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1.  Non-inflammatory destructive periodontal disease: a clinical, microbiological, immunological and genetic investigation 
Journal of Applied Oral Science  2012;20(1):113-121.
Periodontitis comprises a group of multifactorial diseases in which periodontopathogens accumulate in dental plaque and trigger host chronic inflammatory and immune responses against periodontal structures, which are determinant to the disease outcome. Although unusual cases of non-inflammatory destructive periodontal disease (NIDPD) are described, their pathogenesis remains unknown. A unique NIDPD case was investigated by clinical, microbiological, immunological and genetic tools. The patient, a non-smoking dental surgeon with excessive oral hygiene practice, presented a generalized bone resorption and tooth mobility, but not gingival inflammation or occlusion problems. No hematological, immunological or endocrine alterations were found. No periodontopathogens (A. actinomycetemcomitans, P. gingivalis, F. nucleatum and T. denticola) or viruses (HCMV, EBV-1 and HSV-1) were detected, along with levels of IL-1β and TNF-α in GCF compatible with healthy tissues. Conversely ALP, ACP and RANKL GCF levels were similar to diseased periodontal sites. Genetic investigation demonstrated that the patient carried some SNPs, as well HLA-DR4 (*0404) and HLA-B27 alleles, considered risk factors for bone loss. Then, a less vigorous and diminished frequency of toothbrushing was recommended to the patient, resulting in the arrest of alveolar bone loss, associated with the return of ALP, ACP and RANKL in GCF to normality levels. In conclusion, the unusual case presented here is compatible with the previous description of NIDPD, and the results that a possible combination of excessive force and frequency of mechanical stimulation with a potentially bone loss prone genotype could result in the alveolar bone loss seen in NIDPD.
PMCID: PMC3928782  PMID: 22437688
Periodontal diseases; Non-inflammatory destructive periodontal
2.  Dose-Response Met-RANTES Treatment of Experimental Periodontitis: A Narrow Edge between the Disease Severity Attenuation and Infection Control 
PLoS ONE  2011;6(7):e22526.
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.
PMCID: PMC3140528  PMID: 21799885
3.  Cell-Free Antigens from Paracoccidioides brasiliensis Drive IL-4 Production and Increase the Severity of Paracoccidioidomycosis 
PLoS ONE  2011;6(6):e21423.
The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.
PMCID: PMC3120880  PMID: 21731741
4.  The potential role of suppressors of cytokine signaling (SOCS) in the attenuation of inflammatory reaction and alveolar bone loss associated with apical periodontitis 
Journal of endodontics  2008;34(12):1480-1484.
Inflammatory cytokines contribute to periapical tissue destruction. Their activity is potentially regulated by SOCS (suppressors of cytokine signaling), which downregulate signal transduction as part of an inhibitory feedback loop. We investigated the expression of the cytokines TNF-α, IL-10 and RANKL, and SOCS-1, -2 and -3 by Real Time-PCR in 57 periapical granulomas and 38 healthy periapical tissues. Periapical granulomas exhibited significant higher SOCS-1, -2 and -3, TNF-α, IL-10 and RANKL mRNA levels when compared to 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-α, SOCS1 and RANKL, and SOCS3 and TNF-α. Increased SOCS-1, -2 and -3 mRNA levels in periapical granulomas may be related to the downregulation of inflammatory cytokines in these lesions; therefore, SOCS molecules may have a role in the dynamics of periapical granulomas development.
PMCID: PMC2719713  PMID: 19026878
suppressors of cytokine signaling; SOCS; cytokines; TNF-alpha; RANKL; inflammation; periapical granulomas
5.  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▿  
Infection and Immunity  2008;76(8):3725-3734.
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.
PMCID: PMC2493201  PMID: 18541658
6.  Activation and cytokine profile of monocyte derived dendritic cells in leprosy: in vitro stimulation by sonicated Mycobacterium leprae induces decreased level of IL-12p70 in lepromatous leprosy 
Memórias do Instituto Oswaldo Cruz  2015;110(5):655-661.
