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Fibrous dysplasia (FD) is a chronic and progressive disorder of bone growth because of decreased osteoblast formation and osteoclast overactivity. Its main symptoms include pain, fracture, and irregular bone growth. Bisphosphonates are the mainstay of therapy for FD with a primary goal of pain relief. A 50-year-old woman presented to ophthalmology in March 2011 with intermittent proptosis, vertical diplopia, and orbital pain. A computed tomography scan of the head revealed a skull base lesion, which was confirmed to be fibrous dysplasia on bone biopsy. Because of significant headache, she was treated with IV pamidronate monthly starting November 2011, which led to pain reduction. Repeated attempts to decrease the frequency of pamidronate were unsuccessful because of breakthrough pain. Oral alendronate and risedronate did not control her symptoms. She remained on risedronate however because of its convenience. In August 2021, she was diagnosed with metastatic melanoma and started nivolumab. Her headache completely resolved for the first time in 10 years. Although nivolumab, a programmed death-1 blocker, has been used in the treatment of bone malignancy, it has not been previously studied in FD. By suppressing RANK ligand-related osteoclastogenesis, nivolumab decreases cancer-associated bone pain. Our case suggests a potential role for nivolumab in treating FD-associated pain. [ABSTRACT FROM AUTHOR]

Simple Summary: Advances in the treatment of acute lymphoblastic leukemia (ALL) have led to a marked improvement in the survival rate of patients. Nevertheless, these patients may develop adverse effects during and after treatment, such as bone abnormalities and vitamin D deficiency. Bone remodeling allows for bone volume and structure to be maintained, which is controlled by the receptor activator of the nuclear factor-kB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system-determining pathway in the balance between bone formation and resorption. Some reports have explored the role of corticosteroids in modulating the RANKL and OPG levels and RANKL/OPG ratio in pediatric patients. Nevertheless, studies evaluating the role of RANKL and OPG in the bone health of pediatric ALL patients during treatment are limited. During remission, we observed an increase in the RANKL/OPG ratio, increased RANKL levels, and decreased OPG levels in ALL patients. These changes may predispose such patients to the development of bone health disorders in their adult lives. Background: The receptor activator of the nuclear factor-kB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) pathway is a determining pathway in the balance between bone formation and resorption, and disruptions in this complex can affect bone metabolism. Methods: This study analyzes the changes in RANKL, OPG, and 25(OH)D levels; the RANKL/OPG ratio; and other bone turnover markers (BTMs) from diagnosis to complete remission in children with acute lymphoblastic leukemia (ALL). This is a prospective observational cohort study, carried out at the Instituto Mexicano del Seguro Social, Mexico City, including 33 patients (4–17 years) with newly diagnosed B-cell ALL. The patients were treated with the HP09 chemotherapy protocol. Children who had previously been treated with corticosteroids were excluded. A peripheral blood sample at diagnosis and remission was collected to determine the 25(OH)D and BTM concentrations. Results: Increased RANKL (p = 0.001) and osteocalcin (p < 0.001) levels and RANKL/OPG ratio (<0.001) and a decreased OPG level (p = 0.005) were observed at remission, predominantly in the high-risk (HR) relapse and vitamin D deficiency groups. A negative association between RANKL and OPG (r = −0.454, p = 0.008) was observed. Conclusions: we suggest that the RANKL/OPG ratio could serve as a bone remodeling marker in ALL patients. [ABSTRACT FROM AUTHOR]

Osteoclasts are the cells primarily responsible for inflammation‐induced bone loss, as is particularly seen in rheumatoid arthritis. Increasing evidence suggests that osteoclasts formed under homeostatic versus inflammatory conditions may differ in phenotype. While microRNA‐29‐3p family members (miR‐29a‐3p, miR‐29b‐3p, miR‐29c‐3p) promote the function of RANKL‐induced osteoclasts, the role of miR‐29‐3p during inflammatory TNF‐α‐induced osteoclastogenesis is unknown. We used bulk RNA‐seq, histology, qRT‐PCR, reporter assays, and western blot analysis to examine bone marrow monocytic cell cultures and tissue from male mice in which the function of miR‐29‐3p family members was decreased by expression of a miR‐29‐3p tough decoy (TuD) competitive inhibitor in the myeloid lineage (LysM‐cre). We found that RANKL‐treated monocytic cells expressing the miR‐29‐3p TuD developed a hypercytokinemia/proinflammatory gene expression profile in vitro, which is associated with macrophages. These data support the concept that miR‐29‐3p suppresses macrophage lineage commitment and may have anti‐inflammatory effects. In correlation, when miR‐29‐3p activity was decreased, TNF‐α‐induced osteoclast formation was accentuated in an in vivo model of localized osteolysis and in a cell‐autonomous manner in vitro. Further, miR‐29‐3p targets mouse TNF receptor 1 (TNFR1/Tnfrsf1a), an evolutionarily conserved regulatory mechanism, which likely contributes to the increased TNF‐α signaling sensitivity observed in the miR‐29‐3p decoy cells. Whereas our previous studies demonstrated that the miR‐29‐3p family promotes RANKL‐induced bone resorption, the present work shows that miR‐29‐3p dampens TNF‐α‐induced osteoclastogenesis, indicating that miR‐29‐3p has pleiotropic effects in bone homeostasis and inflammatory osteolysis. Our data supports the concept that the knockdown of miR‐29‐3p activity could prime myeloid cells to respond to an inflammatory challenge and potentially shift lineage commitment toward macrophage, making the miR‐29‐3p family a potential therapeutic target for modulating inflammatory response. [ABSTRACT FROM AUTHOR]

The Receptor Activator Nuclear of κB Ligand (RANKL) plays an important function in immune responses, activating osteoclast cells and unchanged bone resorption, which in turn leads to bone erosion and inflammation. Genetic variants in the promoter region of the RANKL gene could lead to a higher risk of rheumatoid arthritis (RA). Objective: To assess the association of rs9533155 (-693C>G) and rs9533156 (-643T>C) genetic variants with RA risk. Methods: A case–control study was carried out. A total of 94 patients with RA (RA group) and 134 subjects without any rheumatologic disease (control group) were included. Genetic DNA was extracted from peripheral white blood cells (leukocytes). Genetic variant rs9533155 (-693C>G) was screened by an approach based on Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP), while rs9533156 (-643T>C) was screened using quantitative polymerase chain reaction (qPCR) with TaqMan probes. RANKL serum levels were measured by ELISA. Results: For rs9533155 (-693C>G), the polymorphic homozygous genotype frequencies (CC) were higher in the RA group (p = 0.006). Individuals carrying the risk genotype presented higher levels of serum RANKL. Carriers of the polymorphic homozygous genotype in the dominant model (CC vs. CG + GG) had an increased risk of developing RA (OR: 1.8, 95% CI 1.04 to 3.1). No association between rs9533156 (-643T>C) and the haplotypes with RA risk was observed. Conclusion: The rs9533155 (-693C>G) genetic variant exhibits a potential role in RA risk. The studied population had no association with the rs9533156 (-643T>C) genetic variant. [ABSTRACT FROM AUTHOR]

Cemento-ossifying fibroma (COF) and juvenile ossifying fibroma (JOF) have been considered distinct entities within the category of fibro-osseous lesions. This study aimed to assess osteoblast and osteoclast activity in COF and JOF by investigating bone resorption markers, specifically receptor activator of nuclear factor-kB (RANK), RANK ligand (RANKL), and its inhibitor osteoprotegerin (OPG). A comparative analysis of these markers was performed on all lesions. Immunohistochemistry was employed to evaluate and quantify the expression of these biomarkers in a sample of 20 cases of cemento-ossifying fibroma (COF), 15 cases of psammomatoid juvenile ossifying fibroma (PsJOF), and 10 cases of trabecular juvenile ossifying fibroma (TrJOF). The expression of osteoprotegerin was significantly higher in cemento-ossifying fibroma (33.9±13.0) compared to trabecular juvenile ossifying fibroma (27.3±9.2) and psammatoid ossifying fibroma (25.2±14.9), with the COF showing the highest expression followed by the latter two (p=0.037). There was a higher percentage (80%) of stromal fibroblast cells that showed positive expression of RANKL in cemento-ossifying fibroma (COF) compared to psammomatoid juvenile ossifying fibroma (PsJOF) (33.3%) and trabecular juvenile ossifying fibroma (TrJOF) (30.0%) when considering a positive expression score of 3 (p=0.024). Cemento-ossifying fibroma demonstrated the highest expression of osteoprotegerin and RANKL-positive stromal fibroblast cells, followed by psammomatoid juvenile ossifying fibroma and trabecular juvenile ossifying fibroma. These findings provide valuable insights into the pathogenesis of these lesions. [ABSTRACT FROM AUTHOR]

