Diagnosis and treatment of Paget's disease of bone: position paper from the Italian Society of Osteoporosis, Mineral Metabolism and Skeletal Diseases (SIOMMMS).

INTRODUCTION: Paget's disease of bone is a focal skeletal disorder causing bone deformities and impairing bone quality. Despite the prevalence of asymptomatic cases is increasing, the progression of the disease can lead to invalidating complications that compromise the quality of life. Doubts on clinical and therapeutic management aspects exist, although beneficial effects of antiresorptive drugs, particularly bisphosphonates are known. However, limited information is available from randomized controlled trials on the prevention of disease complications so that somewhat contrasting positions about treatment indications between expert panels from the main scientific societies of metabolic bone diseases exist. This task force, composed by expert representatives appointed by the Italian Society of Osteoporosis, Mineral Metabolism and Skeletal Diseases and members of the Italian Association of Paget's disease of bone, felt the necessity for more specific and up to date indications for an early diagnosis and clinical management. METHODS: Through selected key questions, we propose evidence-based recommendations for the diagnosis and treatment of the disease. In the lack of good evidence to support clear recommendations, available information from the literature together with expert opinion of the panel was used to provide suggestions for the clinical practice. RESULTS AND CONCLUSION: Description of the evidence quality and support of the strength of the statements was provided on each of the selected key questions. The diagnosis of PDB should be mainly based on symptoms and the typical biochemical and radiological features. While treatment is mandatory to all the symptomatic cases at diagnosis, less evidence is available on treatment indications in asymptomatic as well as in previously treated patients in the presence of biochemical recurrence. However, given the safety and long-term efficacy of potent intravenous bisphosphonates such as zoledronate, a suggestion to treat most if not all cases at the time of diagnosis was released.

Radiofrequency echographic multispectrometry compared with dual X-ray absorptiometry for osteoporosis diagnosis on lumbar spine and femoral neck.

An innovative, non-ionizing technique to diagnose osteoporosis on lumbar spine and femoral neck was evaluated through a multicenter study involving 1914 women. The proposed method showed significant agreement with reference gold standard method and, therefore, a potential for early osteoporosis diagnoses and possibly improved patient management. INTRODUCTION: To assess precision (i.e., short term intra-operator precision) and diagnostic accuracy of an innovative non-ionizing technique, REMS (Radiofrequency Echographic Multi Spectrometry), in comparison with the clinical gold standard reference DXA (dual X-ray absorptiometry), through an observational multicenter clinical study. METHODS: In a multicenter cross-sectional observational study, a total of 1914 postmenopausal women (51-70 years) underwent spinal (n = 1553) and/or femoral (n = 1637) DXA, according to their medical prescription, and echographic scan of the same anatomical sites performed with the REMS approach. All the medical reports (DXA and REMS) were carefully checked to identify possible errors that could have caused inaccurate measurements: erroneous REMS reports were excluded, whereas erroneous DXA reports were re-analyzed where possible and otherwise excluded before assessing REMS accuracy. REMS precision was independently assessed. RESULTS: In the spinal group, quality assessment on medical reports produced the exclusion of 280 patients because of REMS errors and 78 patients because of DXA errors, whereas 296 DXA reports were re-analyzed and corrected. Analogously, in the femoral group there were 205 exclusions for REMS errors, 59 exclusions for DXA errors, and 217 re-analyzed DXA reports. In the resulting dataset (n = 1195 for spine, n = 1373 for femur) REMS outcome showed a good agreement with DXA: the average difference in bone mineral density (BMD, bias ± 2SD) was -0.004 ± 0.088 g/cm2 for spine and - 0.006 ± 0.076 g/cm2 for femur. Linear regression showed also that the two methods were well correlated: standard error of the estimate (SEE) was 5.3% for spine and 5.8% for femur. REMS precision, expressed as RMS-CV, was 0.38% for spine and 0.32% for femur. CONCLUSIONS: The REMS approach can be used for non-ionizing osteoporosis diagnosis directly on lumbar spine and femoral neck with a good level of accuracy and precision. However, a more rigorous operator training is needed to limit the erroneous acquisitions and to ensure the full clinical practicability.

The epidemiology of osteoporosis in Italian postmenopausal women according to the National Bone Health Alliance (NBHA) diagnostic criteria: a multicenter cohort study.

