RANKL/RANK/OPG system and bone status in females with anorexia nervosa

Minimal data exist concerning the relationship between osteokines of the RANKL/RANK/OPG system, especially RANKL, and bone status in females with anorexia nervosa (AN). For this reason we investigated the relationship between bone metabolism (as assessed based on serum levels of OC and CTx), and OPG and sRANKL concentrations in females with AN. Ninety-one female patients with AN and 29 healthy female subjects aged 13 to 18 years of age participated in the study. Serum OC, CTx, OPG and sRANKL were measured by ELISA. The female patients with AN demonstrated an essential suppression of OC and CTx, increased OPG and sRANKL levels, and a reduced OPG/sRANKL ratio. OC, CTx and the OPG/sRANKL ratio correlated positively with body mass and BMI in these patients, whereas in the case of OPG and sRANKL the relationship was negative. A significant positive correlation was observed between OPG and sRANKL and also between bone markers and the OPG/sRANKL ratio, and negative between CTx and sRANKL. In female patients with AN, the OPG/RANKL ratio was a significant and independent predictor of osteocalcin, a bone formation marker — OC (R² = 0.065, p = 0.012) whereas the OPG/sRANKL ratio and BMI were significant and independent predictors of a bone resorption marker — CTx (R² = 0.095; p = 0.012). In conclusion, the body mass, BMI values, and bone markers suppression observed in female patients with AN might be associated with an increase in OPG and sRANKL levels and a significant decrease of the OPG/sRANKL ratio. Although higher OPG levels may compensate for excessive bone resorption in female patients with AN, the lower OPG/sRANKL ratio seems to indicate that some inadequacies exist regarding this compensation effect, which might contribute to low bone density in these patients. The OPG/sRANKL ratio might prove a more relevant marker to predict bone metabolism in female patients with AN than sRANKL and/or OPG alone.     

痩素对成骨细胞骨保护蛋白mRNA表达的影响初探

目的 初步探讨瘦素(Leptin)对成骨细胞骨保护蛋白(OPG)mRNA表达的影响。方法 用SYBR GreenⅠ染料实时监测定量RT-PCR的方法测定骨保护蛋白(OPG)mRNA表达。结果 瘦素(Leptin)使成骨细胞骨保护蛋白OPG mRNA表达呈剂量和时间依赖性降低。结论 瘦素(Leptin)作为一种内分泌因子，可直接与成骨细胞上leptin受体特异结合，通过受体后信号转导下调节成骨细胞OPG mRNA表达，使OPG mRNA表达降低，和其它因子共同参与骨再建调节。     

Human bone marrow stromal cells protect prostate cancer cells from TRAIL-induced apoptosis.

Tumor-derived OPG has recently been shown to protect prostate cancer cells from apoptosis. This study has confirmed that bone marrow stromal cell-derived OPG also suppresses cytokine-induced apoptosis in this tumor type, suggesting that it may be the presence of bone-derived OPG that is responsible for the observed preference of these cells in colonizing the skeleton. INTRODUCTION: Metastasis to the skeleton occurs in around 70% of patients with advanced prostate cancer (CaP), suggesting that the bone microenvironment may provide factors that favor the growth and survival of prostate cancer cells. Osteoprotegerin (OPG) is a molecule involved in bone remodeling, where it acts as an inhibitor of osteoclastogenesis, but it is also a decoy receptor for TRAIL/Apo 2L, a member of the TNF family of pro-apoptotic cytokines. The aim of this study was to determine whether OPG produced by human bone marrow stromal cells could protect prostate cancer cells from TRAIL-induced apoptosis. MATERIALS AND METHODS: Human bone marrow stromal cell cultures were generated from bone biopsies taken from newly diagnosed untreated CaP patients with (M1) or without (M0) bony metastasis. The stromal origin of these cells was confirmed by Western blot analysis using antibodies raised to stromal and epithelial markers. Media were conditioned over the cultures of these cells for 4 days, and levels of OPG were determined using an ELISA. The human prostate cancer cell line PC3 was challenged with TRAIL (50 ng/ml) in fresh media or in media supplemented with 50% conditioned media, and apoptosis was assessed using DAPI stain. The effects of specific removal of OPG activity by immunoprecipitation or by co-treatment of cultures with an alternative ligand for OPG (RANKL) were also tested. RESULTS AND CONCLUSIONS: The presence of stromal cell conditioned media in PC3 culture significantly reduced TRAIL-induced apoptosis. All stromal cell lines isolated were shown to express OPG and to release this protein into the conditioned media. Immunoprecipitation of OPG and co-treatment of cultures with sRANKL reversed the protective effects of the conditioned media. These data suggest that at least part of the survival advantage gained by CaP cells in colonizing bone may be caused by the production of OPG by tumor-associated stromal cells.     