Osteogenesis imperfecta (OI) is an inherited skeletal disorder that leads to bone fragility and multiple fractures. Given advances in the genetic understanding of existing phenotypes and newly discovered mutations, therapeutic management of OI has become challenging. Denosumab, a monoclonal antibody that inhibits the interaction between the receptor activator of nuclear factor kappa B ligand (RANKL) and its receptor RANK, has been approved to treat postmenopausal osteoporosis and emerged as an important therapy for malignancies and other skeletal disorders, including pediatric skeletal conditions such as OI. This review summarizes information about denosumab therapy in OI by exploring its mechanisms of action, main indications, and safety and efficacy. Several case reports and small series have been published about the short-term use of denosumab in children with OI. Denosumab was considered a strong drug candidate for OI patients with bone fragility and a high risk of fracture, particularly for patients with the bisphosphonate (BP)-unresponsive OI-VI subtype. The evidence for denosumab's effects in children with OI indicates that it effectively improves bone mineral density but not fracture rates. A decrease in bone resorption markers was observed after each treatment. Safety was assessed by tracking the effects on calcium homeostasis and reporting side effects. No severe adverse effects were reported. Hypercalciuria and moderate hypercalcemia were reported, suggesting that BPs be used to prevent the bone rebound effect. In other words, denosumab can be used as a targeted intervention in children with OI. The posology and administration protocol require more investigation to achieve secure efficiency. [ABSTRACT FROM AUTHOR]

Periodontal disease is one of the main causes of tooth loss. Clinically, this pathology, mediated by thederegulation of the immune system due to a dysbiosis occurred in the gingival sulcus, begins with the inflammation of the gum and evolves with the irreversible damage of the tissues that surround the tooth. Alveolar bone is one of the most affected tissues by this disease, due to the activation of osteoclasts by the upregulation of RANKL in the host. The aim of this study is to determine the increase of RANKL, in a U2OS tumor cells model, inoculated with Porphyromonas gingivalis and Prevotella intermedia. To identify the level of RANKL, four groups were defined: A control group, not treated; PG group, treated with P.gingivalis; PI group, treated with P. intermedia; and a PG+PI group, treated with both bacteria. The relative level of RANKL was determined in the supernatant and cell extracts independently, using the Western blot technique. In supernatants, the PG group showed higher RANKL levels compared to PI (p < 0.05). In cell extracts the levels were higher in the PG+PI group (p < 0.05.). The PI group showed the lowest levels of RANKL.Polymicrobial infection results in a greater expression of of soluble RANKL was observed. [ABSTRACT FROM AUTHOR]

Introduction: Evidence on second-line agents for osteoporosis and osteopenia associated with glucocorticoid use after first-line bisphosphonate therapy is limited. We, therefore, examine the efficacy of denosumab on bisphosphonate-treated osteoporosis and osteopenia in Japanese systemic rheumatic disease (SRD) patients receiving glucocorticoids. Materials and methods: Glucocorticoid-treated SRD patients with a pre-existing fragility fracture, either lumbar spine (LS) or femoral neck (FN) bone mineral density (BMD) T-score of ≤ −2.5 or of ≤ −1.5 without a significant increase in BMD in the past year despite oral bisphosphonate therapy were enrolled in this study. They were randomized to switch to 60 mg subcutaneous denosumab every six months (switching group) or to continue the bisphosphonate (continuing group). The primary endpoint was the percent change from baseline in BMD at the LS and FN at week 52. Results: Of the 39 subjects, 19 were assigned to the switching group and 20 to the continuing group. The switching group showed significant increases in LS BMD (5.7% vs. 1.1%, p = 0.002) and FN BMD (4.2% vs. −0.3%, p = 0.008) at week 52 than the continuing group, with a significant decrease in serum tartrate-resistant acid phosphatase 5b (−28.1% vs. 7.0%, p < 0.001) and improved patient satisfaction. Conclusion: Switching to denosumab demonstrated greater efficacy than continuing bisphosphonates in increasing BMD, inhibiting osteoclast activation, and enhancing patient satisfaction in Japanese bisphosphonate-treated osteoporosis and osteopenia patients with concomitant SRD receiving glucocorticoids. [ABSTRACT FROM AUTHOR]

Ameloblastoma is a benign, locally invasive epithelial odontogenic neoplasm of the jaw. Treatment of choice is jaw resection, often resulting in significant morbidity. The aim of this study was to recapitulate ameloblastoma in a completely humanised 3D disease model containing ameloblastoma cells, osteoblasts and activated osteoclasts to investigate the RANKL pathway within the ameloblastoma stromal environment and its response to the RANKL antibody denosumab. In vitro bone was engineered by culturing human osteoblasts (hOB) in a biomimetic, dense collagen type I matrix, resulting in extensive mineral deposits by day 21 forming alizarin red positive bone like nodules throughout the 3D model. Activated TRAP + human osteoclasts were confirmed through the differentiation of human CD14+ monocytes after 10 days within the model. Lastly, the ameloblastoma cell lines AM-1 and AM-3 were incorporated into the 3D model. RANKL release was validated through TACE/ADAM17 activation chemically or through hOB co-culture. Denosumab treatment resulted in decreased osteoclast activation in the presence of hOB and ameloblastoma cells. These findings stress the importance of accurately modelling tumour and stromal populations as a preclinical testing platform. [ABSTRACT FROM AUTHOR]

The notion that obesity can be a protective factor for bone health is a topic of ongoing debate. Increased body weight may have a positive impact on bone health due to its mechanical effects and the production of estrogen by adipose tissue. However, recent studies have found a higher risk of bone fracture and delayed bone healing in elderly obese patients, which may be attributed to the heightened risk of bone immune regulation disruption associated with obesity. The balanced functions of bone cells such as osteoclasts, osteoblasts, and osteocytes, would be subverted by aberrant and prolonged immune responses under obese conditions. This review aims to explore the intricate relationship between obesity and bone health from the perspective of osteoimmunology, elucidate the impact of disturbances in bone immune regulation on the functioning of bone cells, including osteoclasts, osteoblasts, and osteocytes, highlighting the deleterious effects of obesity on various diseases development such as rheumatoid arthritis (RA), osteoarthritis (AS), bone fracture, periodontitis. On the one hand, weight loss may achieve significant therapeutic effects on the aforementioned diseases. On the other hand, for patients who have difficulty in losing weight, the osteoimmunological therapies could potentially serve as a viable approach in halting the progression of these disease. Additional research in the field of osteoimmunology is necessary to ascertain the optimal equilibrium between body weight and bone health. [ABSTRACT FROM AUTHOR]

L'ostéoarthropathie neuropathique (ON) ou pied de Charcot est une complication rare mais grave de la neuropathie périphérique car elle conduit à une déformation et à un handicap. Aucun traitement pharmacologique n'est disponible. L'activité ostéoclastique, par l'intermédiaire du receptor activator of nuclear factor ligand (RANK-L), joue un rôle central dans le pied de Charcot actif. L'objectif de ce travail était de décrire l'effet clinique, radiologique et métabolique du dénosumab, un anticorps humanisé anti-RANK-L, dans le traitement de l'ON actif. Dans cette étude ouverte, conduite dans un centre tertiaire, tous les patients consécutifs avec ON réfractaire au traitement usuel ont été inclus. Une évaluation clinique, biologique, structurale (radiologie) et métabolique (imagerie nucléaire) a été effectuée à l'initiation du traitement et après traitement. Sept patients ont été traités par dénosumab entre 2017 et 2020 et suivis en médiane 16 mois [6–39]. Tous les patients ont été améliorés cliniquement. Quatre ont rechuté après une médiane de 4 mois [3–33] et ont été retraités avec une efficacité comparable. L'imagerie de suivi, disponible pour 5 patients a montré une stabilisation des lésions structurales (radiologiques) et une diminution significative de l'activité métabolique en TEP–TDM au FDG pour 4 d'entre eux. Aucun événement indésirable, notamment hypocalcémie, n'a été observé. Chez les patients souffrant d'ON actif, le dénosumab semblait cliniquement efficace, prévenait la destruction ostéoarticulaire et avait un effet métabolique évalué par TEP–TDM au FDG. Ces résultats justifient son évaluation dans l'ON actif au cours d'un essai contrôlé randomisé contre placebo. [ABSTRACT FROM AUTHOR]

Ameloblastoma is a benign, locally invasive epithelial odontogenic neoplasm of the jaw. Treatment of choice is jaw resection, often resulting in significant morbidity. The aim of this study was to recapitulate ameloblastoma in a completely humanised 3D disease model containing ameloblastoma cells, osteoblasts and activated osteoclasts to investigate the RANKL pathway within the ameloblastoma stromal environment and its response to the RANKL antibody denosumab. In vitro bone was engineered by culturing human osteoblasts (hOB) in a biomimetic, dense collagen type I matrix, resulting in extensive mineral deposits by day 21 forming alizarin red positive bone like nodules throughout the 3D model. Activated TRAP + human osteoclasts were confirmed through the differentiation of human CD14+ monocytes after 10 days within the model. Lastly, the ameloblastoma cell lines AM-1 and AM-3 were incorporated into the 3D model. RANKL release was validated through TACE/ADAM17 activation chemically or through hOB co-culture. Denosumab treatment resulted in decreased osteoclast activation in the presence of hOB and ameloblastoma cells. These findings stress the importance of accurately modelling tumour and stromal populations as a preclinical testing platform. [ABSTRACT FROM AUTHOR]

