Role of denosumab in bone erosions in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by systemic inflammation and synovitis which evolve into joint destruction and deformity. Bone abnormalities are represented by marginal bone erosions and iuxta-articular and generalized osteoporosis. Overactivation of osteoclasts along with dysregulation of osteoblasts are the key events. Bone resorption is mediated by the receptor activator of nuclear factor (NF)-κB (RANK) ligand (RANK-L), responsible for the differentiation, proliferation, and activation of osteoclasts. RANK-L binds its receptor RANK, localized on the surface of preosteoclasts and mature osteoclasts promoting osteoclastogenesis. High levels of RANK-L were demonstrated in active RA patients. Denosumab, a fully human monoclonal antibody, binds RANK-L and suppresses the RANK-RANK-L signaling pathway leading to the inhibition of osteoclastogenesis. A retrospective analysis of published studies such as clinical trials evidenced the efficacy of denosumab in preventing bone erosion progression in RA patients. Key messages Key questions to answer in future include the following: Could denosumab be associated with other biologic therapies in RA patients? Could denosumab block the progression of bone damage in RA? Could denosumab be used for the prevention of bone erosion in RA?

Chronic inflammation and extracellular matrix-specific autoimmunity following inadvertent periarticular influenza vaccination.

BACKGROUND: Viral infections may trigger autoimmunity in genetically predisposed individuals. Immunizations mimic viral infections immunologically, but only in rare instances vaccinations coincide with the onset of autoimmunity. Inadvertent vaccine injection into periarticular shoulder tissue can cause inflammatory tissue damage ('shoulder injury related to vaccine administration, SIRVA). Thus, this accident provides a model to study if vaccine-induced pathogen-specific immunity accompanied by a robust inflammatory insult may trigger autoimmunity in specific genetic backgrounds. METHODS: We studied 16 otherwise healthy adults with suspected SIRVA occurring following a single work-related influenza immunization campaign in 2017. We performed ultrasound, immunophenotypic analyses, HLA typing, and influenza- and self-reactivity functional immunoassays. Vaccine-related bone toxicity and T cell/osteoclast interactions were assessed in vitro. FINDINGS: Twelve of the 16 subjects had evidence of inflammatory tissue damage on imaging, including bone erosions in six. Tissue damage was associated with a robust peripheral blood T and B cell activation signature and extracellular matrix-reactive autoantibodies. All subjects with erosions were HLA-DRB1*04 positive and showed extracellular matrix-reactive HLA-DRB1*04 restricted T cell responses targeting heparan sulfate proteoglycan (HSPG). Antigen-specific T cells potently activated osteoclasts via RANK/RANK-L, and the osteoclast activation marker Trap5b was high in sera of patients with an erosive shoulder injury. In vitro, the vaccine component alpha-tocopheryl succinate recapitulated bone toxicity and stimulated osteoclasts. Auto-reactivity was transient, with no evidence of progression to rheumatoid arthritis or overt autoimmune disease. CONCLUSION: Vaccine misapplication, potentially a genetic predisposition, and vaccine components contribute to SIRVA. The association with autoimmunity risk allele HLA-DRB1*04 needs to be further investigated. Despite transient autoimmunity, SIRVA was not associated with progression to autoimmune disease during two years of follow-up.

Regulation of T cell-associated tissues and T cell activation by RANKL-RANK-OPG.

The receptor activator of nuclear factor kappa-B ligand (RANKL)-RANK-osteoprotegerin (OPG) system is critical to bone homeostasis, but genetically deficient mouse models have revealed important roles in the immune system as well. RANKL-RANK-OPG is particularly important to T cell biology because of its organogenic control of thymic development and secondary lymphoid tissues influence central T cell tolerance and peripheral T cell function. RANKL-RANK-OPG cytokine-receptor interactions are often controlled by regulation of expression of RANKL on developing T cells, which interacts with RANK expressed on some lymphoid tissue cells to stimulate key downstream signaling pathways that affect critical tuning functions of the T cell compartment, like cell survival and antigen presentation. Activation of peripheral T cells is regulated by RANKL-enhanced dendritic cell survival, and dysregulation of the RANKL-RANK-OPG system in this context is associated with loss of T cell tolerance and autoimmune disease. Given its broader implications for immune homeostasis and osteoimmunology, it is critical to further understand how the RANKL-RANK-OPG system operates in T cell biology.

