Neutrophil activation induced by ArtinM: Release of inflammatory mediators and enhancement of effector functions

The D-mannose binding lectin ArtinM from Artocarpus integrifolia, previously known as KM+ and artocarpin. is considered a stimulant of Th1-type immunity, which is able to confer resistance to some intracellular pathogens. In addition, ArtinM induces neutrophil migration by haptotaxis through simultaneous interactions of its carbohydrate recognition domains (CRDs) with glycans expressed on the extracellular matrix and the neutrophil surface. In the present study, we have expanded the characterization of ArtinM as a neutrophil activator. Exposure of neutrophils to ArtinM for 15 min resulted in tyrosine phosphorylation of intracellular proteins, a process that was selectively inhibited by D-mannose or mannotriose. Shortly after stimulation, neutrophils secreted high levels of LTB(4) and underwent shedding of L-selectin from their surface. Exposure to ArtinM enhanced neutrophil functions, such as respiratory burst and zymozan and Listeria monocytogenes phagocytosis. In addition, ArtinM-stimulated neutrophils displayed increased CXCL-8 secretion and TLR2 gene transcription. These results demonstrate that ArtinM is able to induce potent neutrophil activation, a feature that should be strongly considered in the assessment of the lectin capacity to confer resistance against infections. (C) 2009 Elsevier B.V. All rights reserved.

Evidence of the presence of T helper type 17 cells in chronic lesions of human periodontal disease

Periodontal disease is a chronic inflammation of the attachment structures of the teeth, triggered by potentially hazardous microorganisms and the consequent immune-inflammatory responses. In humans, the T helper type 17 (Th17) lineage, characterized by interleukin-17 (IL-17) production, develops under transforming growth factor-beta (TGF-beta), IL-1 beta, and IL-6 signaling, while its pool is maintained by IL-23. Although this subset of cells has been implicated in various autoimmune, inflammatory, and bone-destructive conditions, the exact role of T lymphocytes in chronic periodontitis is still controversial. Therefore, in this study we investigated the presence of Th17 cells in human periodontal disease. Gingival and alveolar bone samples from healthy patients and patients with chronic periodontitis were collected and used for the subsequent assays. The messenger RNA expression for the cytokines IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 in gingiva or IL-17 and receptor activator for nuclear factor-kappa B ligand in alveolar bone was evaluated by real-time polymerase chain reaction. The production of IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 proteins was evaluated by immunohistochemistry and the presence of Th17 cells in the inflamed gingiva was confirmed by immunofluorescence confocal microscopy for CD4 and IL-17 colocalization. Our data demonstrated elevated levels of IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 messenger RNA and protein in diseased tissues as well as the presence of Th17 cells in gingiva from patients with periodontitis. Moreover, IL-17 and the bone resorption factor RANKL were abundantly expressed in the alveolar bone of diseased patients, in contrast to low detection in controls. These results provided strong evidence for the presence of Th17 cells in the sites of chronic inflammation in human periodontal disease.

In vitro evidence for immune evasion activity by human plasmin associated to pathogenic Leptospira interrogans

Leptospirosis is a widespread re-emerging zoonosis of human and veterinary concern. It has been shown that virulent leptospires protect themselves against the host`s innate immune system, a strategy that allows the bacteria to reach immunologically safe environments. Although extensive studies on host pathogen interactions have been performed, little is known on how leptospires deal with host immune attack. In a previous work, we demonstrated the ability of leptospires to bind human plasminogen (PLC), that after treatment with activators, conferred plasmin (PLA) activity on the bacteria surface. In this study, we show that the PLA activity associated to the outer surface of Leptospira could interfere with the host immune attack by conferring some evasion advantage during infection. We demonstrate that PLA-coated leptospires interfere with complement Ob and IgG depositions on the bacterial surface, probably through the degradation of these components, thus diminishing opsonization process. Similar decrease on the deposition was observed when normal and immune sera from patients diagnosed with leptospirosis were employed as a source of IgG. We believe that decreasing opsonization by PLA generation might be an important aspect of the leptospiral immune escape strategy and survival. To our knowledge, this is the first proteolytic activity of plasmin associated-Leptospira related to anti-opsonic properties reported to date. (C) 2011 Elsevier Ltd. All rights reserved.