PURPOSE: The study was aimed at evaluating the prevalence of osteoporosis, defined by BMD and the National Bone Health Alliance (NBHA) criteria, and the prevalence of clinical risk factors for fractures in Italian postmenopausal women. METHODS: This is a cross-sectional, multicenter, cohort study evaluating 3247 postmenopausal women aged ≥ 50 and older in different areas of Italy in the period 2012-2014. All the participants were evaluated as far as anthropometrics; questionnaires for FRAX® and DeFRA calculation were administered and bone mineral density was measured at lumbar spine, femoral neck and total hip by DXA. RESULTS: The prevalence of osteoporosis, as assessed by BMD and NBHA criteria was 36.6 and 57%, respectively. Mean ± SD values of FRAX® and DeFRA were: 10.2 ± 7.3 and 11 ± 9.4 for major fractures, and 3.3 ± 4.9 and 3.9 ± 5.9 for hip fractures, respectively. Among clinical risk factors for fracture, the presence of previous fracture, particularly non-spine/non-hip fracture, parental history of hip fracture and current smoking were the most commonly observed. CONCLUSIONS: Our study showed that more that the half of postmenopausal women aged 50 and older in Italy has osteoporosis on the basis of the NBHA criteria. There is a relevant high risk of femur fracture, as assessed by the FRAX® and DeFRA and previous fracture, parental history of hip fracture and current smoking are the most common risk factors. The data should be considered particularly in relation to the need to increase prevention strategies on modifiable risk factors and therapeutic intervention.

Assessment of the 10-year risk of fracture in Italian postmenopausal women using FRAX®: a north Italian multicenter study.

The aim of the study was to estimate the absolute risk of fracture in a sample of postmenopausal women with the Italian version of FRAX®, using femoral neck bone mineral density (BMD) and 3 internationally validated clinical risk factors (CRFs) (history of fragility fracture, family history of hip fracture, current smoking). We retrospectively studied 9586 women (mean age 64.1 yr) examined in three osteoporosis centers from Northern Italy over two years (2001-2002). The risk of major osteoporotic (clinical spine, hip, forearm and humerus) and hip fractures was estimated using the online version of the FRAX algorithm adapted for Italy. The median 10-year risk was 7.5% for osteoporotic fracture and 1.7% for hip fracture. 25% of subjects had a 10-year risk ≥ 12.1% for osteoporotic fracture and ≥ 4.1% for hip fracture. The median 10-year risk of fracture increased with the number of prevalent CRFs. For major osteoporotic fractures risk rose from 6.3% to 10.9%, 21.4% and 40.9% with 1, 2 and 3 prevalent CRFs, respectively. For hip fractures the corresponding figures were: 1.3%, 2.7%, 7.0% and 21.9%, respectively. However, it must be emphasized that in 2 out of 3 women, none of the CRFs examined was present and the assessment of risk was limited to age and BMD. Our data provide the first description of the effect of the combination of BMD, age and CRFs on fracture risk stratification in a large sample of Italian postmenopausal women using FRAX®. The results are a useful starting point to define criteria for the application of FRAX® in clinical practice in Italy.

Validation and further development of the WHO 10-year fracture risk assessment tool in Italian postmenopausal women: project rationale and description.

The threshold for pharmacological intervention for osteoporosis remains controversial. Tools predicting the future risk of new fractures are increasingly used to establish a convenient individual risk/benefit ratio for a long term treatment. FRAX® is likely to become the most widely used tool for assessing fracture risk also for the WHO endorsement. The inevitable limitations will not hamper its value. As for any tool like this a continuous process of validation and further development is highly warranted. The predictive and clinical value of FRAX® has to be tested in individual countries by exploring also the inclusion of additional specific relatively uncommon risk factors. The DeFRA project is intended to validate in a large cohort of postmenopausal women a new algorithm derived from FRAX®. Both, the coefficients of continuous variable and the gradients for clinical risk factors should not be considered as conclusive for the routine clinical use. The new tool will be offered for the routine clinical use only at the completion of the DeFRA project, requiring the prospective collection of at least 60.000 patient-years. Here we report the rational and the design of the project.

Definition of a population-specific DXA reference standard in Italian women: the Densitometric Italian Normative Study (DINS).