Giant Cell Tumor of Bone: An Update.

PURPOSE OF REVIEW: To highlight the new developments in the management of advanced giant cell tumor of bone, a rare locally aggressive benign tumor, which was traditionally managed with surgery alone by either curettage and local adjuvant therapy, wide resection, or marginal excision. Here, we review the current role of systemic therapy for management of locally advanced or metastatic giant cell tumor of bone (GCTB). RECENT FINDINGS: The elucidation of the pathophysiology of giant cell tumor of bone, especially with regards to the role of nuclear factor kappa B ligand (RANKL), has led to the Food and Drug Administration (FDA) approval of denosumab in the management of locally advanced or metastatic GCTB. For advanced giant cell tumor where surgical resection alone can cause severe morbidity, the paradigm has shifted from local treatment alone to multidisciplinary management with the consideration of use of denosumab.

The bad relationship, osteo-decay and diabetes type 2 searching for a link: a literature review.

The diabetes and osteoporotic metabolic diseases are characterized by a wide prevalence of the population worldwide and correlated to alteration of the bone tissues. Several cofactors could influence the clinical course and the biochemistry of the pathologies such as human microbiome, nutrition characteristics, gut microbiota activity and interactions with vitamin K and D across IGF/GH and TP53 signaling pathways and the glucose/energy as mechanism for bone tissue health. Moreover, also the calories and sugar consumption seem to be correlated to an increased inflammatory state with several consequences for hematopoiesis and host tissues response. The aim of the present literature review was to highlight the role of osteoporotic diseases and diabetes type 2 link for the bone metabolism. The literature cases showed that a correlation between bone-gut-kidney-heart-CNS-Immunity crosstalk seems to be linked with bone metabolism and health regulation. Moreover, also the aging process could represent a valuable co-factor for the sustaining of the metabolic disorders upon a multi-systemic level.

Serum levels of osteoprotegerin, RANK-L & vitamin D in different stages of osteoarthritis of the knee.

Background & objectives: Osteoarthritis (OA) is the 11th leading cause of disability in the modern world, but till date, there have been no effective markers for monitoring the progression of OA. The three proteins RANK/RANK-Ligand and Osteoprotegerin (OPG) have been found to be the key regulators of bone metabolism. Interaction of RANK-Ligand with its receptor RANK triggers differentiation of osteoclasts leading to bone resorption. OPG on the other hand is protective as it is expressed by osteoblasts and bind RANKL with higher affinity preventing its interaction with RANK. The levels of these serum proteins are regulated by vitamin D and parathyroid hormones. Therefore, the present study, aimed to study the association of serum RANKL, OPG and vitamin D with disease severity in patients with knee OA. Methods: It was a cross-sectional study where 80 (43 women and 37 men) newly diagnosed subjects with OA knee were recruited. They were classified into four grades based on K-L grading and into two groups as early (grade 1+grade 2) and advanced (grade 3 + grade 4) based on the disease progression. Results: On comparing the biochemical parameters among the four grades decreasing vitamin D levels were seen with increasing severity of knee OA; an increasing trend of RANKL with increase in the severity of OA was seen; OPG was found to be elevated more in the early stages of OA. We also observed a strong association of RANKL/OPG ratio with disease severity. Interpretation & conclusions: Overall the results suggest that OPG may be considered as an early marker of the diseases.

Myokines and Osteokines in the Pathogenesis of Muscle and Bone Diseases.

PURPOSE OF REVIEW: In this review we aim to summarize the latest findings on the network of molecules produced by muscle and bone under physiological and pathological conditions. RECENT FINDINGS: The concomitant onset of osteoporosis and sarcopenia is currently one of the main threats that can increase the risk of falling fractures during aging, generating high health care costs due to hospitalization for bone fracture surgery. With the growing emergence of developing innovative therapies to treat these two age-related conditions that often have common onset, a broader understanding of molecular messengers regulating the communication between muscle and bone tissue became imperative. Recently it has been highlighted that two muscle-derived signals, such as the myokines Irisin and L-BAIBA, positively affect bone tissue. In parallel, there are signals derived from bone that affect either positively the skeletal muscle, such as osteocalcin, or negatively, such as RANKL.