Background: Dickkopf-1 (DKK1) regulates bone formation by inhibiting canonical Wnt/ß-catenin pathway signaling, and indirectly enhances osteoclastic activity by altering the expression ratio of receptor activator of nuclear factor-κB ligand (RANKL) relative to osteoprotegerin (OPG). However, it is difficult to explain continued bone loss after allogeneic stem cell transplantation (allo-SCT) in terms of changes in only RANKL and OPG. Few studies have evaluated changes in DKK1 after allo-SCT. Methods: We prospectively enrolled 36 patients with hematologic malignancies who were scheduled for allo-SCT treatment. Serum DKK1, OPG, and RANKL levels were measured before (baseline), and at 1, 4, 12, 24, and 48 weeks after allo-SCT treatment. Bone mineral density (BMD) was assessed using dual-energy X-ray absorptiometry before (baseline) and 24 and 48 weeks after allo-SCT treatment. Results: After allo-SCT treatment, the DKK1 level decreased rapidly, returned to baseline during the first 4 weeks, and remained elevated for 48 weeks (P<0.0001 for changes observed over time). The serum RANKL/OPG ratio peaked at 4 weeks and then declined (P<0.001 for changes observed over time). BMD decreased relative to the baseline at all timepoints during the study period, and the lumbar spine in female patients had the largest decline (-11.3%±1.6% relative to the baseline at 48 weeks, P<0.05). Conclusion: Serum DKK1 levels rapidly decreased at 1 week and then continued to increase for 48 weeks; bone mass decreased for 48 weeks following engraftment in patients treated with allo-SCT, suggesting that DKK1-mediated inhibition of osteoblast differentiation plays a role in bone loss in patients undergoing allo-SCT. [ABSTRACT FROM AUTHOR]

Denosumab, a monoclonal antibody that specifically targets cytokine receptor activator of nuclear factor-kappa B ligand (RANKL), is a potentially viable option in resistant aneurysmal bone cysts. [ABSTRACT FROM AUTHOR]

Osteoprotegerin (OPG), receptor activator of nuclear factor-kappa B (RANK) and receptor activator of nuclear factor-kappa B ligand (RANKL), the members of the tumor necrosis factor (TNF) family, have multiple effects on bone metabolism, endocrine functions and, as an inflammatory pathway, in the immune system. This study tried to determine the association of the OPG/RANKL/RANK axis with the severity of unstable angina (UA) as an inflammatory condition. Our study involved 50 patients with UA and 50 healthy people. Serum and peripheral blood mononuclear cells were isolated from all participants. Serum levels and gene expression of OPG, RANKL, and RANK in mononuclear cells were measured by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR), respectively. For each patient with UA, the thrombolysis in myocardial infarction (TIMI) and the global registry of acute coronary events (GRACE) scores were determined to evaluate the severity of the disease. Then we analyzed the relation of OPG, RANKL, and RANK levels with TIMI and GRACE scores in patients with UA. Discriminate analysis was used to predict the combinational models of such factors on the prediction of UA. Serum levels of OPG and RANKL (p<0.001) and gene expression of RANKL (p<0.001) were significantly more in patients than those in healthy ones. No relation was seen between the OPG/RANKL/RANK axis and the severity of UA according to TIMI and GRACE scores. Our study shows that serum level, as well as gene expression of OPG/RANKL/RANK axis neither, predicts the occurrence of UA nor shows any relationship with its severity. [ABSTRACT FROM AUTHOR]

Background: Lipopolysaccharide (LPS) is an endotoxin and a vital component of gram-negative bacteria's outer membrane. During gram-negative bacterial sepsis, LPS regulates osteoclast differentiation and activity, in addition to increasing inflammation. This study aimed to investigate how LPS regulates osteoclast differentiation of RAW 264.7 cells in vitro. Results: Herein, we revealed that RAW cells failed to differentiate into mature osteoclasts in vitro in the presence of LPS. However, differentiation occurred in cells primed with receptor activator of nuclear factor-kappa-Β ligand (RANKL) for 24 h and then treated with LPS for 48 h (henceforth, denoted as LPS-treated cells). In cells treated with either RANKL or LPS, an increase in membrane levels of toll-like receptor 4 (TLR4) receptor was observed. Mechanistically, an inhibitor of TLR4 (TAK-242) reduced the number of osteoclasts as well as the secretion of tumor necrosis factor (TNF)-α in LPS-treated cells. RANKL-induced RAW cells secreted a very basal level TNF-α. TAK-242 did not affect RANKL-induced osteoclastogenesis. Increased osteoclast differentiation in LPS-treated osteoclasts was not associated with the RANKL/RANK/OPG axis but connected with the LPS/TLR4/TNF-α tumor necrosis factor receptor (TNFR)-2 axis. We postulate that this is because TAK-242 and a TNF-α antibody suppress osteoclast differentiation. Furthermore, an antibody against TNF-α reduced membrane levels of TNFR-2. Secreted TNF-α appears to function as an autocrine/ paracrine factor in the induction of osteoclastogenesis independent of RANKL. Conclusion: TNF-α secreted via LPS/TLR4 signaling regulates osteoclastogenesis in macrophages primed with RANKL and then treated with LPS. Our findings suggest that TLR4/TNF-α might be a potential target to suppress bone loss associated with inflammatory bone diseases, including periodontitis, rheumatoid arthritis, and osteoporosis. [ABSTRACT FROM AUTHOR]

Background: Giant cell tumor of bone (GCTB) is a rare osteoclastogenic stromal tumor. GCTB can rarely undergo malignant transformation. This post hoc analysis evaluated and classified malignancies in patients with GCTB who received denosumab.Methods: This analysis was conducted on patients with pathologically confirmed GCTB and measurable active disease treated with denosumab 120 mg subcutaneously once every 4 weeks, with loading doses on study days 8 and 15, as part of a phase 2, open-label, multicenter study. We identified potential cases of malignancy related to GCTB through an independent multidisciplinary review or medical history, associated imaging or histopathologic reports, and disease course. The findings were summarized and no statistical analysis was performed.Results: Twenty of five hundred twenty-six patients (3.8%) who received at least one dose of denosumab were misdiagnosed with GCTB that was later discovered to be malignancies: five primary malignant GCTB, five secondary malignant GCTB, four sarcomatous transformations, and six patients with other malignancies (giant cell-rich osteosarcoma, undifferentiated pleomorphic sarcoma, spindle cell sarcoma, osteogenic sarcoma, phosphaturic mesenchymal tumor of mixed connective tissue type, and fibrosarcoma/malignant fibrous histiocytoma). Many malignancies were present before denosumab was initiated (8 definitive cases, 7 likely cases), excluding potential involvement of denosumab in these cases. Signs associated with potential misdiagnoses of GCTB included poor mineralization with denosumab treatment, rapid relapse in pain, or a failure of the typical dramatic improvement in pain normally observed with denosumab.Conclusions: Although rare, GCTB can undergo malignant transformation, and rates in this study were consistent with previous reports. Signs of poor mineralization or lack of response to denosumab treatment may warrant close monitoring.Trial Registration: clinicaltrials.gov , ( NCT00680992 ). Registered May 20, 2008. [ABSTRACT FROM AUTHOR]

Objectives: Evidence shows that lithium, a medication commonly used for bipolar disorder treatment, presents bone anabolic activity. This study evaluated the effects of lithium chloride on periodontitis-induced bone loss (BL) and on intact alveolar bone during estrogen sufficiency and deficiency. Materials and methods: Rats (24/group) received sham surgery plus water (estrogen-sufficient group), ovariectomy plus water (estrogen-deficient group), sham surgery plus lithium chloride (150 mg/kg/every other day) (lithium/estrogen-sufficient group), or ovariectomy plus lithium chloride (lithium/estrogen-deficient group). One first mandibular molar received ligature, while the contralateral molar was left unligated. BL and trabecular bone area (TBA) were assessed in the furcation bone at 10, 20, and 30 days after ligature placement. Histochemical staining for TRAP and immunohistochemical staining for osteocalcin, osteopontin, osteoprotegerin, and RANKL were evaluated at 30 days after ligature placement. Results: At 10 days, the estrogen-deficient group presented the highest BL (0.115 ± 0.026), while the lithium/estrogen-deficient group (0.048 ± 0.024) presented the lowest BL in the ligated teeth (p < 0.05). At 20 and 30 days, the estrogen-deficient group exhibited significantly higher BL than all the other groups (p < 0.05). The ligated teeth of the lithium/estrogen-sufficient group presented the highest TBA while those of the estrogen-deficient group presented the lowest TBA at 10 and 30 days (p < 0.05). Unligated teeth of lithium-treated groups had stronger staining for osteocalcin and osteopontin than the estrogen-deficient group (p < 0.05). Ligated and unligated teeth of the estrogen-deficient group exhibited lower expression of osteoprotegerin than the other groups (p < 0.05). Lithium-treated groups exhibited generally higher staining of RANKL than the untreated groups (p < 0.05). Unligated teeth in both estrogen-sufficient groups presented lower TRAP expression than both estrogen-deficient groups (p < 0.05). Conclusions: Lithium chloride reduced ligature-induced BL in estrogen-deficient rats and yielded an overall greater trabecular area and overexpression of bone markers in alveolar bone under normal and deficient estrogen states. Clinical relevance: Lithium chloride may be a promising agent to assuage alveolar bone loss related to periodontitis, especially in osteoporotic conditions. [ABSTRACT FROM AUTHOR]