Dendritic cells (DCs) play a pivotal role in the connection of innate and adaptive immunity of hosts to mycobacterial infection. Studies on the interaction of monocyte-derived DCs (MO-DCs) using Mycobacterium leprae in leprosy patients are rare. The present study demonstrated that the differentiation of MOs to DCs was similar in all forms of leprosy compared to normal healthy individuals. In vitro stimulation of immature MO-DCs with sonicated M. leprae induced variable degrees of DC maturation as determined by the increased expression of HLA-DR, CD40, CD80 and CD86, but not CD83, in all studied groups. The production of different cytokines by the MO-DCs appeared similar in all of the studied groups under similar conditions. However, the production of interleukin (IL)-12p70 by MO-DCs from lepromatous (LL) leprosy patients after in vitro stimulation with M. leprae was lower than tuberculoid leprosy patients and healthy individuals, even after CD40 ligation with CD40 ligand-transfected cells. The present cumulative findings suggest that the MO-DCs of LL patients are generally a weak producer of IL-12p70 despite the moderate activating properties ofM. leprae. These results may explain the poor M. leprae-specific cell-mediated immunity in the LL type of leprosy.
PMCID: PMC4569830  PMID: 26222022
leprosy; dendritic cells; IL-12p70
7.  Inflammasome Activation Is Critical to the Protective Immune Response during Chemically Induced Squamous Cell Carcinoma 
PLoS ONE  2014;9(9):e107170.
Chronic inflammation affects most stages of tumorigenesis, including initiation, promotion, malignant differentiation, invasion and metastasis. Inflammasomes have been described as involved with persistent inflammation and are known to exert both pro and antitumour effects. We evaluated the influence of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase (CASP)-1 in the antitumor immune response using a multistage model of squamous cell carcinoma (SCC) development. Absence of ASC and CASP-1 resulted in an earlier incidence and increased number of papilloma. Loss of inflammassome function in mice resulted in decreased presence of natural killer (NK), dendritic (DC), CD4+, CD8+ and CD45RB+ T cells in the tumor lesions as well as in lymph nodes (LN) compared with WT mice. Increased percentage of CD4+CD25+Foxp3+ T cells was associated with association with inflammasome loss of function. Moreover, significant differences were also found with neutrophils and macrophage infiltrating the lesions. Myeloperoxidase (MPO), but not elastase (ELA), activity oscillated among the groups during the SCC development. Levels of proinflammatory cytokines IL-1β, IL-18, Tumor Necrosis Factor (TNF)-α and Interferon (IFN)-γ were decreased in the tumor microenvironment in the absence of inflammasome proteins. These observations suggest a link between inflammasome function and SCC tumorigenesis, indicating an important role for inflammasome activation in the control of SCC development.
PMCID: PMC4182037  PMID: 25268644
8.  Randomized controlled clinical trial of long-term chemo-mechanical caries removal using PapacarieTM gel 
Journal of Applied Oral Science  2014;22(4):307-313.
Compare the effectiveness of PapacarieTM gel for the chemo-mechanical removal of carious lesions on primary teeth to conventional caries removal with a low-speed bur with regard to execution time, clinical aspects and radiographic findings.
Material and Methods
A randomized controlled clinical trial with a split-mouth design was carried out. The sample was composed of 20 children aged four to seven years, in whom 40 deciduous teeth were randomly divided into two groups: chemo-mechanical caries removal with PapacarieTM and removal of carious dentin with a low-speed bur. Each child underwent both procedures and served as his/her own control. Restorations were performed with glass ionomer cement. The time required to perform the procedure was also analyzed. The patients underwent longitudinal clinical and radiographic follow-up of the restorations.
No statistically significant difference between groups was found regarding the time required to perform the procedures and the radiographic follow up. Statistically significant differences between groups were found in the clinical evaluation at 6 and 18 months after treatment.
PapacarieTM is as effective as the traditional method for the removal of carious dentin on deciduous teeth, but offers the advantages of the preservation of sound dental tissue as well as the avoidance of sharp rotary instruments and local anesthesia.
PMCID: PMC4126827  PMID: 25141203
Dental caries; Papain; Dental atraumatic restorative treatment
9.  Efficacy of Papacarie® in reduction of residual bacteria in deciduous teeth: a randomized, controlled clinical trial 
Clinics  2014;69(5):319-322.