HIV-1-Infected Human Macrophages, by Secreting RANK-L, Contribute to Enhanced Osteoclast Recruitment.

HIV-1 infection is frequently associated with low bone density, which can progress to osteoporosis leading to a high risk of fractures. Only a few mechanisms have been proposed to explain the enhanced osteolysis in the context of HIV-1 infection. As macrophages are involved in bone homeostasis and are critical host cells for HIV-1, we asked whether HIV-1-infected macrophages could participate in bone degradation. Upon infection, human macrophages acquired some osteoclast features: they became multinucleated, upregulated the osteoclast markers RhoE and ß3 integrin, and organized their podosomes as ring superstructures resembling osteoclast sealing zones. However, HIV-1-infected macrophages were not fully differentiated in osteoclasts as they did not upregulate NFATc-1 transcription factor and were unable to degrade bone. Investigating whether infected macrophages participate indirectly to virus-induced osteolysis, we showed that they produce RANK-L, the key osteoclastogenic cytokine. RANK-L secreted by HIV-1-infected macrophages was not sufficient to stimulate multinucleation, but promoted the protease-dependent migration of osteoclast precursors. In conclusion, we propose that, by stimulating RANK-L secretion, HIV-1-infected macrophages contribute to create a microenvironment that favors the recruitment of osteoclasts, participating in bone disorders observed in HIV-1 infected patients.

Inflammatory markers IL-1ß and RANK-L assessment after non-vital bleaching: A 3-month follow-up.

OBJECTIVE: This study assessed IL-1ß and RANK-L levels in vivo and color stability of non-vital teeth bleached using hydrogen (35%) and carbamide (37%) peroxides 3 months after treatment. MATERIALS AND METHODS: Fifty teeth were randomly divided into two groups(n = 25):35% hydrogen peroxide (HP) or 37% carbamide peroxide (CP). Four sessions of intracoronal walking-bleach procedure were performed. IL-1ß and RANK-L levels were assessed from gingival crevicular fluid samples (from three vestibular and three palatines sites) at eight different time-points: at the beginning of the study (baseline), after four sessions of intracanal bleaching, and at 1 week, 1 month, and 3 months posttreatment. The color variations were visually detected using Vita bleach shade guide (ΔSGU). RESULTS: Significant increases of IL-1ß and RANK-L levels were detected at all time-points (all P < .05) when comparing each time-point to baseline, and a high correlation (>0.8-Spearman) between variables. According the ΔSGU values, a color change of five for HP and four for CP were detected. CONCLUSIONS: Non-vital walking bleach technique promotes an increase in IL-1ß and RANKL levels in periodontal tissues and also, it is maintained until the third-month posttreatment. CLINICAL SIGNIFICANCE: The internal whitening of teeth increases the levels of cytokines associated with inflammation and bone resorption 3 months after the whitening procedure is finished; this should warn of possible harmful effects of this whitening technique.

Clinical and translational pharmacological aspects of the management of fibrous dysplasia of bone.

Fibrous dysplasia (FD) is a genetic, noninheritable rare bone disease caused by a postzygotic activating mutation of the α subunit of the stimulatory G-protein causing increased abnormal bone formation leading to pain, deformity and fractures. To date, no cure has been identified for FD/McCune-Albright syndrome (MAS) and treatment is symptomatic and aimed at decreasing pain and/or local bone turnover. Various drugs have been used to achieve clinical improvement in FD/MAS patients including bisphosphonates and denosumab, however further translational studies are also warranted to address unresolved pathophysiological issues and explore novel pharmacological targets for the management of FD/MAS. In this article, we review literature on the medical treatment of FD/MAS, discuss the unresolved pathophysiological issues and explore novel pharmacological targets for the management of FD/MAS.

Effects of photodynamic therapy in periodontal treatment: A randomized, controlled clinical trial.

