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AIM To investigate the presence, localization and the possible correlation of the fibroblast growth factor receptor-2 (FGFR2) with inflammatory resorption of cementum, periodontal ligament and alveolar bone during development of apical periodontitis in mice. METHODOLOGY Apical periodontitis was experimentally induced in mandibular first molars of mice by pulp exposure to the oral environment. Healthy teeth without pulp exposure were used as controls. At 7, 21 and 42 days following pulp exposure, the animals were euthanized and the jaws were prepared for analysis under conventional and fluorescence microscopy, immunohistochemistry (FGFR2), RT-PCR (RNAm levels of RANK, RANKL, OPG, Runx2 and cathepsin K) and enzyme histochemistry (cementoclasts and osteoclasts). Statistical analysis was performed by Kruskal-Wallis tests and Dunn's post hoc tests for multiple comparisons (α = 0.05) using SAS 9.4 software. RESULTS FGFR2-positive cells were not observed in the tissues surrounding healthy teeth but were observed in teeth with periapical lesions from seven days after root canal contamination. At days 21 and 42 after endodontic infection, the increase in periapical lesion size was accompanied by significantly enhanced expression of FGFR2 (P

Methyl gallate (MG) is a plant-derived phenolic compound known to present remarkable anti-inflammatory effect in different experimental models such as paw oedema, pleurisy, zymosan-induced arthritis and colitis. Herein we investigated the effect of MG in the mice model of antigen-induced arthritis (AIA), a model with complex inflammatory response, driven primally by immune process and that cause bone and cartilage erosion similarly found in rheumatoid arthritis. Arthritis was induced by i.a injection of albumin methylated from bovine serum (mBSA) in C57BL/6 male mice previously immunized. The dose-response analysis of MG (0.7-70 mg/kg; p.o) showed that maximum inhibition was reached with the dose of 7 mg/kg on paw oedema and cell infiltration induced by AIA at 7 h. Treatment with MG (7mg/kg; p.o) or with the reference drug, dexamethasone (Dexa,10 mg/kg, ip) reduced AIA oedema formation, leukocyte infiltration, release of extracellular DNA and cytokine production 7 and 24 h (acute response). Mice treated daily with MG for seven days showed no significant weight loss or liver and kidney toxicity contrary to Dexa that induced some degree of toxicity. Prolonged treatment with MG inhibited the late inflammatory response (28 days) reducing oedema formation, cell infiltration, synovial hyperplasia, pannus formation and cartilage degradation as observed in histopathological analyses. Ultimately, MG reduced bone resorption as evidenced by a decrease in tartrate-resistant acid phosphate (TRAP)-positive cells number in femur histology. Altogether, we demonstrate that MG ameliorates the inflammatory reaction driven primarily by the immune process, suggesting a potential therapeutic application in arthritis treatment.

Made available in DSpace on 2019-10-04T12:15:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-09-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Flavonoid-metal complexes are widely studied because of their interesting luminescent behavior and biological activity. Despite the extensive exploration of flavonoid-metal coordination processes in solution, the formation of complexes using the flavonoid molecule inserted in a lipid membrane has been little investigated. This effect could provide important insight into the biological activity of flavonoids at lipid membranes and could represent an attractive strategy to design supramolecular structures. Here, we studied the complexation between Sr2+ and morin inserted in an octadecylphosphonic acid (OPA) Langmuir monolayer. This is a relevant system due to the synergism imposed by the association of the Sr2+ ability to control bone formation/resorption with the morin antioxidative effect. Morin incorporation into the OPA monolayers and further Sr2+ complexation were monitored by surface pressure isotherms. Electronic absorption spectroscopy and fluorescence techniques showed Srmorin complexation both in solution and at the air-liquid interface. Although morin complexation has been described to occur only at basic pH, the specific thermodynamic properties at the air-liquid interface drove metal complexation. LB films were deposited on Ti surfaces, and the resulting OPA/Sr-morin coatings exhibited high surface free energy and increase on its polar component. This optimized surface feature supported further serum protein adsorption and osteoblast growth and differentiation, indicating that these lipid-based coatings are promising for bioactive coating design. This study paves the way for the use of this lipid-based coating in the design of implants for faster osteointegration. Moreover, flavonoid-metal complexation at membranes could also help to shed light on the biological role played by flavonoids. Univ Sao Paulo, Dept Quim, Fac Filosofia Ciencias & Letras Ribeirao Preto, Sao Paulo, Brazil Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Sao Paulo, Brazil Univ Sao Paulo, Dept Fis, Fac Filosofia Ciencias & Letras Ribeirao Preto, Sao Paulo, Brazil Univ Estadual Paulista, Dept Fis, Fac Ciencias & Tecnol, Sao Paulo, Brazil Univ Estadual Paulista, Dept Fis, Fac Ciencias & Tecnol, Sao Paulo, Brazil FAPESP: 2015/08774-0 FAPESP: 2017/08892-9 FAPESP: 2016/21236-0

Although some glycolytic intermediates have been shown to modulate several cell type formation and activation, the functional role of fructose 1,6-bisphosphate (FBP) on osteoclastogenesis is still unknown. Osteoclastogenesis was evaluated on bone marrow preosteoclasts cultured with M-CSF − 30 ng/ml, RANKL − 10 ng/ml, and two concentrations of FBP (100 and 300 µM). TRAP-positive stained cells were counted, and osteoclastogenic marker genes expression were evaluated by qPCR. Osteoclasts resorption capacity was evaluated by the expression of specific enzymes and capacity to resorb a mineralized matrix. The NF-κB activation was detected using RAW 264.7, stably expressing luciferase on the NF-κB responsive promoter. We show that FBP, the product of the first stage of glycolysis, inhibited RANKL-induced osteoclasts differentiation and TRAP activity. The treatment of preosteoclasts with FBP attenuated osteoclast fusion and formation, without affecting cell viability. Moreover, the inhibition of several osteoclastogenic marker genes expression (TRAP, OSCAR, DC-STAMP, Integrin αv, NFATc1) by FBP correlates with a reduction of mineralized matrix resorption capacity. The mechanism underlying FBP-inhibition of osteoclastogenesis involves NF-κB/NFATc1 signaling pathway inhibition. Altogether these data show a protective role of a natural glycolytic intermediate in bone homeostasis that may have therapeutic benefit for osteolytic diseases.

Background Tooth eruption is a process that is not fully understood. Aim To evaluate whether genetic polymorphisms for RANK/RANKL/OPG are associated with delayed tooth emergence. To evaluate whether the relative expression of this genes is associated with persistent primary teeth. Design To evaluate whether genetic polymorphisms for RANK/RANKL/OPG could be involved in delayed tooth emergence, saliva samples from 160 children, aged 6-13 years old, were analysed. To test if there is correlation between gene expression of RANK/RANKL/OPG in children with delayed tooth emergence and persistent primary teeth, periapical tissue from 15 children with persistent primary teeth and from 15 control subjects were collected for qPCR analysis. Results Fifty-six children with delayed tooth emergence (35%) had at least one permanent tooth with delayed emergence. The T allele in RANKL (rs9594738) increased the risk of delayed tooth emergence (P = 0.02; OR = 1.71, 95%CI 1.09-2.75). The relative gene expression for RANKL and the ratio RANKL/OPG in children with delayed tooth emergence and persistent primary teeth were lower compared to controls (P = 0.02 and P = 0.005, respectively). Conclusions Data suggest that the polymorphism rs9594738 in RANKL is associated with delayed permanent tooth emergence. Moreover, reduced relative gene expression of RANKL in periapical tissue is associated with persistent primary teeth.