Osteoporosis is currently defined on the basis of the T-score by dual-energy X-ray absorptiometry (DXA). Despite its limitations, this definition is applied worldwide. However, the normal values provided by manufacturers may not be fully representative of specific local populations. So far, there are no normative data in the Italian population using Hologic densitometers. The Densitometric Italian Normative Study (DINS) is an ongoing multi-center study that aims to establish reference values for bone densitometry with dual-energy X-ray absorptiometry (DXA) in the male and female Italian population. In this paper we report the results of the lumbar vertebrae (L2-L4) and proximal femur in 1,622 women aged 20-79 years. Bone mineral density (BMD) was determined using dual-energy X-ray absorptiometry (DXA) on Hologic bone densitometers (Hologic, Waltham, Mass.). Most of the subjects were examined with a QDR 4500. The BMD of the lumbar vertebrae was virtually constant between 20 and 49 years (test for trend: P=0.66); the BMD values between 20-45 in premenopausal women (mean 1.036; SD 0.109 g/cm(2)) were thus defined as the peak bone mass values, significantly lower compared to the Hologic reference curve (mean 1.079, SD 0.11 g/cm(2)). The mean BMD values of the femoral neck were virtually identical to those of the NHANES study in the first 3 decades; after the age of 50 the BMD values were slightly greater than those of the NHANES subject. The subject classification according to the WHO criteria was similar using the DINS and NHANES reference values for the femur; for the spine, the Hologic reference values classified a larger proportion of women as osteoporotic (21 vs. 16%) or osteopenic (42 vs. 38%) compared to DINS.

Serum interleukin-6, soluble interleukin-6 receptor and soluble gp130 exhibit different patterns of age- and menopause-related changes.

Growing evidence suggests that interleukin-6 (IL-6) may play a pathogenetic role in postmenopausal bone loss and in other age-related pathological conditions. In this study, we have examined the age-related changes in the serum levels of IL-6 and the soluble receptors that modulate its biological activity--soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130)--in 220 women (from 25 to 104yr old), including 22 centenarians. Serum IL-6 rose exponentially with age (r=0.74, p<0.0001). The median level of IL-6 increased almost ten-fold with age, from 1.16pg/ml in premenopausal women to 10.27pg/ml in centenarians. Serum sIL-6R and sgp130 showed an increase until the seventh decade and a progressive decrease in older ages (r=0.39, p<0.0001 and r=0.26, p=0.008, respectively). IL-6, sIL-6R and sgp130 were significantly higher in women within 10yr of menopause as compared to premenopausal subjects (1.51 vs. 1.16pg/ml, p=0.012; 41.9 vs. 35.7ng/ml, p=0.002; and 253.4 vs. 230.7ng/ml, p=0.008, respectively). In postmenopausal women, a negative correlation was found between sIL-6R and the lumbar bone mineral density (BMD) (r=-0.28, p=0.002) even after adjusting for age and weight. Furthermore, sIL-6R levels were higher in osteoporotic compared to normal women (47.9 vs. 39.5ng/ml, p=0.001). In conclusion, our results show that the serum levels of IL-6, sIL-6R and sgp130 exhibit different patterns of age- and menopause-related changes, and that the biological activity of IL-6 may be increased with age with potential implications in the age-related diseases such as osteoporosis.

Serum concentrations of proinflammatory cytokines in Graves' disease: effect of treatment, thyroid function, ophthalmopathy and cigarette smoking.

