High levels of LIGHT/TNFSF14 in patients with Prader-Willi syndrome.

PURPOSE/METHODS: Prader-Willi syndrome (PWS) is a rare genetic disorder displaying different clinical features, including obesity and bone impairment. LIGHT/TNFSF14 is a cytokine produced by immune cells affecting both fat and bone metabolism. The present study aimed to evaluate LIGHT serum levels in 28 children and 52 adult PWS patients compared to age and sex-matched controls, as well as correlations with parameters of bone and fat metabolism. RESULTS: Median serum LIGHT levels were significantly increased in pediatric PWS with respect to controls [255.82 (284.43) pg/ml vs 168.11 (76.23) pg/ml, p ≤ 0.02] as well as in adult PWS compared to controls [296.85 (895.95) pg/ml vs 134.18 (141.18) pg/ml, p ≤ 0.001]. In pediatric PWS, LIGHT levels were positively correlated with weight-SDS, height-SDS, and glucose levels, and negatively with total 25 (OH) vitamin D, cholesterol, LDL cholesterol and triglycerides. Additionally, LIGHT levels were negatively correlated with total BMD and fat mass. In adult PWS, LIGHT levels were positively correlated with weight, HDL cholesterol and PTH, and negatively with glucose, insulin, HOMA-IR, total cholesterol, LDL cholesterol, triglycerides, calcium, phosphorus, 25(OH)Vitamin D as well as with instrumental parameters of bone and fat quality. Consistently, multiple regression analysis showed that LIGHT serum levels in pediatric and adult PWS were predicted by different parameters including 25 (OH) Vitamin D as well as DXA parameters of bone and fat quality. CONCLUSIONS: In PWS children and adults the high levels of LIGHT could represent a marker of the altered bone and fat metabolism.

The genetic background and vitamin D supplementation can affect irisin levels in Prader-Willi syndrome.

BACKGROUND: Prader-Willi syndrome (PWS) is associated to distinctive clinical symptoms, including obesity, cognitive and behavioral disorders, and bone impairment. Irisin is a myokine that acts on several target organs including brain adipose tissue and bone. The present study was finalized to explore circulating levels of irisin in children and adult PWS patients. METHODS: Seventy-eight subjects with PWS, 26 children (15 females, mean age 9.48 ± 3.6 years) and 52 adults (30 females, mean age 30.6 ± 10.7) were enrolled. Irisin serum levels were measured in patients and controls. Its levels were related with anthropometric and metabolic parameters, cognitive performance and bone mineral density either in pediatric or adult PWS. Multiple regression analysis was also performed. RESULTS: Irisin serum levels in PWS patients did not show different compared with controls. A more in-depth analysis showed that both pediatric and adult PWS with DEL15 displayed significantly reduced irisin levels compared to controls. Otherwise, no differences in irisin concentration were found in UPD15 patients with respect to controls. Our study revealed that in pediatric PWS the 25(OH) vitamin-D levels affected irisin serum concentration. Indeed, patients who were not supplemented with vitamin D showed lower irisin levels than controls and patients performing the supplementation. Multiple regression analysis showed that irisin levels in pediatric and adult PWS were predicted by the genetic background and 25(OH)-vitamin D levels, whereas in a group of 29 adult PWS also by intelligent quotient. CONCLUSION: We demonstrated the possible role of genetic background and vitamin-D supplementation on irisin serum levels in PWS patients.

Vipera snakebite in Europe: a systematic review of a neglected disease.

In 2009, snakebites were included in the list of the World Health Organization (WHO) neglected diseases. Dermatological literature lacks current and up-to-date articles about snakebites and their management, despite the fact that dermatologists, especially from rural hospitals, can be called into the emergency room to consult the management of suspected snakebites. In this systematic review, we highlighted the main clinical and laboratory aspects of snakebites from Vipera spp. in Europe, by reviewing 3574 studies initially retrieved from PubMed, Embase and Cochrane CENTRAL databases. Of these, 78 were finally included in the systematic review. We found that the most involved taxon was V. berus in 63.3% and the most involved anatomic site of the bite was the upper limbs 53.1% with fang marks reported in 90.5%. The mean age of the patients was 32.9 years, and bites were slightly more common among males (58.2%). A wound washing was performed in 86.9% of cases before the hospitalization. The most frequently reported grade of envenomation was G2 (42.2%). In addition to local dermatological symptoms (extended erythema, oedema, cutaneous necrosis, hives, purpura, petechiae, acute compartment syndrome), numerous systemic symptoms have also been reported, including fatigue (14.4%), pain (75.3%), fever (49.2%), direct anaphylactoid reaction (5.3%), anxiety (60.8%), cranial nerve neurotoxicity (14.8%), dysesthesia/paraesthesia (7.9%), vomiting (33.7%), abdominal pain (23.3%), diarrhoea (15.4%), dyspnoea (6.3%), proteinuria (10.6%) and haematuria (9.3%). Secondary infections were present in 3.5% and disseminated intravascular coagulation in 3.1% of cases, and fasciotomy was performed in 4.2% cases, while an amputation in 6.9%. Only 0.9% of patients died. Antivenom was administered in 3053 cases. In conclusion, there is a pressing need for robust multi-centre randomized control trials, standardized protocol for snakebite management and antivenom administration across Europe and a National snakebite register for each European country.