Is leptin the link between fat and bone mass?

Recently, leptin has emerged as a potential candidate responsible for protective effects of fat on bone tissue. However, it remains difficult to draw a clear picture of leptin effects on bone metabolism because published data are sometimes conflicting or apparently contradictory. Beyond differences in models or experimental procedures, it is tempting to hypothesize that leptin exerts dual effects depending on bone tissue, skeletal maturity, and/or signaling pathway. Early in life, leptin could stimulate bone growth and bone size through direct angiogenic and osteogenic effects on stromal precursor cells. Later, it may decrease bone remodeling in the mature skeleton, when trabecular bone turnover is high, by stimulating osteoprotegerin (OPG) expression. Leptin negative effects on bone formation effected through central nervous system pathway could counterbalance these peripheral and positive effects, the latter being predominant when the blood-brain barrier permeability decreases or the serum leptin level rises above a certain threshold. Thus, the sex-dependent specificity of the relationship between leptin and bone mineral density (BMD) in human studies could be, at least in part, caused by serum leptin levels that are two- to threefold higher in women than in men, independent of adiposity. Although these hypotheses remain highly speculative and require further investigations, existing studies consistently support the role of leptin as a link between fat and bone.     

Variations along the 24-hour cycle of circulating osteoprotegerin and soluble RANKL: a rhythmometric analysis

Summary The variability of serum osteoprotegerin (OPG) and soluble RANKL (sRANKL) along the 24-h cycle was assessed in 20 healthy women. No rhythmic variations of serum OPG, sRANKL or sRANKL/OPG ratio were detected as a group phenomenon. Timing of sampling is unlikely to influence the results of measurements of circulating OPG and sRANKL. Introduction Physiological bone turnover shows diurnal variations. The aim of the study was to assess variability of OPG and sRANKL serum levels along the 24-h cycle. Methods Blood was collected from 20 healthy women (median age 31 years, range 25–65 years) at 4-h intervals between 08:00 and 24:00 and at 2-h intervals between 24:00 and 08:00. Serum albumin, cortisol, osteocalcin (OC), C-terminal telopeptide of type I collagen (CTX), OPG and total sRANKL were measured. Temporal variations were assessed by the COSINOR model. Results Circadian rhythms of cortisol and albumin documented a normal synchronization within the circadian structure. Serum OC and CTX showed rhythmic variations, peaking at night-time. Rhythmic variations of serum OPG, sRANKL and sRANKL/OPG ratio were not detected as a group phenomenon. On an individual basis, rhythmic changes were detected in ten patients for OPG and eight patients for sRANKL, with very small amplitudes and heterogeneous acrophases. Conclusions The absence of consistent rhythmic variations of circulating OPG and sRANKL levels may reflect the absence of rhythmic variations of their expression in the bone microenvironment. Were this the case, the nocturnal rise of bone resorption should be accounted for by different, not RANKL/OPG-mediated factors. Since circulating OPG and sRANKL may derive from sources other than bone, rhythmicity could be masked by non-rhythmic or non-synchronized rhythmic expression in these sources. Timing of sampling is unlikely to influence the results of measurements of circulating OPG and sRANKL. Andrea Dovio and Daniele Generali contributed equally to the study.     

Two distinct cellular mechanisms of osteoclast formation and bone resorption in periprosthetic osteolysis.