Qualitative Analysis of the Removal of the Smear Layer in the Apical Third of Curved Roots: Conventional Irrigation versus Activation Systems

Introduction: The aim of this study was to evaluate the effectiveness of different irrigant agitation techniques on smear layer removal in curved root canals. Methods: Mesiobuccal canals of 62 extracted lower molars with a curvature of 33 degrees were used and instrumented up to Pro Taper F2. The samples were divided into 3 experimental groups according to the final irrigation: conventional irrigation, ultrasonic irrigation, and sonic irrigation by using the Endo Activator system. The control group was composed of 2 specimens without any final irrigation. In all of the experimental groups, 5 mL of 17% ethylenediaminetetraacetic acid was used for 1 minute, and 5 mL of 2.5% NaOCl was used for 30 seconds. The analysis of the apical region was performed via scanning electron microscopy by 3 examiners. The data were submitted to the Kruskal-Wallis and Dunn tests (P<.05). Results: The activation systems removed significantly more smear layer than did conventional irrigation. Conclusions: Sonic and ultrasonic irrigation resulted in better removal of the smear layer in the apical third of curved root canals than did conventional irrigation. (J Endod 2011;37:1268-1271)

Cigarette smoke inhalation modulates gene expression in sites of bone healing: a study in rats

Objective. The aim of this study was to assess the effect of cigarette smoke inhalation (CSI) on gene expression in alveolar bone healing sites. Study design. Wistar rats were randomly assigned to the groups: control [animals not exposed to CSI (n = 20)] and test [animals exposed to CSI, starting 3 days before teeth extraction and maintained until killing them (n = 20)]. First mandibular molars were bilaterally extracted, and the expression of alkaline phosphatase, bone morphogenetic protein (BMP) 2 and 7, receptor activator of nuclear factor kappa B ligand, osteoprotegerin, and d2 isoform of vacuolar adenosine triphosphatase V(o) domain were assessed by quantitative polymerase chain reaction in the newly formed tissue in the sockets. Results. Overall, data analysis demonstrated that CSI significantly affected the expression pattern of all of the studied genes except BMP-7. Conclusion. The expression of key genes for bone healing may be affected by CSI in tooth extraction sites. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;110:447-452)

STAT3 expression in salivary gland tumours

The aim of the present study was to evaluate the signal transducer and activator of transcription (STAT3) expression, which is constitutively active in different types of malignant tumours, in salivary gland tumours. Fifty biopsies of salivary gland tumours (9 pleomorphic adenomas, 12 adenoid cystic carcinomas, 7 epithelial-myoepithelial carcinomas, 10 polymorphous low-grade adenocarcinomas and 12 mucoepidermoid carcinomas) and 10 normal. salivary glands were immunohistochemically labeled for STAT3 and Phospho-STAT3 (STAT3P). The labeled sections were quatitatively and quantitatively evaluated. The results showed that, in normal salivary gland, STAT3 was expressed in cytoplasm and STAT3P in nuclei of all tissue cells, except in large mucous acinar cells for which both antibodies were negative. In pleomorphic adenoma, the expression was the same as in normal glands. In malignant tumours, there were variations in the expression of these antibodies. The most important one was the presence of STAT3 in the nuclei of the malignant tumour cells, most evident in the cribriform-type of adenoid cystic carcinoma. Both toss and variation of STAT3P expression were also observed. The presence of STAT3 in the nuclei of malignant salivary gland tumours may represent an important event in oncogenesis probably contributing to tumour cell proliferation white blocking apoptosis. However, further investigation will. be necessary to support this hypothesis. (c) 2007 Pubtished by Elsevier Ltd.