Stemness genes expression in naïve vs. osteodifferentiated human dental-derived stem cells.

OBJECTIVE: Mesenchymal stem cells (MSCs) have been deeply investigated in regenerative medicine because of their crucial role in tissue healing, such as tissue regeneration. Dental-derived stem cells (d-DSCs) are easily available from dental tissues, which can be isolated from all age patients with minimal discomfort. PATIENTS AND METHODS: Normal unerupted third molars tooth buds were collected from adolescents' patients underwent to extractions for orthodontic reasons. The expression of the genes Kruppel-like factor 4 (Klf-4), octamer-binding transcription factor 4 (Oct-4), homeobox transcription factor Nanog (NANOG) was investigated in d-DSCs obtained from dental bud (DBSCs), differentiated toward osteoblastic phenotype and not. RESULTS: Our results showed that DBSCs expressed Oct-4, Nanog, and Klf-4 in undifferentiated conditions and interestingly the expression of such genes increased when the cells were kept in osteogenic medium. CONCLUSIONS: These attractive stemness properties, together with the effortlessly isolation, during common oral and maxillofacial surgical procedures, from undifferentiated tissues such as dental bud, make this kind of d-DSCs a promising tool in regenerative medicine, having the potential for clinical applications, and reinforcing the present challenge to develop new preventive and healing strategies in tissue regeneration.

Analysis of Circulating Mediators of Bone Remodeling in Prader-Willi Syndrome.

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 < 0.01), reaching osteopenic levels in PWS adults. PWS patients showed lower 25(OH)-vitamin D serum levels than controls (P < 0.001). Osteocalcin was increased in PWS children but reduced in adults respect to controls (P < 0.005 and P < 0.01, respectively). RANKL levels were higher in both pediatric and PWS adults than controls (P < 0.004), while OPG levels were significantly reduced (P < 0.004 and P < 0.006, respectively). Sclerostin levels were increased in children (P < 0.04) but reduced in adults compared to controls (P < 0.01). DKK-1 levels did not show significant difference between patients and controls. In PWS patients, RANKL, OPG, and sclerostin significantly correlated with metabolic and bone instrumental parameters. Consistently, with adjustment for age, multiple linear regression analysis showed that BMD and osteocalcin were the most important predictors for RANKL, OPG, and sclerostin in children, and GH and sex steroid replacement treatment in PWS adults. We demonstrated the involvement of RANKL, OPG, and sclerostin in the altered bone turnover of PWS subjects suggesting these molecules as markers of bone disease and new potential pharmacological targets to improve bone health in PWS.

Irisin levels correlate with bone mineral density in soccer players.

Irisin, a novel myokine produced in response to physical exercise by skeletal muscle, displays anabolic effect on bone and can improve the bone-loss-induced osteoporosis in hind limb suspended mice. It is well known that muscles positively impact the skeleton and in different sports, including soccer, total body bone mineral density (TB-BMD) is elevated. Therefore, we have investigated the correlation between irisin serum levels and total and bone sub-regional BMD in soccer players never studied before. In this study, Caucasian football players of Bari team have been enrolled. Their sera were collected to measure by ELISA kit irisin levels and by dual-energy X-ray absorptiometry (DEXA) analysis measurements of BMD (g • cm−2) in the whole body and different bone sub-regions (head, arms, legs, ribs, dorsal vertebrae, lumbar vertebrae, pelvis) were performed. The BMC (g) was measured in the whole body. By means of Pearson's (R) and Cohen's (d) coefficient we investigated the linear association between the irisin serum levels and BMD. In soccer players, we have found a positive correlation between irisin and TB-BMD as demonstrated by the values of Pearson and Cohen's (d) coefficient. Furthermore, linear association was detected between irisin and BMD of different bone-site such as right arm, lumbar vertebrae and head. A positive trend was also observed analyzing circulating levels of irisin and bone mineral content as well as total Z-score. In conclusion, we have demonstrated the correlation between irisin and total or bone sub-regional BMD in soccer players for the first time, an additional systemic effect of the "sport-hormone" defined myokine.