PURPOSE: TNFalpha and IL-1alpha are proinflammatory cytokines that are abundant in periprosthetic tissues. These cytokines stimulate bone resorption and have recently been shown to directly induce osteoclast formation in mouse marrow cultures. We examined whether TNFalpha and IL-1alpha can directly induce osteoclast formation from human arthroplasty-derived (CD14(+)) macrophages by a mechanism independent of RANKL-induced osteoclastogenesis. METHODS: TNFalpha and M-CSF (+/-IL-1alpha) were added to cultures of magnetically sorted (CD14(+)) and unsorted (CD14(+)/CD14(-)) cells isolated from the pseudomembrane of loosened hip arthroplasties. Osteoprotegerin (OPG), RANK:Fc and antibodies to TNF receptors (p55 and p75) were added to these cultures to distinguish the pathway of osteoclastogenesis. Osteoclast differentiation was assessed by expression of tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR) and lacunar resorption. RESULTS: The addition of TNFalpha (+/-IL-1alpha) resulted in differentiation of CD14(+) macrophages into TRAP(+) and VNR(+) multinucleated cells capable of extensive lacunar resorption. Both OPG and RANK:Fc (which inhibit RANKL-induced osteoclastogenesis) did not block osteoclastogenesis. The addition of antibodies directed against the p55 receptor subunit of TNF resulted in significant inhibition of osteoclast formation and lacunar resorption. CONCLUSIONS: Our results indicate that, in the presence of M-CSF, TNFalpha is sufficient for inducing human osteoclast differentiation from arthroplasty macrophages and that TNFalpha acts synergistically with IL-1alpha to stimulate lacunar resorption. This process is distinct from the RANK/RANKL signalling pathway and is likely to operate in periprosthetic tissues when there is heavy wear particle deposition and cytokine production.     

Differential effects of leptin on cancer in vitro

INTRODUCTION: Leptin, a protein produced by adipocytes, is an important signaling molecule in energy regulation and food intake. Many obese patients have leptin resistance associated with increased circulating leptin. Leptin receptor activation downregulates many regulatory genes, including STAT-3 and PAP 1. Certain cancers are associated with obesity, including breast, prostate, and colon. Recent studies have shown that leptin stimulates proliferation of human colon cancer in vitro. We hypothesized that leptin would have stimulatory effects on other human cancers. MATERIALS AND METHODS: Human cancer cell lines from esophagus (KYSE410 and 150), breast (ZR75-1 and MCF-7), prostate (DU145 and PC-3), and pancreas (PANC-1, Mia-PaCa) were cultured using standard techniques. Leptin (0.4 ng/ml and 4.0 ng/ml) was added for 24 h and 48 h. Cell growth was determined by MTT assay. Statistical analysis was performed using analysis of variance. RESULTS: Cancer cell lines demonstrated dose- and time-related responses to treatment. Leptin caused growth potentiation in breast, esophagus, and prostate cancer (P < 0.05). However, in both Mia-PaCa and PANC-1 pancreatic cancer cells, leptin inhibited growth (P < 0.05). This inhibitory effect peaked in PANC-1 at 48 h (78%). CONCLUSIONS: We have shown for the first time that human cancer cells exhibit differential responses to treatment with leptin, depending upon organ of derivation. Both leptin and leptin antagonism have potential efficacy in cancer therapy, based on cellular origin. Further studies are warranted and ongoing.     

Are OPG and RANKL involved in human fracture healing?

Human fracture healing is a complex interaction of several cytokines that regulate osteoblast and osteoclast activity. By monitoring OPG (osteoprotegerin) and sRANKL we aimed to possibly predict normal or impaired fracture healing. In 64 patients with a fracture of a long bone serum level of sRANKL and OPG were evaluated with respect to bony union (n = 57) or pseudarthrosis (n = 7). Measurements were carried out at admission and at 1, 2, 4, 6, 8, 12, 24, and 48 weeks after the injury. Patients' serum levels were compared to 33 healthy controls. Fracture hematoma contained significantly higher sRANKL and OPG concentrations compared to patients serum (p = 0.005, p = 0.028). OPG level in fracture hematoma was higher compared to the unions serum level (p = 0.028). sRANKL was decreased in unions during the observation period. In non‐unions sRANKL and OPG levels showed a variable course, with no statistical significance. This is the first study to document the course of OPG and sRANKL in normal and delayed human fracture healing emphasizing its local and systemic involvement. We provide evidence of strongly enhanced OPG levels in patients with a long bone fracture compared to healthy controls. Further, levels of free sRANKL were decreased during regular fracture repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1557–1561, 2014.     

