Samuel Barros Ferreira

The broad effects of the functional IL-10 promoter-592 polymorphism : modulation of IL-10, TIMP-3, and OPG expression and their association with periodontal disease outcome

Periodontal diseases are infectious diseases, in which periodontopathogens trigger chronic inflammatory and immune responses that lead to tissue destruction. It occurs through the generation of metalloproteinases and the activation of bone resorption mechanisms. Anti‐inflammatory cytokines such as IL‐10 seem to attenuate periodontal tissue destruction through the induction of tissue inhibitors of metalloproteinases (TIMPs) and the inhibitor of osteoclastogenesis osteoprotegerin (OPG). A high individual variation in levels of IL‐10 mRNA is verified in periodontitis patients, which is possibly determined by genetic polymorphisms. In this study, the IL‐10 promoter ‐592C/A single nucleotide polymorphism (SNP), which is associated with a decrease in IL‐10 production, was analyzed by RFLP in 116 chronic periodontitis (CP) patients and 173 control (C) subjects, and the IL‐10, TIMPs, and OPG mRNA expression levels in diseased gingival tissues were determined by real‐time‐PCR. The IL‐10‐592 SNP CA (P=0.0012/OR=2.4/CI:1.4‐4.1), AA (P=0.0458/OR=2.3/CI:1.1‐4.9), and CA+AA (P=0.0006/OR=2.4/CI:1.4‐3.4) genotypes and the allele A (P=0.0036/OR=1.7/CI:1.2‐2.4) were found to be significantly more prevalent in the CP group when compared with control subjects. Both CA and AA genotypes were associated with lower levels of IL‐10, TIMP‐3, and OPG mRNA expression in diseased periodontal tissues and were also associated with disease severity as mean pocket depth. Taken together, the results presented here demonstrate that IL10‐592 SNP is functional in CP, being associated with lower levels of IL‐10 mRNA expression, which is supposed to consequently decrease the expression of the downstream genes TIMP‐3 and OPG, and influence periodontal disease outcome.

Evidences of the cooperative role of the chemokines CCL3, CCL4 and CCL5 and its receptors CCR1+ and CCR5+ in RANKL+ cell migration throughout experimental periodontitis in mice.

Periodontal disease (PD) is characterized by the inflammatory bone resorption in response to the bacterial challenge, in a host response that involves a series of chemokines supposed to control cell influx into periodontal tissues and determine disease outcome. In this study, we investigated the role of chemokines and its receptors in the immunoregulation of experimental PD in mice. Aggregatibacter actinomycetemcomitans-infected C57Bl/6 (WT) mice developed an intense inflammatory reaction and severe alveolar bone resorption, associated with a high expression of CCL3 and the migration of CCR5+, CCR1+ and RANKL+ cells to periodontal tissues. However, CCL3KO-infected mice developed a similar disease phenotype than WT strain, characterized by the similar expression of cytokines (TNF-alpha, IFN-gamma and IL-10), osteoclastogenic factors (RANKL and OPG) and MMPs (MMP-1, MMP-2, MMP-3, TIMP-1 and TIMP-3), and similar patterns of CCR1+, CCR5+ and RANKL+ cell migration. The apparent lack of function for CCL3 is possible due the relative redundancy of chemokine system, since chemokines such as CCL4 and CCL5, which share the receptors CCR1 and CCR5 with CCL3, present a similar kinetics of expression than CCL3. Accordingly, CCL4 and CCL5 kinetics of expression after experimental periodontal infection remain unaltered regardless the presence/absence of CCL3. Conversely, the individual absence of CCR1 and CCR5 resulted in a decrease of leukocyte infiltration and alveolar bone loss. When CCR1 and CCR5 were simultaneously inhibited by met-RANTES treatment a significantly more effective attenuation of periodontitis progression was verified, associated with lower values of bone loss and decreased counts of leukocytes in periodontal tissues. Our results suggest that the absence of CCL3 does not affect the development of experimental PD in mice, probably due to the presence of homologous chemokines CCL4 and CCL5 that overcome the absence of this chemokine. In addition, our data demonstrate that the absence of chemokine receptors CCR1+ and CCR5+ attenuate of inflammatory bone resorption. Finally, our data shows data the simultaneous blockade of CCR1 and CCR5 with MetRANTEs presents a more pronounced effect in the arrest of disease progression, demonstrating the cooperative role of such receptors in the inflammatory bone resorption process throughout experimental PD.