Impaired bone remodeling in children with osteogenesis imperfecta treated and untreated with bisphosphonates: the role of DKK1, RANKL, and TNF-α.

UNLABELLED: 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. INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

CELLULAR MECHANISMS OF BONE REGENERATION: ROLE OF WNT-1 IN BONE-MUSCLE INTERACTION DURING PHYSICAL ACTIVITY39.

Wnt1 is one of the several glycoproteins activating Wnt signaling, critical for normal skeletal development and bone homeostasis. Wnt1 was previously believed to solely regulate central nervous system development, in particular in midbrain and cerebellum. However, remarkable findings have recently shown that several patients affected by severe form of Osteogenesis Imperfecta (OI) display a Wnt1 mutation thereby revealing a possible role of Wnt1 in bone metabolism. Here, we show that recombinant Wnt1 (r-Wnt1) strongly increases differentiation of bone marrow stromal cells into mature osteoblasts, as demonstrated by the enhanced number of cells positively stained for alkaline phosphatase, one of the osteoblastic marker genes, whose mRNA levels are also significantly up-regulated. Furthermore, other osteogenic master genes such as Collagen I and Osteopontin are also enhanced when bone marrow precursors were differentiated toward osteoblastic phenotype in the presence of r-Wnt1. Intriguingly, by in vivo and in vitro findings, we report that in the bone marrow of mice subjected to physical activity there is a high endogenous Wnt1 synthesis compared to mice kept in resting conditions. Moreover, conditioned medium collected from ex vivo myoblasts, harvested from exercised mice, up-regulates Wnt1 expression in osteoblast cell cultures obtained from control mice. Overall our findings support the role of Wnt1 in regulating bone metabolism and suggest that this molecule could be one of the mediators through which physical activity may exert beneficial effect on bone.

Evaluation of impact of steroid replacement treatment on bone health in children with 21-hydroxylase deficiency.

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.

Myeloma cells suppress osteoblasts through sclerostin secretion.

Wingless-type (Wnt) signaling through the secretion of Wnt inhibitors Dickkopf1, soluble frizzled-related protein-2 and -3 has a key role in the decreased osteoblast (OB) activity associated with multiple myeloma (MM) bone disease. We provide evidence that another Wnt antagonist, sclerostin, an osteocyte-expressed negative regulator of bone formation, is expressed by myeloma cells, that is, human myeloma cell lines (HMCLs) and plasma cells (CD138+ cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. We demonstrated that BM stromal cells (BMSCs), differentiated into OBs and co-cultured with HMCLs showed, compared with BMSCs alone, reduced expression of major osteoblastic-specific proteins, decreased mineralized nodule formation and attenuated the expression of members of the activator protein 1 transcription factor family (Fra-1, Fra-2 and Jun-D). Moreover, in the same co-culture system, the addition of neutralizing anti-sclerostin antibodies restored OB functions by inducing nuclear accumulation of ß-catenin. We further demonstrated that the upregulation of receptor activator of nuclear factor κ-B ligand and the downregulation of osteoprotegerin in OBs were also sclerostin mediated. Our data indicated that sclerostin secretion by myeloma cells contribute to the suppression of bone formation in the osteolytic bone disease associated to MM.

Osteogenic properties of human dental pulp stem cells.