血液透析患者血管钙化、骨密度与骨保护素及其配体的相互关系

目的 研究维持性血液透析（MHD）患者血管钙化、骨密度与血清骨保护素（OPG）及其配体sRANKL的相互关系。 方法 采用酶联免疫吸附法测定血清OPG、sRANKL水平；X线平片检测腹主动脉、股动脉及桡动脉部位血管钙化，计算血管钙化积分；双能X线骨密度仪测定腰椎及股骨骨密度。对各参数的相互关系进行统计分析。 结果 （1）39例MHD患者中25例（64.1%）在不同部位有不同程度的血管钙化，其中轻度钙化16例（41.0%），中重度钙化9例（23.1%）。中重度钙化者血清OPG水平、OPG/sRANKL比值显著高于轻度钙化者[（342.50±171.53） ng/L比（206.21±137.88） ng/L，t = -2.253，P = 0.025；454.65±455.63比135.31±136.81，t = 59，P = 0.035]，而sRANKL水平差异无统计学意义[（0.10±0.08） pmol/L比(0.12±0.08) pmol/L，t = 0.534，P > 0.05]。多元线性回归分析显示 OPG/sRANKL是血管钙化评分的独立影响因素。（2）与骨量正常者比较，骨量异常者血清OPG水平升高[（249.05±137.66） ng/L比（226.67±170.12） ng/L]，sRANKL水平降低[（0.11±0.08） pmol/L比（0.12±0.02） pmol/L]，OPG/sRANKL比值升高（202.31±219.24比148.08±210.10），但差异均无统计学意义。多元线性回归分析显示OPG/sRANKL是腰椎T值的独立影响因素。（3）多元线性回归分析显示血管钙化评分是腰椎和股骨T值的独立影响因素。 结论 MHD患者血管钙化程度是腰椎及股骨骨密度的独立影响因素， OPG/sRANKL可能在血管钙化和骨密度的关系中起了纽带作用。     

Hyperleptinemia and its relation with peripheral C34(+)CD7(+) stem cells in renal transplant recipients

OBJECTIVES: Leptin, the Ob gene product, centrally regulates weight control. Transplant recipients are exposed to many factors affecting body mass. Leptin has been reported to activate the peripheral immune system. In this study, we evaluated serum leptin levels and factors contributing to hyperleptinemia; the relationship between serum leptin levels and lymphoid stem and mature cells; and their role in the rejection process in renal transplant recipients. MATERIALS AND METHODS: Sixty-three renal transplant recipients were included in the study. Patients were grouped according to serum leptin percentiles as hypoleptinemic (n=17), normoleptinemic (n=32), and hyperleptinemic (n=14). We determined serum leptin levels by radioimmunoassay and absolute number of CD34(+), CD7(+), CD34(+)CD7(+) lymphoid stem cells, CD4(+) and CD8(+) lymphocytes in peripheral blood by flow cytometry. RESULTS: The hyperleptinemic patients constituted 22.3% of the transplant patient. The mean peripheral blood CD34(+)CD7(+) lymphocyte count was significantly higher in hyperleptinemic patients than in normo- or hypoleptinemic patients (6.9, 6.1, and 44.3 cells/mm(3), respectively, P<0.05). There were no significant differences in the mean CD34(+), CD7(+), CD8(+), and CD4(+) lymphocyte count and CD4/CD8 ratio among the groups with respect to serum leptin levels. CD34(+)CD7(+) lymphocyte count was positively correlated with serum leptin levels (r=0.416, P<0.05). CONCLUSIONS: Hyperleptinemia is not rare during the posttransplant period. Our data support the results of previous experimental studies that have demonstrated the effect of the leptin hormone on lymphoid stem cells. The central and peripheral effects of leptin may differ on lymphoid stem cells and a serum threshold level may apply for the central effects.     

Osteoclast formation and bone resorption are inhibited by megakaryocytes

It has been previously reported that addition of megakaryocytes (MKs) to osteoblasts in vitro results in increased osteoblastic collagen and osteoprotegerin (OPG) production, suggesting a role for MKs in bone formation. To further investigate this role, we have studied the effects of MKs on osteoclast formation and activity. Human osteoclasts were generated from CD14 monocytes isolated from peripheral blood and cultured in the presence of M-CSF and sRANKL on dentine and calcium phosphate substrates. MKs were generated from CD34+ cells isolated from either human peripheral blood or cord blood and cultured in liquid medium for 6 days, after which time maturing MKs (CD61-positive cells) were isolated and added to monocyte cultures. After 6 and 9 days of culture, the number of osteoclasts identified morphologically and by TRAP staining was counted. Cells were removed and the area of resorption was identified by von Kossa staining and quantitatively assessed by image analysis. The addition of MKs to osteoclast cultures at day 0 inhibited the number of osteoclasts formed 1.9-fold (p>0.003), whereas addition at 3 days had no effect on osteoclast number. The presence of MKs inhibited resorption 8.7-fold when co-cultured with osteoclasts from day 0 (p>0.004), but only by 3.1-fold when co-cultured from day 3 (p>0.01). In dose-response experiments, it was found that 1-10% of MKs added to monocyte cultures elicited the greatest inhibition of resorption. Similar osteoclast cultures were treated with CD61-negative cells (non-MKs) to confirm that the inhibition of osteoclast formation and activity was specifically due to MKs. Experiments with a cell-impermeable membrane indicated that both cell to cell contact and release of soluble factor(s) were involved in mediating these effects. These results show that MKs inhibit osteoclast formation and activity. The most pronounced effects were seen when MKs and osteoclasts were co-cultured from day 0, suggesting that MKs act primarily on osteoclast precursors.     