An Interleukin-1β (IL-1β) Single-Nucleotide Polymorphism at Position 3954 and Red Complex Periodontopathogens Independently and Additively Modulate the Levels of IL-1β in Diseased Periodontal Tissues

ABSTRACT Inflammatory cytokines such as interleukin-1β (IL-1β) are involved in the pathogenesis of periodontal diseases. A high individual variation in the levels of IL-1β mRNA has been verified, which is possibly determined by genetic polymorphisms and/or by the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans. In this study, we investigated the role of an IL-1β promoter single-nucleotide polymorphism at position 3954 [IL-1β(3954) SNP] and the presence of the periodontopathogens in the determination of the IL-1β levels in the periodontal tissues of nonsmoking chronic periodontitis (CP) patients (n = 117) and control (C) subjects (n = 175) and the possible correlations with the clinical parameters of the disease. IL-1β(3954) SNP was investigated by restriction fragment length polymorphism, while the IL-1β levels and the presence of the periodontopathogens were determined by real-time PCR. Similar frequencies of IL-1β(3954) SNP were found in the C and CP groups, in spite of a trend toward a higher incidence of T alleles in the CP group. The IL-1β(3954) SNP CT and TT genotypes, as well as P. gingivalis, T. forsythia, and T. denticola, were associated with higher IL-1β levels and with higher values of the clinical parameters of disease severity. Concomitant analyses demonstrate that IL-1β(3954) and the red complex periodontopathogens were found to independently and additively modulate the levels of IL-1β in periodontal tissues. Similarly, the concurrent presence of both factors was associated with increased scores of disease severity. IL-1β(3954) genotypes and red complex periodontopathogens, individually and additively, modulate the levels of IL-1β in the diseased tissues of nonsmoking CP patients and, consequently, are potentially involved in the determination of the disease outcome.

Experimental periodontitis in mice selected for maximal or minimal inflammatory reactions: increased inflammatory immune responsiveness drives increased alveolar bone loss without enhancing the control of periodontal infection.

BACKGROUND AND OBJECTIVE Inflammatory immune reactions that occur in response to periodontopathogens are thought to protect the host against infection, but may trigger periodontal destruction. However, the molecular and genetic mechanisms underlying host susceptibility to periodontal infection and to periodontitis development have still not been established in detail. MATERIAL AND METHODS In this study, we examined the mechanisms that modulate the outcome of Aggregatibacter (Actinobacillus) actinomycetemcomitans-induced periodontal disease in mice mouse strains selected for maximal (AIRmax) or minimal (AIRmin) inflammatory reactions. RESULTS Our results showed that AIRmax mice developed a more severe periodontitis than AIRmin mice in response to A. actinomycetemcomitans infection, and this periodontitis was characterized by increased alveolar bone loss and inflammatory cell migration to periodontal tissues. In addition, enzyme-linked immunosorbent assays demonstrated that the levels of the cytokines interleukin-1beta, tumor necrosis factor-alpha and interleukin-17 were higher in AIRmax mice, as were the levels of matrix metalloproteinase (MMP)-2, MMP-13 and receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA levels. However, the more intense inflammatory immune reaction raised by the AIRmax strain, in spite of the higher levels of antimicrobial mediators myeloperoxidase and inducible nitric oxide synthase, did not enhance the protective immunity to A. actinomycetemcomitans infection, because both AIRmax and AIRmin strains presented similar bacterial loads in periodontal tissues. In addition, the AIRmax strain presented a trend towards higher levels of serum C-reactive protein during the course of disease. CONCLUSION Our results demonstrate that the intensity of the inflammatory immune reaction is associated with the severity of experimental periodontitis, but not with the control of A. actinomycetemcomitans periodontal infection, suggesting that the occurrence of hyperinflammatory genotypes may not be an evolutionary advantage in the complex host-pathogen interaction observed in periodontal diseases.