Stem cells are a promising tool for bone tissue regeneration. Dental pulp stem cells (DPSCs) can be easily obtained even in human young adults. In this study we investigated the capability of DPSCs, to express the osteoblastic phenotype when cultured with osteogenic medium. DPSCs isolated from the dental pulp of impacted third molar teeth were cultured with appropriate medium to induce osteoblast differentiation. Using Western-Blot, RT-PCR and microarray analysis, we studied the expression of osteoblastic parameter, and by Von Kossa staining we evaluated the production of mineralized matrix nodules. The results were compared with controls represented by undifferentiated DPSCs. DPSCs, differentiated into osteoblast-like cells, express large amount of alkaline phosphatase (ALP), collagen I (Coll I), osteopontin (OPN) and osteocalcin (OCN), all these parameters characterizing the osteoblastic phenotype. Differentiated DPSCs express Runx2 and JunB, a member of the AP-1 complex; both the transcription factors are associated with osteoblast differentiation and skeletal morphogenesis. Moreover, DPSCs express insulin growth factor-binding protein 5 (IGFBP-5), one of the regulating proteins of IGFs function. Finally, DPSCs can form mineralized matrix nodules that are a feature exclusive to osteoblasts. DPSCs could represent a potential source of osteoblasts to be used for bone regeneration.

Soluble decoy receptor 3 modulates the survival and formation of osteoclasts from multiple myeloma bone disease patients.

Decoy receptor 3 (DcR3), a member of the tumor necrosis factor (TNF) receptor superfamily, is known to be involved in cell survival and osteoclast (OC) formation. In this study, we show that malignant plasma cells and T lymphocytes from multiple myeloma (MM) bone disease patients, as well as Karpas 909, a human myeloma cell line, directly produce DcR3. By interacting with FasL, this molecule could inhibit OC apoptosis. In fact, the use of a neutralizing anti-DcR3 antibody induces a reduction of cell viability with a consequent increase of apoptotic cell number, the activation of caspase-8 and -3, and DNA fragmentation. Furthermore, we show that DcR3 supports OC formation in samples from MM patients through the upregulation of RANKL and TNFalpha by T lymphocytes and only TNFalpha by CD14+ cells. In conclusion, our data provide the first evidence of the expression of DcR3 in MM, and the involvement of this molecule in supporting the survival and formation of OCs from MM bone disease patients. The production of DcR3 by T lymphocytes confers these cells a role in the pathogenesis of bone disease associated with MM.

Chlamydia pneumoniae and atherosclerosis: the role of mast cells.

Chlamydia pneumoniae (C. pneumoniae), a respiratory pathogen, has been implicated in the pathogenesis of atherosclerosis, an inflammatory progressive disease, characterized by the formation of atherosclerotic plaques. Among several types of inflammatory cells involved in the atherogenesis process, recently particular attention has been directed toward the mast cells. Experimental studies have provided several mechanisms by which C. pneumoniae and mast cells could play a role in all stages of atherosclerosis, from initial inflammatory lesions to plaque rupture. C. pneumoniae, as well as mast cells, may actively participate both through the production of cytokines and matrix-degrading metalloproteinases and by provoking apoptosis of atheroma-associated vascular cells, key events in plaque rupture. This mini-review provides a brief overview on adventitial inflammatory effects of C. pneumoniae and mast cells and their potential role in plaque instability. In addition, in this paper we review the role of mast cells in innate immunity.

Osteoblast apoptosis in periodontal disease: role of TNF-related apoptosis-inducing ligand.

Periodontal disease (Pd) is characterized by an increased osteoclast resorption and a decreased osteoblast (OB) bone formation. OBs obtained from alveolar bone of Periodontitis patients (Pp) undergo apoptosis in the presence of TNF-related apoptosis-inducing ligand (TRAIL). We studied the intracellular apoptotic pathway induced by TRAIL; TRAIL death (DR4, DR5) and decoy (DcR1, DcR2) receptors expression in Periodontitis patients OBs (PpOBs), and we measured the concentration of TRAIL in the serum of Pp. We demonstrated that DNA fragmentation and activation of caspase-8 and caspase-3 in PpOBs, following TRAIL stimulation, occurred in shorter time; moreover, a higher amount of both caspases was activated in order to direct OBs. Down-regulation of DcR2 in PpOBs was demonstrated and high TRAIL levels were detected in the serum of Pp. In conclusion, our data suggest that PpOBs are more sensitive to TRAIL-induced apoptosis when compared to the control group. The down-regulation of DcR2 possibly leads to an imbalanced ratio between death and decoy receptors. Our findings highlight a role of TRAIL in the pathogenesis of Pd.

Normal and osteoporotic human osteoblast behaviour after 1,25-dihydroxy-vitamin D(3) stimulation.