Objective To verify the association between 25(OH)D level and polymorphisms in the vitamin D receptor gene (VDR) with the disturbance in the dental development and eruption. Design A total of 183 children from two datasets were evaluated. The first dataset was a case-control (15:15) designed to assess if persistent primary tooth (PPT) is associate with serum 25(OH)D level and with genetic polymorphisms in VDR. The second dataset of genomic DNA samples from 54 children with delayed tooth eruption (DTE) and 99 controls were analysed to verify if genetic polymorphisms in VDR (rs2228570 and rs739837) are associated with DTE. The 25(OH)D and the genotyping/allele distribution were analysed using the T-test and chi-square test, respectively. Results The level of 25(OH)D in the PPT group (24.9 ± 6.4 mg/mL) was significantly lower than the control (30.0 ± 7.0 mg/mL) (p=.047). Our data show that children with 25(OH)D deficiency are more likely to present PPT (OR = 2.36; 95%CI: 1.51, 3.70). The rs739837 and rs2228570 polymorphisms were not associated with DTE (OR = 1.44; 95%CI: 0.87, 2.39 and OR = 0.80; 95%CI: 0.45, 1.44, respectively). Conclusions Vitamin D deficiency is a risk factor for PPT.

Objectives Neutrophil extracellular traps (NETs) play a role in the pathogenesis of periodontitis and rheumatoid arthritis (RA). However, it remains poorly understood whether NETs participate in the cross-talk between periodontitis and RA. Herein, we investigated the production of NETs in individuals with periodontitis and RA and its association with clinical parameters. The impact of periodontal therapy on RA and NET release was also assessed. Methods The concentration of NETs and cytokines was determined in the saliva and plasma of individuals with early RA (n = 24), established RA (n = 64) and individuals without RA (n = 76). The influence of periodontitis on the production of NETs and cytokines was also evaluated. Results Individuals with early RA had a higher concentration of NETs in saliva and plasma than individuals with established RA or without RA. Periodontitis resulted in an increase in the concentration of NETs of groups of individuals without RA and with early RA. The proportion of individuals with high concentrations of IL-6, IL-10 and GM-CSF was higher among individuals with periodontitis than among individuals without periodontitis. The concentrations of TNF-α, IL-6, IL-17/IL-25 and IL-28A were particularly high in individuals with early RA. Worse periodontal clinical parameters, RA onset and RA activity were significantly associated with circulating NETs. Periodontal therapy was associated with a reduction in the concentration of NETs and inflammatory cytokines and amelioration in periodontitis and RA. Conclusion This study reveals that NETs are a possible link between periodontitis and RA, with periodontal therapy resulting in a dramatic switch in circulating NET levels.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint destruction and severe morbidity. Cigarette smoking (CS) can exacerbate the incidence and severity of RA. Although Th17 cells and the Aryl hydrocarbon receptor (AhR) have been implicated, the mechanism by which CS induces RA development remains unclear. Here, using transcriptomic analysis, we show that microRNA-132 is specifically induced in Th17 cells in the presence of either AhR agonist or CS-enriched medium. miRNA-132 thus induced is packaged into extracellular vesicles produced by Th17 and acts as a proinflammatory mediator increasing osteoclastogenesis through the down-regulation of COX2. In vivo, articular knockdown of miR-132 in murine arthritis models reduces the number of osteoclasts in the joints. Clinically, RA patients express higher levels of miR-132 than do healthy individuals. This increase is further elevated by cigarette smoking. Together, these results reveal a hitherto unrecognized mechanism by which CS could exacerbate RA and further advance understanding of the impact of environmental factors on the pathogenesis of chronic inflammatory diseases.

Objective To evaluate the role of neutrophil extracellular traps (NETs) in the genesis of joint hyperalgesia using an experimental model of arthritis and transpose the findings to clinical investigation. Methods C57BL/6 mice were subjected to antigen-induced arthritis (AIA) and treated with Pulmozyme (PLZ) to degrade NETs or Cl-amidine to inhibit NET production. Oedema formation, the histopathological score and mechanical hyperalgesia were evaluated. NETs were injected intra-articularly in wild type (WT), Tlr4−/−, Tlr9−/−, Tnfr1−/− and Il1r−/− mice, and the levels of cytokines and Cox2 expression were quantified. NETs were also quantified from human neutrophils isolated from RA patients and individual controls. Results AIA mice had increased NET concentration in joints, accompanied by increased Padi4 gene expression in the joint cells. Treatment of AIA mice with a peptidyl arginine deiminase 4 inhibitor or with PLZ inhibited the joint hyperalgesia. Moreover, the injection of NETs into joints of naïve animals generated a dose-dependent reduction of mechanical threshold, an increase of articular oedema, inflammatory cytokine production and cyclooxygenase-2 expression. In mice deficient for Tnfr1, Il1r, Tlr4 and Tlr9, joint hyperalgesia induced by NETs was prevented. Last, we found that neutrophils from RA patients were more likely to release NETs, and the increase in synovial fluid NET concentration correlated with an increase in joint pain. Conclusion The findings indicate that NETs cause hyperalgesia possibly through Toll-like receptor (TLR)-4 and TLR-9. These data support the idea that NETs contribute to articular pain, and this pathway can be an alternative target for the treatment of pain in RA.

We report the first observation of room temperature spontaneous electric polarization in an electronic ferroelectric material, a YbFe2O4 single crystal. The observation was based on second harmonic generation (SHG), a nonlinear optical process. Tensor analysis of the SHG signal revealed that this material has a polar charge superstructure with Cm symmetry. This result settles the long-term discussion on the uncertainty about electronic ferroelectric properties, including the charge order structure. We present a complete picture of the polar charge ordering of this material via consistent results from two different characterization methods. The SHG signal shows the same temperature dependence as the superlattice signal observed in neutron diffraction experiments. These results prove ferroelectric coupling to electron ordering in YbFe2O4, which results in electronic ferroelectricity which is enabled by the real space ordering of iron cations with different valences. The existence of electronic ferroelectricity holds promise for future electronics technologies where devices run a thousand times faster than frequency of the present CPU (a few gigahertz) embedded in smartphones, etc.

Calcium phosphate (CaP)-based ceramics are the most investigated materials for bone repairing and regeneration. However, the clinical performance of commercial ceramics is still far from that of the native tissue, which remains as the gold standard. Thus, reproducing the structural architecture and composition of bone matrix should trigger biomimetic response in synthetic materials. Here, we propose an innovative strategy based on the use of track-etched membranes as physical confinement to produce collagen-free strontium-substituted CaP nanotubes that tend to mimic the building block of bone, i.e., the mineralized collagen fibrils. A combination of high-resolution microscopic and spectroscopic techniques revealed the underlying mechanisms driving the nanotube formation. Under confinement, poorly crystalline apatite platelets assembled into tubes that resembled the mineralized collagen fibrils in terms of diameter and structure of bioapatite. Furthermore, the synergetic effect of Sr2+ and confinement gave rise to the stabilization of amorphous strontium CaP nanotubes. The nanotubes were tested in long-term culture of osteoblasts, supporting their maturation and mineralization without eliciting any cytotoxicity. Sr2+ released from the particles reduced the differentiation and activity of osteoclasts in a Sr2+ concentration-dependent manner. Their bioactivity was evaluated in a serum-like solution, showing that the particles spatially guided the biomimetic remineralization. Further, these effects were achieved at strikingly low concentrations of Sr2+ that is crucial to avoid side effects. Overall, these results open simple and promising pathways to develop a new generation of CaP multifunctional ceramics that are active in tissue regeneration and able to simultaneously induce biomimetic remineralization and control the imbalanced osteoclast activity responsible for bone density loss.