Tumor necrosis factor-alpha −308G/A single nucleotide polymorphism and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-α in diseased periodontal tissues

BACKGROUND AND OBJECTIVE Inflammatory cytokines such as tumor necrosis factor-alpha are involved in the pathogenesis of periodontal diseases. A high between-subject variation in the level of tumor necrosis factor-alpha mRNA has been verified, which may be a result of genetic polymorphisms and/or the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola (called the red complex) and Aggregatibacter actinomycetemcomitans. In this study, we investigated the effect of the tumor necrosis factor-alpha (TNFA) -308G/A gene polymorphism and of periodontopathogens on the tumor necrosis factor-alpha levels in the periodontal tissues of nonsmoking patients with chronic periodontitis (n = 127) and in control subjects (n = 177). MATERIAL AND METHODS The TNFA -308G/A single nucleotide polymorphism was investigated using polymerase chain reaction-restriction fragment length polymorphism analysis, whereas the tumor necrosis factor-alpha levels and the periodontopathogen load were determined using real-time polymerase chain reaction. RESULTS No statistically significant differences were found in the frequency of the TNFA -308 single nucleotide polymorphism in control and chronic periodontitis groups, in spite of the higher frequency of the A allele in the chronic periodontitis group. The concomitant analyses of genotypes and periodontopathogens demonstrated that TNFA -308 GA/AA genotypes and the red-complex periodontopathogens were independently associated with increased levels of tumor necrosis factor-alpha in periodontal tissues, and no additive effect was seen when both factors were present. P. gingivalis, T. forsythia and T. denticola counts were positively correlated with the level of tumor necrosis factor-alpha. TNFA -308 genotypes were not associated with the periodontopathogen detection odds or with the bacterial load. CONCLUSION Our results demonstrate that the TNFA -308 A allele and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-alpha in diseased tissues of nonsmoking chronic periodontitis patients and consequently are potentially involved in determining the disease outcome.

Strong and persistent microbial and inflammatory stimuli overcome the genetic predisposition to higher matrix metalloproteinase‐1 (MMP‐1) expression: a mechanistic explanation for the lack of association of MMP1‐1607 single‐nucleotide polymorphism genotypes with MMP‐1 expression in chronic periodontitis lesions

AIMS Our objective was to evaluate the association between the MMP1-1607 single-nucleotide polymorphism (SNP), periodontopathogens and inflammatory cytokines with matrix metalloproteinase-1 (MMP-1) mRNA levels in vitro and in vivo. MATERIALS AND METHODS This study investigated the influence of genetic (MMP1-1607 SNP), microbial (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Actinobacillus actinomycetemcomitans) and inflammatory [tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta)] factors on the determination of MMP-1 mRNA levels in periodontal tissues of non-smoker chronic periodontitis (CP, N=178) and control (C, N=190) groups. The effects of single and repeated lipopolysaccharide (LPS) and inflammatory cytokine stimulation of macrophages with distinct MMP1-1607 SNP genotypes were also investigated. RESULTS In healthy tissues, the MMP1-1607 2G allele was associated with higher MMP-1 levels while in CP MMP-1 levels were associated with the presence and load of periodontopathogens, and also with TNF-alpha and IL-1beta expression irrespective of the MMP1-1607 genotype. In vitro data demonstrate that in 2G macrophages low- and intermediate-dose LPS and TNF-alpha+IL-1beta stimulation was associated with increased MMP-1 expression, while strong and repeated stimulation resulted in higher MMP-1 levels irrespective of the MMP1-1607 genotype. CONCLUSION Our data demonstrate a limited role for MMP1-1607 SNP in periodontitis, where the extensive chronic antigenic challenge exposure overcomes the genetic control and plays a major role in the determination of MMP-1 expression.

Dose-Response Met-RANTES Treatment of Experimental Periodontitis: A Narrow Edge between the Disease Severity Attenuation and Infection Control

Chemokines and chemokine receptors have been implicated in the selective migration of leukocyte subsets to periodontal tissues, which consequently influences the disease outcome. Among these chemoattractants, the chemokines CCL3, CCL4 and CCL5 and its receptors, CCR1 and CCR5, have been associated with increased disease severity in mice and humans. Therefore, in this study we investigated the modulation of experimental periodontitis outcome by the treatment with a specific antagonist of CCR1 and 5 receptors, called met-RANTES. C57Bl/6 mice was orally infected with Aggregatibacter actinomycetemcomitans and treated with 0.05, 0.1, 0.5, 1.5 and 5 mg doses of met-RANTES on alternate days, and evaluated by morphometric, cellular, enzymatic and molecular methods. At 0.5 mg up to 5 mg doses, a strong reduction in the alveolar bone loss and inflammatory cell migration were observed. Interestingly, 5 mg dose treatment resulted in the maximum inhibition of inflammatory cell migration, but resulted in a similar inhibition of bone loss when compared with the lower doses, and also resulted in increased bacterial load and CRP response. When 0.5 and 5 mg therapy regimens were compared it was observed that both therapeutic protocols were able to downregulate the levels of pro-inflammatory, Th1-type and osteoclastogenic cytokines, and CD3+ and F4/80+ cells migration to periodontal tissues, but the high dose modulates host response in a more pronounced and unspecific and excessive way, interfering also with the production of antimicrobial mediators such as MPO, iNOS and IgG, and with GR1+ and CD19+ cells migration. Our results demonstrate a thin line between beneficial immunoregulation and impaired host defense during experimental periodontitis, and the determination of the exact equilibrium point is mandatory for the improvement of immune-targeted therapy of periodontitis.