The aim of the present study was to analyze the efficacy of Papacarie® gel compared with the traditional method (low-speed bur) in reducing the counts of total bacteria, Lactobacillus, total Streptococcus and Streptococcus mutans group.
A randomized, controlled clinical trial with a split-mouth design was performed. The sample comprised 40 deciduous teeth in 20 children (10 males and 10 females) aged four to seven years. The teeth were randomly allocated to two groups: G1, or chemomechanical caries removal with Papacarie Duo®, and G2, or the removal of carious dentin tissue with a low-speed bur. Infected dentin was collected prior to the procedure, and the remaining dentin was collected immediately following the removal of the carious tissue. Initial and final counts of bacterial colonies were performed to determine whether there was a reduction in the number of colony-forming units (CFUs) of each microorganism studied. NCT01811420.
Reductions were found in the numbers of total bacteria, total Streptococcus and Streptococcus mutans group following either of the caries removal methods (p<0.05). A reduction was also noted in the number of Lactobacillus CFUs; however, this difference did not achieve statistical significance (p>0.05).
Papacarie® is an excellent option for the minimally invasive removal of carious tissue, achieving significant reductions in total bacteria, total Streptococcus and S. mutans with the same effectiveness as the traditional caries removal method.
PMCID: PMC4012231  PMID: 24838896
Dental Caries; Papain; Streptococcus mutans; Bacteria
10.  Pain during Removal of Carious Lesions in Children: A Randomized Controlled Clinical Trial 
The aim of the present study was to assess pain and the need for anesthesia during chemomechanical caries removal with Papacarie gel and the traditional method (low-speed bur) in pediatric patients. A randomized, controlled, clinical trial with a “split-mouth” design was carried out involving 20 children (10 girls and 10 boys) aged four to seven years. Forty primary teeth (two per child) were randomly allocated to either Group 1 (G1: chemomechanical caries removal with Papacarie gel) or Group 2 (G2: removal of carious dentin with low-speed bur). A face scale was used to classify the sensation of pain during the procedure (1: absence of pain; 2: mild pain; 3: moderate pain; 4: moderately intense pain; 5: intense pain; and 6: extremely intense pain). Statistical analysis of the data was performed using the Wilcoxon-Mann-Whitney (U) test. Pain scores were higher in G2, with statistically significant differences in comparison to G1 (U = 148.0; W = 358.0; P = 0.041). Chemomechanical caries removal with Papacarie provides a lesser degree of pain in comparison to conventional caries removal and does not require the use of local anesthesia. The clinical trial registration number is NCT01811420.
PMCID: PMC3865729  PMID: 24363672
11.  CCL3 and CXCL12 production in vitro by dental pulp fibroblasts from permanent and deciduous teeth stimulated by Porphyromonas gingivalis LPS 
Journal of Applied Oral Science  2013;21(2):99-105.
The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS).
Material and Methods:
Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0 - 10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results.
Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF.
These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation.
PMCID: PMC3881878  PMID: 23739851
CCL3 chemokine; Chemokines; CXCL12 chemokine; Dental pulp; Fibroblasts; Dental pulp inflammation
12.  Inflammatory events during murine squamous cell carcinoma development 
Squamous cell carcinoma (SCC) is one of the most common human cancers worldwide. In SCC, tumour development is accompanied by an immune response that leads to massive tumour infiltration by inflammatory cells, and consequently, local and systemic production of cytokines, chemokines and other mediators. Studies in both humans and animal models indicate that imbalances in these inflammatory mediators are associated with cancer development.
We used a multistage model of SCC to examine the involvement of elastase (ELA), myeloperoxidase (MPO), nitric oxide (NO), cytokines (IL-6, IL-10, IL-13, IL-17, TGF-β and TNF-α), and neutrophils and macrophages in tumour development. ELA and MPO activity and NO, IL-10, IL −17, TNF-α and TGF-β levels were increased in the precancerous microenvironment.