Interleukin-4 and interleukin-13: bidirectional effects on human osteoclast formation

Osteoclasts are cells that resorb bone; they derive from macrophage colony-stimulating factor (M-CSF)-dependent hematopoietic precursors in the presence of soluble activator of NFkappaB ligand (sRANKL). Because transforming growth factor (TGF)-beta, a macrophage deactivator, enhances osteoclast formation we hypothesized that interleukin (IL)-4 and IL-13, also macrophage deactivators, should exert a similar effect. However, IL-4 and IL-13 have been reported as suppressors of murine osteoclast formation. In contrast to the effect of these molecules on murine osteoclast formation, IL-4 and IL-13 were found to be powerful inducers of osteoclast formation and bone resorption when added to human peripheral blood mononuclear cell (PBMC) cultures for 4 days. This stimulatory effect was only observed in cultures containing nonadherent PBMCs. In contrast, both molecules significantly suppressed osteoclast formation in lymphocyte-depleted cultures. These data demonstrate that the cytokine milieu and/or state of cell activation determines how cells of the osteoclast precursor respond to IL-4 and IL-13.     

Regulation of synthesis of osteoprotegerin and soluble receptor activator of nuclear factor-κB ligand in normal human osteoblasts via the p38 mitogen-activated protein kinase pathway by the application of cyclic tensile strain

Mechanical stress is thought to play an important role in bone remodeling. However, the correlation between mechanical stress and bone remodeling is poorly understood. In this context, using a model of cyclic tensile strain (CTS) toward human osteoblasts, synthesis of osteoprotegerin (OPG) and soluble receptor activator of nuclear factor-κB ligand (sRANKL), and the activation of mitogen-activated protein kinases (MAPKs) were examined. The application of 7%, 0.25-Hz CTS once a day for 4 h for 3 successive days simultaneously caused an increase of OPG synthesis and a decrease of sRANKL release and RANKL mRNA expression in osteoblasts. As for MAPKs activation in osteoblasts with the application of CTS, p38 MAPK was activated 10–20 min after the application of CTS, but extracellular signal-regulated kinase (ERK1/2) and c-Jun NH₂-terminal kinase (JNK) were not activated by such application. Furthermore, when CTS was applied once a day for 4 h for 1, 2, or 3 successive days to osteoblasts, p38 MAPK activation was maintained during the 3-day period but ERK1/2 activation was downregulated from day to day, simultaneously. Then, when CTS was applied once a day for 4 h for 3 successive days to osteoblasts pretreated with the p38 MAPK inhibitor SB203580 for 1 h, OPG synthesis was dose-dependently suppressed and inhibition of sRANKL release and RANKL mRNA expression was abrogated. These results indicate that biological responses of OPG and sRANKL synthesis in osteoblasts to the application of CTS are regulated via the p38 MAPK pathway and suggest that CTS might modulate and regulate bone metabolism.     

Metabolic activity of osteoblasts retrieved from osteoarthritic patients after stimulation with mediators involved in periprosthetic loosening