CCR5 Mediates Pro-osteoclastic and Osteoclastogenic Leukocyte Chemoattraction

Periodontal disease (PD) progression involves the selective leukocyte infiltration into periodontium, supposedly mediated by the chemokine/chemokine receptor system. In this study, we investigated the role of chemokine receptor CCR5 in the immunoregulation of experimental PD in C57BL/6 (WT) and CCR5KO mice. Aggregatibacter actinomycetem comitans infection triggered the chemoattraction of distinct CCR5+ leukocyte subpopulations (determined by flow cytometry): CCR5+F4/80+ leukocytes, which co-express CD14 , CCR2, TNF-α, and IL-1β, indicative of activated macrophages; and CCR5+CD4+ cells, which co-express CXCR3, IFN-γ, and RANKL, indicative of Th1 lymphocytes, therefore comprising pro-osteoclastic and osteoclastogenic cell subsets, respectively. CCR5KO mice presented a lower PD severity (lower inflammation and alveolar bone loss) when compared with the WT strain, since the migration of F4/80+, TNF-α+, CD4+, and RANKL+ cells specifically decreased due to the lack of CCR5. Also, ELISA analysis demonstrated that the production of TNF-α, IL-1β, IL-6, IFN-γ, and RANKL in periodontal tissues was significantly decreased in the CCR5KO strain. The periodontal bacterial load and antimicrobial patterns were unaltered in CCR5KO mice. Our results demonstrate that the chemokine receptor is involved in the migration of distinct leukocyte subpopulations throughout experimental PD, being a potential target for therapeutic intervention in PD.

The role of keratinized mucosa in peri-implant health.

Objective To evaluate the role of keratinized mucosa around dental implants, correlating with other clinical parameters related to the success of dental implants. Design Cross-section. Setting Institutional tertiary referral hospital. Patients A total of 202 dental implants fixed in the cleft area of 109 patients with cleft lip and/or palate were evaluated. Interventions The evaluated clinical parameters were probing depth and gingival and plaque indexes on the buccal surface (three sites). Main Outcome Measures All clinical parameters were correlated with the width of keratinized mucosa around the implants. Results The largest probing depths were detected when the width of keratinized mucosa was 2 mm or more, with a statistically significant difference between the means of the probing depth and keratinized mucosa width. Conclusion Even though the present results suggest that peri-implant health can be observed in areas with keratinized mucosa width under 2 mm provided an adequate oral hygiene control is performed, longitudinal randomized studies are necessary to analyze the relationship between the width of keratinized mucosa and the health of peri-implant tissues.

Survival of Dental Implants in the Cleft Area—A Retrospective Study

Objective To evaluate the survival rate of dental implants placed in the cleft area. Design Retrospective study. Setting Hospital for Rehabilitation of Craniofacial Anomalies, Brazil. Institutional Tertiary Healthcare Center. Patients 120 patients who received dental implants in the grafted cleft area in the years 1999 to 2005. Interventions Clinical data were evaluated from the records of 120 patients according to the following criteria: placement grafted, cleft area, and age at surgery; age at placement of dental implants; site and dimension of implants; interval between placement of implants and the last clinical follow-up; and interval between placement and removal or indication for removal of implants. Main Outcome Measures Percentage of survival rate of implants. Results Mean age at placement of the bone graft was 17.6 years and 21 years at placement of implants. A total of 123 cleft areas received secondary bone graft and bone graft to install implants (regraft). The mean survival rate was 34 months since placement of the implant to the last clinical follow-up and 26 months since placement of the prosthesis. Seven dental implants were removed. The survival rate since placement to the last clinical follow-up was 94.3%. Conclusion Rehabilitation of the cleft area with dental implants is a viable and secure alternative, with good prognosis.