AIM: To evaluate the effects of photodynamic therapy (PDT) in the nonsurgical treatment of chronic periodontitis. MATERIALS AND METHODS: A randomized, single-blind, controlled, parallel-group clinical trial was performed. Sixty patients were enrolled: 20 healthy controls and 40 patients with periodontitis. The 40 patients were randomized for scaling and root planing (SRP) or SRP + PDT. Periodontal (plaque index, probing depth, clinical recession, clinical attachment level, bleeding on probing and gingival crevicular fluid volume, corresponding to 381 versus 428 critical sites), microbiological (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia and Campylobacter rectus presence, 18 versus 19 samples) and biochemical (interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α, receptor activator of nuclear factor-kappaB ligand (RANK-L) and osteoprotegerin (OPG) levels, 18 versus 19 samples) parameters were recorded. RESULTS: Within each group, significant improvements were found for clinical parameters, though without significant differences between groups. RANK-L was significantly decreased at week 13 in the SRP + PDT group compared with the SRP group. SRP + PDT, but not SRP alone, significantly reduced the abundance of A. actinomycetemcomitans. CONCLUSIONS: Except for a significant decrease in the pathogenic burden of A. actinomycetemcomitans, coadjuvant PDT resulted in no additional improvement compared with SRP alone in patients diagnosed with moderate-to-advanced chronic periodontitis.

A Type-2 Block-Component-Decomposition Based 2D AOA Estimation Algorithm for an Electromagnetic Vector Sensor Array.

This paper investigates a two-dimensional angle of arrival (2D AOA) estimation algorithm for the electromagnetic vector sensor (EMVS) array based on Type-2 block component decomposition (BCD) tensor modeling. Such a tensor decomposition method can take full advantage of the multidimensional structural information of electromagnetic signals to accomplish blind estimation for array parameters with higher resolution. However, existing tensor decomposition methods encounter many restrictions in applications of the EMVS array, such as the strict requirement for uniqueness conditions of decomposition, the inability to handle partially-polarized signals, etc. To solve these problems, this paper investigates tensor modeling for partially-polarized signals of an L-shaped EMVS array. The 2D AOA estimation algorithm based on rank- ( L 1 , L 2 , · ) BCD is developed, and the uniqueness condition of decomposition is analyzed. By means of the estimated steering matrix, the proposed algorithm can automatically achieve angle pair-matching. Numerical experiments demonstrate that the present algorithm has the advantages of both accuracy and robustness of parameter estimation. Even under the conditions of lower SNR, small angular separation and limited snapshots, the proposed algorithm still possesses better performance than subspace methods and the canonical polyadic decomposition (CPD) method.

Reduced bone volumetric density and weak correlation between infection and bone markers in cystic fibrosis adult patients.

UNLABELLED: In our current adult CF population, low BMD prevalence was only 20 %, lower than that historically described. We found a mild increase of serum RANK-L levels, independent from the bone resorption level. The increased fracture risk in CF may be explained by a lower tibial cortical thickness and total vBMD. INTRODUCTION: Bone disease is now well described in cystic fibrosis (CF) adult patients. CF bone disease is multifactorial but many studies suggested the crucial role of inflammation. The objectives of this study were, in a current adult CF population, to assess the prevalence of bone disease, to examine its relationship with infections and inflammation, and to characterize the bone microarchitecture using high resolution peripheral scanner (HR-pQCT). METHODS: Fifty-six patients (52 % men, 26 ± 7 years) were assessed in clinically stable period, during a respiratory infection, and finally 14 days after the end of antibiotic therapy. At each time points, we performed a clinical evaluation, lung function tests, and biochemical tests. Absorptiometry and dorso-lumbar radiographs were also performed. A subgroup of 40 CF patients (63 % men, 29 ± 6 years) underwent bone microarchitecture assessment and was age- and gender-matched with 80 healthy controls. RESULTS: Among the 56 CF patients, the prevalence of low areal BMD (T-score < -2 at any site), was 20 % (95 % CI: [10.2 %; 32.4 %]). After infections, serum RANK-L (+24 %, p = 0.08) and OPG (+13 %, p = 0.04) were increased with a stable ratio. Microarchitectural differences were mostly observed at the distal tibia, with lower total and cortical vBMD and trabecular thickness (respectively -9.9, -3.0, and -5 %, p < 0.05) in CF patients compared to controls, after adjustment for age, gender, weight, and height. CONCLUSIONS: In this study, bone disease among adult CF patients was less severe than that previously described with only 20 % of CF patients with low BMD. We found a mild increase of biological marker levels and an impaired volumetric density of the tibia that may explain the increased fracture risk in CF population.

Osteoprotegerin activates osteosarcoma cells that co-express RANK and RANKL.

