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Background and Aims: Coronavirus disease 2019 (COVID-19) lockdown has represented an inedited model of increased metabolic risk in all age groups, due to negative changes in dietary habits, physical activity, lifestyle. These effects have been generally explored at a population level in distinct age groups. Potential intra-familial, specific effects in adults and children sharing the same socio-economic, cultural level and living habits have been scarcely explored. We aimed to characterize changes of anthropometric indices in parents and in their children during COVID-19 lockdown. Methods: A cohort of 149 couple parent/children were prospectively enrolled. By a validated questionnaire we explored changes of body mass index (BMI) and individual lifestyle during a 2-month lockdown (May- July 2020). Results: BMI increased in 70.5% of parents and in 67.8% of their children, with a Δ-BMI of 1.44+0.09 kg/ m 2 and 0.36+0.02 Kg/m 2 , respectively. BMI increments, however, were only significant in adults and did not correlate in the couple parents/children. Most adults (80.5%) and children (71.4%) did not perform regular physical activity during the lockdown. Direct correlations between dietary changes and BMI variations became evident in children, mainly in terms of a decreased consumption of fresh fruit, pulses, fish, and an increased consumption of cereals, carbohydrates, dairy products, olive oil. In normal weight, overweight and obese children, but not in adults, the increase in sleep hours increased with BMI. Conclusions: Despite marked lifestyle changes imposed by the COVID-19 lockdown, BMI variations in parents were independent from those observed in their children, pointing to different outcomes in response to the same external, critical event. Thus, primary prevention measures aimed at maintaining a healthy lifestyle require different approaches according to age.

To evaluate the use of QUS for the bone status assessment in children cared because of a chronic disease such as: inherited metabolic disorder, kidney disease and endocrine defect and considered by the attending physician as at specific risk.QUS outputs were calculated for each disorder and compared to: sex, age, Tanner stage, Z-score for height, weight and BMI (body mass index).One-hundred-sixty-eight subjects aged between 3.5 and 18 years met the inclusion criteria. The overall bone quality indexes were under the normal range in all the groups considered. Impairment of bone quality parameters was more evident in the group of patients with inherited metabolic disorders, in which 65% of patients in charge were studied by QUS. Older age and sexual development were associated with less pronounced bone quality impairment, as measured by QUS, in the vast majority of conditions. Overall, the diseases for which the prediction of outcome was the strongest were: hyperphenylalaninemia, nephrotic syndrome and insulin dependent diabetes mellitus.QUS is capable to provide information on skeletal status in children. Initial evaluation by QUS may allow defining patients with chronic disorders who deserve further, more invasive diagnostic studies. Inherited metabolic disorders warrant specific attention and strict monitoring for their potential effect on bone.

Subjects affected with Turner Syndrome (TS) suffer low bone mineral density and high risk of fracture from a young age. Estrogen deficiency is considered the main risk factor but other factors, such as intrinsic bone abnormalities, enhanced osteoclastogenesis, vitamin D deficiency and other comorbidities may contribute to the exalted bone fragility.The authors performed a literature search in PubMed and EMBASE, using selected key words. They focused their search on pathogenetic mechanisms of osteoporosis in TS and updated the diagnosis, prevention and therapeutic interventions.Bone health is a concern in subjects with TS, and strategies to prevent osteoporosis and fractures should be considered from childhood. Advice on how to live a healthy lifestyle, including physical activity and correct nutrition, should be given during childhood in order to prevent bone impairment later in life. The screening for vitamin D deficiency should be performed between the ages of 9 and 11, and every 2-3 years thereafter. Early initiation of estrogen replacement therapy (ERT) between 11-12 years of age, prompt titration to the adult dose after 2 years, and long-term follow-up to guarantee compliance with ERT, are the key points of osteoporosis prevention in women with TS.

Bone loss induced by ovariectomy is due to the direct activity on bone cells and mesenchymal cells and to the dysregulated activity of bone marrow cells, including immune cells and stromal cells, but the underlying mechanisms are not completely known. Here, we demonstrate that ovariectomy induces the T-cell co-stimulatory cytokine LIGHT, which stimulates both osteoblastogenesis and osteoclastogenesis by modulating osteoclastogenic cytokine expression, including TNF, osteoprotegerin, and the receptor activator of nuclear factor-κB ligand (RANKL). Predictably, LIGHT-deficient (Tnfsf14-/- ) mice are protected from ovariectomy-dependent bone loss, whereas trabecular bone mass increases in mice deficient in both LIGHT and T and B lymphocytes (Rag -/- Tnfsf14 -/- ) and is associated with an inversion of the TNF and RANKL/OPG ratio. Furthermore, women with postmenopausal osteoporosis display high levels of LIGHT in circulating T cells and monocytes. Taken together, these results indicate that LIGHT mediates bone loss induced by ovariectomy, suggesting that patients with postmenopausal osteoporosis may benefit from LIGHT antagonism. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tumor necrosis factor superfamily member 14 (TNFSF14), LIGHT, is a component of the cytokine network that regulates innate and adaptive immune responses, which promote homeostasis of lymphoid organs, liver, and bone. Metastatic tumors often disrupt the tissue microenvironment, thus altering the homeostasis of the invaded organ; however, the underlying mechanisms required further studies. We investigated the role of LIGHT in osteolytic bone disease induced by metastatic non-small cell lung cancer (NSCLC). Patients diagnosed with NSCLC bone metastasis show significantly higher levels of LIGHT expressed in monocytes compared with non-bone metastatic tumors and healthy controls. Serum LIGHT levels were also higher in patients with bone metastases than in controls, suggesting a role for LIGHT in stimulating osteoclast precursors. In bone metastatic patients, we also detected increased RNA expression and serum RANKL levels, thus by adding anti-LIGHT or RANK-fragment crystallizable region (RANK-Fc) in PBMC cultures, a significant inhibition of osteoclastogenesis was observed. To model this observation in mice, we used the mouse lung cancer cell line LLC-1. After intratibial implantation, wild-type mice showed an increased number of osteoclasts but reduced numbers of osteoblasts and decreased osteoid formation. In contrast, Tnfsf14-/- mice showed no significant bone loss or other changes in bone homeostasis associated with this model. These data indicate LIGHT is a key control mechanism for regulating bone homeostasis during metastatic invasion. Thus, LIGHT may be a novel therapeutic target in osteolytic bone metastases. © 2019 American Society for Bone and Mineral Research.

Introduction Multiple daily injections (MDI) and continuous subcutaneous insulin infusion (CSII) are two modalities of treating type 1 diabetes mellitus (T1DM). The benefits of CSII on long-term metabolic control and outcomes compared to those of MDI are still debated. We investigated both vascular function and myocardial performance in T1DM adolescents on MDI or CSII treatment. Methods One hundred twenty-three T1DM subjects (mean age 14.16±2.55 years), 63 on MDI regimen, 60 on CSII, and 57 controls were enrolled. Anthropometric and biochemical characteristics were evaluated. Ultrasound assessments of carotid intima-media thickness (cIMT), flow-mediated dilatation of brachial artery, anteroposterior diameter of the infrarenal abdominal aorta (APAO), and transthoracic echocardiography were performed. Results T1DM subjects on the CSII regimen showed better glycemic control than those on MDI, expressed as glycated haemoglobin (HbA1c). c-IMT and APAO were higher in MDI than CSII patients (0.61±0.11 mm vs. 0.56±0.07 mm, p=0.04; 13.61±3.29 mm vs. 11.65±1.84 mm, p=0.01, respectively). Left and right Tei index and left E/e’ ratio were higher in MDI than CSII subjects (0.82±0.40 vs. 0.52±0.19, p=0.002; 0.86±0.41 vs. 0.64±0.1, p=0.02; 5.89±2.0 vs. 4.73±1.59, p=0.02, respectively). Multiple regression analyses showed that glucose level, HbA1c and diabetes onset were significantly related to vascular and echocardiographic parameters in MDI and CSII patients. Conclusions CSII regimen in T1DM adolescents improves glycemic control and seems to ameliorate endothelial function and global myocardial performance as compared to MDI therapy.