Strontium ranelate (SrR) has been used as the ultimate choice for osteoporosis treatment. However, the development of more tolerable and bioactive Sr2+ carriers is still a need. The design of Sr2+-based platforms has moved towards the obtention of anion carriers that can also exhibit a positive effect on bone metabolism. In this sense, we used morin, a natural flavonoid, as a new arrangement for Sr2+ carriage in the synthesis of an Sr2+ complex. It has been claimed that phenolic compounds promote bone health. Therefore, we hypothesized that the association of Sr2+ with morin could improve its anabolic effects. Complexes with the general formula [(C15H9O7)Sr(H2O)2]Cl·3H2O were synthesized and characterized by elemental analysis, thermogravimetry, UV-Vis and infrared absorption spectroscopies and 1H-nuclear magnetic resonance. We showed that the complexation between morin and Sr2+ occurred among the 3-OH and 4C[double bond, length as m-dash]O groups of morin. Preosteoclasts cultures with the Sr-morin complex exhibited a reduced osteoclast differentiation rate and sustained osteoblast mineralization ability. The response of Sr-morin was higher than that observed for SrR at the same concentration range. Considering the above-mentioned observations, the Sr-morin complex could be an interesting approach to be further exploited not only as an alternative treatment for osteoporosis but also in the design of materials for faster osteointegration.

The present study examined the hemodynamics [arterial pressure (AP), AP variability (APV), heart rate (HR), and heart rate variability (HRV)], cardiac function (echocardiographycally), and myocardial inflammation in Balb/c mice submitted to Periodontitis, through the ligation of the left first molar, or Sham surgical procedure. The first protocol indicated that the AP was similar (136 ± 2 vs. 132 ± 3 mmHg in Sham), while the HR was higher in mice with Periodontitis (475 ± 20 vs. 412 ± 18 bpm in Sham), compared to their Sham counterparts. The APV was higher in mice with Periodontitis when evaluated in the time domain (4.5 ± 0.3 vs. 3.4 ± 0.2 mmHg in Sham), frequency domain (power of the LF band of systolic AP), or through symbolic analysis (patterns 0V + 1V), indicating a sympathetic overactivity. The HRV was similar in the mice with Periodontitis, as compared to their Sham counterparts. In the second protocol, the mice with Periodontitis showed decreased cardiac output (10 ± 0.8 vs. 15 ± 1.4 mL/min in Sham) and ejection fraction (37 ± 3 vs. 47 ± 2% in Sham) associated with increased myocardial cytokines (Interleukin-17, Interleukin-6, and Interleukin-4). This study shows that experimental Periodontitis caused cardiac dysfunction, increased heart cytokines, and sympathetic overactivity, in line with epidemiological studies indicating an increased risk of cardiovascular events in clinical Periodontitis.

Introduction Anticoagulants are widely used in orthopedic surgery to decrease the risk of deep vein thrombosis. While significant bone impairment is induced by long-term heparin therapy, little is known about the effects of direct oral anticoagulants (DOACs). Herein, we investigated the effects of dabigatran etexilate (Pradaxa®), a DOAC inhibitor of thrombin, on bone cells using in vitro and ex vivo cell culture models. Materials and methods Osteoblasts and osteoclasts exposed to different concentrations of dabigatran etexilate and untreated cells were assayed for cell differentiation and activity. Favorable osteogenic conditions for osteoblasts were tested using titanium with nanotopography (Ti-Nano). In addition, mice treated with a dabigatran etexilate solution had bone marrow cells analyzed for the ability to generate osteoclasts. Results Dabigatran etexilate at concentrations of 1 μg/mL and 2 μg/mL did not impact osteoclast or osteoblast viability. The drug inhibited osteoclast differentiation and activity as observed by the reduction of TRAP+ cells, resorption pits and gene and protein expression of cathepsin K. Consistently, osteoclasts from mice treated with dabigatran showed decreased area, resorptive activity, as well as gene and protein expression of cathepsin K. In osteoblast cultures, grown both on polystyrene and Ti-Nano, dabigatran etexilate reduced alkaline phosphatase (ALP) activity, matrix mineralization, gene expression of ALP and osteocalcin. Conclusions Dabigatran etexilate inhibited osteoclast differentiation in ex vivo and in vitro models in a dose-dependent manner. Moreover, the drug reduced osteoblast activity even under optimal osteogenic conditions. This study provides new evidence regarding the negative overall impact of DOACs on bone cells.

Introduction Probiotics are live microorganisms that, when consumed in appropriate amount, can provide health benefits. Although many studies have shown positive results with the use of probiotics in bone loss control, as in periodontal disease, the effect of probiotics on a mechanical force-induced alveolar bone resorption is still unknown. Therefore, this study aimed to investigate the impact of the specific probiotic Bifidobacterium animalis subsp. lactis on bone remodeling induced by orthodontic tooth movement. Methods For this study, thirty C57BL6/J male mice were used and divided into two groups: 1- Mice were orally treated with the probiotic; 2- Mice were treated with vehicle. All mice were submitted to the experimental model of orthodontic tooth movement (OTM). Bone parameters and OTM was evaluated by MicroCT. OTM and TRAP positive cells were analysed by histomorphometric analysis. Osteoclasts markers were evaluated by qPCR and short chain fatty acids were measured in feces. Results Micro-CT analysis showed that probiotic treatment did not modify the alveolar bone parameters. However, supplementation with probiotics restrained the tooth movement, as demonstrated by the reduced distance of OTM. Probiotic-treated mice presented down-regulation of Trap expression and reduced osteoclast numbers compared to the control. Accordingly, probiotics supplemented mice exhibited a higher concentration of short-chain fatty acid in their feces. Conclusions The supplementation with Bifidobacterium animalis subsp. lactis impaired tooth movement without altering the alveolar bone microarchitecture. The effect on bone remodeling induced by Bifidobacterium animalis subsp. lactis may be associated with the short-chain fatty acids' production.

To evaluate the effect and mechanisms of naringenin in TiO2-induced chronic arthritis in mice, a model resembling prosthesis and implant inflammation. Flavonoids are antioxidant and anti-inflammatory molecules with important anti-inflammatory effect. Mice were daily treated with the flavonoid naringenin (16.7–150 mg/kg, orally) for 30 days starting 24 h after intra-articular knee injection of 3 mg of TiO2. TiO2-induced arthritis resembles cases of aseptic inflammation induced by prosthesis and/or implants. Mice were stimulated with 3 mg of TiO2 and after 24 h mice started to be treated with naringenin. The disease phenotype, treatment toxicity, histopathological damage, oxidative stress, cytokine expression and NFκB were evaluated after 30 days of treatment. Naringenin inhibited TiO2-induced mechanical hyperalgesia (96%), edema (77%) and leukocyte recruitment (74%) without inducing toxicity. Naringenin inhibited histopathological index (HE, 49%), cartilage damage (Toluidine blue tibial staining 49%, and proteoglycan 98%), and bone resorption (TRAP-stained 73%). These effects were accompanied by inhibition of oxidative stress (gp91phox 93%, NBT 83%, and TBARS 41%) cytokine mRNA expression (IL-33 82%, TNFα 76%, pro-IL-1β 100%, and IL-6 61%), and NFκB activation (100%). Naringenin ameliorates TiO2-induced chronic arthritis inducing analgesic and anti-inflammatory responses with improvement in the histopathological index, cartilage damage, and bone resorption.