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 down-regulate 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. (J Endod 2008;34:1480-1484)

Expression of bone resorption regulators (RANK, RANKL, and OPG) in odontogenic tumors

Objective. To investigate the expression of bone resorption regulators (receptor activator of nuclear factor kappa B [RANK], RANK ligand [RANKL], and osteoprotegerin [OPG]) in calcifying cystic odontogenic tumor (CCOT), adenomatoid odontogenic tumor (AOT), calcifying epithelial odontogenic tumor (CEOT), odontogenic myxoma (OM), and ameloblastic fibroma (AF). Study design. The expression of these mediators was evaluated by means of immunohistochemistry. Results. All specimens demonstrated positive immunoreactivity to RANK, RANKL, and OPG. The quantification of these mediators in epithelium revealed a similar pattern of expression for RANKL and OPG in CCOT, AOT, CEOT, and AF. With regard to stromal/mesenchymal cells, the majority of AOT and CCOT cases showed a higher content of OPG than RANKL, whereas CEOT, OM, and especially AF had a tendency to present a greater content of RANKL than OPG. Conclusion. Our data indicate that the CCOT, AOT, CEOT, OM, and AF cell constituents express key regulators of bone metabolism that might locally modulate tumor-associated bone resorption. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:548-55)

Occlusal outcomes and efficiency of 1-and 2-phase protocols in the treatment of Class II Division 1 malocclusion

Introduction: The purpose of this study was to compare the occlusal outcomes and the efficiency of 1-phase and 2-phase treatment protocols in Class II Division 1 malocclusions. Treatment efficiency was defined as a change in the occlusal characteristics in a shorter treatment time. Methods: Class II Division 1 subjects ( n = 139) were divided into 2 groups according to the treatment protocol for Class II correction. Group 1 comprised 78 patients treated with a 1-phase treatment protocol at initial and final mean ages of 12.51 and 14.68 years. Group 2 comprised 61 patients treated with a 2-phase treatment protocol at initial and final mean ages of 11.21 and 14.70 years. Lateral cephalometric radiographs were taken at the pretreatment stage to evaluate morphological differences in the groups. The initial and final study models of the patients were evaluated by using the peer assessment rating index. Chi-square tests were used to test for differences between the 2 groups for categorical variables. Variables regarding occlusal results were compared by using independent t tests. A linear regression analysis was completed, with total treatment time as the dependent variable, to identify clinical factors that predict treatment length for patients with Class II malocclusions. Results: Similar occlusal outcomes were obtained between the 1-phase and the 2-phase treatment protocols, but the duration of treatment was significantly shorter in the 1-phase treatment protocol group. Conclusions: Treatment of Class II Division 1 malocclusions is more efficient with the 1-phase than the 2-phase treatment protocol.

Immunolocalization of matrix metalloproteinases-2 and-9 during apical periodontitis development

Objective: The objective of this study was to determine the expression of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) during apical periodontitis development. Methods: Using an experimental design of induced periapical lesions in rats and immunohistochemistry assay as investigative tool, the MMP-2 and MMP-9 expression and distribution were evaluated at 3, 7,14, 21, 30,60 and 90 days after coronary access and pulp exposure of the first left mandibular molar to the oral environment. Two blind observers scored the immunoreactivity. A semi-quantitative analysis was performed. Results: Except at day 3, MMP-2 and MMP-9 immunostaining was observed in all experimental periods. The MMP-2 (p = 0.004) and MMP-9 (p = 0.005) immunostaining was higher in the period between 7 and 21 days. They were mainly observed in cells surrounding the apical foramen and adjacent periapical areas. Cells into the hypercementosis areas were strongly stained while both osteoblasts and osteoclasts; presented discrete staining along of this study. No staining was observed on epithelial walls. At 30, 60 and 90 days, the subjacent connective tissue presented intense MMP-2 and MMP-9 immunostaining in mononuclear cells (suggestive of fibroblasts, macrophages, infiltrating neutrophils and lymphocytes). Conclusion: The results observed in this study suggest that MMP-2 and MMP-9 play a critical role in the development of inflammatory periapical lesions, probably involved in the extracellular matrix (ECM) degradation during the initial phase of the lesion development. (C) 2009 Elsevier Ltd. All rights reserved.