OBJECTIVE: In the present study we have measured the concentrations of interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and IL-1 receptor antagonist (IL-1Ra) in the serum of patients with Graves' disease (GD). By multivariate analysis, we have evaluated the effect of antithyroid treatment, thyroid function, the presence or absence of active thyroid-associated ophthalmopathy (TAO), the patient's smoking habits and the relation to circulating anti-thyrotropin (TSH) receptor (TRAb) and anti-thyroperoxidase antibodies (TPOAb). SUBJECTS: We studied 84 GD patients, 51 untreated and 33 receiving methimazole (MMI) therapy. Twenty-three (45%) untreated patients and 18 (54%) patients on MMI had active TAO. We also studied 67 normal subjects as controls. Thirty-one GD patients (43%) and 16 controls (36%) were smokers. RESULTS: Serum IL-6 concentrations were significantly higher in both untreated patients (P<0.001) and treated patients (P<0.006), when compared with controls. Serum sIL-6R concentrations were significantly affected by treatment (P=0.001). Serum IL-1Ra concentrations were not different in GD patients, whether treated or untreated, compared with controls. Serum IL-6 concentrations were not influenced by thyroid function and there was a significant interaction between treatment and the presence of active TAO (P=0.003). In hyperthyroid patients with active TAO serum, sIL-6R concentrations were significantly higher than in those with inactive TAO (P=0.003). In untreated GD patients there was no significant effect of thyroid function and TAO activity on the serum concentrations of TNF-alpha and IL-1 beta. Serum IL-1Ra concentrations were not affected by the presence of TAO. Smoking had no effect on serum IL-6, sIL-6R, TNF-alpha, IL-1 beta and IL-1Ra concentrations, even in the presence of an active TAO. Serum concentrations of IL-6, sIL-6R, TNF-alpha and IL-1 beta and IL-1Ra were not different in patients with and without TRAb or TPOAb, in relation to either thyroid function, TAO activity or smoking. CONCLUSIONS: Our work shows that: (i) the proinflammatory cytokine pattern in GD is greatly influenced by antithyroid drug treatment; (ii) the increased circulating IL-6/sIL-6R concentrations observed in patients with active TAO may derive from the activation of humoral reactions in sites other than the thyroid; and, (iii) cigarette smoking has no effect on serum IL-1/IL-1Ra concentrations in TAO.

Ethanol and acetaldehyde inhibit the formation of early osteoblast progenitors in murine and human bone marrow cultures.

Alcohol abuse is commonly associated with reduced bone mass and osteoporosis as a consequence of both systemic and direct cellular effects. To clarify some of the pathways by which alcohol exerts its actions directly on bone cells, we investigated the formation of early osteoblast progenitors (colony-forming units for fibroblasts; CFU-F) in long-term murine and human bone marrow cultures exposed to ethanol and to its main metabolite, acetaldehyde. In murine bone marrow cultures, obtained from Swiss female mice, ethanol inhibited CFU-F formation (maximal reduction +/- SEM: 50 +/- 2%; p < 0.01) at concentrations ranging from 0.04% to 0.6% that are similar to those reached in vivo in alcoholics. Acetaldehyde strongly reduced CFU-F formation at concentrations of 0.004% and 0.02%, and completely abolished it at the dose of 0.06%. Similarly, ethanol (at concentrations > or =0.02%) and acetaldehyde (from 0.004% to 0.06%) significantly decreased the number of CFU-F in human bone marrow cultures; the mean reduction observed with ethanol was 63 +/- 12% (p < 0.05), whereas acetaldehyde completely prevented CFU-F formation at the concentration of 0.06%. These in vitro observations were confirmed by the in vivo findings that the CFU-F formation in bone marrow cultures from nine young, chronic, noncirrhotic alcoholics was significantly reduced (70 +/- 15%), compared with seven age-matched normal subjects (p < 0.01). In addition, acetaldehyde inhibited cell proliferation in human osteoblastic cells (MG-63 and HOBIT cell lines), whereas ethanol reduced proliferation only in MG-63 cells. Our results indicate that ethanol and acetaldehyde may directly inhibit the osteoblastogenic potential of the bone marrow, and this effect may contribute to the decreased bone formation observed in alcoholics.

Oestrogens prevent the increase of human serum soluble interleukin-6 receptor induced by ovariectomy in vivo and decrease its release in human osteoblastic cells in vitro.