BACKGROUND: Bone loss-related diseases such as rheumatoid arthritis, osteomyelitis, osteoporosis and periodontitis are associated with high rates of morbidity worldwide. These disorders are characterized by an imbalance between the formation and activity of osteoblasts and osteoclasts, leading to bone loss. In this context, we evaluated the effect of cinnamoyloxy-mammeisin (CNM), an anti-inflammatory coumarin found in Melipona scutellaris geopropolis, on key targets related to bone remodeling. METHODS: We investigated the in vitro effects of CNM on osteoclast differentiation and M-CSF+RANKL-induced osteoclastogenic markers expression. Additionally, the interference of CNM treatment on osteoclast activity was evaluated by zymography and resorption area. Finally, we assessed the capacity of the compound to mitigate alveolar bone loss in vivo in experimental murine periodontitis induced by Porphyromonas gingivalis. RESULTS: Treatment with CNM impaired osteoclast differentiation, as evidenced by a reduced number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells as well as the expression of osteoclastogenic markers upon M-CSF+RANKL-induced stimulation. Similarly, we observed reduced gelatinolytic and resorption capacity in M-CSF+RANKL-induced cells in vitro. Lastly, CNM attenuated alveolar bone loss in experimental murine periodontitis model. CONCLUSION: CNM reduced M-CSF+RANKL-induced osteoclast differentiation and bone resorption, down regulating the expression of markers involved in osteoclast activity. These results were also observed in vivo, suggesting that CNM may be a promising treatment for bone loss diseases.

Chemerin is an adipokine that regulates adipogenesis and metabolic functions of mature adipocytes mainly through the activation of chemokine-like receptor 1 (CMKLR1). Elevated levels of chemerin have been found in individuals with obesity, type 2 diabetes, and osteoporosis. This adipokine was identified as an inflammatory and metabolic syndrome marker. Considering that the association between metabolic syndrome and bone health remains unclear, the present study aimed to clarify the role of chemerin in the pathophysiology of bone loss induced by dyslipidemia, particularly modulating osteoclastogenesis. In vitro analyses showed a downregulation of CMKLR1 at the early stage of differentiation and a gradual increase at late stages. Strikingly, chemerin did not modify osteoclast differentiation markers or osteoclast formation; however, it increased the actin-ring formation and bone resorption activity in mature osteoclasts. The increased bone resorption activity induced by chemerin was effectively inhibited by CMKLR1 antagonist (CCX832). Chemerin boosting mature osteoclast activity involves ERK5 phosphorylation. Moreover, two models of dyslipidemia (high-fat diet [HFD]-treated C57/BL6 and db/db mice) exhibited significantly increased level of chemerin in the serum and gingival tissue. Morphometric analysis showed that HFD-treated and db/db mice exhibited increased alveolar bone loss compared to respective control mice, which was associated with an up-regulation of chemerin, CMKLR1 and cathepsin K mRNA expression in the gingival tissue. The treatment of db/db mice with CCX832 effectively inhibited bone loss. Antagonism of chemerin receptor also inhibited the expression of cathepsin K in the gingival tissue. Our results show that chemerin not only increases osteoclasts activity in vitro, but also that increased level of chemerin in dyslipidemic mice plays a critical role in bone homeostasis. © 2016 American Society for Bone and Mineral Research.

Background The genus Uncaria (Rubiaceae) has several biological properties significant to human health. However, the mechanisms underlying the protective effect of this plant on bone diseases are uncertain. Purpose The present study investigated the role of Uncaria tomentosa extract (UTE) on alveolar bone loss in rats and on osteoclastogenesis in vitro. Materials UTE was characterized by an Acquity UPLC (Waters) system, coupled to an Electrospray Ionization (ESI) interface and Quadrupole/Flight Time (QTOF, Waters) Mass Spectrometry system (MS). The effect of UTE treatment for 11 days on the ligature-induced bone loss was assessed focusing on several aspects: macroscopic and histological analysis of bone loss, neutrophil and osteoclast infiltration, and anabolic effect. The effect of UTE on bone marrow cell differentiation to osteoclasts was assessed in vitro. Results The analysis of UTE by UPLC-ESI-QTOF-MS/MS identified 24 compounds, among pentacyclic or tetracyclic oxindole alkaloids and phenols. The administration of UTE for 11 days on ligature-induced rat attenuated the periodontal attachment loss and alveolar bone resorption. It also diminished neutrophil migration to the gingiva tissue, demonstrated by a lower level of MPO. UTE treatment also decreased the level of RANKL/OPG ratio, the main osteoclast differentiation-related genes, followed by reduced TRAP-positive cell number lining the alveolar bone. Additionally, the level of bone-specific alkaline phosphatase, an anabolic bone marker, was elevated in the plasma of UTE treated rats. Next, we determined a possible direct effect of UTE on osteoclast differentiation in vitro. The incubation of primary osteoclast with UTE decreased RANKL-induced osteoclast differentiation without affecting cell viability. This effect was supported by downregulation of the nuclear factor activated T-cells, cytoplasmic 1 expression, a master regulator of osteoclast differentiation, and other osteoclast-specific activity markers, such as cathepsin K and TRAP. Conclusion UTE exhibited an effective anti-resorptive and anabolic effects, which highlight it as a potential natural product for the treatment of certain osteolytic diseases, such as periodontitis.

Short-chain fatty acids (SCFAs) exert a variety of immune and metabolic functions by binding to G-protein-coupled receptors, mainly free fatty acid receptor 2 (FFAR2). However, the effects of SCFAs and FFARs on bone remodeling, especially in alveolar bone, have been less explored. In this study, we investigated the influence of the SCFA/FFAR2 axis on alveolar bone. Bone samples from wild-type (WT) and FFAR2-deficient mice (FFAR2-/-) were analyzed using micro-CT, histology and qPCR. WT and FFAR2-/- animals received a high-fiber diet (HFD) reported to increase circulating levels of SCFAs. Additionally, we analyzed the effects of SCFAs and a synthetic FFAR2 agonist, phenylacetamide-1 (CTMB), on bone cell differentiation. The participation of histone deacetylase inhibitors (iHDACs) in the effects of SCFAs was further assessed in vitro. CTMB treatment was also evaluated in vivo during orthodontic tooth movement (OTM). FFAR2-/- mice exhibited deterioration of maxillary bone parameters. Consistent with this, FFAR2-/- mice exhibited a significant increase of OTM and changes in bone cell numbers and in the expression of remodeling markers. The HFD partially reversed bone loss in the maxillae of FFAR2-/- mice. In WT mice, the HFD induced changes in the bone markers apparently favoring a bone formation scenario. In vitro, bone marrow cells from FFAR2-/- mice exhibited increased differentiation into osteoclasts, while no changes in osteoblasts were observed. In line with this, differentiation of osteoclasts was diminished by SCFAs and CTMB. Moreover, CTMB treatment significantly reduced OTM. Pretreatment of osteoclasts with iHDACs did not modify the effects of SCFAs on these cells. In conclusion, SCFAs function as regulators of bone resorption. The effects of SCFAs on osteoclasts are dependent on FFAR2 activation and are independent of the inhibition of HDACs. FFAR2 agonists may be useful to control bone osteolysis.

Anthropomorphic measures among type 1 diabetic patients are changing as the obesity epidemic continues. Excess fat mass may impact bone density and ultimately fracture risk. We studied the interaction between bone and adipose tissue in type 1 diabetes subjects submitted to two different clinical managements: (I) conventional insulin therapy or (II) autologous nonmyeloablative hematopoietic stem-cell transplantation (AHST). The study comprised 3 groups matched by age, gender, height and weight: control (C = 24), type 1 diabetes (T1D = 23) and type 1 diabetes treated with AHST (T1D-AHST = 9). Bone mineral density (BMD) and trabecular bone score (TBS) were assessed by dual X-ray absorptiometry (DXA). 1 H Magnetic resonance spectroscopy was used to assess bone marrow adipose tissue (BMAT) in the L3 vertebra, and abdominal magnetic resonance imaging was used to assess intrahepatic lipids (IHL), visceral (VAT) and subcutaneous adipose tissue (SAT). Individuals conventionally treated for T1D were more likely to be overweight (C = 23.8 ± 3.7; T1D=25.3 ± 3.4; T1D-AHST = 22.5 ± 2.2 Kg/m 2 ; p > 0.05), but there was no excessive lipid accumulation in VAT or liver. Areal BMD of the three groups were similar at all sites; lumbar spine TBS (L3) was lower in type 1 diabetes ( p