Immune and bone cells cross talk has been established by different years; however the underlying mechanisms require continuous investigation. To this end both in vivo and in vitro models have been realized and some of this are described in this chapter. In particular, here we described the animal models used for the understanding of lymphocyte role in bone homeostasis, together with some in vitro models.

To assess ductal size correlated to spontaneous closure, pharmacological or surgical treatment; to index ductal diameter to body weight and body surface area; to evaluate the morbidities.Retrospective study on preterms ≤32 weeks, birth weight ≤1500 g, extremely low birth weight (ELBW) and very low birth weight (VLBW). Inclusion criteria: patent ductus arteriosus (PDA) with a diameter ≥1 millimeter (mm) at 72 h from birth; need for ibuprofen treatment on the basis of a hemodynamically significant ductus arteriosus (HsPDA).One hundred infants with the diagnosis of PDA have been included. We observed a prevalence of spontaneous closure in 34% of newborns (41.3% VLBW versus 26.7% ELBW). The percentage of response to a single course of ibuprofen was of 62% (68.5% ELBW versus 54.3% VLBW). The mean of absolute ductal diameter was of 2.26 ± 0.62 mm in ELBW and 2.18 ± 0.42 mm in VLBW. The indexing of ductus size to body weight demonstrated a higher value in ELBW than VLBW (2.76 ± 0.97 mm/kg versus 1.84 ± 0.40 mm/kg).Our results confirmed that HsPDA can develop in presence of a ductus1.5 mm as absolute value or1.4 mm/kg as indexed to body weight. In ELBW infants the ductal size indexed for body weight and body surface area could be more predictive of spontaneous closure or need for pharmacological treatment compared to the absolute value of ductal size. A strong association between HsPDA and short- or long-term morbidities was confirmed particularly in ELBW.

Bone is an active tissue that remodels continuously throughout life [...]

Obesity may affect bone health, but literature reports are contradictory about the correlation of body mass index (BMI) and bone markers. LIGHT, one of the immunostimulatory cytokines regulating the homeostasis of bone and adipose tissue, could be involved in obesity. The study involved 111 obese subjects (12.21 &plusmn, 3.71 years) and 45 controls. Patients underwent the evaluation of bone status by quantitative ultrasonography (QUS). LIGHT amounts were evaluated in sera by ELISA, whereas its expression on peripheral blood cells was evaluated by flow cytometry. Osteoclastogenesis was performed by culturing peripheral blood mononuclear cells (PBMCs) with or without anti-LIGHT antibodies. Obese patients showed significant high BMI-standard deviation score (SDS), weight-SDS, and Homeostatic model assessment for insulin resistance (HOMA-IR) that negatively correlated with the reduced Amplitude Dependent Speed of Sound (AD-SoS)-Z-score and Bone Transmission Time (BTT-Z)-score. They displayed significantly higher serum levels of LIGHT compared with controls (497.30 &plusmn, 363.45 pg/mL vs. 186.06 &plusmn, 101.41 pg/mL, p &lt, 0.001). LIGHT expression on monocytes, CD3+-T-cells, and neutrophils was also higher in obese patients than in the controls. Finally, in PBMC cultures, the addition of anti-LIGHT antibodies induced a significant osteoclastogenesis inhibition. Our study highlighted the high serum levels of LIGHT in obese children and adolescents, and its relationship with both the grade of obesity and bone impairment.

Short stature is a common clinical presentation, thus it is widely accepted that it is a polygenic trait. However, genome wide association and next generation sequencing studies have recently challenged this view, suggesting that many of the children classified as idiopathic short stature could instead have monogenic defects. Linear growth is determined primarily by chondrogenesis at the growth plate. This process results from chondrocyte proliferation, hypertrophy, and extracellular matrix secretion, and it is perfectly coordinated by complex networks of local paracrine and endocrine factors. Alterations in genes which control growth plate development can explain a large number of cases of isolated short stature, allowing an etiological diagnosis. We reviewed recent data on the genetic alterations in fundamental cellular processes, paracrine signaling, and cartilage matrix formation associated with impaired growth plate chondrogenesis. In particular we focused on growth plate gene involvement in nonsyndromic short stature. The identification of genetic basis of growth failure will have a significant impact on the care of children affected with short stature.

Alkaptonuria (AKU) is a rare disorder characterized by the deficiency of the enzyme homogentisate 1,2-dioxygenase and consequent homogentisate accumulation, which leads to progressive and severe osteoarthopathy starting from the second decade of life. Thus, in AKU patients, bone involvement represents an important clinical issue, which we investigated. Serum levels of receptor activator of NF-κB ligand (RANKL), osteoprotegerin, sclerostin, Dickkopf-1, and bone remodeling markers were measured in nine AKU patients (two children and seven adults) and 22 controls, together with lumbar spine bone mineral density (LS-BMD) and femoral-BMD. In the two AKU children, the average of LS-BMD and femoral-BMD Z-scores were within the normal range, but reduced with respect to the controls. Otherwise, in the adult AKU patients, LS-BMD T-score was inside the normal range, but femoral-BMD T-score reached osteopenic levels. Consistently, in AKU adults, higher RANKL and C-terminal telopeptide of collagen type 1 and lower osteoprotegerin levels were observed than in controls. Otherwise, spontaneous osteoclastogenesis was already evident in peripheral blood mononuclear cell cultures from AKU children, together with a high percentage of circulating osteoclast precursors. Osteoclastogenesis was sustained by the high levels of tumor necrosis factor-α, RANK, RANKL, and LIGHT. In conclusion, the altered osteoclastogenesis was observed already in AKU children, despite the absence of evident injury. Thus, a preventive approach in young patients, targeting osteoclast activity, may prevent the macroscopic bone disease that appears in adult AKU.