ELA and MPO activity and NO, IL-10, IL −17, TNF-α and TGF-β levels were increased in the precancerous microenvironment. Significantly higher levels of IL-6 and lower levels of IL-10 were detected at 4 weeks following 7,12-Dimethylbenz(a)anthracene (DMBA) treatment. Similar levels of IL-13 were detected in the precancerous microenvironment compared with control tissue. We identified significant increases in the number of GR-1+ neutrophils and F4/80+/GR-1- infiltrating cells in tissues at 4 and 8 weeks following treatment and a higher percentage of tumour-associated macrophages (TAM) expressing both GR-1 and F4/80, an activated phenotype, at 16 weeks. We found a significant correlation between levels of IL-10, IL-17, ELA, and activated TAMs and the lesions. Additionally, neutrophil infiltrate was positively correlated with MPO and NO levels in the lesions.
Our results indicate an imbalance of inflammatory mediators in precancerous SCC caused by neutrophils and macrophages and culminating in pro-tumour local tissue alterations.
PMCID: PMC3542019  PMID: 23176085
Elastase; Nitric oxide; Myeloperoxidase; Inflammatory cells; Cytokines
13.  Differential patterns of RANKL/OPG expression in human periapical granulomas: possible association with progressive or stable nature of the lesions 
Journal of endodontics  2008;34(8):932-938.
RANKL and OPG are expressed in apical periodontitis, suggesting a role for these molecules during lesion development. However, the profiles of RANKL/OPG expression in periapical lesions remain unknown. In this study we investigated the patterns of RANKL and OPG mRNA expression by RealTime-PCR in human periapical granulomas (N=44), and compared them with sites presenting characteristic bone resorpting activity: healthy (n=14) and orthodontically stretched and compressed periodontal ligament (n=26), healthy gingiva (n=24), chronic gingivitis (n=32) and chronic periodontitis (n=34) samples. Both RANKL and OPG mRNA expression was higher in periapical granulomas when compared to healthy periodontal ligament. Distinct patterns of RANKL and OPG expression ratio were found in the granulomas and in different physiological and pathological conditions with characteristic bone resorption activity potentially being an indicative of the stable or progressive nature of the lesions. Lesions with radiographic image smaller than 5mm demonstrated higher RANKL/OPG expression than images greater than 5mm. Periapical granulomas presented heterogeneous patterns of RANKL and OPG expression, ranging from samples with RANKL/OPG ratio similar to that seen in sites with minimal or absent bone resorption, until samples with RANKL/OPG expression pattern comparable with active bone resorption sites.
PMCID: PMC2719712  PMID: 18634923
RANKL; OPG; periapical granulomas; bone resorption; Real Time-PCR
14.  Ageing exacerbates damage of systemic and salivary neutrophils from patients presenting Candida-related denture stomatitis 
Ageing leads to a decline in the function of the immune system, increasing the body's susceptibility to infections through the impairment of T-cells, macrophages, neutrophils and dendritic cells Denture stomatitis is a primary oral disease affecting elderly denture wearers. The major etiologic factor involved in this pathology is the infection by Candida albicans, an opportunistic pathogen that causes local and disseminated diseases in immunosuppressed humans. Neutrophils play a critical role in the immune response against C. albicans and are continually present in the salivary fluid and in the blood. The aim of this study was to determine ageing-related changes in salivary and blood neutrophils and their potential implications in Candida-related denture stomatitis.
Our results showed a lower number of neutrophils in the saliva from patients presenting Candida-related denture stomatitis in comparison to their matched controls. Furthermore, fewer neutrophils were isolated from the saliva of aged control individuals in comparison to matched younger subjects. CXCR1, CD62L and CD11b expression were significantly greater on systemic neutrophils from younger control individuals. Elderly individuals showed more apoptotic salivary neutrophils and lower GM-CSF levels than younger ones, regardless of the occurrence of Candida infection. On the other hand, CXCL-8 concentrations were higher in the saliva from elderly individuals. Besides, TNF-α was detected at elevated levels in the saliva from infected elderly subjects. Salivary neutrophils from elderly and young patients presented impaired phagocytic activity against C. albicans. However, just systemic neutrophils from elderly showed decreased phagocytosis when compared to the younger ones, regardless of the occurrence of infection. In addition, neutrophils from aged individuals and young patients presented low fungicidal activity.
The data suggests that the Candida related-denture stomatitis is associated to neutrophils function deficiency, and ageing drastically appears to alter important characteristics of such cells, facilitating the establishment of this infection.
PMCID: PMC2669447  PMID: 19327169

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