In order to examine the effects of vitamin D on osteoblast function and to evaluate if osteoporotic and normal osteoblasts show a different behaviour in response to vitamin D, this report investigates the changes in osteocalcin production, after 1,25-dihydroxy-vitamin D(3) stimulation of cultured osteoblasts derived from osteoporotic patients. Our results indicate an inadequate osteoblastic function in osteoporosis and demostrate that 1,25-dihydroxy-vitamin D(3) can stimulate the metabolic activity of human osteoblasts in vitro. Considering that osteoporotic bone samples were representative of senile osteoporosis, our results may indicate a different metabolic phenotype in osteoporotic osteoblasts compared with normal osteoblasts. The increased osteocalcin production after 1,25-dihydroxy-vitamin D(3) stimulation of osteoporotic osteoblasts suggests a reduced, but not absent, anabolic function in senile osteoporotic osteoblasts. The results of this study confirm the validity of vitamin D(3) to treat senile osteoporosis and suggest the need of higher vitamin D(3) intake in senile osteoporotic patients than in younger subjects.

L: -carnitine fumarate and isovaleryl-L: -carnitine fumarate accelerate the recovery of bone volume/total volume ratio after experimetally induced osteoporosis in pregnant mice.

Anabolic skeletal agents have recently broadened the therapeutic options for osteoporosis by directly stimulating bone formation and improving bone turnover, bone density, bone size, and bone microarchitecture. We recently demonstrated that two new L: -carnitine derivatives, L: -carnitine fumarate (LC) and isovaleryl-L: -carnitine fumarate (Iso-V-LC), stimulated osteoblast proliferation and differentiation. We here investigated, by histomorphometry in a mouse model of osteoporosis, the impact of these compounds on the repair of trabecular bone and the osteoblast involvement in this process. Fifty-nine inbred adult female CD1 mice in pregnancy were assigned to four treatment groups: (1) controls, mice fed a standard normocalcemic pre- and postpartal diet; (2) Hypo, mice fed a low-calcium isocaloric prepartal diet and a standard postpartal diet; (3) LC, mice fed a group 2-type diet supplemented post-partum with LC; (4) Iso-V-LC, mice fed a group 2-type diet supplemented post-partum with Iso-V-LC. Bone volume/total volume ratio (BV/TV), bone perimeter, osteoblast surface/bone surface, and osteoblast number/bone surface were measured from sections of L3 and L4 vertebral bodies obtained from animals killed on the day of delivery (controls and Hypo) and on days 7, 14, and 21 after delivery (all groups). BV/TV and all osteoblast-based indexes were significantly higher in LC and Iso-V-LC than in Hypo mice at each time point, and Iso-V-LC at the end of the treatment attained levels observed in controls. In conclusion, Iso-V-LC and, to a lesser extent, LC accelerated the recovery of normal BV/TV level after a hypocalcemic diet.

In vitro stem cell cultures from human dental pulp and periodontal ligament: new prospects in dentistry.

In spite of the vast knowledge of tooth development and of the various kinds of specialized bone/tooth-associated cells, the characteristics and properties of their precursor cell populations present in the postnatal organism are little known, as is their possible therapeutic use. Taken together dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) possess stem-cell-like qualities, including self-renewal capability and multi-lineage differentiation. Regenerative medicine is based on stem cells, signals and scaffolds. Transplantation of those cells, which can be obtained from an easily accessible tissue resource and expanded in vitro, holds promise as a therapeutic approach for reconstruction of tissues and bone in vivo.

Lymphocytes and synovial fluid fibroblasts support osteoclastogenesis through RANKL, TNFalpha, and IL-7 in an in vitro model derived from human psoriatic arthritis.

Psoriatic arthritis (PsA) is an inflammatory joint disease, characterized by extensive bone resorption, whose mechanisms have not been fully elucidated. Thus, in the present study we investigated the involvement of RANKL, TNFalpha, and IL-7 in the osteoclastogenesis of PsA patients. In vitro osteoclastogenesis models, consisting of unfractionated and T-cell-depleted mononuclear cells from peripheral blood (PBMCs) and synovial fluid (SFMCs) of 20 PsA patients as well as from healthy donors were studied. Freshly isolated T and B cells from PBMCs and T cells and fibroblasts from SFMCs of PsA patients were subjected to RT-PCR to detect the levels of RANKL, TNFalpha, and IL-7. Osteoclastogenesis was studied in the presence of RANK-Fc, anti-TNFalpha, and anti IL-7 functional antibodies. We demonstrate that lymphocytes and fibroblasts support osteoclast (OC) formation in PsA patients through the production of osteoclastogenic cytokines. In particular, OC formation was completely abolished in unstimulated T cell-depleted PBMC cultures, and reduced by approximately 70% in unstimulated T cell-depleted SFMC cultures. Freshly isolated T cells from PBMCs and SFMCs of PsA patients overexpressed RANKL and TNFalpha, while fibroblasts from synovial fluid produced only RANKL. We show that the presence of RANK-Fc and/or anti-TNFalpha functional antibodies reduced OC formation. Moreover, T and B cells from PBMCs as well as T cells and fibroblasts from SFMCs expressed IL-7 mRNA. Finally, the anti-IL-7 functional antibody significantly reduced osteoclastogenesis. Our results suggest that fibroblasts, B and T lymphocytes support OC formation by producing RANKL, TNFalpha, and IL-7, contributing to the aggressive bone resorption in PsA patients.