OBJECTIVE: Interleukin-6 (IL-6) seems to be a key mediator of the increased bone loss that follows loss of ovarian function. Based on this and on evidence that oestrogen deficiency may also increase cell sensitivity to IL-6, we studied the effects of ovariectomy and of oestrogen replacement therapy on the serum levels of IL-6 and of soluble IL-6 receptor (sIL-6R) in vivo. DESIGN AND PATIENTS: Thirty-seven fertile women undergoing surgery for benign uterine diseases were divided into 3 groups and monitored for 12 months: hysterectomized women (n = 9), ovariectomized untreated women (n = 12) and ovariectomized women starting treatment with transdermal estradiol (E2, 50 microg/d) 1 month after surgery (n = 16). RESULTS: Hysterectomy alone caused no significant changes of sIL6R whereas serum levels of sIL-6R rose progressively after ovariectomy (mean +/- SEM: 31 +/- 9% and 38 +/- 7% over baseline, at 6 and 12 months, respectively; P < 0.01). Oestrogen replacement therapy prevented the increase of sIL6R over a 1-year period. A similar pattern was also found for serum IL-6 but the changes did not reach statistical significance. In ovariectomized (OVX) women there were significant correlations between serum sIL-6R levels and FSH (r = 0.59; P < 0. 01), oestradiol (r = - 0.43; P < 0.01), testosterone (r = - 0.41; P < 0.05), osteocalcin (r = 0.42; P < 0.05) and bone alkaline phosphatase (r = 0.44; P < 0.05). To examine whether oestrogen directly regulates sIL-6R secretion by bone cells, we studied in vitro the basal and phorbol ester (PMA) stimulated release of sIL-6R in a human osteoblastic cell-line (MG-63) and in a tumour-derived osteoclastic cell line (GCT-51). Osteoblastic (but not osteoclastic) cells spontaneously produced considerable amounts of sIL-6R and the protein kinase-C activator PMA (10-8 M) increased the release of sIL-6R by osteoblasts more than 3-fold. More strikingly, 17beta E2 (but not 17alpha) significantly inhibited both the spontaneous- and PMA-induced release of sIL-6R by osteoblastic cells (P < 0.05). CONCLUSIONS: These results indicate that oestrogen loss causes alterations of the IL-6 system, and that sIL-6R is under the direct inhibitory control of oestrogens both in vivo and in vitro.

[Bisphosphonates stimulate the production of basic fibroblast growth factor and the formation of bone marrow precursors of osteoblasts. New findings about their mechanism of action]. / I bisfosfonati stimolano la produzione di "basic fibroblast growth factor" e la formazione dei precursori midollari degli osteoblasti. Nuove acquisizioni sul meccanismo di azione.

BACKGROUND: It has been recently shown that bisphosphonates may affect bone resorption either directly on osteoclast activity or through the mediation of osteoblasts activity. In this experimental study the potential effects of etidronate and alendronate on osteoblastic bone formation are investigated. METHODS: The number of fibroblastic colony forming units (CFU-F) and mineralized nodules (CFU-OB) in murine and human bone marrow cultures, assessed. In addition, the basic-FGF production by human osteoblastic cells MG-63 measured. RESULTS: In murine bone marrow cultures etidronate and alendronate stimulate CFU-F formation with a mean increases vs control of 106 +/- 17% at 10(-5) M and 78 +/- 5% at 10(-7) M respectively (p < 0.001). The formation of bone nodules is inhibited by bisphosphonates at high concentrations (> 10(-6) M) and stimulated at lower concentrations (from 10(-7) M to 10(-9) M for etidronate and from 10(-7) M to 10(-10) M for alendronate; p < 0.001). Similarly, in human bone marrow cultures alendronate increases CFU-F formation with a maximal effect at 10(-10) M (+161 +/- 12% vs control; p < 0.01) and the formation of CFU-OB with a maximal effect at 10(-10) M (+133 +/- 34%; p < 0.001). Finally, etidronate (from 10(-9) to 10(-11) M) and alendronate (from 10(-9) to 10(-12) M) stimulate the b-FGF production by human osteoblastic cells (p < 0.01). CONCLUSIONS: In line with previous histomorphometric and clinical observations, the results obtained indicate that bisphosphonates directly affect osteoblastic cells with a positive effect on bone formation, probably via the stimulation of growth factors.

Bisphosphonates stimulate formation of osteoblast precursors and mineralized nodules in murine and human bone marrow cultures in vitro and promote early osteoblastogenesis in young and aged mice in vivo.