We tested the hypothesis that the levels of bone remodeling mediators may be altered in Prader–Willi syndrome (PWS). We assessed RANKL, OPG, sclerostin, DKK-1 serum levels, and bone metabolism markers in 12 PWS children (7.8 ± 4.3 years), 14 PWS adults (29.5 ± 7.2 years), and 31 healthy controls matched for sex and age. Instrumental parameters of bone mineral density (BMD) were also evaluated. Lumbar spine BMD Z-scores were reduced in PWS children (P

Childhood obesity is associated with the development of severe comorbidities, such as diabetes, cardiovascular diseases, and increased risk of osteopenia/osteoporosis and fractures. The status of low-grade inflammation associated to obesity can be reversed through an enhanced physical activity and by consumption of food enrich of anti-inflammatory compounds, such as omega-3 fatty acids and polyphenols. The aim of this study was to deepen the mechanisms of bone impairment in obese children and adolescents through the evaluation of the osteoclastogenic potential of peripheral blood mononuclear cells (PBMCs), and the assessment of the serum levels of RANKL and osteoprotegerin (OPG). Furthermore, we aimed to evaluate the in vitro effects of polyphenol cherry extracts on osteoclastogenesis, as possible dietary treatment to improve bone health in obese subjects. High RANKL levels were measured in obese with respect to controls (115.48 ± 35.20 pg/ml vs. 87.18 ± 17.82 pg/ml; p < 0.01), while OPG levels were significantly reduced in obese than controls (378.02 ± 61.15 pg/ml vs. 436.75 ± 95.53 pg/ml, respectively, p < 0.01). Lower Ad-SoS- and BTT Z-scores were measured in obese compared to controls (p < 0.05). A significant elevated number of multinucleated TRAP+ osteoclasts (OCs) were observed in the un-stimulated cultures of obese subjects compared to the controls. Interestingly, obese subjects displayed a higher percentage of CD14+/CD16+ than controls. Furthermore, in the mRNA extracts of obese subjects we detected a 2.5- and 2-fold increase of TNFα and RANKL transcripts compared to controls, respectively. Each extract of sweet cherries determined a dose-dependent reduction in the formation of multinucleated TRAP+ OCs. Consistently, 24 h treatment of obese PBMCs with sweet cherry extracts from the three cultivars resulted in a significant reduction of the expression of TNFα. In conclusion, the bone impairment in obese children and adolescents is sustained by a spontaneous osteoclastogenesis that can be inhibited in vitro by the polyphenol content of sweet cherries. Thus, our study opens future perspectives for the use of sweet cherry extracts, appropriately formulated as nutraceutical food, as preventive in healthy children and therapeutic in obese ones.

BackgroundBone remodeling is a lifelong process due to the balanced activity of osteoclasts (OCs), the bone-reabsorbing cells, and osteoblasts (OBs), and the bone-forming cells. This equilibrium is regulated by numerous cytokines, but it has been largely demonstrated that the RANK/RANKL/osteoprotegerin and Wnt/-catenin pathways play a key role in the control of osteoclastogenesis and osteoblastogenesis, respectively. The pro-osteoblastogenic activity of the Wnt/-catenin can be inhibited by sclerostin and Dickkopf-1 (DKK-1). RANKL, sclerostin and DKKs-1 are often up-regulated in bone diseases, and they are the target of new monoclonal antibodies.Data sourcesThe authors performed a systematic literature search in PubMed and EMBASE to June 2018, reviewed and selected articles, based on pre-determined selection criteria.ResultsWe re-evaluated the role of RANKL, osteoprotegerin, sclerostin and DKK-1 in altered bone remodeling associated with some inherited and acquired pediatric diseases, such as type 1 diabetes mellitus (T1DM), alkaptonuria (AKU), hemophilia A, osteogenesis imperfecta (OI), 21-hydroxylase deficiency (21OH-D) and Prader-Willi syndrome (PWS). To do so, we considered recent clinical studies done on pediatric patients in which the roles of RANKL-RANK/osteoprotegerin and WNT-ss-catenin signaling pathways have been investigated, and for which innovative therapies for the treatment of osteopenia/osteoporosis are being developed.ConclusionsThe case studies taken into account for this review demonstrated that quite frequently both bone reabsorbing and bone deposition are impaired in pediatric diseases. Furthermore, for some of them, bone damage began in childhood but only manifested with age. The use of denosumab could represent a valid alternative therapeutic approach to improve bone health in children, although further studies need to be carried out.

In this study, we investigated the bone cell activity in patients with osteogenesis imperfecta (OI) treated and untreated with neridronate. We demonstrated the key role of Dickkopf-1 (DKK1), receptor activator of nuclear factor-κB ligand (RANKL), and tumor necrosis factor alpha (TNF-α) in regulating bone cell of untreated and treated OI subjects. These cytokines could represent new pharmacological targets for OI. Bisphosphonates are widely used in the treatment of children with osteogenesis imperfecta (OI) with the objective of reducing the risk of fractures. Although bisphosphonates increase bone mineral density in OI subjects, the effects on fracture incidence are conflicting. The aim of this study was to investigate the mechanisms underlying bone cell activity in subjects with mild untreated forms of OI and in a group of subjects with severe OI treated with cycles of intravenous neridronate. Sclerostin, DKK1, TNF-α, RANKL, osteoprotegerin (OPG), and bone turnover markers were quantified in serum of 18 OI patients (12 females, mean age 8.86 ± 3.90), 8 of which were receiving cyclic intravenous neridronate, and 21 sex- and age-matched controls. The effects on osteoblastogenesis and OPG expression of media conditioned by the serum of OI patients and anti-DKK1 neutralizing antibody were evaluated. Osteoclastogenesis was assessed in cultures from patients and controls. DKK1 and RANKL levels were significantly increased both in untreated and in treated OI subjects with respect to controls. The serum from patients with high DKK1 levels inhibited both osteoblast differentiation and OPG expression in vitro. High RANKL and low OPG messenger RNA (mRNA) levels were found in lymphomonocytes from patients. High amounts of TNF-α were expressed by monocytes, and an elevated percentage of circulating CD11b-CD51/CD61+ osteoclast precursors was observed in patients. Our study demonstrated the key role of DKK1, RANKL, and TNF-α in regulating bone cell activity of subjects with OI untreated and treated with bisphosphonates. These cytokines could represent new pharmacological targets for OI patients.

BACKGROUND Small-for-gestational-age (SGA) children have increased cardiovascular risk, but the mediating factors are poorly understood. We hypothesized that birth size could affect the cardiovascular system since childhood in the absence of other risk factors. We investigated endothelial and myocardial function in SGA children with regular catch-up growth. METHODS AND RESULTS Biochemical markers, blood pressure, flow-mediated vasodilation (FMD), common carotid intima-media thickness (cIMT), anteroposterior diameter of the infrarenal abdominal aorta (APAO) and echocardiographic parameters of left and right ventricular (LV and RV) function were studied in 27 SGA and 25 appropriate-for-gestational-age (AGA) subjects. SGA subjects had a higher homeostasis model assessment index than controls (2.61±1.27 vs. 1.56±0.40, P=0.01), higher cIMT (0.51±0.04 mm vs. 0.45±0.07 mm, P=0.007) and APAO (1.31±1.35 cm vs. 1.30±0.16 cm, P=0.005), and lower FMD (10.11±4.17% vs. 12.34±4.28, P=0.04) than controls. On echocardiography SGA had higher Tei index both at LV and RV than controls (P=0.001). Reduced RV systolic function was also observed in SGA subjects. CONCLUSIONS SGA subjects had vascular morphological and function abnormalities compared with AGA, which increase their cardiovascular risk profile. Furthermore, a subtle cardiac alteration in both RV and LV functions was seen in SGA patients compared with AGA.