Objectives: Miniscrew has been frequently used, considering that anchorage control is a critical point in orthodontic treatment, and its failure, the main adverse problem. Using two groups of stable (successful) and unstable (failed) mini-implants, this in vivo study aimed to quantify proinflammatory cytokines IL-1 α, IL-6, IL-17, and TNF-α and osteoclastogenesis marker RANK, RANKL, and OPG in gingival tissue, using the real-time polymerase chain reaction technique. Methodology: Thirteen patients of both sexes (11-49 years old) under orthodontic treatment were selected, obtaining 11 successful and 7 failed mini-implants. The mini-implants were placed and removed by the same surgeon, in both jaws. The mean time of permanence in the mouth was 29.4 months for successful and 7.6 months for failed mini-implants. At removal time, peri-mini-implant gingival tissue samples were collected and processed for quantification of the proinflammatory cytokines and osteoclastogenesis markers. Nonparametric Wilcoxon rank-sum test considering the clusters and Kruskal-Wallis test were used for statistical analysis (α=0.05). Results: No significant difference (p>0.05) was observed between the groups for either quantification of cytokines or osteoclastogenesis markers, except for IL-6 (p

Apical periodontitis is an inflammatory disorder that results from the host immune response to microbial infection through the dental pulp, leading to alveolar bone destruction. The nod-like receptor 12 (NLRP12) is an atypical intracellular sensor of the NLR family that is involved in the negative regulation of several inflammatory conditions and also osteoclastogenesis. However, the role of NLRP12 in the regulation of immune response and bone loss induced by bacterial infection remains unclear. Here we investigated the development of apical periodontitis in wild-type (WT) and NLRP12 knockout (NLRP12–/–) mice by using micro–computed tomography together with histological, immunohistochemical, and molecular analyses. We found that NLRP12–/– mice are highly susceptible to apical periodontitis induced by bacterial infection, which is associated with an elevated infiltration of neutrophils and macrophages, periapical lesion extension, and alveolar bone destruction. Furthermore, NLRP12–/– mice showed a high expression of inflammatory cytokines ( Il1b, Il6, and Tnfa) and the osteoclastogenic markers ( Rankl and Acp5) in the periapical tissues. Consistent with this observation, NLRP12–/– mice showed an increased number of tartrate-resistant acid phosphatase–positive cells lining the apical periodontitis site, which was associated with augmented expression of the osteoclast effector genes, Ctsk and Mmp9. Mechanistically, NLRP12-deficient preosteoclasts showed elevated IκB-α degradation and p65 phosphorylation when stimulated with receptor activator of nuclear factor (NF)–κB ligand (RANKL). Similarly, increased IκB-α degradation was observed in the periapical tissue of NLRP12–/– mice. Furthermore, our in vitro study showed that preosteoclasts from NLRP12–/– mice exhibited higher RANKL-induced osteoclastogenesis, which was synergistically amplified by interleukin-1β and tumor necrosis factor α (mimicking an inflammatory periapical milieu). In conclusion, our data show that NLRP12 exhibits a protective role in the periapical bone destruction by attenuating inflammation and osteoclastogenesis through negative regulation of the NF-κB pathway.

Reactive oxygen species (ROS) are produced by NADPH oxidase (NOX), an enzyme that reduces oxygen by using NADPH as a substrate. Apocynin (APO) is a catechol that is used as a NOX inhibitor, and N-acetyl-cysteine ​​(NAC) can reduce intracellular ROS levels. In this work, the effect of APO and NAC on osteoclast formation were evaluated. APO and NAC significantly decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive cells and the osteoclast area. We analyzed bone-marrow derived monocyte-macrophages (BMMs) that differentiated into osteoclasts after RANKL stimulation. Stimulation was associated with either APO or NAC treatment and osteoclastogenesis marker expression, including NFATc1, MMP-9, and DC-STAMP, was evaluated. APO decreased the intracellular calcium concentration by calcium channels other than ITPR1 and TPC2. On the other hand, APO reduced Tnfrsf11a (RANK) expression and did not alter Fam102a (EEIG1) expression. Therefore, our results demonstrate that APO inhibits osteoclastogenesis by the RANK-RANKL-related signaling pathways, decreases osteoclast markers, and reduces intracellular calcium concentration.

To evaluate the effect and mechanisms of naringenin in TiOFlavonoids are antioxidant and anti-inflammatory molecules with important anti-inflammatory effect. Mice were daily treated with the flavonoid naringenin (16.7-150 mg/kg, orally) for 30 days starting 24 h after intra-articular knee injection of 3 mg of TiOTiONaringenin inhibited TiONaringenin ameliorates TiO

A high-fat (HF) diet leads to detrimental effects on alveolar bone (AB); however, the mechanisms linking adiposity to bone loss are poorly understood. This study investigated if AB resorption induced by an HF diet is associated with the regulation of inflammatory gene expression and if adipocytes can directly interfere with osteoclastogenesis. We also evaluated the effects of diet restriction (DR) on bone phenotype. C57BL6/J mice were fed normal chow or an HF diet for 12 weeks. Samples of maxillae, femur, blood and white adipose tissue were analyzed. In vitro co-culture of bone marrow-derived osteoclasts and mature adipocytes was carried out. The results revealed an increased number of osteoclasts and fewer osteoblasts in animals fed the HF diet, which led to the disruption of trabecular bone and horizontal AB loss. Similar effects were observed in the femur. The metabolic parameters and the deleterious effects of the HF diet on AB and the femur were reversed after DR. The HF diet modulated the expression of 30 inflammatory genes in AB such as Fam3c, InhBa, Tnfs11, Ackr2, Pxmp2 and Chil3, which are related to the inflammatory response and bone remodeling. In vitro, mature adipocytes produced increased levels of adipokines, and co-culture with osteoclasts resulted in augmented osteoclastogenesis. The results indicate that the mechanisms by which an HF diet affects bone involve induction of osteoclastogenesis and inflammatory gene expression. Adipokines apparently are key molecules in this process. Strategies to control diet-induced bone loss might be beneficial in patients with preexisting bone inflammatory conditions.

Titanium dioxide (TiO2) is a common component of orthopedic prosthesis. However, prosthesis wear releases TiO2, which induces inflammation and osteolysis in peri-prosthetic tissues. Quercetin is a flavonoid widely present in human diet, which presents biological activities such as antinociceptive, anti-inflammatory and antioxidant effects. Therefore, the effect of intraperitoneal treatment with quercetin in TiO2-induced arthritis model was evaluated. In the first set of experiments, mice received injection of TiO2 (0.1-3 mg/knee joint) and articular mechanical hyperalgesia, edema and histopathology analysis were performed in a 30 days protocol. The dose of 3 mg of TiO2 showed the most harmful effect, and was chosen to the following experiments. Subsequently, mice received 3 mg of TiO2 followed by post-treatment with quercetin during 30 days. Quercetin (10-100 mg/kg) inhibited in a dose-dependent manner TiO2-induced knee joint mechanical hyperalgesia, edema and leukocyte recruitment and did not induce damage in major organs such as liver, kidney and stomach. The dose of 30 mg/kg was chosen for the subsequent analysis, and reduced histopathological changes such as leukocyte infiltration, vascular proliferation and synovial hyperplasia (pannus formation) on day 30 after TiO2 challenge. The protective analgesic and anti-inflammatory mechanisms of quercetin included the inhibition of TiO2-induced neutrophil and macrophage recruitment, proteoglycan degradation, oxidative stress, cytokine production (TNF-α, IL-1β, IL-6, and IL-10), COX-2 mRNA expression, and bone resorption as well as activation of Nrf2/HO-1 signaling pathway. These results demonstrate the potential therapeutic applicability of the dietary flavonoid quercetin to reduce pain and inflammatory damages associated with prosthesis wear process-induced arthritis.