Recent in vitro findings suggest that bisphosphonates, potent inhibitors of osteoclastic bone resorption, may also have a direct action on osteoblasts. The purpose of this study was to search for potential effects of etidronate and alendronate on the formation of early and late osteoblastic cell precursors by measuring the number of colony-forming units for fibroblasts (CFU-F) and colony-forming units for osteoblasts (CFU-OB) in murine and human bone marrow cultures. In murine marrow cultures, etidronate (10(-5) to 10(-9) mol/L) significantly stimulated the formation of CFU-F with a maximal effect at 10(-5) mol/L (mean increase over control values+/-SD: 106+/-17%;p < 0.001), whereas alendronate had a biphasic effect, being stimulatory at concentrations below 10(-7) mol/L (78+/-5%; p < 0.001), and inhibitory at higher doses. The formation of CFU-OB was also inhibited by both bisphosphonates at the highest concentrations (10(-5) mol/L and 10(-6) mol/L), but it was significantly stimulated at lower concentrations (from 10(-7) to 10(-9) mol/L for etidronate and 10(-7) to 10(-10) mol/I, for alendronate; p < 0.001). In human bone marrow cultures, alendronate (10(-8) to 10-(12) mol/L) increased CFU-F formation with a maximal effect at 10(-10) mol/L (161+/-12 %; p < 0.01). CFU-OB formation, observed only in the presence of dexamethasone (10(-8) mol/L), was markedly stimulated by alendronate at the above concentrations with a maximal increase at 10(-10) mol/L (133+/-34%; p < 0.001). The in vivo short-term effects of bisphosphonates on the formation of early osteoblast precursors were also studied in bone marrow cultures from young female mice treated with weekly subcutaneous injections of etidronate (0.3, 3, and 30 mg/kg) or alendronate (0.3, 3, and 30 microg/kg) and from aging female mice treated with the two lowest doses of both drugs. After 1 month of treatment, etidronate (0.3 and 3 mg/kg) and alendronate (0.3 and 3 microg/kg) significantly increased the number of CFU-F colonies in the bone marrow from young and old animals, whereas the highest dose of both drugs had no effect in young mice. Our results, together with previously reported observations of bone-forming effects in osteoporosis, suggest that bisphosphonates may have, in vivo, a potentially relevant influence on cells of the osteoblastic lineage, distinct from their inhibitory action on osteoclasts.

Bisphosphonates inhibit IL-6 production by human osteoblast-like cells.

Since bisphosphonates prevent bone loss in osteoporosis and rheumatoid arthritis, diseases in which the osteoclastogenic and inflammatory cytokine interleukin-6 plays a pathophysiologic role, we studied whether these drugs regulate the production of this cytokine by osteoblasts. Spontaneous and IL-1 + TNF-alpha stimulated IL-6 release was measured in supernatants of cultures of human osteoblastic osteosarcoma cells MG-63, pretreated for 4 hours with different doses of etidronate, clodronate or alendronate using a specific bioassay. Etidronate [from 10(-4) to 10(-8) M] or alendronate [from 10(-6) to 10(-11) M] inhibited in a dose-dependent manner the cytokine-induced IL-6 secretion [60+/-9.5% at 10(-5) M and 65+/-12% at 10(-7) M, respectively; p < 0.01]. Though significant, the inhibitory effect of clodronate was less [35+/-7% at 10(-5) M, p < 0.05]. These in vitro observations might have in vivo relevance in explaining at least in part the mechanisms by which bisphosphonates inhibit systemic and periarticular bone resorption.

The possible role of hematin and Mössbauer effect in the inhibition of osteosarcoma cell proliferation.

Hematin, like many hematoporphyrines and porphyrin derivatives, shows a higher affinity and preferential localization for tumor cells. These properties are particularly interesting in view of their possible applications in tumor treatment. The aim of the present work was to test the capability of porphyrines and resonant low energy gamma rays to produce some kind of selective inhibition of tumor cell proliferation. We investigated the role of hematin to potentiate the killing capability of 14.4 keV resonant gamma rays from a 57Co Mössbauer source. Human osteosarcoma cell cultures "MG-63" were incubated in the presence or absence of hematin (10(-4) to 10(-5) M) for 24 hours. They were successively irradiated for 4 hours with 14.4 keV gamma rays from a 3.7 GBq 57Co source. The combined effects of hematin and resonant gamma radiation were then examined and tested statistically for significance. Different degrees of growth inhibition were observed when hematin alone, radiation alone, and hematin plus gamma rays were administered to the cultures. Hematin, at the concentrations of 10(-4) M is capable of inhibiting tumor cells growth. While no significant effect is attributable to irradiation alone, hematin plus irradiation show a larger inhibition than that expected for purely additive effects.