Subjects with hypergonadotropic hypogonadism due to Turner's syndrome show low cortical mineral density, osteoporosis and risk of fractures. It is not clear if this bone fragility derives from chromosomal abnormalities or is the result of inadequate bone formation due to estrogen deficiency. The aim of this study was to investigate the cellular mechanisms underlying bone fragility in subjects with Turner's syndrome before induction of puberty and after hormonal replacement therapy (HRT). For this purpose, we have evaluated the osteoclastogenic potential of non-fractioned and T-cell depleted cultures of peripheral blood mononuclear cells (PBMCs) belonging to girls with Turner's syndrome who had not been treated with HRT yet, girls and young women who were on HRT and age-matched controls. Untreated subjects showed high FSH serum levels, whereas the other subjects displayed normal FSH serum levels. T-cell immunophenotype was analyzed through flow cytometry. Biochemical and DXA analyses were performed. Spontaneous osteoclastogenesis in non-fractioned and T-cell depleted cultures of PBMC belonging to girls with high FSH levels was more evident than in cultures of subjects with normal FSH levels. In the former, osteoclastogenesis was sustained by monocytes expressing high levels of c-fms, TNF-α and RANK, and T-cells producing high RANKL and TNF-α; in the latter it was supported by T-cells expressing high RANKL levels. CD4+CD25high T-cells were reduced in all subjects, whereas CD3+/CD16+/CD56+ NKT-cells were increased in those with high FSH levels. High RANKL and CTX levels were detected in the sera. Bone impairment was already detectable by DXA in subjects aged under 10, although it became more evident with aging. In conclusion, our results demonstrated that bone fragility in subjects with Turner's syndrome is associated to enhanced osteoclastogenesis. This process seems to be due to high FSH serum levels before HRT, whereas it is caused by high RANKL during HRT.

We have previously shown that through the production of high LIGHT levels, immune cells contribute to both osteoclastogenesis and bone destruction in Multiple Myeloma (MM)-related bone disease. With the aim of further exploring the mechanisms underlying the development of MM-related bone disease, here we focused on a possible role of LIGHT in MM patients with active bone disease despite the treatment received. We detected LIGHT over-expression by circulating CD14+ monocytes from MM patients still showing active bone disease, despite the treatment. In addition, we found over-expression of receptor activator of nuclear factor kappa-B ligand (RANKL), whose pro-osteoclastogenic role is well-known, in T-lymphocytes isolated from the same patients. Although the percentages of circulating osteoclast progenitors, CD14+CD16+ monocytes, were higher in all the MM patients than in the controls spontaneous osteoclastogenesis occurred only in the cultures derived from PBMCs of MM patients with unresponsive bone disease. Of note, in the same cultures osteoclastogenesis was partially or completely inhibited, in a dose-dependent manner, by the addition of RANK-Fc or anti-LIGHT neutralizing antibody, demonstrating the contribution of both LIGHT and RANKL to the enhanced osteoclast formation observed. In addition, high serum levels of TRAP5b and CTX, the two markers of osteoclast activity, were detected in MM patients with bone disease not responsive to treatment. In conclusion, our study indicates a prominent role of LIGHT in the crosstalk among osteoclasts and immune cells, co-involved together with RANKL in the pathophysiological mechanisms leading to MM-related bone disease. This TNF superfamily member may thus be a possible new therapeutic target in MM-related bone disease.

Cells from dental tissues have a mesenchymal stem cell (MSC) phenotype, are multipotent and can differentiate into osteoblastic cells, as we have previously found. MSCs, due to their tumor‑homing ability, are currently being used as cell‑based delivery systems for cancer protein therapeutics, such as the TNF‑related apoptosis‑inducing ligand (TRAIL). In the present study we revealed that dental pulp stem cells (DPSCs) expressed TRAIL to a greater extent when they were differentiated into the osteoblastic lineage. TRAIL affected the viability of undifferentiated DPSCs, while osteoblastic differentiated DPSCs were not sensitive to TRAIL. The expression trend of TRAIL receptors underwent changes during the osteoblastic differentiation of DPSCs exhibiting low DcR2 and high DR5 levels in the undifferentiated DPSCs and an opposite scenario was presented in the differentiated cells. The sensitivity of the undifferentiated DPSCs to the TRAIL‑apoptotic effect was also associated with low levels of intracellular anti‑apoptotic proteins, such as c‑FLIP, XIAP and the activation of caspase‑8 and ‑3. DPSC‑differentiated osteoblasts expressing high TRAIL levels were capable to affect the cell viability of the human myeloma cell line H929, thus representing an effective anticancer therapeutic method.

Irisin is a new peptide produced mainly by the skeletal muscle playing an important role both in glucose/energy homeostasis and bone metabolism. Childhood type 1 diabetes mellitus (T1DM) is associated with decreased bone mass. We aimed to evaluate irisin levels in TD1M children and their correlation with bone metabolism and glycaemic control.Ninety-six T1DM subjects (12.2 ± 4 years), 56 on multiple daily injections (MDI), 40 on continuous subcutaneous insulin infusion (CSII), and 34 controls were included in the study. Irisin and bone remodeling markers were quantified in sera from patients and controls. Bone mineral density (BMD) was evaluated by QUS.Increased irisin levels were found in T1DM patients respect to controls (p 0.001). With adjustment for age, irisin levels significantly correlated negatively with HbA1c% (r = -0.105, p 0.001), years of diabetes (r = -0.07, p 0.04), 25(OH)-Vitamin D (r = -0.175, p 0.0001), and positively with BTT-Z-score (r = 0.088, p = 0.016), and osteocalcin (r = 0.059, p 0.04). We detected the highest levels of irisin in CSII patients compared to MDI and controls (p 0.001 and p 0.007 respectively).We demonstrated high irisin levels in T1DM children and the association of highest irisin amounts to a better glycaemic control and bone health in TDM1 subjects on CSII.

Background Children born small for gestational age (SGA) are at increased risk of metabolic dysfunction. Dysregulation of specific microRNAs (miRNAs) contributes to aberrant gene expression patterns underlying metabolic dysfunction. Objective We aimed to determine and compare circulating miRNA (c-miRNA) profile of SGA and appropriate for gestational age (AGA) children with obesity and with normal weight, in order to identify biomarkers for early detection of increased risk of developing metabolic dysfunction in SGA and AGA children with obesity. Methods Small non-coding RNAs from serum of 15 SGA children with obesity (OB-SGA), 10 SGA children with normal weight (NW-SGA), 17 AGA children with obesity (OB-AGA) and 12 AGA children with normal weight (NW-AGA) (mean age 11.2 ± 2.6) have been extracted and sequenced in order to detect and quantify miRNA expression profiles. Results RNA-seq analyses showed 28 miRNAs dysregulated in OB-SGA vs. NW-SGA and 19 miRNAs dysregulated in OB-AGA vs. NW-AGA. Among these, miR-92a-3p, miR-122-5p, miR-423-5p, miR-484, miR-486-3p and miR-532-5p were up regulated, and miR-181b-5p was down regulated in both OB-SGA and OB-AGA compared with normal weight counterparts. Pathway analysis and miRNA target prediction suggested that these miRNAs were particularly involved in insulin signalling, glucose transport, insulin resistance, cholesterol and lipid metabolism. Conclusion We identified a specific profile of c-miRNAs in SGA and AGA children with obesity compared with SGA and AGA children with normal weight. These c-miRNAs could represent specific biomarkers for early detection of increased risk of developing metabolic dysfunction in SGA and AGA children with obesity.