Interleukin-33 (IL-33) and its receptor, ST2, are implicated in bone remodeling. The lack of estrogen after menopause results in an accelerated bone loss. Here we investigated the role of ST2 in the bone loss induced by estrogen deficiency. ST2-deficient mice (ST2-/- ) and their littermates (wildtype [WT]) were ovariectomized (OVX), while ovary-intact mice were used as controls. Bone sites were analyzed by microcomputed tomography, histomorphometry, and quantitative real-time polymerase chain reaction (qPCR). Deletion of IL-33 or ST2 resulted in a similar bone loss in the femur and maxilla. Ovariectomy in WT mice caused bone loss in the same areas. The lack of ST2 in OVX mice did not alter bone remodeling in the femur but prevented bone loss in the maxilla. Consistently, ovariectomy increased the IL-33 messenger RNA (mRNA) levels in the maxilla but not in the femur. Under mechanical stimulation, ovariectomy and ST2 deletion independently increased bone remodeling induced by orthodontic tooth movement, which was also associated with a greater number of osteoclasts and a reduced number of osteoblasts in the maxillary bone. ST2-/- OVX mice, however, displayed twice as many osteoblasts as that of WT OVX mice. Ovariectomy and ST2 deletion differently altered the cytokine mRNA levels in the maxilla. Remarkably, interleukin-10 expression was decreased in both WT OVX and ST2-/- mice, and this reduction was completely restored in ST2-/- OVX mice. The results demonstrate that estrogen and IL33/ST2 independently protect against bone loss. However, the ovariectomy-induced bone loss is IL-33/ST2-dependent in the maxilla but not in the femur, indicating a bimodal and site-specific role of ST2 in bone remodeling.

OBJECTIVES: The present study was designed to evaluate the bone phenotypes and mechanisms involved in bone disorders associated with hepatic osteodystrophy. Hepatocellular disease was induced by carbon tetrachloride (CCl4). In addition, the effects of disodium pamidronate on bone tissue were evaluated. METHODS: The study included 4 groups of 15 mice: a) C = mice subjected to vehicle injections; b) C+P = mice subjected to vehicle and pamidronate injections; c) CCl4+V = mice subjected to CCl4 and vehicle injections; and d) CCl4+P = mice subjected to CCl4 and pamidronate injections. CCl4 or vehicle was administered for 8 weeks, while pamidronate or vehicle was injected at the end of the fourth week. Bone histomorphometry and biomechanical analysis were performed in tibiae, while femora were used for micro-computed tomography and gene expression. RESULTS: CCl4 mice exhibited decreased bone volume/trabecular volume and trabecular numbers, as well as increased trabecular separation, as determined by bone histomorphometry and micro-computed tomography, but these changes were not detected in the group treated with pamidronate. CCl4 mice showed increased numbers of osteoclasts and resorption surface. High serum levels of receptor activator of nuclear factor-κB ligand and the increased expression of tartrate-resistant acid phosphatase in the bones of CCl4 mice supported the enhancement of bone resorption in these mice. CONCLUSION: Taken together, these results suggest that bone resorption is the main mechanism of bone loss in chronic hepatocellular disease in mice.

Aim To evaluate the absence of IL-22 on the progression of periapical lesions in wild-type (WT) and IL-22 knockout (IL-22 KO) mice. Methodology The evaluation of the oral microbial profile of mice was performed by Checkerboard DNA–DNA hybridization from saliva samples. Periapical lesions were induced in manbibular first molars by pulpal exposure and evaluated after 7, 21 and 42 days (n = 15). Haematoxylin-eosin-stained sections were analysed under conventional and fluorescence microscopy to evaluate the tissue features and size of periapical lesions and tartrate-resistant acid phosphatase histoenzymology (TRAP), Brown & Brenn staining and immunohistochemistry. The scores of the number of bacterial cells present in the oral cavity were analysed by the Mann–Whitney test, and the results and comparisons for periapical lesion size and number of osteoclasts were subjected to one-way anova and Bonferroni's post-test (α = 0.05). Results Significant differences were observed for bacterial load between the groups of animals for 6 bacterial species (P

NO is a free radical with pleiotropic functions. We have shown earlier that NO induces a population of CD4+CD25+Foxp3− regulatory T cells (NO-Tregs) that suppress the functions of CD4+CD25− effector T cells in vitro and in vivo. We report in this study an unexpected finding that NO-Tregs suppressed Th17 but not Th1 cell differentiation and function. In contrast, natural Tregs (nTregs), which suppressed Th1 cells, failed to suppress Th17 cells. Consistent with this observation, NO-Tregs inhibited the expression of retinoic acid–related orphan receptor γt but not T-bet, whereas nTregs suppressed T-bet but not retinoic acid–related orphan receptor γt expression. The NO-Treg–mediated suppression of Th17 was partially cell contact–dependent and was associated with IL-10. In vivo, adoptively transferred NO-Tregs potently attenuated experimental autoimmune encephalomyelitis. The disease suppression was accompanied by a reduction of Th17, but not Th1 cells in the draining lymph nodes, and a decrease in the production of IL-17, but an increase in IL-10 synthesis. Our results therefore demonstrate the differential suppressive function between NO-Tregs and nTregs and indicate specialization of the regulatory mechanism of the immune system.

Titanium dioxide (TiO

Introduction Bone remodeling is a tightly regulated process influenced by chemokines. ACKR2 is a decoy receptor for CC chemokines functioning as regulator of inflammatory response. In this study we investigated whether the absence of ACKR2 would affect bone phenotype and remodeling induced by mechanical loading. Methods An orthodontic appliance was placed between incisors and first molar of ACKR2 deficient (ACKR2 −/− ) and C57BL6/J (wild-type/WT) mice. Microtomography, histology and qPCR were performed to evaluate bone parameters, orthodontic tooth movement (OTM), bone cells counts and the expression of ACKR2, bone remodeling markers, CC chemokines and chemokines receptors. Bone marrow cells (BMC) from WT and ACKR2 −/− mice were differentiated in osteoclasts and osteoblasts for analysis of activity and expression of specific markers. Results Mechanical stimulus induced ACKR2 production in periodontium. The expression of ACKR2 in vitro was mostly detected in mature osteoclasts and early-differentiated osteoblasts. Although ACKR2 −/− mice exhibited regular phenotype in maxillary bone, the amount of OTM, osteoclasts counts and the expression of pro-resorptive markers were increased in this group. In contrast, the number of osteoblasts and related markers were decreased. OTM resulted in augmented expression of CC chemokines and receptors CCR5 and CCR1 in periodontium, which was higher in ACKR2 −/− than WT mice. In vitro experiments demonstrated an augmented formation of osteoclasts and diminished differentiation of osteoblasts in ACKR2 −/− mice. Conclusions These data suggests that ACKR2 functions as a regulator of mechanically-induced bone remodeling by affecting the differentiation and activity of bone cells and the availability of CC chemokines at periodontal microenvironment. Therapeutic strategies based on increase of ACKR2 might be useful to hinder bone loss in inflammatory conditions.