There have been new advances in understanding bone remodeling on a molecular level including the RANKL-OPG pathway, leading to advancements in targeted therapeutic intervention. There is however limited data in pediatrics with little known on normative values in healthy children. This is the largest cohort to quantify RANKL, OPG, and RANKL: OPG levels in healthy children as well as study the influence of age, gender, Tanner stage, and BMI in this population. Healthy subjects, 1–21 years of age, were recruited from general pediatric clinics affiliated with CHLA and in collaboration with samples stored from a previously completed study. Healthy children were defined as those with no chronic disease, daily medication, or fractures in the past six months. Free soluble RANKL and OPG levels were quantified using a sandwich ELISA. Three hundred samples were collected with overall serum concentrations of RANKL, OPG and RANKL: OPG of 0.28 pmol/L, 3.56 pmol/L and 0.08 pmol/L, respectively. Serum RANKL and RANKL: OPG concentrations were significantly different by age (p = 0.0001 and 0.0027, respectively). There was an overall downward trend by age except in the 11–15-year age group where a slight increase was noted. RANKL concentrations were also significantly different between Tanner stages, with highest concentrations seen at Tanner 3 (p = 0.0481), and zBMI (p = 0.001). OPG was inversely correlated with zBMI, but not influenced by gender, age, or Tanner stage. We showed significant difference in RANKL levels by age, Tanner stage, and zBMI. OPG was inversely correlated with zBMI. Insight into circulating levels of RANKL, OPG and RANKL: OPG in healthy children may be a potential tool to better understand disease states in pediatrics. Future studies are needed to evaluate the clinical significance of RANKL and OPG levels for diagnostic and therapeutic purposes in this population. • Normative data on RANKL and OPG levels in children is limited. • We quantify RANKL and OPG levels in the largest, healthy, pediatric cohort to date. • RANKL levels were significantly different by age, Tanner stage, and zBMI. • OPG was inversely related to zBMI. [ABSTRACT FROM AUTHOR]

Background: Tissue engineering using Stem cell from Human Exfoliated Deciduous Teeth (SHED) and a natural biomaterials biomaterial scaffold has become a promising therapy for the alveolar bone defect. The aim of this study was to analyze the Osteoprotegerin (OPG) and Receptor Activator of NF-κb ligand (RANKL) expression after the application of Hydroxyapatite scaffold and SHED. Methods: A laboratory experimental research with a post test-only control group. 14 male Wistar rats weighing from 260 to 280 g were used as the animal study. The animals were randomly assigned to an experimental group Hydroxyapatite scaffold (group I) and Hydroxyapatite scaffold combined with SHED (group II). The alveolar bone defect in the animal study model was affected by extracting anterior mandible teeth. Immunostaining was performed after 8 weeks in order to facilitate the examination of OPG and RANKL expression. Data were analyzed by independent t test. The correlation between OPG and RANKL expression were analyzed using Pearson's correlation test (P<0.05). Statistical analysis was performed using R statistical software version 3.4.0. Results: The independent t test showed that the differences were statistically significant. OPG expression in Group I (6.0±1.00) was lower than in Group II (11.6±1.14) (P=0.0004). The independent t test showed that the differences were statistically significant. RANKL expression for Group I (12.67±2.08) and Group II (4.80±1.304) showed a statistically significant difference (P=0.0005). Conclusion: Hydroxyapatite scaffold and SHED increase Osteoprotegerin and decrease Receptor Activator of NF-κB Ligand expression with high potential as an effective agent in alveolar bone defect regeneration. Please cite this article as: Prahasanti Ch, Subrata LH, Saskianti T, Suardita K, Ernawati DS. Combined Hydroxyapatite Scaffold and Stem Cell from Human Exfoliated Deciduous Teeth Modulating Alveolar Bone Regeneration via Regulating Receptor Activator of Nuclear Factor-κb and Osteoprotegerin System. [ABSTRACT FROM AUTHOR]

Apical periodontitis caused by root canal infection is the most frequent pathological lesion in the jaws, mainly manifested as periapical granulomas and cysts. Understanding of the formation and progression of apical periodontitis as well as the identification of inflammatory biomarkers can help increase the knowledge of pathogenic mechanisms, improve the diagnosis and provide support for different therapeutic strategies. The objective of the present article is to review inflammatory biomarkers such as cytokines, chemokines, inflammatory cells, neuropeptides, RANK/RANKL/OPG system and other inflammatory markers and to relate these systems to the development and progression of pathological conditions related to apical periodontitis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Different degrees of bone resorption and granulation tissues appear in the apical region of chronic periapical periodontitis. RANK-RANKL-osteoprotegerin signaling pathway has been shown to be the key pathway for regulating bone resorption. OBJECTIVE: To detect the expression and role of RANKL in mouse apical periodontitis. METHODS: C57BL/6J mice were allocated into experimental and control groups. In the experimental group, were used to open the mandibular first molars exposed to the oral environment were open to create the model of periapical periodontitis. The mandibular tissue was collected at 1, 2, 3 and 4 weeks after modeling, and the periapical tissue changes were observed by hematoxylin-eosin staining. The expression level of RANKL at each time point was detected by immunohistochemical staining. RESULTS AND CONCLUSION: Hematoxylin-eosin staining results showed that the mouse model of chronic periapical periodontitis was successfully established. In the control group, only a few inflammatory cells were found in the apical area of mandibular first molars, and the periodontal tissue was intact. The extent of inflammatory infiltration was gradually increased from 1 week to 4 weeks after modeling, and the alveolar bone destruction gradually increased. The results of immunohistochemistry showed that the expression level of RANKL in the experimental group was significantly higher than that in the control group, the level increased significantly from 1 week to 2 weeks after modeling, continued to increase at 3 weeks, and reduced at 4 weeks (all P < 0.05). To conclude, the model of chronic periapical periodontitis is successfully established in mice. The expression level of RANKL in mouse chronic apical periodontitis is high and shows a clear trend, which may promote the progress of chronic apical periodontitis and bone destruction. [ABSTRACT FROM AUTHOR]

BACKGROUND: Myeloma patients have less movement because of bone pain and osteolytic destruction, which result in bone loss and disease aggravation. OBJECTIVE: To explore the effect of myeloma cells (U266 cells) on osteocytes (Y4 cells), osteoblasts and osteoclasts as well as RANKL secreted by osteocytes under fluid shear stress simulating human mechanical movement by a fluid flow devise. METHODS: A flow and no-flow model was established. U266 cell culture fluid was used in the experimental group and Y4 standard culture fluid was used in the flow and control groups. Myeloma cell line U266 and mouse osteocytes cell line Y4 were cultured and passaged in vitro. Y4 cells were allotted and cultured for 48 hours, and the morphology of osteocytes and osteoclasts was observed and counted under microscope. The level of RANKL was quantitatively determined by ELISA and western blot assay. The RANKL, sample and standard culture system was established, RAW264.7 cells were cultured in vitro, and induced by RANKL. Number and morphology of RAW264.7 cells differentiating into osteoclasts were observed by TRAP staining. RESULTS AND CONCLUSION: Compared with the control group, Y4 cells in U266 culture supernatant exhibited morphological changes and their number was significantly decreased. The number of TRAP-positive osteoclasts was increased and the expression of RANKL protein in Y4 cells was significantly up-regulated (P < 0.05). Comparison between the flow model and the no-flow model showed that the number of Y4 cells was significantly increased, the expression of RANKL protein decreased and the number of TRAP-positive osteoclasts was significantly reduced (P < 0.05). Our study indicates that myeloma cells can inhibit the growth of normal osteocyte and might promote the expression of RANKL protein and the growth of osteoclasts. Compared with static state, fluid shear stress may promote the proliferation of osteocytes and might inhibit the expression of RANKL protein and the growth of osteoclasts. So, we hypothesize that mild exercise may prevent the progression of myeloma bone disease. [ABSTRACT FROM AUTHOR]