Osteoporosis is the most widespread skeletal disease requiring innovative therapeutic strategies for its management. The understanding of receptor activator of nuclear factor kappa-B ligand (RANKL) and sclerostin's role in bone cell biology is completely changing the therapeutic landscape. RANKL supports osteoclast formation and activity and is mainly produced by cells of osteoblastic lineage. Sclerostin, an antagonist of the Wnt pathway, has a key role in bone formation and is mainly secreted by osteocytes. High levels of RANKL and sclerostin have been detected in osteoporosis, leading to the production of antibodies able to neutralize their activity.In this review, the authors give an overview and discuss the literature and data on denosumab and romosozumab to treat osteoporosis. Clinical studies indicate that long-term treatment with denosumab causes a continuous increase in bone mineral density with low incidence of adverse effects. Romosozumab treatment gives increases bone formation and improves bone mineral density (BMD) though further studies are needed to better evaluate the adverse effects.Denosumab and romosozumab show promise in the treatment of osteoporosis. Furthermore, their different mechanisms of action compared to existing anti-osteoporotic drugs may permit alternative strategies for osteoporosis treatment down the line

We previously showed that Irisin, a myokine released from skeletal muscle after physical exercise, plays a central role in the control of bone mass. Here we report that treatment with recombinant Irisin prevented bone loss in hind-limb suspended mice when administered during suspension (preventive protocol) and induced recovery of bone mass when mice were injected after bone loss due to a suspension period of 4 weeks (curative protocol). MicroCT analysis of femurs showed that r-Irisin preserved both cortical and trabecular bone mineral density, and prevented a dramatic decrease of the trabecular bone volume fraction. Moreover, r-Irisin protected against muscle mass decline in the hind-limb suspended mice, and maintained the fiber cross-sectional area. Notably, the decrease of myosin type II expression in unloaded mice was completely prevented by r-Irisin administration. Our data reveal for the first time that Irisin retrieves disuse‐induced bone loss and muscle atrophy. These findings may lead to development of an Irisin-based therapy for elderly immobile osteoporotic and physically disable patients, and might represent a countermeasure for astronauts subjected to microgravity-induced bone and muscle losses.

Background/Objectives Calcific aortic valvular disease (CAVD) is an actively regulated process characterized by the activation of specific osteogenic signaling pathways and apoptosis. We evaluated the involvement in CAVD of the TNF-related apoptosis-inducing ligand (TRAIL), an apoptotic molecule which induces apoptosis by interacting with the death receptor (DR)-4 and DR5, and whose activity is modulated by the decoy receptor (DcR)-1 and DcR2. Methods Sections of calcific and normal aortic valves, obtained at surgery time, were subjected to immunohistochemistry and confocal microscopy for TRAIL immunostaining. Valvular interstitial cells (VICs) isolated from calcific (C-VICs) and normal (N-VICs) aortic valves were investigated for the gene and protein expression of TRAIL receptors. Cell viability was assayed by MTT. Von Kossa staining was performed to verify C-VIC ability to produce mineralized nodules. TRAIL serum levels were detected by ELISA. Results Higher levels of TRAIL were detected in calcific aortic valves and in sera from the same patients respect to controls. C-VICs express significantly higher mRNA and protein levels of DR4, DR5, DcR1, DcR2 and Runx2 compared to N-VICs. C-VICs and N-VICs, cultured in osteogenic medium, express significantly higher mRNA levels of DR4, Runx2 and Osteocalcin compared to baseline. C-VICs and N-VICs were sensitive to TRAIL-apoptotic effect at baseline and after osteogenic differentiation, as demonstrated by MTT assay and caspase-3 activation. TRAIL enhanced mineralized matrix nodule synthesis by C-VICs cultured in osteogenic medium. Conclusions TRAIL is characteristically present within calcific aortic valves, and mediates the calcification of aortic valve interstitial cells in culture through mechanism involving apoptosis.

Keywords: sclerostin; haemophilia A; bone mineral density; receptor activator of nuclear factor-κB ligand; osteoprotegerin; DKK1

Apoptosis can occur throughout the life span of osteoblasts (OBs), beginning from the early stages of differentiation and continuing throughout all stages of their working life. Here, we investigated the effects of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal human OBs showing for the first time that the expression of TRAIL receptors is modulated during OB differentiation. In particular, the TRAIL receptor ratio was in favor of the deaths because of the low expression of DcR2 in undifferentiated OBs, differently it was shifted toward the decoys in differentiated ones. Undifferentiated OBs treated with TRAIL showed reduced cell viability, whereas differentiated OBs displayed TRAIL resistance. The OB sensitiveness to TRAIL was due to the up-regulation of DR5 and the down-regulation of DcR2. The main death receptor involved in TRAIL-reduced OB viability was DR5 as demonstrated by the rescue of cell viability observed in the presence of anti-DR5 neutralizing antibody. Besides the ratio of TRAIL receptors, the sensitivity of undifferentiated OBs to TRAIL-cytotoxic effect was also associated with low mRNA levels of intracellular anti-apoptotic proteins, such as cFLIP, the activation of caspase-8 and -3, as well as the DNA fragmentation. This study suggests that apoptotic effect exerted by TRAIL/TRAIL-receptor system on normal human OB is strictly dependent upon cell differentiation status.

Sclerostin, negative regulator of bone formation, has been originally known as an osteocyte product. Recently, it has been also detected in hypertrophic chondrocytes, distinctive cells of avascular cartilage which is invaded by capillaries and then replaced by vascularized bone. Thus, we hypothesized that sclerostin, in addition to its role already known, may exert an angiogenic activity. We first proved that sclerostin increased the proliferation of human umbilical vein endothelial cells (HUVECs), and next, by using the chicken chorioallantoic membrane (CAM) in vivo assay, we demonstrated that it exerts an angiogenic activity similar to that of vascular endothelial growth factor (VEGF). This last finding was reinforced by several in vitro approaches. Indeed, we showed that sclerostin induced the formation of a network of anastomosing tubules, a significant increase in the percentage of tubule number, total tubule length and number of junctions, as well as the ability of sclerostin-stimulated HUVECs to organize capillary-like structures and closed-meshes similar to VEGF. The angiogenic response elicited by the protein may be due to the binding to its receptor, LRP6, which is highly expressed at mRNA and protein levels by sclerostin treated HUVECs and through the production of two well-known pro-angiogenic cytokines, VEGF and placental growth factor (PlGF). Finally, we demonstrated that sclerostin was also responsible for the recruitment of osteoclasts and their circulating monocyte progenitors. Overall, these findings showed for the first time the new angiogenic in vitro role of sclerostin which could be also considered as a novel molecule in angiogenesis-osteogenesis coupling.

