Ana Paula Favaro Trombone

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.

Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis

BackgroundmRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease.ResultsWe produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 μg of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7).ConclusionsTaken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

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.

Periodontitis and arthritis interaction in mice involves a shared hyper-inflammatory genotype and functional immunological interferences

Periodontitis (PD) and rheumatoid arthritis (RA) have been found to be clinically associated and to share the chronic nature of the inflammatory reaction associated with bone resorption activity. However, the mechanisms underlying such association are unknown. Therefore, we examined the basis of Actinobacillus actinomycetemcomitans- and Porphyromonas gingivalis-induced PD and pristane-induced arthritis (PIA) interaction in mice. Higher severity PD in the genetically inflammation prone acute inflammatory reactivity maximum (AIRmax) mice strain was associated with higher levels of TNF-α, IL-1β, IL-17, matrix metalloproteinase (MMP)-13, and RANKL, whereas PD/PIA co-induction resulted in even higher levels of IL-1β, IFN-γ, IL-17, RANKL, and MMP-13 levels. Conversely, PD/PIA co-induction in AIRmin strain did not alter the course of both pathologies. PIA/PD co-induction resulted in altered expression of T-cell subsets transcription factors expression, with T-bet and RORγ levels being upregulated, whereas GATA-3 levels were unaltered. Interestingly, PIA induction resulted in alveolar bone loss, such response being highly dependent on the presence of commensal oral bacteria. No differences were found in PIA severity parameters by PD co-induction. Our results show that the interaction between experimental PD and arthritis in mice involves a shared hyper-inflammatory genotype and functional interferences in innate and adaptive immune responses.

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.

The potential role of suppressors of cytokine signaling in the attenuation of inflammatory reaction and alveolar bone loss associated with apical periodontitis.

Inflammatory cytokines contribute to periapical tissue destruction. Their activity is potentially regulated by suppressors of cytokine signaling (SOCS), which downregulate signal transduction as part of an inhibitory feedback loop. We investigated the expression of the cytokines tumor necrosis factor alpha (TNF-alpha); interleukin (IL)-10 and RANKL; and SOCS-1, -2, and -3 by real-time polymerase chain reaction in 57 periapical granulomas and 38 healthy periapical tissues. Periapical granulomas exhibited significantly higher SOCS-1, -2, and -3, TNF-alpha, IL-10, and RANKL messenger RNA levels when compared with healthy controls. Significant positive correlations were found between SOCS1 and IL-10 and between SOCS3 and IL-10. Significant inverse correlations were observed between SOCS1 and TNF-alpha, SOCS1 and RANKL, and SOCS3 and TNF-alpha. Increased SOCS-1, -2, and -3 messenger RNA levels in periapical granulomas may be related to the downregulation of inflammatory cytokines in these lesions; therefore, SOCS molecules may play a role in the dynamics of periapical granulomas development.

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.

The use of chronic gingivitis as reference status increases the power and odds of periodontitis genetic studies – a proposal based in the exposure concept and clearer resistance and susceptibility phenotypes definition

AIM Current literature on chronic periodontitis genetics encompasses numerous single nucleotide polymorphisms-focused case-control studies with inconsistent and controversial results, which typically disregards the exposure concept embraced by case-control definition. Herein, we propose a case-control design reappraisal by clear phenotype selection, where chronic gingivitis represents a genetically resistant phenotype/genotype opposing the susceptible cohort. MATERIAL AND METHODS The hypothesis was tested in healthy, chronic periodontitis and gingivitis groups through Real-time PCR-based allelic discrimination of classic variants IL1B-3954, IL6-174, TNFA-308, IL10-592 and TLR4-299. RESULTS Observed allele/genotype frequencies characterize the healthy group with an intermediate genetic profile between periodontitis and gingivitis cohorts. When comparing genotype/allele frequencies in periodontitis versus healthy and periodontitis versus gingivitis scenarios, the number of positive associations (2-4) and the degree of association (p and odds ratio values) were significantly increased by the new approach proposed (periodontitis versus gingivitis), suggesting the association of IL1B-3954, TNFA-308, IL10-592 and TLR4-299 with periodontitis risk. Power study was also significantly improved by the new study design proposed when compared to the traditional approach. CONCLUSIONS The data presented herein support the use of new case-control study design based on the case-control definition and clear resistance/susceptibility phenotypes selection, which can significantly impact the study power and odds of identification of genetic factors involved in PD.

Expression Analysis of Wound Healing Genes in Human Periapical Granulomas of Progressive and Stable Nature

INTRODUCTION Wound healing process involves the activation of extracellular matrix components, remodeling enzymes, cellular adhesion molecules, growth factors, cytokines and chemokines genes. However, the molecular patterns underlying the healing process at the periapical environment remain unclear. Here we hypothesized that endodontic infection might result in an imbalance in the expression of wound healing genes involved in the pathogenesis of periapical lesions. Furthermore, we suggest that differential expression of wound healing markers in active and latent granulomas could account for different clinical outcomes for such lesions. METHODS Study samples consisted of 93 periapical granulomas collected after endodontic surgeries and 24 healthy periodontal ligament tissues collected from premolars extracted for orthodontic purposes as control samples. Of these, 10 periapical granulomas and 5 healthy periapical tissues were used for expression analysis of 84 wound healing genes by using a pathway-specific real-time polymerase chain reaction array. The remaining 83 granulomas and all 24 control specimens were used to validate the obtained array data by real-time polymerase chain reaction. Observed variations in expression of wound healing genes were analyzed according to the classification of periapical granulomas as active/progressive versus inactive/stable (as determined by receptor activator for nuclear factor kappa B ligand/osteoprotegerin expression ratio). RESULTS We observed a marked increase of 5-fold or greater in SERPINE1, TIMP1, COL1A1, COL5A1, VTN, CTGF, FGF7, TGFB1, TNF, CXCL11, ITGA4, and ITGA5 genes in the periapical granulomas when compared with control samples. SERPINE1, TIMP1, COL1A1, TGFB1, and ITGA4 mRNA expression was significantly higher in inactive compared with active periapical granulomas (P < .001), whereas TNF and CXCL11 mRNA expression was higher in active lesions (P < .001). CONCLUSIONS The identification of novel gene targets that curb the progression status of periapical lesions might contribute to a more accurate diagnosis and lead to treatment modalities more conducive to endodontic success.