Purpose: We investigated the expression of the proinflammatory and proangiogenic factor osteoprotegerin (OPG) and its ligands, receptor activator of nuclear factor-κB ligand (RANKL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and the receptor RANK in proliferative diabetic retinopathy (PDR).Materials and Methods: Vitreous samples from PDR and nondiabetic control patients and epiretinal membranes from PDR patients were studied by enzyme-linked immunosorbent assay, immunohistochemistry, and Western blot analysis.Results: Vascular endothelial growth factor, OPG, and soluble RANK levels in vitreous samples from PDR patients were significantly higher than that in nondiabetic controls. Soluble TRAIL levels were significantly lower in PDR patients than that in nondiabetic control, whereas soluble RANKL levels did not differ significantly. RANKL, RANK, and TRAIL were expressed in vascular endothelial cells, myofibroblasts, and CD45-expressing leukocytes in PDR epiretinal membranes.Conclusions: Dysregulated expression of OPG/RANKL/RANK pathway and TRAIL might be related to inflammation and angiogenesis in PDR. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC) is the most common malignancy among oral cancers and shows potent activity for local bone invasion. Receptor activator of nuclear factor κB (RANK) ligand (RANKL) is critical for bone‐resorbing osteoclast formation. We previously demonstrated that OSCC tumor cells express high levels of RANKL. In this study, confocal microscopy demonstrated RANKL specific receptor, RANK expression in OSCC tumor cell lines (SCC1, SCC12, and SCC14a). We also confirmed the expression of RANK and RANKL in primary human OSCC tumor specimens. However, regulatory mechanisms of RANKL expression and a functional role in OSCC tumor progression are unclear. Interestingly, we identified that RANKL expression is autoregulated in OSCC tumor cells. The RANKL specific inhibitor osteoprotegerin (OPG) treatment to OSCC cells inhibits autoregulation of RANKL expression. Further, we showed conditioned media from RANKL CRISPR‐Cas9 knockout OSCC cells significantly decreased osteoclast formation and bone resorption activity. In addition, RANKL increases OSCC tumor cell proliferation. RANKL treatment to OSCC cells demonstrated a dose‐dependent increase in RANK intracellular adaptor protein, TRAF6 expression, and activation of IKK and IκB signaling molecules. We further identified that transcription factor NFATc2 mediates autoregulation of RANKL expression in OSCC cells. Thus, our results implicate RANKL autoregulation as a novel mechanism that facilitates OSCC tumor cell growth and osteoclast differentiation/bone destruction. [ABSTRACT FROM AUTHOR]

BACKGROUND: It is generally believed that various cytokines, such as receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG), which regulate osteoclast and osteoblast formation and differentiation, are mainly secreted by osteoblasts and bone marrow stromal cells. Whereas in the periodontal tissues, homologous periodontal ligament stem cells (PDLSCs) also play a significant role in the regulation of inflammation and immunity. Thus, virulence factors are very likely to mediate alveolar bone resorption by involvement in the regulation of RANKL and OPG. OBJECTIVE: To explore whether the inhibition of nuclear factor-κB activity by pyrrolidine dithiocarbamate (PDTC) can relieve the imbalance of RANKL/RANK/OPG system in PDLSCs in inflammatory state. METHODS: After isolation, culture and differentiation in vitro, passage 4 PDLSCs were divided into four group: control group, cultured in the DMEM medium containing 10% fetal bovine serum; lipopolysaccharide (LPS) group, cultured in the DMEM medium containing 10 mg/L LPS; 10 μmol/L PDTC group, pretreated with 10 μmol/L PDTC for 30 minutes followed by removal of the medium and addition of the medium containing 10 mg/L LPS; 100 μmol/L PDTC group, pretreated with 100 μmol/L PDTC for 30 minutes followed by removal of the medium and addition of the medium containing 10 mg/L LPS. In the latter two groups, cell morphology was observed under inverted microscope at 30 minutes after PDTC pretreatment. After 24 hours of culture, expression of OPG and RANKL mRNA and the ratio of RANKL/OPG in cells were detected in each group. RESULTS AND CONCLUSION: (1) PDTC pretreatment at a concentration of 100 μmol/L could cause cell morphological changes: some cells were adherent and retracted, and became round with the cell body shrinking in size. (2) Compared with the control group, the expression of RANKL in the PDLSCs was significantly increased in the LPS group (P < 0.05), and the expression of RANKL in the PDLSCs pretreated with 10 μmol/L PDTC had no significant change (P > 0.05), while compared with the LPS group, the expression of RANKL in the PDLSCs pretreated with 100 μmol/L PDTC was significantly lowered (P < 0.05). (3) Compared with the control group, the expression of OPG in the PDLSCs was slightly lowered in the LPS group (P > 0.05). After pretreatment with 10 μmol/L PDTC, the expression of OPG in the PDLSCs was significantly increased (P <0.05) compared with the LPS group, while the expression of OPG in the PDLSCs pretreated with 100 μmol/L PDTC had no significant difference from that in the LPS group (P > 0.05). (4) Compared with the control group, the ratios of RANKL/OPG in the other groups were significantly increased (P < 0.05), especially in the LPS group. With the increase of PDTC concentration, the ratio of RANKL/OPG decreased gradually(P < 0.05). These results reveal that PDTC can relieve the imbalance of RANK/RANKL/OPG system in the PDLSC in inflammatory state by inhibiting nuclear factor-κB activity. [ABSTRACT FROM AUTHOR]

Abstract: Aim: To investigate whether apical periodontitis lesions infected by Epstein–Barr virus (EBV) exhibit higher levels of oxidative stress biomarkers [8‐hydroxydeoxyguanosine (8‐OHdG) and oxidized glutathione (GSSG)] and bone resorption regulators [receptor activator of nuclear factor (NF‐κB) ligand (RANKL) and osteoprotegerin (OPG)] compared to EBV‐negative periapical lesions and healthy pulp tissues. Methodology: The experimental group consisted of 30 EBV‐positive and 30 EBV‐negative periapical lesions collected in conjunction with apicoectomy. The pulp tissues of 20 impacted third molars were used as healthy controls. The qualitative and quantitative analysis of EBV was performed by nested and real‐time polymerase chain reaction (PCR), respectively. The levels of RANKL and OPG were analysed by reverse transcriptase real‐time PCR. The levels of 8‐OHdG and GSSG were determined by enzyme‐linked immunosorbent assay (ELISA). Mann–Whitney U‐test and Spearman's correlation were used for statistical analysis. Results: The levels of RANKL, OPG, 8‐OHdG and GSSG were significantly higher in apical periodontitis lesions compared to healthy pulp controls (P = 0.001, P < 0.001, P < 0.001 and P < 0.05, respectively). RANKL and OPG mRNA expression was significantly higher in EBV‐positive compared to EBV‐negative periapical lesions (P < 0.05). There was no significant correlation between EBV copy numbers and levels of RANKL, OPG, 8OH‐dG and GSSG in apical periodontitis. Conclusion: Levels of bone resorption regulators and oxidative stress biomarkers were increased in apical periodontitis compared to healthy pulp tissues. EBV‐positive periapical lesions exhibited higher levels of RANKL and OPG compared to EBV‐negative periapical lesions. EBV may contribute to progression of apical periodontitis via enhanced production of bone resorption regulators. [ABSTRACT FROM AUTHOR]

Exposure to fluoride from environmental sources remains an overlooked, but serious public health risk. In this study, we looked into the role osteocytes play on the mechanism underlying fluoride induced osteopathology. We analyzed bone formation and resorption related genes generated by osteocytes that were exposed to varied doses of fluoride with and without PTH in vitro. Correspondingly, osteogenesis and osteoclastogenesis related genes were also investigated in rats exposed to fluoride for 8 weeks, and the PTH(1–34)was applied at the last 3 weeks to observe its role in regulating bone turnover upon fluoride treatment. The data in vitro indicated that fluoride treatment inhibited Sost expression of mRNA and protein and stimulated RANKL mRNA protein expression as well as the RANKL/OPG ratio in the primary osteocytes. Single PTH treatment played the similar role on expression of these genes and proteins. The PTH combined administration enhanced the action of fluoride treatment on RNAKL/OPG and SOST/Sclerostin. The up-regulation of RANKL and decreasing of Sost induced by fluoride and/or PTH treatment was validated in vivo and suggests that osteocytes are a major source of RANKL and Sost, both of which play essential roles in fluoride affecting osteogenesis and osteoclastogenesis. Expression of Wnt/β-catenin was up-regulated in both in vitro osteocytes treated with high dose of fluoride and bone tissue of rats in the presence of fluoride and PTH. In vivo, fluoride and single PTH stimulated bone turnover respectively, furthermore, PTH combined with low dose of fluoride treatment reinforced the osteogenesis and osteoclastogenesis genes expression, however, co-treatment of PTH reversed the effect of high dose of fluoride on osteogenesis and osteoclastogenensis related factors. In conclusion, this study demonstrated that osteocytes play a key role in fluoride activated bone turnover, and PTH participates in the process of fluoride modulating SOST/Sclerostin and RANKL expression. [ABSTRACT FROM AUTHOR]

Microfold cells (M cells), which are located in the follicle-associated epithelium (FAE) covering mucosal lymphoid follicles, are specialized epithelial cells that initiate mucosal immune responses. These cells take luminal antigens and transport them via transcytosis across the FAE to the antigen-presenting cells underneath. Several intestinal pathogens exploit M cells as their portal for entry to invade the host and cause disease conditions. Recent studies have revealed that the uptake of antigens by M cells is essential for efficient antigen-specific IgA production and that this process likely maintains the homeostasis of mucosal tissues. The present article reviews recent advances in understanding the molecular mechanism of M-cell differentiation and describes the molecules expressed by M cells that are associated with antigen uptake and/or the transcytosis process. Current efforts to augment M-cell-mediated uptake for use in the development of effective mucosal vaccines are also discussed. [ABSTRACT FROM AUTHOR]