Context: Childhood type 1 diabetes mellitus (T1DM) is associated with decreased bone mass. Sclerostin and dickkopf-1 (DKK-1) are Wnt inhibitors that regulate bone formation. Objective: To evaluate sclerostin and DKK-1 levels in T1DM children and to analyze the influence of glycemic control on bone health. Design and setting: Cross-sectional study conducted at a clinical research center. Participants: One hundred and six T1DM subjects (12.2 ± 4 years), 66 on multiple daily injections (MDIs) and 40 on continuous subcutaneous infusion of insulin (CSII), and 80 controls. Results: The average bone transmission time (BTT) and amplitude-dependent speed of sound (AD-SoS) z scores were lower in patients with diabetes than in controls. Significantly increased DKK-1 (3593 ± 1172 vs 2652 ± 689 pg/mL; P < 0.006) and sclerostin (29.45 ± 12.32 vs 22.53 ± 8.29; P < 0.001) levels were found in patients with diabetes with respect to controls, particularly in patients on MDI compared with ones on CSII. Glycemic control was improved in CSII patients compared with MDI ones (P < 0.001) and was also associated with significantly higher BMI-SDS (P < 0.002) and BTT z scores (P < 0.02). With adjustment for age, multiple linear regression analysis of DKK-1 and sclerostin as dependent variables showed that levels of glycated hemoglobin, glucose, 25(OH) vitamin D, osteocalcin, and parathyroid hormone; years of diabetes; and BMI-SDS and AD-SoS z score were the most important predictors (P < 0.0001). Conclusions: Our study highlighted (1) the high serum levels of DKK-1 and sclerostin in T1DM children and their relationship with altered glycemic control and (2) the effect of CSII on improvement of glycemic control and bone health in T1DM children.

Background: Stem cells are defined as clonogenic cells capable of self-renewal and multi-lineage differentiation. A population of these cells has been identified in human Dental Follicle (DF). Dental Follicle Stem Cells (DFSCs) were found in pediatric unerupted wisdom teeth and have been shown to differentiate, under particular conditions, into various cell types of the mesenchymal tissues. Aim: The aim of this study was to investigate if cells isolated from DF show stem features, differentiate toward osteoblastic phenotype and express osteoblastic markers. Methods: We studied the immunophenotype of DFSCs by flow cytometric analysis, the osteoblastic markers of differentiated DFSCs were assayed by histochemical methods and real-time PCR. Results: We demonstrated that DFSCs expressed a heterogeneous assortment of makers associated with stemness. Moreover DFSCs differentiated into osteoblast-like cells, producing mineralized matrix nodules and expressed the typical osteoblastic markers, Alkaline Phosphatase (ALP) and Collagen I (Coll I). Conclusion: This study suggests that DFSCs may provide a cell source for tissue engineering of bone.

Control of osteoclast (OC) apoptosis has been recognized as a critical regulatory factor in bone remodeling. TRAIL, a member of the TNF superfamily, induces apoptosis in neoplastic and normal cells. However, few data are available on the effects of TRAIL on bone cells, thus in the present study we investigated TRAIL role on the apoptosis of human mature OCs. We show that TRAIL treatment causes reduced cell viability, loss of nuclei integrity, and derangement of the actin microfilament in OCs. We also demonstrated that the death receptor DR5, upregulated by TRAIL, could be the mediator of TRAIL-induced OC apoptosis.

High levels of interleukin-7 (IL-7) have been associated with bone loss due to its stimulatory osteoclastogenic activity. Osteolytic patients' peripheral blood mononuclear cells (PBMCs) differentiate into osteoclasts without adding stimulating factors. Now, we investigated the potential role of IL-7 in the spontaneous osteoclastogenesis occurring in these patients. We identified significant differences in serum IL-7 levels between patients with/without bone metastases, suggesting that IL-7 might be effective as a clinical marker of disease progression. In patients' PBMC cultures we demonstrated that IL-7 stimulates osteoclastogenesis by inducing TNF-alpha release by T and B cells. These findings add further details to the disclosure of the mechanisms controlling bone metastases in solid tumors.

Several studies have reported the beneficial effects of mesenchymal stem cells (MSCs) in tissue repair and regeneration. New sources of stem cells in adult organisms are continuously emerging; dental tissues have been identified as a source of postnatal MSCs. Dental bud is the immature precursor of the tooth, is easy to access and we show in this study that it can yield a high number of cells with ≥95% expression of mesenchymal stemness makers and osteogenic capacity. Thus, these cells can be defined as Dental Bud Stem Cells (DBSCs) representing a promising source for bone regeneration of stomatognathic as well as other systems. Cell interactions with the extracellular matrix (ECM) and neighboring cells are critical for tissue morphogenesis and architecture; such interactions are mediated by integrins and cadherins respectively. We characterized DBSCs for the expression of these adhesion receptors and examined their pattern during osteogenic differentiation. Our data indicate that N-cadherin and cadherin-11 were expressed in undifferentiated DBSCs and their expression underwent changes during the osteogenic process (decreasing and increasing respectively), while expression of E-cadherin and P-cadherin was very low in DBSCs and did not change during the differentiation steps. Such expression pattern reflected the mesenchymal origin of DBSCs and confirmed their osteoblast-like features. On the other hand, osteogenic stimulation induced the upregulation of single subunits, αV, β3, α5, and the formation of integrin receptors α5β1 and αVβ3. DBSCs differentiation toward osteoblastic lineage was enhanced when cells were grown on fibronectin (FN), vitronectin (VTN), and osteopontin (OPN), ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. In addition we established that integrin αVβ3 plays a crucial role during the commitment of MSCs to osteoblast lineage, whereas integrin α5β1 seems to be dispensable. These data suggest that functionalization of biomaterials with such ECM proteins would improve bone reconstruction therapies starting from dental stem cells.

The aim of the present study was to evaluate the effect of bone pate on human osteoblast differentiation by measuring cell viability, alkaline phosphatase activity and expression of the transcription factors and of the major components of the extracellular matrix. Although bone pate has been used in ear surgery for many years and when placed in contact with mastoid and external auditory canal bone become viable, the cellular mechanisms that lead to its osteointegration have never been described. Bone pate taken from four patients subjected to mastoidectomy and affected by middle ear and mastoid cholesteatoma was placed in contact with osteoblast-like cell cultures. Four experimental conditions were obtained: cell cultures treated with bone pate, with bone pate mixed with fibrin glue, with fibrin glue and untreated. After 24 h, the viability of the cells was evaluated; after 1 week, alkaline phosphatase activity and the expression of transcription factors and bone matrix proteins were assessed by quantitative polymerase chain reaction. After 24 h osteoblasts showed increased viability when treated with bone pate (19 % increase) and bone pate mixed with fibrin glue (34 % increase). After 1 week, the number of alkaline phosphatase positive cells increased by 97 and 94 % in cultures treated with bone pate alone and bone pate mixed with fibrin glue. Treatment with bone pate upregulated transcription factors and components of the extracellular matrix. The present data show that bone pate has a high osteoinductive potential on human osteoblasts, enhancing their activity.

CD99 is a transmembrane glycoprotein expressed in physiological conditions by cells of different tissues, including osteoblasts (OBs). High or low CD99 levels have been detected in various pathological conditions, and the supernatant of some carcinoma cell lines can modulate CD99 expression in OB-like cells. In the present work we demonstrate for the first time that two different human myeloma cell lines (H929 and U266) and, in a less degree, their conditioned media significantly downregulate CD99 expression in normal human OBs during the differentiation process. In the same experimental conditions the OBs display a less differentiated phenotype as demonstrated by the decreased expression of RUNX2 and Collagen I. On the contrary, when CD99 was activated by using a specific agonist antibody, the OBs become more active as demonstrated by the upregulation of Alkaline Phosphatase, Collagen I, RUNX2, and JUND expression. Furthermore, we demonstrate that the activation of CD99 is able to induce the phosphorylation of ERK 1/2 and AKT intracellular signal transduction molecules in the OBs.