OBJECTIVE: To compare the effects of polymethylmetacrylate (PMMA), interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) on osteoarthritic (OA) human osteoblasts (Ob). METHODS: Ob cell cultures were prepared from metaphyseal trabecular bone of OA patients (n=9). Their cellular metabolic activity was characterized by measuring alkaline phosphatase (ALPase), osteocalcin (Oc), osteoprotegerin (OPG), prostaglandin E2 (PGE2), IL-6, macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of NF-kappaB ligand (sRANKL) in supernatants after Ob stimulation with PMMA, IL-1beta, IL-6 or TNF-alpha. RESULTS: IL-6 and PGE2 production by Ob was significantly increased by all mediators used. PMMA and TNF-alpha both stimulated M-CSF and sRANKL production whereas PMMA decreased and TNF-alpha augmented OPG release by Ob. The ratio sRANKL/OPG was significantly increased with PMMA and TNF-alpha, and treatment with anti-IL-6 antibodies did not alter this ratio. ALPase levels remained constant while only PMMA affected Oc production. Subclassification of patients into high or low endogenous producers of IL-6 and PGE2 before stimulation of Ob indicated that the two subgroups maintained similar responses to stimulation by all mediators. CONCLUSION: PMMA and TNF-alpha have comparable dual action on Ob, lowering their anabolic capacity and favoring production of bone resorption activators, an action not mediated by IL-6. This study confirms the important role of PMMA in implant loosening. However, the pertinence of high and low metabolic activities in OA Ob remains unclear, but could be involved in the pathophysiology of bone disease and aseptic loosening.     

Serum osteoprotegerin and receptor activator of nuclear factor-κB ligand (RANKL) concentrations in allogeneic stem cell transplant-recipients: a role in bone loss?

Purpose: Osteoporosis is a long-term complication of allogeneic stem cell transplantation (SCT). Receptor activator of nuclear factor-κB ligand (RANKL) increases osteoclast activity, while osteoprotegerin (OPG) neutralizes RANKL. A deficiency of OPG or an excess of RANKL may contribute to post-SCT bone loss. Methods: Serum OPG and soluble RANKL (sRANKL) concentrations were determined in 30 patients who received calcium, vitamin D and sex steroids – with or without pamidronate – prior to SCT and 1, 3, 6, and 12 months post-SCT and compared to those in healthy controls. Results: Despite all treatments patients lost bone at the hip. At baseline, serum OPG was similar in patients and controls; in the two patient groups it increased by 26–27% at 6 months post-SCT (p=0.002–0.028) and over the control level (p=0.002). Serum sRANKL concentrations were also similar in patients and controls at baseline. In those patients receiving pamidronate sRANKL concentrations decreased by 42% (p=0.0007) at 3 months post-SCT. The findings on the effect of SCT on OPG and sRANKL serum levels were ascertained in 28 additional patients who did not receive pamidronate, at a median of 122 days after SCT. In this latter group, OPG but not sRANKL concentrations were clearly elevated (p<0.001) in comparison to healthy controls. In conclusion, the present study fails to support the view that an excess of sRANKL or a deficiency of OPG would have a substantial impact on bone loss in SCT-recipients. Conclusion: Serum sRANKL concentrations may be modulated by bisphosphonates.     

OPG and sRANKL Serum Concentrations in Osteopenic,Postmenopausal Women After 2‐Year Genistein Administration

Introduction : RANKL and its decoy receptor osteoprotegerin (OPG) constitute a complex physiological mediator system involved in the regulation of bone resorption and may be responsible for the homeostatic mechanism of normal bone remodeling. Genistein, an isoflavone representing 1–5% of total phytoestrogen content in soybean products, may positively regulate cellular bone metabolism, but its mechanism of action on bone is not yet fully understood. Materials and Methods : We studied the serum levels of both soluble RANKL (sRANKL) and OPG and the sRANKL/OPG ratio in 389 postmenopausal women (age, 49–67 yr) with a femoral neck BMD <0.795 g/cm² and no significant comorbid conditions after 24‐mo therapy with genistein, (n = 198; 54 mg/d) or placebo (n = 191). Both intervention and placebo contained calcium and vitamin D₃. All patients received dietary instruction in an isocaloric fat‐reduced diet. Results : In comparison with placebo, sRANKL level was lower (p < 0.001 versus placebo) and OPG higher in genistein recipients (p < 0.001 versus placebo) at 1 and 2 yr, respectively. Moreover, at the end of 24 mo, genistein produced a significant reduction in the sRANKL/OPG ratio compared with placebo (genistein = ?0.021, 95% CI, ?0.020 to ?0.022; placebo = +0.004, 95% CI, 0.003–0.005; difference = ?0.020, 95% CI, ?0.015 to ?0.025, p < 0.001). Conclusions : Our findings suggest that genistein plus calcium and vitamin D₃ as part of a healthy diet is able to positively modulate bone turnover in a cohort of osteopenic, postmenopausal women and improve sRANKL‐OPG balance after 24 mo of treatment.     

