of the maturation markers CD80 and CD86 from purified human dendritic cells by flow cytometry. We also evaluated the mRNA expression levels for the cytokines interleukin-1beta, interleukin-2, interleukin-5, interleukin-6, interleukin-10, interleukin-12, interleukin-13, interferon-gamma, tumor necrosis factor-alpha and tumor necrosis factor-beta by quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Both P. gingivalis and A. actinomycetemcomitans led to dendritic cell maturation, but the expression of CD80 was higher when the dendritic cells were stimulated with A. actinomycetemcomitans. Although both pathogens induced a T helper 1 pattern of cytokine expression, A. actinomycetemcomitans-stimulated dendritic cells expressed interleukin-1beta, interleukin-12, interferon-gamma, tumor necrosis factor-alpha and tumor necrosis factor-beta at lower bacterial concentrations than P. gingivalis. While 10(6) bacteria/mL of P. gingivalis or 10(4) bacteria/mL of A. actinomycetemcomitans induced expression of interleukin-12p40, the expression of other cytokines required 10 to 100-fold higher concentrations of bacteria. CONCLUSION: These results demonstrate that A. actinomycetemcomitans is a more potent immunogen than P. gingivalis because, at least in vitro, it induces stronger differentiation and activation of dendritic cells. In addition, our data also show that for a given strain, the bacterial load determines the pattern of cytokines that are expressed.

Stimulatory response of neutrophils from periodontitis patients with periodontal pathogens.

OBJECTIVE: Neutrophils play a crucial role in the defense of invading bacteria by releasing biologically active molecules. The response of peripheral blood neutrophils was studied in periodontitis-affected patients and in healthy controls towards stimulation to Porphyromonas gingivalis (Pg) and Actinobacillus actinomycetemcomitans (Aa) extracts. MATERIALS AND METHODS: Peripheral venous blood was drawn from 23 adult patients with moderate to advanced chronic periodontitis (probing depth >or=5 mm, attachment loss >or=3 mm), and 30 healthy volunteers. Neutrophil response followed by metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8) secretion was assayed by zymography and enzyme-linked immunosorbent assay, respectively, on both whole blood and purified neutrophils. In addition to periodontal pathogen extracts, known stimulating agents were tested, such as Escherichia coli-lipopolysaccharide (LPS), phytohemagglutinin, and zymosan A. RESULTS: Neutrophil response, expressed as a secretion ratio under stimulated and non-stimulated conditions, measured in whole blood, showed no differences between periodontitis and healthy controls. Instead, in purified neutrophils from patients, MMP-9 exhibited a significantly higher secretion ratio with LPS and Pg (1.5- to 2-fold), whereas IL-8 showed a larger increase in secretion ratio (3- to 7-fold) in the presence of Pg, Aa, LPS, and zymosan A. CONCLUSION: Peripheral neutrophils of periodontitis-affected patients are more reactive as suggested by their significantly higher response toward periodontal pathogen extracts and other stimulating agents.

Respuesta inmune Th1 en la osteoartritis de la articulación temporomandibular / Th1 immune response in osteoarthritis of the temporomandibular joint

Propósito. La osteoartritis temporomandibular es una enfermedad articular degenerativa, caracterizada por la destrucción del cartílago y hueso articular consecutiva a la respuesta inflamatoria e inmune desarrollada. En el presente trabajo se evalúa la expresión a nivel de ARN mensajero de diversas citoquinas proinflamatorias en los sinoviocitos articulares de individuos afectados por osteoartritis temporomandibular. Material y métodos. En 12 pacientes afectados de osteoartritis temporomandibular y en 6 sujetos sanos se evaluó la expresión de citoquinas en sinoviocitos de la articulación temporomandibular mediante la técnica de PCR cuantitativa en tiempo real. Resultados. Significativamente mayores niveles de IL-1Beta, IL-2, IL-12, IL-17, TNFalpha, TNFBeta e IFNGamma fueron observados en pacientes afectados de osteoartritis temporomandibular en comparación a sujetos sanos. En los sujetos enfermos la citoquina predominante fue IL-12 y en los sanos fue IL-10. Conclusión. Tomados en conjunto, nuestros datos demuestran una asociación de elevados niveles de citoquinas propias de una respuesta inmune Th1 y la destrucción observada durante la osteoartritis temporomandibular (AU)

Background. Temporomandibular osteoarthritis is a degenerative disease that affects the temporomandibular joint and is characterized by the cartilage and bone destruction caused by the inflammatory and immune response. This communication reports on the expression of proinflammatory cytokines in synovial cells of patients with temporomandibular osteoarthritis. Methods. In twelve temporomandibular osteoarthritis patients and six healthy control subjects, cytokine expression in synovial cells was evaluated by quantitative PCR. Results. Levels of IL-1Beta, IL-2, IL-12, IL-17, TNFalpha, TNFBeta e IFNGamma were significantly higher in synovial cells of patients than in controls. In particular, IL-12 was the predominant cytokine in patients and IL-12 in healthy subjects. Conclusion. Taken together, these data suggest that a Th1 immune response is associated to the destruction observed during the temporomandibular osteoarthritis (AU)