Bone diseases are associated with great morbidity; thus, the understanding of the mechanisms leading to their development represents a great challenge to improve bone health. Recent reports suggest that a large number of molecules produced by immune cells affect bone cell activity. However, the mechanisms are incompletely understood. This review aims to shed new lights into the mechanisms of bone diseases involving immune cells. In particular, we focused our attention on the major pathogenic mechanism underlying periodontal disease, psoriatic arthritis, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, metastatic solid tumors, and multiple myeloma.

The development of multiple myeloma (MM) bone disease is mediated by increased number and activity of osteoclasts (OCs). Using an in vitro osteoclastogenesis model consisting of unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs) from patients with MM, we showed that T cells support the formation of OCs with longer survival. Different from T-cell–depleted MM PBMC cultures, exogenous macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) were necessary for the formation of OCs; however, they did not exhibit longer survival. We found up-regulated production of RANKL, osteoprotegerin (OPG), and TNF-related apoptosis-inducing ligand (TRAIL) by fresh MM T cells. Despite high OPG levels, the persistence of osteoclastogenesis can be related to the formation of the OPG/TRAIL complex demonstrated by immunoprecipitation experiments and the addition of anti-TRAIL antibody which decreases OC formation. OCs overexpressed TRAIL decoy receptor DcR2 in the presence of MM T cells and death receptor DR4 in T-cell–depleted cultures. In addition, increased Bcl-2/Bax (B-cell lymphoma-2/Bcl2-associated protein X) ratio, following Bcl-2 up-regulation, was detected in OCs generated in the presence of T cells. Our results highlight that MM T cells support OC formation and survival, possibly involving OPG/TRAIL interaction and unbalanced OC expression of TRAIL death and decoy receptors.

Bone metastases represents a common cause of morbidity in patients suffering many types of cancer: breast, lung, kidney, prostate, and multiple myeloma. Osteolytic metastases often cause severe pain, pathologic fractures, hypercalcemia, spinal cord compression, and other nerve-compression syndromes. Osteoclasts (OCs), cells deriving from granulocitic-macrophagic lineage, are responsible for osteolysis, which may be reduced by inhibiting both OCs formation and activity. By studying bone osteolytic metastases mechanism in solid tumors, we report here our findings that cancer patients with bone involvement display an increase in osteoclasts precursors, compared with both healthy controls and cancer patients without bone metastases. Peripheral blood mononuclear cells (PBMCs) from patients with osteolytic lesions show osteoclastogenesis without adding M-CSF, RANKL, or TNF-alpha. However, these factors are necessary to generate OCs from healthy donors, non-osteolytic patient PBMCs and T-cell depleted PBMCs. OCs derived from cancer patients show more resorption pits than OCs from healthy donors and express genes involved in osteoclastogenesis. Our data show that a spontaneous osteoclastogenesis occurs in patients affected by osteolytic lesions and may be supported by factors released by T lymphocytes. These factors could give a priming to osteoclast precursors and promote osteoclastogenesis. In fact, T-cell depleted PBMCs do not differentiate into OCs without adding M-CSF and RANKL. Moreover, we do not obtain a higher number of OCs by increasing RANKL doses in cultures, and OCs and T lymphocytes mRNA level are detected for TNF-alpha but not for RANKL. The addition of OPG to PBMCs cultures do not modify spontaneous osteoclastogenesis. A neutralizing anti-TNF-alpha antibody in unstimulated PBMC cultures of osteolytic cancer patients induces an inhibition of osteoclastogenesis. These data suggest that TNF-alpha may be responsible for osteoclastogenesis in these tumors.

A significant number of long-term complications have been described in childhood leukemia survivors. In particular, these patients may present features of metabolic syndrome (MetS), and therefore increased risk for cardiovascular diseases. The aim of this meta-analysis is to evaluate the prevalence and the risk of MetS in survivors of childhood leukemia. Two authors independently performed a systematic literature search in PubMed and EMBASE to March 2014, reviewed and selected articles, based on pre-determined selection criteria. Twelve articles, comprising 2,337 participants (1,462 cases and 875 controls), were included in the meta-analysis. Only three of them were case-control studies eligible for the meta-analysis. The childhood leukemia survivors showed an increased risk of MetS as compared to healthy controls (OR = 4.36; 95 % CI 1.19-16.22). The risk was significantly increased only in patients treated with chemotherapy and radiotherapy (OR = 7.79; 95 % CI 1.27-47.77), and not in patients treated with only chemotherapy (OR = 2.35; 95 % CI 0.40-13.78). Childhood leukemia survivors, in particular if treated also with radiotherapy, are prone to develop MetS more than healthy controls. Monitoring of MetS components in these patients is necessary to avoid cardiovascular consequences later in life.

There are conflicting data regarding the potential impact of chronic glucocorticoid (GC) therapy on the bone mineral density of patients with congenital adrenal hyperplasia (CAH). Previous studies performed by dual-energy X-ray absorptiometry reported conflicting results. The purpose of this study was to assess the impact of chronic GC replacement treatment in children with classical and non classical CAH due to 21-hydroxylase deficiency (21-OHD) by quantitative ultrasonometry (QUS), an easy, cheap, and radiation-free technique. The study population consisted of nineteen 21-OHD patients (nine males) on lifelong GC treatment. Anthropometric, hormonal, and treatment data were recorded for each patient, and bone quality was assessed by QUS measurements. QUS findings (amplitude-dependent speed of sound and bone transmission time) were normal in 21-OHD patients and did not correlate with duration of treatment, daily, total, and yearly hydrocortisone dose. Furthermore, no significant correlation was found between QUS findings and 17α-hydroxy progesterone, Δ4-androstenedione, and testosterone levels. In conclusion, our results provide reassurance that currently used replacement doses of GC do not have a major impact on bone in patients with CAH. QUS seems to be a reliable tool for screening of bone health in children with 21-OHD.

Bone is a metabolically active tissue that undergoes continuous remodeling by two sequential events, bone formation and resorption. These events are strongly linked and tightly regulated to maintain skeletal homeostasis. The bone cells responsible for the dual events include the bone-resorbing cells, the osteoclasts, arising from monocyte-macrophage precursors, and the bone forming cells, the osteoblasts, having a mesenchymal origin. Immune and bone cell activities are linked by several pathways [1] and the former can promote bone building or destruction. Further, immune cells can be involved in the mineralization process occurring in extra-skeletal sites. In this special issue different authors highlighted these items both through research articles and reviews. In detail, interaction between osteoblast precursors, the mesenchymal stem cells (MSCs) [2], and immune cells during fracture repair acts as one of the key factors governing successful bone healing. Additionally, bone damage following immune deregulation may be local as in arthritis and periodontal disease (PD) or systemic as in osteoporosis [3] and osteotropic cancers [4, 5]. It could be multifactorial and thus due to genetic modifications (i.e., Gaucher disease) as well as to lipopolysaccharide- (LPS-) mediated release of inflammatory cytokines (i.e., PD, osteomyelitis, and arthritis), and so forth. New insights suggest that, in immune-mediated bone diseases, bone resorption active phases are characterized by increased levels of immunoreceptor tyrosine-based activation motifs (ITAMs); these molecules together with OSCAR could be indicative of disease progression. Further, osteotropic cancer-related immune alterations showed distinct immune cell phenotype as observed in chronic myeloid leukemia, multiple myeloma, and bone metastatic solid tumors. State-of-the-art and new mechanisms are clearly described in this special issue; they can be useful for the identification of new therapeutic targets and bone disease markers. Giacomina Brunetti Giorgio Mori Patrizia D'Amelio Roberta Faccio