Objectives. RANK ligand (RANKL) is involved in destruction and osteoporosis in RA. In this study, the relationships between RANKL and ACPA, anti-carbamylated protein antibodies (anti-CarP), cytokines and chemokines were analysed in individuals before the onset of RA symptoms, and their associations with radiological findings at disease onset were assessed. Methods. This was a case-control study performed within the Medical Biobank of Northern Sweden that included 470 pre-symptomatic individuals [334 women and 136 men; mean (S.D.) age 52.3 (9.4) years] using blood samples donated before symptom onset (pre-dating time; 5.0 years) and 96 controls (60 women and 36 men). Plasma was analysed for RANKL (BioVendor, Karasek, Brno, Czech Republic), anti-CCP2 antibodies (Eurodiagnostics, Malmö, Sweden), anti-CarP antibodies (in-house ELISA), ACPA specificities (ISAC-platform, Phadia AB, Uppsala, Sweden) and cytokines/chemokines (Meso Scale Discovery methods, Rockville, MD, USA). Radiographs of hands and feet were graded using the Larsen score. Results. The concentration of RANKL was higher in the pre-symptomatic individuals compared with controls; mean (S.E.M.): 0.50 (0.03) vs 0.22 (0.02) nmol/l (P<0.001). The concentration increased gradually over time until symptom onset but appeared later than ACPA/RF/anti-CarP antibodies. Positivity for these antibodies yielded higher levels of RANKL compared with seronegativity (P<0.001). RANKL concentrations were significantly associated with IL-6 and IL-10 concentrations. The combination of positivity for RANKL and anti-CarP antibodies resulted in a higher Larsen score at diagnosis β = 6.18 (95% CI: 0.93, 11.43; P = 0.022). Conclusion. RANKL concentrations were increased several years before symptom onset for RA, particularly in ACPA/RF/anti-CarP-positive individuals, all detectable earlier than RANKL. Positivity for RANKL and anti-CarP antibodies yielded the highest Larsen score at disease onset. [ABSTRACT FROM AUTHOR]

• Anti-CarP level has potential as a novel disease biomarker. • Anti-CarP antibodies may play a critical role in bone loss associated with RA. • Serum CarP antibody is significantly positively correlated with RANKL. We investigated the association of serum anti-carbamylated protein (anti-CarP) antibodies with disease activity and bone loss in rheumatoid arthritis (RA). The serum anti-CarP antibody concentrations of RA and non-RA patients and healthy controls were determined by enzyme-linked immunosorbent assay (ELISA) and then compared. The diagnostic value of anti-CarP antibodies in RA was determined by the receiver operating characteristic curve. Patients with RA and bone erosions were evaluated using ultrasound examinations. Ultrasonography was performed using a semiquantitative scale. The serum receptor activator of nuclear factor Κ-Β ligand (RANKL) concentrations were measured by ELISA to focus on bone loss. Peripheral serum anti-CarP antibody concentrations in patients with RA were significantly higher than those in patients without RA and in healthy controls and were positively correlated with disease activity. Anti-CarP antibody concentrations were significantly increased in patients with anti-CCP-positive RA. Positive correlation were found between anti-CarP and RANKL. Increased serum anti-CarP antibodies in women with postmenopausal osteoporosis (OP). Anti-CarP antibodies are associated with RA disease activity and may play an important role in bone loss associated with RA. The concentration of anti-CarP antibodies may be beneficial in the early diagnosis of RA, thus supporting its potential as a novel disease biomarker. [ABSTRACT FROM AUTHOR]

Objective To evaluate the effects of metformin on bone healing around titanium implants inserted in non-diabetic rats. Methods Twenty Wistar rats were randomly assigned to one of the following groups: control group ( n = 10): rats without metformin treatment; MT group ( n = 10): rats treated with metformin (40 mg/kg/day by gavage). At thirty days after implant placement, animals were euthanized. Histometric measurements of bone-to-implant contact ( BIC) and bone area ( BA), in addition to immunohistochemical analysis of the number of cells stained for RANKL and OPG, were assessed in the cortical and medullary areas around implants. Results The percentages of BIC and BA in the cortical bone were significantly lower in the MT group than in the control group ( P < 0.05). Furthermore, the medullary bone around the implants inserted in the metformin-treated animals exhibited an increased number of RANKL-stained cells than that around the implants inserted in the control animals ( P < 0.05). Conclusions Metformin negatively affected osseointegration by reducing the percentages of BIC and BA and increasing the expression of RANKL around titanium implants inserted in non-diabetic rats. [ABSTRACT FROM AUTHOR]

Summary: There is marked bone loss after spinal cord injury (SCI); however, its pathogenesis and clinical management remain unclear. The increased circulating levels of receptor activator of nuclear factor kB ligand (RANKL) associated with bone loss shortly after SCI and the prevention of bone loss with denosumab treatment suggest a contributory role of RANKL in SCI-induced osteoporosis. Introduction: Bone turnover and bone loss are markedly increased shortly after SCI. However, the pathogenesis and clinical management of this process remain unclear, especially the role of the osteoprotegerin (OPG)/RANKL system in this disorder. The aim of this study was to analyze serum levels of OPG and RANKL in bone loss associated with recent SCI and the effect of denosumab treatment on these mediators. Methods: Twenty-three males with recent complete SCI were prospectively included. Serum OPG and RANKL levels, bone turnover markers (PINP, bone ALP, CTX), and bone mineral density (BMD) were assessed at baseline, at 6 months of follow-up, prior to initiating denosumab, and 6 months after treatment. The results were compared with a healthy control group. Results: At baseline, SCI patients showed higher RANKL levels vs. controls which were correlated with days-since-SCI and total hip BMD loss at 6 months. OPG levels were similar to controls at baseline. After denosumab treatment, OPG showed no changes, whereas RANKL levels became undetectable in 67% of patients. Patients with undetectable RANKL during treatment showed better response in femoral BMD and bone markers vs. patients with detectable RANKL at 6 months of denosumab. Increased parathormone (PTH) levels during treatment were negatively correlated with RANKL changes. Conclusions: RANKL levels are increased after SCI and related to BMD loss at the proximal femur, becoming undetectable after denosumab treatment. The effect of denosumab on preventing sublesional bone loss, especially in patients with undetectable levels during treatment, suggests a contributory role of RANKL in this process. [ABSTRACT FROM AUTHOR]

Objectives. Mitochondrial DNA (mtDNA) contains sequestered damage-associated molecular patterns that might be involved in osteoimmunological pathogenesis of RA. Here, we aimed to investigate the cellular source of mtDNA and its role in RANK ligand (RANKL) expression by RA SF neutrophils. Methods. The gene expression signature of SF neutrophils was examined by proteomic quantitative analysis. Levels of mtDNA in circulating and SF neutrophils from RA patients and OA control subjects were assessed by real-time PCR. Purified neutrophils were challenged in vitro with Toll-like receptor agonists as well as mtDNA. RANKL expression by neutrophils was studied by flow cytometry. Results. SF neutrophils from RA patients displayed a gene expression signature of oxidative stress. This stress signature was associated with the release of mtDNA in SF as observed by a significant increase of mtDNA in the SF of RA patients compared with OA patients. mtDNA in RA SF was correlated with systemic inflammation as assessed by CRP concentrations. We also showed that mtDNA drives neutrophil RANKL expression to the same extent as Toll-like receptor agonists. Conclusion. Our data identify SF neutrophils as a cellular source of mtDNA that leads to a subsequent expression of RANKL. This highlights the important role of neutrophils in RA osteoimmunology. [ABSTRACT FROM AUTHOR]

Giant cell tumour of bone (GCTB) is an intermediate locally aggressive primary bone tumour, occurring mostly at the meta-epiphysis of long bones. Overexpression of receptor activator of nuclear factor kappa-B ligand (RANKL) by mononuclear neoplastic stromal cells promotes recruitment of numerous reactive multinucleated osteoclast-like giant cells, causing lacunar bone resorption. Preferential treatment is curettage with local adjuvants such as phenol, alcohol or liquid nitrogen. The remaining cavity may be filled with bone graft or polymethylmethacrylate (PMMA) bone cement; benefits of the latter are a lower risk of recurrence, possibility of direct weight bearing and early radiographic detection of recurrences. Reported recurrence rates are comparable for the different local adjuvants (27–31%). Factors increasing the local recurrence risk include soft tissue extension and anatomically difficult localisations such as the sacrum. When joint salvage is impossible, en-bloc resection and endoprosthetic joint replacement may be performed. Local tumour control on the one hand and maintenance of a functional native joint and quality of life on the other hand are the main pillars of surgical treatment for this disease. Current knowledge and development in the fields of imaging, functional biology and systemic therapy are forcing us into a paradigm shift from a purely surgical approach towards a multidisciplinary approach. Systemic therapy with denosumab (RANKL inhibitor) or zoledronic acid (bisphosphonates) blocks, respectively inhibits, bone resorption by osteoclast-like giant cells. After use of zoledronic acid, stabilisation of local and metastatic disease has been reported, although the level of evidence is low. Denosumab is more extensively studied in two prospective trials, and appears effective for the optimisation of surgical treatment. Denosumab should be considered in the standard multidisciplinary treatment of advanced GCTB (e.g. cortical destruction, soft tissue extension, joint involvement or sacral localisation) to facilitate surgery at a later stage, and thereby aiming at immediate local control. Even though several questions concerning optimal treatment dose, duration and interval and drug safety remain unanswered, denosumab is among the most effective drug therapies in oncology. [ABSTRACT FROM AUTHOR]