Chemerin is an adipokine that regulates adipogenesis and metabolic functions of mature adipocytes mainly through the activation of chemokine-like receptor 1 (CMKLR1). Elevated levels of chemerin have been found in individuals with obesity, type 2 diabetes, and osteoporosis. This adipokine was identified as an inflammatory and metabolic syndrome marker. Considering that the association between metabolic syndrome and bone health remains unclear, the present study aimed to clarify the role of chemerin in the pathophysiology of bone loss induced by dyslipidemia, particularly modulating osteoclastogenesis. In vitro analyses showed a downregulation of CMKLR1 at the early stage of differentiation and a gradual increase at late stages. Strikingly, chemerin did not modify osteoclast differentiation markers or osteoclast formation; however, it increased the actin-ring formation and bone resorption activity in mature osteoclasts. The increased bone resorption activity induced by chemerin was effectively inhibited by CMKLR1 antagonist (CCX832). Chemerin boosting mature osteoclast activity involves ERK5 phosphorylation. Moreover, two models of dyslipidemia (high-fat diet [HFD]-treated C57/BL6 and db/db mice) exhibited significantly increased level of chemerin in the serum and gingival tissue. Morphometric analysis showed that HFD-treated and db/db mice exhibited increased alveolar bone loss compared to respective control mice, which was associated with an up-regulation of chemerin, CMKLR1 and cathepsin K mRNA expression in the gingival tissue. The treatment of db/db mice with CCX832 effectively inhibited bone loss. Antagonism of chemerin receptor also inhibited the expression of cathepsin K in the gingival tissue. Our results show that chemerin not only increases osteoclasts activity in vitro, but also that increased level of chemerin in dyslipidemic mice plays a critical role in bone homeostasis. © 2016 American Society for Bone and Mineral Research.

Introduction Although studies have recently shown that osteocytes embedded in mineralized bone matrix play an important role in bone diseases, the participation of cementocytes in apical periodontitis has not been evaluated. The aim of the present study was to evaluate the possible involvement of cementocytes in the development of apical periodontitis. Methods Apical periodontitis was experimentally induced in the lower first molars of wild-type mice by pulp exposure to the oral environment. At 0, 7, 21, and 42 days after pulp infection, the animals were euthanized, and the jaws were prepared for analysis under conventional and fluorescence microscopy (morphologic and morphometric analysis), immunohistochemistry and immunofluorescence (receptor activator of nuclear factor kappa-B [RANK], receptor activator of the nuclear factor kappa-B ligand [RANKL], and osteoprotegerin [OPG]), enzyme histochemistry (osteoclasts and cementoclasts), and real-time polymerase chain reaction (RANK, RANKL, OPG, and cathepsin K). Results At 7, 21, and 42 days after pulp exposure, there was a progressive increase in periodontal ligament, cementum and bone resorption areas, osteoclasts, and cementoclast counts as well as higher messenger RNA levels of RANK, RANKL, OPG, and cathepsin K. In intact teeth, cementocytes and osteocytes did not express RANKL. After infection, RANKL was strongly expressed in cementocytes, but not in osteocytes, and its expression increased with lesion progression. Conclusions Our findings show that cementocytes express RANKL in response to endodontic infection and may be involved in the pathogenesis of apical periodontitis.

Interleukin (IL)-33, a member of the IL-1 cytokine family, is an important modulator of the immune system associated with several immune-mediated disorders. High levels of IL-33 are expressed by the central nervous system (CNS) suggesting a potential role of IL-33 in autoimmune CNS diseases. We have investigated the expression and function of IL-33 in the development of experimental autoimmune encephalomyelitis (EAE) in mice. We report here that IL-33 and its receptor ST2 (IL-33Rα) are highly expressed in spinal cord tissue, and ST2 expression is markedly increased in the spinal cords of mice with EAE. Furthermore, ST2-deficient (ST2(-/-) ) mice developed exacerbated EAE compared with wild-type (WT) mice while WT, but not ST2(-/-) EAE mice treated with IL-33 developed significantly attenuated disease. IL-33-treated mice had reduced levels of IL-17 and IFN-γ but produced increased amounts of IL-5 and IL-13. Lymph node and splenic macrophages of IL-33-treated mice showed polarization toward an alternatively activated macrophage (M2) phenotype with significantly increased frequency of MR(+) PD-L2(+) cells. Importantly, adoptive transfer of these IL-33-treated macrophages attenuated EAE development. Our data therefore demonstrate that IL-33 plays a therapeutic role in autoimmune CNS disease by switching a predominantly pathogenic Th17/Th1 response to Th2 activity, and by polarization of anti-inflammatory M2 macrophages.

To investigate the role of regulatory T cells in the modulation of long-term immune dysfunction during experimental sepsis. It is well established that sepsis predisposes to development of a pronounced immunosuppression. Nevertheless, the mechanisms underlying the immune dysfunction after sepsis are still not well understood.Prospective experimental study.University research laboratory.Wild-type mice underwent cecal ligation and puncture and were treated with antibiotic during 3 days after surgery. On days 1, 7, or 15 after cecal ligation and puncture, the frequency of regulatory T cells, proliferation of CD4 T cells and bacterial counts were evaluated. Fifteen days after cecal ligation and puncture, surviving mice underwent secondary pulmonary infection by intranasal inoculation of nonlethal dose of Legionella pneumophila. Some mice received agonistic glucocorticoid-induced tumor necrosis factor receptor antibody (DTA-1) before induction of secondary infection.Mice surviving cecal ligation and puncture showed a markedly increased frequency of regulatory T cells in thymus and spleen, which was associated with reduced proliferation of CD4 T cells. Fifteen days after cecal ligation and puncture, all sepsis-surviving mice succumbed to nonlethal injection of L. pneumophila. Treatment of mice with DTA-1 antibody reduced frequency of regulatory T cells, restored CD4 T cell proliferation, reduced the levels of bacteria in spleen, and markedly improved survival of L. pneumophila infection.These findings suggest that regulatory T cells play an important role in the progression and establishment of immune dysfunction observed in experimental sepsis.

Objectives Interleukin 33 (IL-33) is a new member of the IL-1 family of cytokines which signals via its receptor, ST2 (IL-33R), and has an important role in Th2 and mast cell responses. This study shows that IL-33 orchestrates neutrophil migration in arthritis. Methods and results Methylated bovine serum albumin (mBSA) challenge in the knee joint of mBSA-immunised mice induced local neutrophil migration accompanied by increased IL-33R and IL-33 mRNA expression. Cell migration was inhibited by systemic and local treatments with soluble (s)IL-33R, an IL-33 decoy receptor, and was not evident in IL-33R-deficient mice. IL-33 injection also induced IL-33R-dependent neutrophil migration. Antigen- and IL-33-induced neutrophil migration in the joint was dependent on CXCL1, CCL3, tumour necrosis factor α (TNFα) and IL-1β synthesis. Synovial tissue, macrophages and activated neutrophils expressed IL-33R. IL-33 induces neutrophil migration by activating macrophages to produce chemokines and cytokines and by directly acting on neutrophils. Importantly, neutrophils from patients with rheumatoid arthritis successfully treated with anti-TNFα antibody (infliximab) expressed significantly lower levels of IL-33R than patients treated with methotrexate alone. Only neutrophils from patients treated with methotrexate alone or from normal donors stimulated with TNFα responded to IL-33 in chemotaxis. Conclusions These results suggest that suppression of IL-33R expression in neutrophils, preventing IL-33-induced neutrophil migration, may be an important mechanism of anti-TNFα therapy of inflammation.

Homocysteine is an independent risk factor for coronary heart disease, as well as for cerebrovascular and peripheral vascular diseases. The purpose of this study was to investigate the effects of hyperhomocysteinemia (HHcy) on vascular reactivity within carotid artery segments isolated from ovariectomized female rats. Treatment with DL-Hcy thiolactone (1 g/kg body weight per day) reduced the phenylephrine-induced contraction of denuded rings. However, the treatment did not alter KCl-induced contractions, or relaxations induced by sodium nitroprusside or acetylcholine. We report elevated expressions of iNOS, eNOS, and nitrotyrosine in homocysteine-treated rat artery sections. Moreover, the inhibition of NOS by L-NAME, 1,400 W, or L-NNA restored phenylephrine-induced vasoconstriction in carotid artery segments from Hcy-treated rats. In conclusion, our findings show that severe HHCy can promote an acute decrease in the endothelium-independent contractile responses of carotid arteries to adrenergic agonists. This effect was restored by nitric oxide synthase inhibitors, which further supports the involvement of nitric oxide in HHcy-derived vascular dysfunction.