Regulation of the gp80 and gp130 subunits of the IL-6 receptor by sex steroids in the murine bone marrow.

Both estrogen and androgen exert their antiosteoporotic effects, at least in part, by inhibiting IL-6 production, thereby suppressing osteoclastogenesis. Several observations, however, suggest that besides increased IL-6 production, sensitivity of the osteoclastogenic process to this cytokine is altered after ovariectomy. Based on this and evidence that the ligand-binding subunit of the IL-6 receptor (gp80) is a limiting factor for the actions of IL-6 on bone, we hypothesized that sex steroids regulate expression of the IL-6 receptor as well. We report that 17beta-estradiol or dihydrotestosterone in vitro decreased the abundance of the gp80 mRNA as well as the mRNA of the signal-transducing subunit of the IL-6 receptor (gp130) in cells of the bone marrow stromal/osteoblastic lineage, and also decreased gp130 protein levels. These effects did not require new protein synthesis. In contrast to sex steroids, parathyroid hormone stimulated gp130 expression; this effect was opposed by sex steroids. Consistent with these findings, ovariectomy in mice caused an increase in expression of gp80, gp130, and IL-6 mRNAs in ex vivo bone marrow cell cultures as determined by quantitative reverse transcription (RT)-PCR, and confirmed on an individual cell basis using in situ RT-PCR. The demonstration of increased expression of the IL-6 receptor after loss of sex steroids provides an explanation for why IL-6 is important for skeletal homeostasis in the sex steroid-deficient, but not replete, state.

Clinical observations with a new specific assay for bone alkaline phosphatase: a cross-sectional study in osteoporotic and pagetic subjects and a longitudinal evaluation of the response to ovariectomy, estrogens, and bisphosphonates.

The purpose of this study was to examine the serum levels of bone alkaline phosphatase (BALP) measured with a new assay in normal and in osteoporotic women, and to evaluate prospectively its responsiveness to changes of bone metabolism. The following groups of subjects were studied: (1) 95 healthy women (44-75 years) (22 pre- and 73 postmenopausal) and 35 osteoporotic women [vertebral bone mineral density (BMD) more than 2.5 SD below the normal adult mean]; (2) 10 women (44-50 years) ovariectomized (OVX) for benign uterine diseases, examined before and 12 months after surgery; (3) 16 OVX women (36-54 years), examined before and after 12 months of transdermal estrogen replacement therapy (50 microg/day); (4) 12 previously untreated pagetic patients (4 women and 8 men, 50-80 years), examined before and 3 months after the I.V. administration of clodronate (600 mg) or alendronate (5 mg) for 2 consecutive days. The median BALP value was 11.6 U/liter (25-75th percentiles: 10.5-12. 7; range 7.7-19.3) in healthy premenopausal (PreMP) women and significantly higher (median: 16.8 U/liter; 25-75th percentile: 13. 8-21.8; P < 0.01) in postmenopausal (PostMP) women. There was a clear age-related increase in normal subjects (r = 0.43; P < 0.001). In the osteoporotic group, BALP levels, as well as other biochemical parameters of bone turnover, were not significantly different from those of normal women when adjusted for age. In OVX women, BALP levels showed a marked increase 12 months after surgery (median: 113%; 25-75th percentile: 87-139%), significantly higher than the increase of total ALP (median: 43%; 25-75th percentile: 25-66%; P < 0.001), and similar to the increases of serum osteocalcin and urinary hydroxyproline. Transdermal estrogen treatment prevented the BALP increase, even if no reduction was observed; total ALP showed a similar behavior. The basal levels of BALP were significantly elevated in pagetic patients (median: 91 U/liter; range 18-610 U/liter) and correlated to the scintigraphic extent of the disease (r = 0.76; P < 0.01). Three months after the I.V. administration of bisphosphonates, the decrease of BALP was more marked than that of total ALP (median: -54% versus -41%; P < 0.05). In conclusion, these results suggest that BALP measurement with this immunoassay may be clinically useful, and more sensitive than total ALP, in the assessment of bone turnover during changes of the estrogen status as well as in monitoring the effects of treatments that modify the metabolic activity of the skeleton.