The management of steroid-sensitive nephrotic syndrome (SSNS) requires treatment with high-dose glucocorticoids (GCs), but GC usage causes the most frequent form of drug-induced osteoporosis. The aim of our study was to evaluate the impact of GCs on bone mineralization in patients with SSNS using two diagnostic tools, dual-energy X-ray densitometry (DXA) and quantitative ultrasound (QUS), and to compare the diagnostic efficacy of these two imaging tools. A total of 30 children with SSNS (age 5.20 ± 2.20 years) were evaluated at the start (T0) and after 1 (T1), 2.44 ± 0.75 (T2, 18 patients) and 5.96 ± 2.33 years (T4, 12 patients) of GC treatment. Patients who stopped at T2 were also evaluated at the 1-year timepoint after ceasing GC treatment (T3). Of the patients assessed at T2, 11 had bone mineralization at the lower limit of normal versus those at T0 and T1, with bone mineralization rescue at the 1-year timepoint after GC discontinuation. At T4, 6/12 patients had densitometric parameters at the lower limit of normal values, and 3/12 patients showed reduced bone mineralization. The parameters derived from measurements of DXA and QUS were significantly related to each timepoint. Patients with SSNS receiving GC therapy undergo bone status alteration related to the dosage and duration of the therapy. In terms of diagnostic efficacy, DXA and QUS were comparable, indicating that QUS is a reliable tool to evaluate bone health in children with SSNS.

Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal dysplasia characterized by hypoplastic or aplastic clavicles, dental abnormalities, and delayed closure of the cranial sutures. In addition, mid-face hypoplasia, short stature, skeletal anomalies and osteoporosis are common. We aimed to evaluate osteoclastogenesis in a child (4 years old), who presented with clinical signs of CCD and who have been diagnosed as affected by deletion of RUNX2, master gene in osteoblast differentiation, but also affecting T cell development and indirectly osteoclastogenesis. The results of this study may help to understand whether in this disease is present an alteration in the bone-resorptive cells, the osteoclasts (OCs). Unfractionated and T cell-depleted Peripheral Blood Mononuclear Cells (PBMCs) from patient were cultured in presence/absence of recombinant human M-CSF and RANKL. At the end of the culture period, OCs only developed following the addition of M-CSF and RANKL. Moreover, real-time PCR experiment showed that freshly isolated T cells expressed the osteoclastogenic cytokines (RANKL and TNFα) at very low level, as in controls. This is in accordance with results arising from flow cytometry experiments demonstrating an high percentage of circulating CD4(+)CD28(+) and CD4(+)CD27(+) T cells, not able to produce osteoclastogenic cytokines. Also RANKL, OPG and CTX serum levels in CCD patient are similar to controls, whereas QUS measurements showed an osteoporotic status (BTT-Z score -3.09) in the patient. In conclusions, our findings suggest that the heterozygous deletion of RUNX2 in this CCD patient did not alter the osteoclastogenic potential of PBMCs in vitro.

Calcific aortic valve disease (CAVD) represents a slowly progressive pathologic process associated with major morbidity and mortality. The process is characterized by multiple steps: inflammation, fibrosis, and calcification. Numerous studies focalized on its physiopathology highlighting different “actors” for the multiple “acts.” This paper focuses on the role of the tumor necrosis factor superfamily (TNFSF) members in the pathogenesis of CAVD. In particular, we discuss the clinical and experimental studies providing evidence of the involvement of tumor necrosis factor-alpha (TNF-α), receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL), its membrane receptor RANK and its decoy receptor osteoprotegerin (OPG), and TNF-related apoptosis-inducing ligand (TRAIL) in valvular calcification.

OBJECTIVES: The receptor activator of the nuclear factor kappa-B (NF-κB) ligand (RANKL), its membrane receptor RANK and its decoy receptor osteoprotegerin (OPG) are all members of the tumour necrosis factor family involved in bone metabolism and immune response. We evaluated the activation of the OPG/RANKL/RANK pathway in patients undergoing cardiac surgery with and without cardiopulmonary bypass (CPB). METHODS: Twenty consecutive patients undergoing elective coronary artery surgery were enrolled in the study and assigned either to the on-pump or to the off-pump group. Pre- and postoperative serum levels of OPG and RANKL were evaluated by enzyme-linked immunosorbent assay; gene expression of OPG, RANKL, RANK and NF-κB p50 subunits were determined by real-time polymerase chain reaction in peripheral blood T-cells and monocytes. RESULTS: Serum levels of OPG significantly increased after surgery in both groups, whereas serum levels of RANKL did not differ over time. T-cells from the on-pump group showed increased gene expression of OPG, RANKL and RANK after the intervention, whereas no mRNA variation for these genes was detected in T-cells from off-pump patients. Gene expression of p50 subunit increased in T-cells and monocytes from both groups. CONCLUSIONS: Cardiac surgery induces the activation of the OPG/RANKL/RANK pathway; both on- and off-pump procedures are associated with increased postoperative OPG serum levels and up-regulation of the NF-κB p50 subunit.

Children with 21-hydroxylase deficiency (21-OHD) need chronic glucocorticoid (cGC) therapy to replace congenital deficit of cortisol synthesis, and this therapy is the most frequent and severe form of drug-induced osteoporosis. In this study, we enrolled 18 patients (9 females) and 18 sex- and age-matched controls. We found in 21-OHD patients high serum and leukocyte levels of dickkopf-1 (DKK1), a secreted antagonist of the Wnt/β-catenin signaling pathway known to be a key regulator of bone mass. In particular, we demonstrated by flow cytometry, confocal microscopy, and real-time PCR that monocytes, T lymphocytes, and neutrophils from patients expressed high levels of DKK1, which may be related to the cGC therapy. In fact, we showed that dexamethasone treatment markedly induced the expression of DKK1 in a dose- and time-dependent manner in leukocytes. The serum from patients containing elevated levels of DKK1 can directly inhibit in vitro osteoblast differentiation and receptor activator of NF-κB ligand (RANKL) expression. We also found a correlation between both DKK1 and RANKL or COOH-terminal telopeptides of type I collagen (CTX) serum levels in 21-OHD patients on cGC treatment. Our data indicated that DKK1, produced by leukocytes, may contribute to the alteration of bone remodeling in 21-OHD patients on cGC treatment.

In the last two decades, numerous scientists have highlighted the interactions between bone and immune cells as well as their overlapping regulatory mechanisms. For example, osteoclasts, the bone-resorbing cells, are derived from the same myeloid precursor cells that give rise to macrophages and myeloid dendritic cells. On the other hand, osteoblasts, the bone-forming cells, regulate hematopoietic stem cell niches from which all blood and immune cells are derived. Furthermore, many of the soluble mediators of immune cells, including cytokines and growth factors, regulate the activities of osteoblasts and osteoclasts. This increased recognition of the complex interactions between the immune system and bone led to the development of the interdisciplinary osteoimmunology field. Research in this field has great potential to provide a better understanding of the pathogenesis of several diseases affecting both the bone and immune systems, thus providing the molecular basis for novel therapeutic strategies. In these review, we reported the latest findings about the reciprocal regulation of bone and immune cells.