Osteoporosis in hemodialysis patients revisited by bone histomorphometry: a new insight into an old problem

Osteoporosis in hemodialysis patients is associated with high morbidity and mortality and, although extensively studied by noninvasive methods, has never been assessed through bone biopsy. The aim of this study was to use histomorphometry to evaluate osteoporosis and identify factors related to its development in hemodialysis patients. We conducted a cross-sectional study involving 98 patients (35 women and 63 men; mean age: 48.4 +/- 13 years) on hemodialysis for 36.9 +/- 24.7 months. Patients were submitted to transiliac bone biopsy with double tetracycline labeling. The bone metabolism factors ionized calcium, phosphorus, bone alkaline phosphatase, deoxypyridinoline, intact parathyroid hormone, and 25(OH) vitamin D were evaluated, as were the bone remodeling cytokines osteoprotegerin (OPG), soluble receptor-activator of NF-kappabeta ligand (sRANKL) and tumor necrosis factor-alpha (TNF)alpha. Osteoporosis was defined as trabecular bone volume (BV/TV) greater than 1 s.d. below normal (men <17.4%; women <14.7%). Forty-five patients (46%) presented osteoporosis, which was correlated with white race. We found BV/TV to correlate with age, OPG/sRANKL ratio, TNFalpha levels, and length of amenorrhea. In multiple regression analysis adjusted for sex and age, length of amenorrhea, white race, and OPG/sRANKL ratio were independent determinants of BV/TV. Histomorphometric analysis demonstrated that osteoporotic patients presented normal eroded surface and low bone formation rate (BFR/BS). Osteoporosis is prevalent in hemodialysis patients. Low BFR/BS could be involved in its development, even when bone resorption is normal. Cytokines may also play a role as may traditional risk factors such as advanced age, hypogonadism, and white race.     

Xenogeneic human anti-pig cytotoxicity mediated by activated natural killer cells

Abstract: Even if hyperacute rejection, which is mediated by human natural antibodies (nAb) and complement, could be prevented, xenoreactive human anti-pig cellular responses may lead to delayed and/or chronic xenograft rejection. Among the cell populations participating in such rejection, NK cells have been proposed as an important component. In this study we report the in vitro cytotoxic activity of natural killer (NK) cells obtained from healthy human donors against porcine target cells. Freshly isolated peripheral blood mononuclear cells (PBMC) and purified NK cells (CD16+/CD56+, CD3-, CD20-, CD33-) exhibited little or no cytotoxic activity when tested on porcine phytohemagglutinin (PHA)-stimulated lymphoblasts or bone marrow- or aortic-derived endothelial cell lines in the presence of serum-free medium. Killing was considerably higher in the presence of human decomplemented plasma, containing xenoreactive nAb, or purified Gal(α1,3)Gal-reactive antibodies, suggesting that antibody dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells is an important mechanism involved in xenogeneic cytotoxicity. After incubation of human PBMC for 6 days in the presence of irradiated xenogeneic porcine or allogeneic stimulator cells, or in the presence of exogenous interleukin 2 (IL-2), the cytotoxic activity of the bulk cultures as well as that of isolated NK cells (separated from stimulated bulk cultures) against xenogeneic targets increased considerably, and corresponded to an increased NK-specific lysis of K562 target cells. Cell surface staining and flow cytometry showed that CD16+/CD56+, CD3- NK cells composed ca. 25% of short-term (6 days) xenogeneic, allogeneic, or IL-2 stimulated bulk cultures. In summary, these data suggest that, in contrast to allogeneic cell- mediated killing, xenogeneic human anti-porcine cytotoxicity includes an important contribution from NK cells.     