BACKGROUND: Osteosarcoma (OS) is an aggressive and often fatal cancer that afflicts over 1000 humans and 10,000 dogs per year in the United States. Recent evidence suggests deregulation in the signaling triad, receptor activator of nuclear factor kappa B (RANK), its activating ligand (RANKL), and the RANKL inhibitor, osteoprotegerin (OPG) plays a key role in the pathogenesis of OS. This study investigated the expression of RANK and RANKL in osteosarcoma tumors and cell lines and describes an activating effect of OPG on OS cells in vitro. RESULTS: Canine OS tumors and cell lines co-express mRNA for both RANK and RANKL. Expression of these proteins in OS cell lines was confirmed by Western blot and immunofluorescence microscopy. Expression of the soluble form of RANKL was not detected in media from OS cells. OPG-Fc incubation increased the phosphorylation status of ERK, AKT and the p65 subunit of nuclear factor kappa B (NFκB) and induced NFκB translocation from the cytoplasm to the nucleus in canine OS cells. OPG increased proliferation in both canine and human derived OS cell lines. CONCLUSION: RANKL is produced by OS tumors and cell lines that also express RANK. This data provides preliminary evidence for a potential autocrine and or paracrine activation pathway in canine OS. An activating effect of exogenous OPG on signal transduction proteins, NFκB and proliferation in OS is described. These data provide new information concerning aberrant signaling in OS and could be important to those considering OPG as a therapeutic agent for osteosarcoma.

Preventive oral surgery before bisphosphonate administration to reduce osteonecrosis of the jaws.

OBJECTIVES: The intravenous injection of bisphosphonates, currently used for osteoporosis, myeloma, or bone metastases, can cause ONJ especially in consequence of trauma. To avoid trauma during bisphosphonate treatment, preventive oral surgery is recommended. The research aimed to evidence whether inflammatory and osteoclastogenic factors are not induced in oral mucosa after bisphosphonate treatment in patients receiving oral preventive surgery procedure and whether proliferation factors are not inhibited. PATIENTS AND METHODS: Specimens of oral mucosa were removed from healthy subjects and from patients undergoing preventive oral surgery before bisphosphonate treatment. The expression of cytokines and factors involved in osteoclast activity, cell proliferation, and angiogenesis were examined. RESULTS: Cytokines and RANK-L levels decreased significantly in mucosa from patients undergoing preventive oral surgery procedure before bisphosphonate treatment in comparison with their levels at the beginning of procedure and also in comparison with the level in patients treated only with bisphosphonates and not developing ONJ; conversely, osteoprotegerin and hydroxymethylglutaryl coenzyme A reductase significantly increased or not changed. CONCLUSIONS: The results suggest that preventive oral surgery could be able to prevent ONJ due to bisphosphonate treatment: The mucosa is not stimulated by bisphosphonates to cause ONJ, as bisphosphonates are probably not released from the bone.

Efficacy of denosumab treatment for lung metastasis secondary to proximal humerus chondroblastoma.

Chondroblastoma is a rare benign cartilaginous tumor that accounts for approximately 1% of bone tumors, but it can be associated with lung metastasis in extremely rare cases, leading to a poor prognosis and death. Herein, we report the case of a 19-year-old male patient who presented with an aggressive chondroblastoma of the proximal humerus and bilateral lung metastasis. The patient was treated with wide local resection, partial metastasectomy, and denosumab. Denosumab treatment was effective in controlling metastatic progression and preventing local recurrence.

Molecular and Biological Aspects of Orthodontic Tooth Movement: Possibilities for Bioengineering Intervention: A Narrative Review.

BACKGROUND: The current review's goal is to examine, with a critical eye, the effect of various biomedical parameters on orthodontic tooth movement in an attempt to provide the reader with related mechanisms of this issue focusing on certain key points. METHODS: This critical review was conducted using the following keywords in the search strategy: "biomedical molecules", "biomarkers", "orthodontics", "orthodontic tooth movement", "acceleration", "gene therapy", and "stem cells". Cochrane Library, Medline (PubMed), and Scopus were the databases that were used for the electronic search. Studies published until June 2023 were considered. RESULTS: The use of biomedical approaches in orthodontic tooth movement has been investigated via different procedures and applications. Surgical approaches, biomarkers affecting orthodontic tooth movement, different biological events and mechanisms, RANK, RANK-L, OPG molecular triad, and vibration methods are the basic parameters of biomedical interventions that are examined in the present review. CONCLUSIONS: The biomedical approach seems to offer a variety of applications to control orthodontic tooth movement. The scarcity of human studies, as well as the high cost and complexity of these methods, currently limit the available accurate data concerning this issue.