In Vitro Proliferation and Osteoblastic Phenotype Expression of Cells Derived From Human Vertebral Lamina and Iliac Crest

Study Design. Osteoblastic cells derived from vertebral lamina and iliac crest were isolated and cultured under the same conditions (osteogenic medium, pH, temperature, and CO(2) levels). Objective. To compare proliferation and expression of osteoblastic phenotype of cells derived from vertebral lamina and iliac grafting. Summary of Background Data. Many factors play a role in the success of bone graft in spinal fusion including osteoblastic cell population. Two common sources of graft are vertebral lamina and iliac crest, however, differences in proliferation and osteoblastic phenotype expression between cells from these sites have not been investigated. Methods. Cells obtained from cancellous bone of both vertebral lamina and iliac crest were cultured and proliferation was evaluated by direct cell counting and viability detected by Trypan blue. Alkaline phosphatase (ALP) activity was evaluated by thymolphthalein release from thymolphthalein monophosphate and matrix mineralization by staining with alizarin red S. Gene expression of ALP, osteocalcin, runt-related transcription factor 2, Msh homeobox 2, bone morphogenetic protein 7, intercellular adhesion molecule 1 precursor, osteoprotegerin, and receptor activator of NF-kB ligand was analyzed by real-time PCR. All comparisons were donor-matched. Results. Proliferation was greater at days 7 and 10 in cells from vertebral lamina compared with ones from iliac crest without difference in cell viability. ALP activity was higher in cells from vertebral lamina compared with cells from iliac crest at days 7 and 10. At 21 days, mineralized matrix was higher in cells derived from vertebral lamina than from iliac crest. At day 7, gene expression of ALP, osteocalcin, runt-related transcription factor 2, Msh homeobox 2, bone morphogenetic protein 7, intercellular adhesion molecule 1 precursor, receptor activator of NF-kB ligand, and osteoprotegerin was higher in cells derived from vertebral lamina compared with iliac crest. Conclusion. Cell proliferation and osteoblastic phenotype development in cells derived from cancellous bone were more exuberant in cultures of vertebral lamina than of iliac crest.

Development of the osteoblastic phenotype in human alveolar bone-derived cells grown on a collagen type I-coated titanium surface

The aim of this study was to evaluate the development of the osteoblastic phenotype in human alveolar bone-derived cells grown on collagen type I-coated titanium (Ti) surface (Col-Ti) obtained by plasma deposition acrylic acid grafting compared with machined Ti (M-Ti). Osteoblastic cells were cultured until subconfluence and subcultured on Col-Ti and M-Ti for periods of up to 21 days. Cultures grown on Col-Ti and M-Ti exhibited similar cell morphology. Cell adhesion, total protein content, and alkaline phosphatase (ALP) activity were not affected by Ti surface modification in all evaluated periods. Growth analyses indicated that there were significantly more cells in cultures grown on Col-Ti at day 3. Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteoprotegerin (OPG) mRNA expression of cells subcultured on Col-Ti was higher, whereas collagen type I (COL) was lower compared with M-Ti. Ti surface modification neither affected the osteocalcin (OC), ALP and receptor activator of NF-kappa B ligand (RANKL) mRNA expression nor the calcium content extracted from mineralized matrix. These results demonstrated that Col-Ti favours cell growth during the proliferative phase (day 3) and osteoblastic differentiation, as demonstrated by changes in mRNA expression profile during the matrix mineralization phase (day 14), suggesting that this Ti surface modification may affect the processes of bone healing and remodelling. To cite this article:Assis AF, Beloti MM, Crippa GE, de Oliveira PT, Morra M, Rosa AL. Development of the osteoblastic phenotype in human alveolar bone-derived cells grown on a collagen type I-coated titanium surface.Clin. Oral Impl. Res. 20, 2009; 240-246.doi: 10.1111/j.1600-0501.2008.01641.x.

The Effect of TAK-778 on Gene Expression of Osteoblastic Cells Is Mediated Through Estrogen Receptor