Differential Bone Metabolism Between Postmenopausal Women With Osteoarthritis and Osteoporosis

A comparative study of bone metabolism between postmenopausal women with osteoarthritis and osteoporosis showed that differential levels of bone remodeling markers, leptin, free leptin index, and osteoprotegerin might partly contribute to the proposed inverse relationship in bone mass between postmenopausal women with osteoarthritis and osteoporosis. Introduction : Osteoarthritis (OA) and osteoporosis (OP) are two common disorders affecting the quality of life in the elderly. The association between OA and OP has always been debated. The objective of this study was to compare bone metabolism between postmenopausal women with OA and OP. Materials and Methods : A total of 120 postmenopausal women with OA and OP (n = 60, respectively) were included in this comparative study. Anthropometric parameters and BMD at the spine and the proximal femur were measured. Serum leptin, soluble leptin receptor (sLR), osteoprotegerin (OPG), and bone remodeling markers, including bone‐specific alkaline phosphatase (BALP), osteocalcin (OC), deoxypyridinoline cross‐links (DPD), and cross‐linked N‐telopeptides of type I collagen (NTX), were quantified with commercial ELISA or EIA kits. Free leptin index (FLI) was also calculated by the ratio between serum leptin and sLR levels. Results : Postmenopausal women with OA had higher body weight, body mass index, fat mass, and percentage of fat than those suffered from OP. Compared with the patients in OP group, the patients in OA group had significantly higher BMD values at all sites measured. Higher serum leptin and FLI and lower OPG levels were shown in the OA group (leptin: 31.22 ± 6.4 versus 26.50 ± 9.27 ng/ml, p < 0.001; FLI: 3.20 ± 1.02 versus 2.50 ± 0.95, p < 0.05; OPG: 4.75 ± 1.97 versus 6.96 ± 2.75 pM, p < 0.001), whereas lower serum OC and higher urine DPD were noted in the OP group (OC: 16.45 ± 8.45 versus 13.06 ± 6.25 ng/ml, p < 0.05; DPD: 10.83 ± 7.12 versus 15.29 ± 6.65 nM BCE/mM Cr, p < 0.001). Serum OPG levels negatively correlated with BMD at all sites assessed. However, no correlation was found between leptin and BMD. Only in the OA group di positive correlations exist between FLI and Z‐score at the femoral neck and Ward's triangle region. After stepwise regression analysis, it was found that differential factors were able to predict the variance of BMD at different sites to a certain extent. Conclusions : Our study suggests that there are significant differences in bone metabolism between postmenopausal women with OA and OP and provides evidence for the inverse relationship between OA and OP. Differential levels of bone remodeling markers, leptin, FLI, and OPG may partly contribute to the proposed inverse relationship. Roles of leptin and its soluble receptor in bone metabolism regulation should be explored further.     

Megakaryocytes modulate osteoblast synthesis of type-l collagen, osteoprotegerin, and RANKL

We have previously reported evidence that megakaryocytes may play a role in bone remodeling, possibly by interactions with cells at the bone surface. To investigate the direct effects of megakaryocytes on osteoblasts, maturing megakaryocytes (CD61 positive cells) were isolated and added to cultures of human osteoblasts. Osteoblasts alone and osteoblasts treated with CD61-negative (non-megakaryocytic) cells were used as control cultures. After 48 h in culture, megakaryocytes were removed and osteoblasts immunolocalized for type-1 collagen, osteoprotegerin (OPG), and RANKL expression. Similar cultures were used for RNA extraction with mRNA for Col 1A1, OPG, and RANKL in osteoblasts measured quantitatively by RT-PCR. Osteoblasts cultured alone showed high levels of expression of collagen with 74% (+/-7) of cells staining positively. When cultured with megakaryocytes, the number of positively staining cells remained similar but the intensity of expression was increased 1.54-fold (P < 0.02). OPG was expressed by 32% (+/-6.3) of osteoblasts increasing to 51% (+/-5.5) when cultured in the presence of megakaryocytes (P < 0.01) with a 1.63-fold increase in intensity of expression (P < 0.01). In contrast, osteoblasts cultured with megakaryocytes showed suppression of RANKL expression; 35.6% (+/-5.8) of osteoblasts cultured alone stained positively decreasing to 24.3% (+/-5.3) with a 1.6-fold diminished intensity of expression (P < 0.02). Osteoblasts co-cultured with CD61-negative cells showed no differences in collagen, OPG, or RANKL expression levels compared to osteoblasts cultured alone. mRNA data supported these findings with a 3.1-fold increase in Col 1A1 expression in megakaryocyte-treated cultures compared to controls (P < 0.02). Low-level OPG mRNA expression increased 8.14-fold in osteoblasts cultured in the presence of megakaryocytes (P < 0.01), while RANKL expression was suppressed 3.3-fold (P < 0.02). These results demonstrate that in vitro, megakaryocytes have direct effects on osteoblastic production of factors affecting both bone formation and resorption. These data provide further evidence that megakaryocytes may play an important role in bone remodeling.