Background and purpose: We investigated the effect of nitric oxide synthase (NOS) inhibition on polymorphonuclear cell (PMN) influx in zymosan or lipopolysaccharide (LPS)-induced arthritis and peritonitis. Experimental approach: Wistar rats received intra-articular (i.art.) zymosan (30–1000 µg) or LPS (1–10 µg). Swiss C57/Bl6 mice genetically deficient in intercellular adhesion molecule-1 (ICAM-1−/−) or in β2-integrin (β2-integrin−/−) received zymosan either i.art. or i.p. PMN counts, leukotriene B4 (LTB4), tumour necrosis factor-α (TNF-α) and interleukin-10 (IL-10) levels were measured in joint and peritoneal exudates. Groups received the NOS inhibitors NG-nitro-L-arginine methyl ester (LN), nitro-L-arginine, N-[3-(aminomemethyl)benzyl] acetamide or aminoguanidine, prior to zymosan or LPS, given i.p. or s.c. in the arthritis and peritonitis experiments respectively. A group of rats received LN locally (i.art. or i.p.), 30 min prior to 1 mg zymosan i.art. Key results: Systemic or local NOS inhibition significantly prevented PMN migration in arthritis while increasing it in peritonitis, regardless of stimuli, concentration of NOS inhibitors and species. NOS inhibition did not alter TNF-α and IL-10 but decreased LTB4 in zymosan-induced arthritis. LN administration significantly inhibited PMN influx into the joints of ICAM-1−/− and β2-integrin−/− mice with zymosan-arthritis, while not altering PMN influx into the peritoneum of mice with zymosan-peritonitis. Conclusions and implications: Nitric oxide has a dual modulatory role on PMN influx into joint and peritoneal cavities that is stimulus- and species-independent. Differences in local release of LTB4 and in expression of ICAM-1 and β2-integrin account for this dual role of NO on PMN migration.

Introduction: Periapical chronic lesion formation involves activation of the immune response and alveolar bone resorption around the tooth apex. However, the overall roles of T helper type 1 (Th1), Th2, and T-regulatory cell (Treg) responses and osteoclast regulatory factors in periapical cysts and granulomas have not been fully determined. This study aimed to investigate whether different forms of apical periodontitis, namely cysts and granulomas, show different balances of Th1, Th2 regulators, Treg markers, and factors involved in osteoclast chemotaxis and activation. Methods: Gene expression of these factors was assessed using quantitative real-time polymerase chain reaction, in samples obtained from healthy gingiva (n = 8), periapical granulomas (n = 20), and cysts (n = 10). Results: Periapical cysts exhibited a greater expression of GATA-3, while a greater expression of T-bet, Foxp3, and interleukin-10 (IL-10) was seen in granulomas. The expression of interferon-γ, IL-4, and transforming growth factor-β was similar in both lesions. Regarding osteoclastic factors, while the expression of SDF-1α/CXCL12 and CCR1 was higher in cysts, the expression of RANKL was significantly higher in granulomas. Both lesions exhibited similar expression of CXCR4, CKβ8/CCL23, and osteoprotegerin, which were significantly higher than in control. Conclusion: Our results showed a predominance of osteoclast activity in granulomas that was correlated with the Th1 response. The concomitant expression of Treg cell markers suggests a possible suppression of the Th1 response in granulomas. On the other hand, in cysts the Th2 activity is augmented. The mechanisms of periradicular lesion development are still not fully understood but the imbalance of immune and osteoclastic cell activity in cysts and granulomas seems to be critically regulated by Treg cells.

Galectin-3 (Gal-3) is a member of the β-galactoside-binding lectin family and plays an important role in inflammation. However, the precise role of Gal-3 in autoimmune diseases remains obscure. We have investigated the functional role of Gal-3 in experimental autoimmune encephalomyelitis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG)35–55 peptide. Gal-3 deficient (Gal-3−/−) mice developed significantly milder EAE and markedly reduced leukocyte infiltration in the CNS compared with similarly treated wild-type (WT) mice. Gal-3−/− mice also contained fewer monocytes and macrophages but more apoptotic cells in the CNS than did WT mice. Following Ag stimulation in vitro, lymph node cells from the immunized Gal-3−/− mice produced less IL-17 and IFN-γ than did those of the WT mice. In contrast, Gal-3−/− mice produced more serum IL-10, IL-5, and IL-13 and contained higher frequency of Foxp3+ regulatory T cells in the CNS than did the WT mice. Furthermore, bone marrow-derived dendritic cells from Gal-3−/− mice produced more IL-10 in response to LPS or bacterial lipoprotein than did WT marrow-derived dendritic cells. Moreover, Gal-3−/− dendritic cells induced Ag-specific T cells to produce more IL-10, IL-5, and IL-12, but less IL-17, than did WT dendritic cells. Taken together, our data demonstrate that Gal-3 plays an important disease-exacerbating role in EAE through its multifunctional roles in preventing cell apoptosis and increasing IL-17 and IFN-γ synthesis, but decreasing IL-10 production.

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in host defense, as well as in inflammation-induced tissue lesions. Here we evaluated the role of NO in bone loss in bacterial infection-induced apical periodontitis by using iNOS-deficient mice (iNOS−/−). The iNOS−/− mice developed greater inflammatory cell recruitment and osteolytic lesions than WT mice. Moreover, tartrate-resistant acid-phosphatase-positive (TRAP+) osteoclasts were significantly more numerous in iNOS−/− mice. Furthermore, the increased bone resorption in iNOS−/− mice also correlated with the increased expression of receptor activator NF-kappaB (RANK), stromal-cell-derived factor-1α (SDF-1α/CXCL12), and reduced expression of osteoprotegerin (OPG). These results show that NO deficiency was associated with an imbalance of bone-resorption-modulating factors, leading to severe infection-stimulated bone loss.

Introduction: p27Kip1 is a cyclin kinase inhibitor that induces cell cycle arrest. In this study, the efficacy of fusion protein TAT- p27Kip1 to inhibit cell proliferation in rat perivascular injured carotid arteries was tested. Methods: The cDNA of p27Kip1 and GFP (green fluorescein protein) fused to the TAT epitope, which allows cell penetration, yielded TAT-p27 Kip1 and TAT-GFP fusion proteins. In vitro biological activity on cell proliferation was evaluated by [3H] thymidine DNA incorporation in rabbit aortic endothelial cells (REC). An in vivo model used a silicone collar filled with saline positioned around the carotid vessel for 14 days to produce an increased adventitia cross-sectional area. Results: TAT-p27Kip1 inhibited REC proliferation in vitro using either 100, 200, and 500 nM compared to control (88.2 ± 4.4, 81.3 ± 7, 71.9 ± 4.2 vs. 100 ± 6.7%, N = 3, respectively, p < 0 .05). This response was stable for purified proteins stored at -20•C for at least 23 days. In vivo , TAT-p27Kip1 solution reduced adventitia cross-sectional area in a dose-dependent manner compared to TAT-GFP (area in mm2 — TAT-p27Kip1: 200 nM, 0.160 ± 0.018; 500 nM, 0.050 ± 0.005 vs. TAT-GFP: 500 nM, 0.595 ± 0.066 vs. the contralateral: 0.047 ± 0.005, N = 7, p < 0 .01). Conclusion: Taken together, these results provide evidence that TAT-p27Kip1 can inhibit vascular cells proliferation. It is the first successful demonstration that the cell permeable TAT-p27Kip1 has potential as a vascular anti-proliferative agent.