Papel de los linfocitos T CD4+ en la destrucción ósea observada durante la periodontitis crónica / Role of CD4+ T cells in bone destruction during chronic periodontitis

Propósito: La periodontitis crónica es una enfermedad de naturaleza inflamatoria y etiología infecciosa, caracterizada por la destrucción del aparato de inserción del diente, cemento radicular, ligamento periodontal y hueso alveolar, y que causa la pérdida de los dientes. Durante su desarrollo se establece un denso infiltrado inflamatorio celular, constituido principalmente por linfocitos T, con la capacidad de secretar una serie de citoquinas que participan en los eventos patogénicos de la enfermedad, regulando la inflamación de los tejidos periodontales y la destrucción del hueso alveolar. En la artritis reumatoide, la citoquina recientemente identificada RANKL (Ligando del receptor activador del factor nuclear κB) es expresada por los linfocitos T CD4+, participando directamente en los procesos de estimulación de la diferenciación osteoclástica y en la activación de osteoclastos maduros, y por lo tanto, en la destrucción ósea articular. El objetivo del presente estudio es determinar si mayores niveles de RANKL se encuentran asociados a la periodontitis crónica y si estos son sintetizados por los linfocitos T CD4+ reclutados en los sitios con periodontitis crónica. Material y métodos: En 33 pacientes mayores de 35 años de edad afectados de periodontitis crónica y 20 individuos controles sanos, se determinaron los niveles de mensajero de ácido desoxiribonucleico (mARN) de RANKL en biopsias de encía mediante transcriptasa reversa-reacción en cadena de la polimerasa (RT-PCR) en tiempo real. A partir de las mismas biopsias, células gingivales totales fueron aisladas para inmunotipificar y cuantificar los leucocitos infiltrantes gingivales e identificar las células responsables de la expresión de RANKL mediante doble tinción por citometría de flujo. Resultados: Los pacientes con periodontitis mostraron mayores niveles de mARN de RANKL en comparación a individuos sanos, observándose que la expresión de RANKL se incrementó en 238,3 veces con relación a los sujetos controles. Los individuos con periodontitis mostraron mayores porcentajes de linfocitos T CD4+ y CD8+. Además, una asociación entre RANKL y los linfocitos T CD4+ fue determinada mediante doble tinción por citometría de flujo. Conclusión: Estos datos demuestran que mayores niveles de RANKL se encuentran asociados a la periodontitis y que estos mayores niveles se pueden explicar en parte a la actividad de los linfocitos T CD4+ en el sitio de la infección. La determinación de la asociación entre la periodontitis crónica y la síntesis de RANKL constituye un interesante mecanismo molecular que contribuye a explicar en parte la destrucción tisular asociada y permite proyectar posibles nuevas estrategias inmunoterapéuticas que ayudarían a controlar la pérdida de tejido característica de la enfermedad periodontal (AU)

Background: Chronic periodontitis is an infectious disease, characterized by alveolar bone destruction and teeth loss. Receptor activator of nuclear factor kB- ligand (RANKL) is an osteoclastogenic cytokine, central regulatory factor in the osteoclasts life-span and physiological and pathological bone resorption. Gingival T cells synthesize RANKL contributing to molecular local unbalance that entail to the alveolar bone resorption seen in periodontitis. Our study was aimed at associating the levels of RANKL with the CD4+ T cell activity present in gingival tissues of chronic periodontitis patients. Methods: Gingival biopsies were obtained from 33 chronic periodontitis patients and 20 healthy controls. Specimens were either formalin fixed and paraffin embedded for real-time reverse transcription polymerase chain reaction (RT-PCR) and histological analysis, or tissue digestion processed for cell culture and flow cytometry analysis. RANKL mRNA and protein levels were determined by quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) in gingival cells culture supernatants. Gingival leukocytes were quantified by flow cytometry. RANKL and CD4 immunoreactivity was analyzed by flow cytometry and confocal microscopy. Results: RANKL mRNA levels were higher in periodontitis than in healthy subjects and spontaneous and LPSand PHA-stimulated RANKL synthesis were higher also in patients than controls. CD4+ T lymphocytes were the predominant infiltrate cell subset present in gingival tissues of periodontitis patients. Furthermore, an association between RANKL and CD4+ T cells was determined by double-staining flow cytometry and confocal microscopy. Conclusion: Taken together, these data demonstrate that gingival CD4+ T cells are the main cells responsible for the higher levels of RANKL observed in chronic periodontitis human patients (AU)