Lack of increased serum interleukin-6 and soluble IL-6 receptor concentrations in patients with thyroid diseases following recombinant human interferon alpha therapy.

BACKGROUND: Serum interleukin-6 (IL-6) concentrations are frequently elevated in inflammatory thyroid diseases, such as subacute thyroiditis and amiodarone induced thyroiditis. We and others have recently observed that recombinant interferon-alpha (rIFN-alpha) therapy for chronic, active viral hepatitis and malignant disorders may induce thyroid dysfunction, including thyrotoxicosis secondary to thyroiditis. Serum IL-6 and its soluble receptor (sIL-6R) have been measured for the first time in patients with chronic active hepatitis receiving rIFN-alpha therapy. METHODS: Studies were carried out in 37 patients treated with rIFN-alpha for chronic, active viral hepatitis. Thyroid function tests and serum IL-6 and sIL-6R were measured before and during rIFN-alpha therapy. RESULTS: Six patients developed inflammatory or destructive thyrotoxicosis confirmed by elevated serum free T4 or free T3 concentrations, suppressed serum thyroid-stimulating hormone (TSH) values, and a low thyroid radioactive iodine uptake. Serum IL-6 and sIL-6R concentrations were not elevated in these patients with rIFN-alpha-induced thyroiditis. CONCLUSIONS: These results suggest that serum IL-6 concentrations are not useful in differentiating between inflammatory thyrotoxicosis and hyperthyroidism induced by rIFN-alpha therapy as is the case in amiodarone-induced thyrotoxicosis. It is possible that rIFN-alpha therapy could be associated with an inhibitory effect of rIFN-alpha on the release of IL-6 from damaged thyroid cells and not on the basal secretion of IL-6.

Increased serum concentrations of interleukin-6 (IL-6) and soluble IL-6 receptor in patients with Graves' disease.

Increased serum interleukin-6 (IL-6) concentrations have been reported in patients with thyroid destructive processes. In the present study we measured IL-6 and soluble IL-6 receptor (sIL-6R) concentrations in the serum of normal subjects and patients with Graves' disease using a high sensitivity sandwich enzyme-linked immunoassay. We found increased serum IL-6 and sIL-6R concentrations (69.3 fmol/L, and 964 pmol/L, respectively) in 49 hyperthyroid patients with Graves' disease (GD) compared to those in controls [55.8 fmol/L (P = 0.019) and 772 pmol/L (P = 0.007), respectively]. In 31 newly diagnosed GD patients, serum concentrations of IL-6 and sIL-6R during the hyperthyroid phase were elevated, and after therapy with methimazole only, serum sIL-6R concentrations returned to normal (940 vs. 726 pmol/L; P < 0.001) but serum IL-6 did not. Serum sIL-6R concentrations (mean +/- 2 SD) were higher in GD patients with active inflammatory thyroid-associated ophthalmopathy than those in patients with inactive or absent thyroid-associated ophthalmopathy (P < 0.05). In conclusion, we have demonstrated activation of the IL-6 system in GD and, for the first time, have measured and found increased serum sIL-6R concentrations in hyperthyroid GD patients.

[The physiopathology of osteoporosis: the role of local factors]. / Fisiopatologia dell'osteoporosi: ruolo dei fattori locali.

Bone remodelling continues throughout life and depends on two processes that are tightly coupled: resorption of old bone by osteoclasts and subsequent new bone formation by osteoblasts. Evidence accumulated during the last few years has clearly indicated that bone marrow microenvironment plays an essential role in regulating bone remodelling. Indeed major advances in our understanding of the ontogeny of osteoclasts and osteoblasts indicate that both cell types derive from progenitors present in the marrow, and that systemic and local factors regulate their development and the coupling of their function. In particular, a network of cytokines and growth factors is essential for the regulation of both osteoclastogenesis and osteoblast formation: these factors play a pivotal role in the paracrine regulatory control of bone turnover under physiological conditions. Among this array of local factors, IL-6, IL-1, TNF, GM-CSF, cytokines with osteoclastogenic and bone resorptive properties, have been implicated in the pathophysiology of osteoporosis since their production is increased in vivo in humans and animals with estrogen deficiency. These findings provide emerging insights into the pathophysiology of osteoporosis and deserve further investigation.