Osteoblasts, originating from mesenchymal cells, make the receptor activator of the nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) in order to control differentiation of activated osteoclasts, originating from hematopoietic stem cells. When the RANKL binds to the RANK of the pre-osteoclasts or mature osteoclasts, bone resorption increases. On the contrary, when OPG binds to the RANK, bone resorption decreases. Denosumab (AMG 162), like OPG (a decoy receptor), binds to the RANKL, and reduces binding between the RANK and the RANKL resulting in inhibition of osteoclastogenesis and reduction of bone resorption. Bisphosphonates (BPs), which bind to the bone mineral and occupy the site of resorption performed by activated osteoclasts, are still the drugs of choice to prevent and treat osteoporosis. The merits of denosumab are reversibility targeting the RANKL, lack of adverse gastrointestinal events, improved adherence due to convenient biannual subcutaneous administration, and potential use with impaired renal function. The known adverse reactions are musculoskeletal pain, increased infections with adverse dermatologic reactions, osteonecrosis of the jaw, hypersensitivity reaction, and hypocalcemia. Treatment with 60 mg of denosumab reduces the bone resorption marker, serum type 1 C-telopeptide, by 3 days, with maximum reduction occurring by 1 month. The mean time to maximum denosumab concentration is 10 days with a mean half-life of 25.4 days. In conclusion, the convenient biannual subcutaneous administration of 60 mg of denosumab can be considered as a first-line treatment for osteoporosis in cases of low compliance with BPs due to gastrointestinal trouble and impaired renal function. [ABSTRACT FROM AUTHOR]

A disequilibrium of tumor necrosis superfamily (TNF) members, including the serum osteoprotegerin, soluble receptor activator of nuclear factor-κB ligand, soluble TNF-related apoptosis-inducing ligand and TNF-α, was associated with the occurrence of a reduced skeletal mass and osteoporosis in male patients with end-stage chronic obstructive pulmonary disease (COPD). The purpose of this study was to explore the associations between serum biomarkers of tumor necrosis factor (TNF) superfamily and body and bone compositions in end-stage COPD males. Pulmonary function, T-score at the lumbar spine and femoral neck, lean mass, serum osteoprotegerin (OPG), soluble receptor activator of nuclear factor-κB ligand (sRANKL), TNF-α and its receptors (sTNFR-I, sTNFR-II) and soluble TNF-related apoptosis-inducing ligand (sTRAIL) levels were evaluated in 48 male patients with end-stage COPD and 36 healthy male volunteers. OPG was lower in male COPD patients than in control subjects, whereas sRANKL, TNF-α and its receptors were higher. The serum sTRAIL level showed a tendency to increase compared with that of healthy subjects (P = 0.062). Serum OPG showed a positive correlation with bone density. In contrast, serum TNF-α, sRANKL and sTRAIL were inversely associated with pretransplant bone density. We have noted the appearance of statistically significant inverse relationships between lean mass values and TNF-α, sTNFR-I and II and sRANKL levels in male COPD patients. Moreover, there was a negative correlation between sTRAIL levels with airway obstruction (P = 0.005) and hypercapnia (P = 0.042) in advanced COPD patients. Through a multiple linear regression analysis, our study revealed that a disequilibrium of TNF family members was strongly associated with the occurrence of a reduced skeletal mass and osteoporosis. These results provide further evidence that abnormal levels of TNF superfamily molecules may cause not only a decrease in BMD, but also lower muscle mass in end-stage COPD. [ABSTRACT FROM AUTHOR]

Objective: Receptor activator of nuclear factor-kappa B ligand (RANKL) appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells (HSCs). This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor (CTR) on cord blood HSC surface. Materials and Methods: In this experimental study, CD133+ hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase (TRAP) staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction (RT-PCR) assay for specific genes. Results: Hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells. Conclusion: Presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast. [ABSTRACT FROM AUTHOR]

Objective The objective of this in vitro study was to evaluate the effect of low-intensity pulsed ultrasound on the resorption activity of osteoclast cell cultures. Design RAW 264.7 cells were cultured and seeded over plates that were pre-coated with a synthetic carbonate apatite, and marked with fluoresceinamine-labeled sodium chondroitin polysulfate. Plates were randomly divided into 4 groups according to the treatment assigned to each one of them: NO RANKL (no RANK-L addition and no ultrasound application), NO LIPUS (addition of RANK-L and no ultrasound application), LIPUS 10 (addition of RANK-L and 10 min of ultrasound application per day), and LIPUS 20 (addition of RANK-L and 20 min of ultrasound application per day). The ultrasound device produced 1.5 MHz pulses with a repetition rate of 1 kHz and intensity of 30 mW/cm 2 . The experiment extended for one week and afterwards, resorption activity was evaluated according to the fluorescence intensity analysis and pit resorption measurements (number of pits and mean area). Results Our experiment consistently demonstrated that low-intensity pulsed ultrasound application enhanced osteoclasts resorptive activity. In addition, it was demonstrated that when daily ultrasound application lasted longer (20 min) the resorption was the highest. Results obtained from both evaluation methods were reasonably coherent. Conclusions Low-intensity pulsed ultrasound increases osteoclast resorptive activity in the absence of osteoblasts. This effect seems to be influenced by ultrasound treatment time. Future research might be directed to investigate osteoclast response to different ultrasound application protocols (frequencies and intensities) and potential cellular mechanisms. [ABSTRACT FROM AUTHOR]

Osteoclastogenesis is the major cellular event responsible for bone loss and is triggered by inflammation. Acai-berry has proven anti-inflammatory effects. However, there is a lack of evidence for its effects on osteoclastogenesis. Thus, the aim of this study was to determine whether acai-berry extract (ABE) could inhibit osteoclastogenesis and osteoclast activity in vitro . The secretion of cytokines by osteoclasts has been also evaluated. RAW 264.7 cells were stimulated with RANKL (50 ng/mL) and treated with various concentrations of ABE (25–100 μg/mL) to verify: cell viability (MTT), total protein concentration (BCA), osteoclast differentiation and activity, and cytokine secretion. Cell viability and protein assays showed no toxicity to RAW cells for the tested ABE concentrations (p > 0.05). ABE also showed a dose-dependent inhibition of osteoclastogenesis and osteoclast activity evaluated by tartrate-resistant acid phosphatase (TRAP) and hydroxylapatite resorption assay, respectively (p < 0.05). ABE decreased the secretion of interleukin (IL)-1α, -6 and tumor necrosis factor alpha while increasing the secretion of IL-3, -4, -13 and interferon gamma when compared to the control group (p < 0.05). Results of this study showed that acai-berry extract inhibits osteoclast differentiation and activity possibly due to the modulation of a vast number of cytokines produced by osteoclast precursor cells. [ABSTRACT FROM AUTHOR]

During periodontitis, alveolar bone resorption is associated with activation of T helper type 17 (Th17) lymphocytes and receptor activator of nuclear factor-κB ligand (RANKL) -induced osteoclasts. We previously reported that serotype b of Aggregatibacter actinomycetemcomitans has a higher capacity to trigger Th17-type differentiation and function in activated T lymphocytes and its lipopolysaccharide is a more potent immunogen compared with the other serotypes. This study aimed to investigate whether serotype b of A. actinomycetemcomitans induces higher Th17-associated RANKL production, RANKL-induced osteoclast activation, and antigen-specific memory T lymphocyte proliferation. On naive CD4(+) T lymphocytes stimulated with autologous dendritic cells primed with different A. actinomycetemcomitans serotypes, RANKL production, T-bet, GATA-3, RORC2 and Foxp3 expression, RORC2/RANKL intracellular double-expression, TRAP(+) osteoclast activation, and bone resorption were quantified. The frequency of proliferating memory T lymphocytes in response to A. actinomycetemcomitans serotypes was determined in periodontitis and healthy subjects. Naive CD4(+) T lymphocytes stimulated by serotype b-primed dendritic cells elicited higher levels of RANKL, RORC2, TRAP(+) osteoclasts, and bone resorption than the same cells stimulated with the other serotypes. RANKL positively correlated and co-expressed with RORC2. Memory T lymphocytes responding to serotype b were more frequently detected in periodontitis patients than healthy subjects. These results indicate that serotype b of A. actinomycetemcomitans is associated with higher production of RANKL and these increased levels are associated with Th17 lymphocyte induction, osteoclast activation, and bone resorption. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare the use of antimicrobial photodynamic therapy (aPDT) as an adjunct to scaling and root planing (SRP) in the treatment of experimentally induced periodontitis in female rats that were systemically treated with or without nicotine. Female rats (n = 180) were divided into two groups: vehicle administration (Veh) and nicotine administration (Nic). Mini-pumps containing either vehicle or nicotine were implanted in the rats 30 days before the induction of experimental periodontitis (EP). EP was induced by placing a cotton ligature around the left mandibular first molar. After 7 days, the ligature was removed, and the rats were randomly divided into three treatment subgroups: SRP (only SRP), DL (SRP plus diode laser), and aPDT (SRP plus aPDT). The aPDT consisted of phenothiazine photosensitizer deposition followed by diode laser irradiation. Ten rats from each subgroup were euthanized at 7, 15, and 30 days after treatment. Alveolar bone loss (ABL) in the furcation region was evaluated using histological, histometric, and immunohistochemical analyses. The rats that were treated with nicotine showed more ABL compared to those treated with vehicle. In both the Veh and Nic groups, SRP plus aPDT treatment resulted in reduced ABL, smaller numbers of both TRAP- and RANKL-positive cells, and higher numbers of PCNA-positive cells compared to SRP treatment alone. aPDT was an effective adjunctive therapy for the treatment of periodontitis in female rats regardless of whether they received nicotine. [ABSTRACT FROM AUTHOR]