Alteration of activity and survival of osteoblasts obtained from human periodontitis patients: role of TRAIL.

Periodontal disease (Pd) is characterized by extensive alveolar bone loss, that occurs as a consequence of the impairment of the normal bone remodelling. Bone remodelling is regulated by the correct balance between osteoclast and osteoblast formation and activity. Alveolar bone loss could be due to an increased bone resorption by osteoclasts or a decreased bone formation by osteoblasts (OBs) or both. Although the role played by osteoclasts in increasing bone resorption in Pd is already known, the behaviour of OBs in this disease is poorly understood. In the present study we hypothesized that activity and survival of OBs, locally present in alveolar bone of Pd patients, are altered. Thus, we studied the activity and survival of OBs obtained from alveolar bone fragments of Pd patients. The results, obtained in OBs from the patients were compared with those from OBs obtained from healthy donors. We demonstrated that OBs from Pd patients weakly express OB phenotype in respect to the control cells. In particular, the alkaline phosphatase activity and the collagen type I production, as well as the formation of mineralized nodules, typical markers of differentiated OBs, were significantly lower in Pd patients. Interestingly, we also demonstrated that OBs from the patients were more sensitive to the apoptotic effect induced by TNF-related apoptosis-inducing ligand (TRAIL). TRAIL, a member of the TNF superfamily, induces apoptosis by interacting with its death receptors, (DR4, DR5). However, its activity can be modulated by two decoy receptors, DcR1 and DcR2. Thus, the sensitiveness of TRAIL induced apoptosis is determined by the ratio of death and decoy receptor. We demonstrated that OBs from Pd patients showed an imbalanced ratio between death and decoy TRAIL receptors due to the down-regulation of DcR2 expression. Furthermore, the levels of TRAIL in the serum of the same patients were significantly higher than those detected in the controls. In conclusion, we show for the first time that the alveolar bone loss in Pd patients could be due to the increased TRAIL-mediated apoptosis of OBs.

T cells support osteoclastogenesis in an in vitro model derived from human periodontitis patients.

BACKGROUND: Periodontitis is characterized by alveolar bone destruction; however, the mechanisms responsible for bone damage are poorly understood. It has been reported that T cells are implicated in the pathogenesis of periodontitis. It has been also demonstrated that activated T lymphocytes secrete receptor activator of nuclear factor-kappa B ligand (RANKL) and can support the differentiation of monocytes into resorbing osteoclasts (OCs). Therefore, the purpose of this study was to examine the OC formation in periodontitis patients (PP) and the role of T cells in osteoclastogenesis. METHODS: To study OC formation, we used an in vitro model consisting of unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs) from PP and controls. In parallel, T-cell-depleted PBMCs from the same patients were also established. The expression of RANKL and tumor necrosis factor-alpha (TNF-alpha) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot in fresh T cells isolated from PP and controls. Functional antibodies, anti-RANKL and anti-TNF-alpha, were utilized to study osteoclastogenesis in PBMC cultures from PP. RESULTS: We showed that, in unfractionated PBMCs from PP, the OCs spontaneously developed in a T-cell-dependent way. The addition of macrophage colony stimulating factor (MCSF) and RANKL was necessary to promote the osteoclastogenesis in T-cell-depleted PBMC cultures from PP and in unfractionated PBMCs from periodontally healthy controls. Moreover, freshly isolated T cells from PBMCs of PP overexpressed RANKL and TNF-alpha. Finally, functional anti-RANKL and anti-TNF-alpha antibodies significantly inhibited osteoclastogenesis. CONCLUSION: Our data suggest that T cells support spontaneous osteoclastogenesis in PP via RANKL and TNF-alpha overexpression.