Clinical Implications of Immune Checkpoints and the RANK/RANK-L Signaling Pathway in High-Grade Canine Mast Cell Tumors.

Mast cell tumors (MCTs) are the most common malignant cutaneous tumors in dogs, and they present extremely variable biological behavior. The interaction between RANK, RANK-L, and immune checkpoints is frequently detected in the tumor microenvironment, and, together, they participate in every stage of cancer development. Thus, the aim of this study was to characterize the molecular profiles of PD-L1, CTLA-4, RANK/RANK-L signaling pathway, and IFN-γ in primary tumors and lymph node metastases. Formalin-fixed, paraffin-embedded slides of MCTs and metastatic lymph nodes of ten dogs were submitted to immunohistochemical investigations. The results demonstrated that the tumor microenvironment of the high-grade mast cell tumors showed moderate or intense immunolabeling of all proteins, and the lymph node metastases also showed moderate or intense immunolabeling of checkpoint proteins. In addition, MCTs larger than 3 cm were associated with intensified PD-L1 (p = 0.03) in metastatic lymph nodes and RANK-L (p = 0.049) immunoreactivity in the tumor. Furthermore, dogs with a survival time of less than 6 months showed higher PD-L1 immunoreactivity (p = 0.042). In conclusion, high-grade MCT is associated with an immunosuppressive microenvironment that exhibits elevated RANK/RANK-L signaling and enhanced immune checkpoint immunoreactivity, potentially facilitating intratumorally immune escape. These biomarkers show promise as clinical indicators of disease progression and might response to immunotherapy in dogs with high-grade MCTs, thus emphasizing their importance for guiding treatment decisions and improving outcomes.

RANK pathway in cancer: underlying resistance and therapeutic approaches.

Cancer remains one of the deadliest diseases despite advances in treatment. Metastatic cancers are the leading cause of death for advanced cancer patients. Those with advanced cancer with osteolytic-type bone metastases have a significantly lower quality of life. A novel treatment plan is needed now more than ever for breast cancer patients with bone metastases. There are shreds of evidence that cancer cells in the bloodstream interact with the bone microenvironment and that this interaction is a contributing component to breast cancer progression. Preventing any stage of this cycle can result in anti-metastasis effects. Since RANKL interacts with its receptor RANK and plays an important role in the vicious cycle, it has proven to be a successful therapeutic target in cancer treatment. As a result, we have presented a complete overview of the RANK pathway in cancer and discussed RANK signaling and tumor microenvironment, and potential therapeutic approaches in this review.

Analysis of Salivary Levels of IL-1ß, IL17A, OPG and RANK-L in Periodontitis Using the 2017 Classification of Periodontal Diseases-An Exploratory Observational Study.

Periodontitis is a chronic disease with a high overall prevalence. It involves a complex interplay between the immune-inflammatory pathways and biofilm changes, leading to periodontal attachment loss. The aims of this study were (i) to assess whether the salivary IL-1ß, IL-17A, RANK-L and OPG levels have the potential to discriminate between the mild and severe periodontitis conditions; and (ii) to enable diagnostic/prognostic actions to differentiate between distinct levels of the disease. The analysis of the clinical parameters and the evaluation of the salivary immunomediators levels by means of a multiplex flow assay revealed a statistically significantly higher level of IL-1ß in the periodontitis III/IV patients, as well as a higher level of RANK-L in the periodontitis III/IV and I/II patients, when compared to the healthy controls. Furthermore, the grade C periodontitis patients presented a significantly higher level of RANK-L compared to the grade B and grade A patients. In the grade C patients, IL-1ß had a positive correlation with the PPD and CAL indices and RANK_L had a positive correlation with CAL. The evidence emerging from this study associates the salivary IL-1ß and RANK-L levels with an advanced stage of periodontitis, stage III/IV, and with grade C, suggesting the possible cooperative action of both in the inflammatory and bone loss events. In addition to IL-1ß, RANK-L could be considered a combined diagnostic biomarker for periodontitis.