This study evaluated the effect of TAK-778 [(2R, 4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide)] on in vitro osteogenic events and on gene expression of osteoblastic cells derived from human alveolar bone and the participation of estrogen receptors (ERs) on such effect. Osteoblastic cells were subcultured, with or without TAK-778 (10(-5) M), to evaluate cell growth and viability, total protein content, and alkaline phosphatase (ALP) activity at 7, 14, and 21 days; bone-like formation at 21 days; and gene expression, using cDNA microarray, at 7 days. Also, osteoblastic cells were exposed to TAK-778 (10-5 M) combined to ICI182,780, a nonspecific ER antagonist (10(-6) M), and gene expression was evaluated by real-time polymerase chain reaction (PCR) at 7 days. TAK-778 induced a reduction in culture growth and an increase in cell synthesis, ALP activity, and bone-like formation. The cDNA microarray showed genes associated with cell adhesion and differentiation, skeletal development, ossification, and transforming growth factor-P receptor signaling pathway, with a tendency to be higher expressed in cells exposed to TAK-778. The gene expression of ALP, osteocalcin, Msh homeobox 2, receptor activator of NF-kappa B ligand, and intercellular adhesion molecule 1 was increased by TAK-778 as demonstrated by real-time PCR, and this effect was antagonized by ICI182,780. The present results demonstrated that TAK-778 acts at a transcriptional level to enhance the in vitro osteogenic process and that its effect on gene expression of osteoblastic cells is mediated, at least partially, through ERs. Based on these findings, TAK-778 could be considered in the treatment of bone metabolic disorders. Exp Biol Med 234:190-199, 2009

Antimicrobial Photodynamic Therapy in the Non-Surgical Treatment of Aggressive Periodontitis: Cytokine Profile in Gingival Crevicular Fluid, Preliminary Results

Background: Aggressive periodontitis is a specific form of periodontal disease that is characterized by rapid attachment loss and bone destruction. Cytokine profiles are of considerable value when studying disease course during treatment. The aim of this trial was to investigate cytokine levels in the gingival crevicular fluid (GCF) of patients with aggressive periodontitis, after treatment with photodynamic therapy (PDT) or scaling and root planing (SRP), in a split-mouth design on -7, 0, +1, +7, +30, and +90 days. Methods: Ten patients were randomly treated with PDT using a laser source associated with a photosensitizer or SRP with hand instruments. GCF samples were collected, and the concentrations of tumor necrosis factor-alpha (TNF-alpha) and receptor activator of nuclear factor-kappa B ligand (RANKL) were determined by enzyme-linked immunosorbent assays. The data were analyzed using generalized estimating equations to test the associations among treatments, evaluated parameters, and experimental times (alpha = 0.05). Results: Non-surgical periodontal treatment with PDT or SRP led to statistically significant reductions in TNF-alpha level 30 days following treatment. There were similar levels of TNF-alpha and RANKL at the different time points in both groups, with no statistically significant differences. Conclusion: SRP and PDT had similar effects on crevicular TNF-alpha and RANKL levels in patients with aggressive periodontitis. J Periodontol 2009;80:98-105.

The effect of a single episode of antimicrobial photodynamic therapy in the treatment of experimental periodontitis. Microbiological profile and cytokine pattern in the dog mandible

The purpose of this study was to evaluate the effect of a single application of antimicrobial photodynamic therapy (aPDT) on microbiological profile and cytokine pattern in dogs. Periodontal disease was induced by placing 3.0 silk ligatures around the mandibular pre-molars bilaterally during 8 weeks. The dogs were randomly treated with aPDT using a dye/laser system, scaling and root planning (SRP), or with the association of treatments (SRP + aPDT). Plaque samples were collected at baseline, 1, 3, and 4 weeks, and the mean counts of 40 species were determined using DNA-DNA hybridization. Gingival biopsies were removed and the expression of tumor necrosis factor alpha (TNF-alpha), receptor activator of NF-kB ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase (MMP-1), interleukin (IL) 6, IL-10 and total bacterial load by analysis of 16 S rRNA gene were evaluated through real-time PCR. The results shows that the levels of the majority of the species were reduced 1 week post-therapy for all treatments, however, an increase in counts of Prevotella intermedia (p = 0.00), Prevotella. nigrescens (p = 0.00) and Tannerella forsythia (p = 0.00) was observed for aPDT and SRP + aPDT. After 4 weeks, a regrowth of Porphyromonas gingivalis (p = 0.00) and Treponema denticola (p = 0.00), was observed for all treatments. Also, a strikingly reduction of counts on counts of Aggregatibacter actinomycetemcomitans was observed for the aPDT (p = 0.00). For the cytokine pattern, the results were similar for all treatments, and a reduction in the expression of cytokines and bacterial load was observed throughout the study. Our results suggest that SRP, aPDT in a single application, and SRP + aPDT affects different bacterial species and have similar effects on the expression of cytokines evaluated during the treatment of ligature-induced periodontitis.