Osteoclasts are multinucleated cells of hematopoietic origin that are responsible for the degradation of old bone matrix. Osteoclast differentiation and activity are controlled by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-kB ligand (RANKL). M-CSF and RANKL bind to their respective receptors c-Fms and RANK to stimulate osteoclast differentiation through regulation of delicate signaling systems. Here, we summarize the critical or essential signaling pathways for osteoclast differentiation including M-CSF-c-Fms signaling, RANKL-RANK signaling, and costimulatory signaling for RANK. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) play an integral role in cellular cascade that initiate and maintain Th2 responses in allergy. In this study, we examined the interaction between platelets and DCs to determine the role of platelets in the intervention of immune responses through modulation of DC functions. Blood-purified myeloid DCs, which had been stimulated with thymic stromal lymphopoietin (TSLP-DCs), formed aggregates with activated platelets. TSLP-DC maturation was induced after the interaction with TRAP6-activated platelets as indicated by an increase in the expression of CD86, CD40, and CD83. In addition, production of a Th2 cell-attracting chemokine, CCL17, was clearly upregulated by coculture of TSLP-DCs with TRAP6-activated platelets. We further found that an expression of RANK ligand (RANKL) on platelets was upregulated by the TRAP6 activation, and that, using the neutralizing antibody against RANKL, the platelet-derived RANKL induces the activation of TSLP-DCs. Thus, activated platelets can intervene in adaptive immune responses through induction of functional modulation of TSLP-DCs. Platelets have the ability to enhance the DC-mediated Th2 response and may contribute to the allergic inflammation. In conclusion, our study provides new insights in platelet functions and the possible mechanism of allergic responses that stem from DCs. [ABSTRACT FROM AUTHOR]

Tmem173 was identified as a growth inhibitor associated with major histocompatibility complex (MHC) class II and a potential stimulator for IFN-β, an innate immune inducer and a negative feedback controller for RANKL-induced osteoclast differentiation of monocytic macrophage cells. In this study, we confirmed that transmembrane protein 173 (Tmem173) overexpression inhibited the expression of osteoclast-specific genes, tartrate-resistant acid phosphatase (TRAP), cathepsin K, and matrix metalloproteinase-9 (MMP-9), as well as bone resorption pit formation in RANKL-treated RAW 264.7 cells. Activation of osteoclast-specific transcription factors, c-Fos and nuclear factor of activated T cells cytoplasmic-1 (NFATc1), and RANKL-induced activation of ERK were also down-regulated by Tmem173 overexpression. Collectively, these results suggest that Tmem173 plays a regulatory role in RANKL–RANK-mediated signaling in osteoclastogenesis. [ABSTRACT FROM AUTHOR]

Summary Objectives We aimed to determine the prognostic significance of receptor activator of nuclear factor kappa-B ligand (RANKL), RANK and osteoprotegerin (OPG) in patients with oral squamous cell carcinoma (OSCC). Materials and methods The protein expression of RANKL, RANK and OPG was assessed by immunohistochemistry on pretreatment biopsies of 93 patients with locally advanced OSCC who received preoperative chemoradiotherapy (CRT). The primary endpoint was cancer-specific survival. Secondary endpoints were correlation of biomarkers with bone invasion and pathological tumor response. Kaplan–Meier curves and Cox regression models were used for survival analyses. Results A significantly higher OPG expression was demonstrated in patients with malignant bone invasion and non-responders to CRT as compared to patients without bone invasion and responders ( p = 0.032 and p = 0.033, respectively). Multivariate analysis revealed that higher OPG expression was independently associated with shorter cancer-specific survival ( p = 0.04). The expression status of RANKL and RANK was not significantly related to clinicopathological characteristics and had no impact on survival of OSCC patients. Conclusion Upregulation of OPG expression is associated with bone invasion, poor pathological tumor regression to neoadjuvant CRT, and worse long-term cancer-specific survival in patients with locally advanced OSCC. Our results indicate that OPG may be a novel prognostic biomarker in oral cancer. [ABSTRACT FROM AUTHOR]

Osteoclasts are multinucleated cells formed mainly on bone surfaces in response to cytokines by fusion of bone marrow-derived myeloid lineage precursors that circulate in the blood. Major advances in understanding of the molecular mechanisms regulating osteoclast formation and functions have been made in the past 20 years since the discovery that their formation requires nuclear factor-kappa B (NF-κB) signaling and that this is activated in response to the essential osteoclastogenic cytokine, receptor activator of NF-κB ligand (RANKL), which also controls osteoclast activation to resorb (degrade) bone. These studies have revealed that RANKL and some pro-inflammatory cytokines, including tumor necrosis factor, activate NF-κB and downstream signaling, including c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATcl), and inhibition of repressors of NFATcl signaling, to positively regulate osteoclast formation and functions. However, these cytokines also activate NF-κB signaling that can limit osteoclast formation through the NF-κB signaling proteins, TRAF3 and p100, and the suppressors of c-Fos/NFATc1 signaling, IRF8, and RBP-J. This paper reviews current understanding of how NF-κB signaling is involved in the positive and negative regulation of cytokine-mediated osteoclast formation and activation. [ABSTRACT FROM AUTHOR]

Background: There is no established systemic treatment option for unresectable osteosarcoma progressing after standard chemotherapy. A recently published clinical trial has demonstrated some activity of sorafenib in this situation. Preclinical research suggests a role for the inhibition of the receptor activator of nuclear factor-ĸB ligand (RANKL), but no clinical data have been reported so far. Case Report: A 37-year-old man was diagnosed with unresectable osteoblastic, osteoblastoma-like osteosarcoma in the C7/Th1 vertebra. The tumour progressed locally despite two lines of chemotherapy and stereotactic radiotherapy. On treatment with sorafenib and denosumab, a complete metabolic remission was achieved and is ongoing for over 18 months. Immunohistochemistry revealed an overexpression of RANK and RANKL in the patient's primary tumour. Discussion: This is the first report of activity achieved by the combination of the tyrosine kinase inhibitor sorafenib and the RANKL inhibitor denosumab in a patient with osteosarcoma. It confirms preclinical data on RANK/RANKL inhibition in osteosarcoma and could serve as a hypothesis-generating approach for clinical trials in this patient population. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Receptor activator of nuclear factor kappa-B ligand (RANKL) is a critical osteoclastogenic factor expressed in bone marrow stromal/osteoblast lineage cells. Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) levels are elevated in pathologic conditions such as multiple myeloma and inflammatory arthritis, and have been positively correlated with osteolytic markers. Osteoprotegerin (OPG) which inhibits osteoclastogenesis is a decoy receptor for RANKL and also known to interact with TRAIL. Herein, we show that TRAIL increases DR5 and DcR1 receptors but no change in the levels of DR4 and DcR2 expression in human bone marrow derived stromal/preosteoblast (SAKA-T) cell line. We further demonstrated that TRAIL treatment significantly decreased OPG mRNA expression. Interestingly, TRAIL treatment induced RANKL mRNA expression in these cells. In addition, TRAIL significantly increased NF-kB and c-Jun N-terminal kinase (JNK) activity. Human transcription factor array screening by real-time RT-PCR identified TRAIL up-regulation of the signal transducers and activators of the transcription (STAT)-6 expression in SAKA-T cells. TRAIL stimulation induced p-STAT-6 expression in human bone marrow derived primary stromal/preosteoblast cells. Confocal microscopy analysis further revealed p-STAT-6 nuclear localization in SAKA-T cells. Chromatin immunoprecipitation (ChIP) assay confirmed p-STAT-6 binding to the hRANKL gene distal promoter region. In addition, siRNA suppression of STAT-6 expression inhibits TRAIL increased hRANKL gene promoter activity. Thus, our results suggest that TRAIL induces RANKL expression through a STAT-6 dependent transcriptional regulatory mechanism in bone marrow stromal/preosteoblast cells. [ABSTRACT FROM AUTHOR]