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A hydroxytyrosol (HTyr)-enriched fraction containing HTyr 6% w/w, derived from Olea europaea L. byproducts and obtained using an environmentally and economically sustainable technology, was lipophilized under green chemistry conditions. The effects of three fractions containing hydroxytyrosyl butanoate, octanoate, and oleate, named, respectively, lipophilic fractions 5, 6, and 7, and unreacted HTyr on the human colon cancer cell line HCT8-β8 engineered to overexpress estrogen receptor β (ERβ) were evaluated and compared to those of pure HTyr. The experimental data demonstrated that HTyr and all fractions showed an antiproliferative effect, as had been observed by the evaluation of the cellular doubling time under these different conditions (mean control, 32 ± 4 h; HTyr 1, 65 ± 9 h; fraction 5, 64 ± 11 h; fraction 6, 62 ± 14 h; fraction 7, 133 ± 30 h). As evidenced, fraction 7 containing hydroxytyrosyl oleate showed the highest activity. These results were related to the link with ER-β, which was assessed through simultaneous treatment with an inhibitor of ERβ.

Optimal peak bone mass and bone health later in life are favored by a sufficient calcium intake in infancy, childhood and adolescence. The purpose of this study was to test a new educational program created to monitor and to improve calcium and vitamin D intake in children. Nutritional habits in children were evaluated through a food frequency questionnaire (FFQ) to assess the intake of calcium, vitamin D, dairy products, and total caloric energy at baseline and after seven months of exposure to a unique educational program applied between November 2013 and May 2014 in 176 schoolchildren (48% male, 52% female) attending the fourth and fifth grades of two selected primary schools in Florence, Italy. A significant increase of calcium (from 870 ± 190 to 1100 ± 200 mg/day, p &lt, 0.05), and vitamin D (from 3.6 ± 1.53 to 4.1 ± 2 µg/day) intake in children was documented after the educational program. The amount of specific foods important for bone health consumed, such as milk and vegetables, increased significantly, both in male and female children (p &lt, 0.05). The proposed educational program appears to be effective in modifying calcium intake in children, with a significant increase in the consumption of dairy products and vegetables, but without a significant change in the total caloric intake.

The Mediterranean diet is associated with a lower incidence of atherosclerosis, cardiovascular diseases, and some types of cancer. Recent interest has been focused on the biological activity of phenolic compounds present in extra virgin olive oils (EVOOs). Both in vivo and in vitro studies have shown that EVOO components have positive effects on metabolic parameters, such as plasma lipoproteins, oxidative damage, inflammatory markers, platelet function, and antimicrobial activity. We have investigated the possible interactions between 2 extracts of extra virgin olive oil and estrogen receptor β (ERβ) in an in vitro model of colon cancer. The qualification and quantification of the components of the 2 samples tested showed that phenolic compounds-hydroxytyrosol, secoiridoids, and lignans-are the major represented compounds. EVOO extracts were tested on a colon cancer cell line engineered to overexpress ERβ (HCT8-β8). By using custom made Oligo microarray, gene expression profiles of colon cancer cells challenged with EVOO-T extracts when compared with those of cells exposed to 17β-estradiol (17β-E2). This study demonstrated that the EVOO extracts tested showed an antiproliferative effect on colon cancer cells through the interaction with estrogen-dependent signals involved in tumor cell growth. Specifically, the ability of EVOO extracts to inhibit cell proliferation was superimposable to the activation of the ERβ receptor, similar to what was observed after 17β-E2 challenge.

The lack of a continuous cell line of epithelial parathyroid cells able to produce parathyroid hormone (PTH) has hampered the studies on in vitro evaluation of the mechanisms involved in the control of parathyroid cell function and proliferation. The PT-r cell line was first established from rat parathyroid tissue in 1987, but these cells were known to express the parathyroid hormone-related peptide (Pthrp) gene, but not the Pth gene. In an attempt to subclone the PT-r cell line, a rat parathyroid cell strain was isolated and named PTH-C1. During 3 years, in culture, PTH-C1 cells maintained an epithelioid morphology, displaying a diploid chromosome number, a doubling time around 15 h during the exponential phase of growth, and parathyroid functional features. PTH-C1 cell line produces PTH and expresses the calcium sensing receptor (Casr) gene and other genes known to be involved in parathyroid function. Most importantly, the PTH-C1 cells also exhibit an in vitro secretory response to calcium. Altogether these findings indicate the uniqueness of the PTH-C1 cell line as an in vitro model for cellular and molecular studies on parathyroid physiopathology.

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed "cellular spheres". Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

Background and aims: There is evidence that distinct genetic polymorphisms of LRP5 are associated with low Bone Mineral Density (BMD) and the risk of fracture. However, relationships between LRP5 polymorphisms and micro- and macroarchitectural bone characteristics assessed by pQCT have not been studied. The aim of the present study was to investigate the association of Ala1330Val and Val667Met polymorphisms in LRP5 gene with volumetric BMD (υBMD) and macro-architectural bone parameters in a population-based sample of men and women. Methods: We studied 959 participants of the InCHIANTI study (451 men and 508 women, age range: 21–94 yrs). Trabecular υBMD (υBMDt, mg/cm3), cortical υBMD (υBMDc, mg/cm3), cortical bone area (CBA, mm2) and cortical thickness (Ct.Th, mm) at the level of the tibia were assessed by peripheral quantitative computed tomography (pQCT). Ala1330Val and Val667Met genotypes were determined on genomic DNA by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: In age-adjusted analyses both LRP 1330-valine and LRP 667-metionin variants were associated with lower υBMDt in men (p

Background and objectives: Medullary sponge kidney (MSK) is a rare nephropathy characterized by cystic anomalies of precalyceal ducts, nephrocalcinosis, renal stones, and tubule dysfunctions. Its association with various malformations and cases of familial aggregation supports the conviction that genetic factors are involved, but no genetic studies have been conducted to date. It is hypothesized that MSK is due to a disruption at the “ureteric bud/metanephric blastema” interface caused by critical developmental genes functioning abnormally. Design, setting, participants, & measurements: Fifty-five apparently sporadic MSK patients were analyzed by direct DNA sequencing of all exons and exon-intron boundaries of glial cell-derived neurotrophic factor (GDNF) gene and rearranged during transfection (RET) gene, which have a leading role in renal development. Results: Two novel variants were found in heterozygosity in the MSK case population: GDNF{ENST00000344622}:c.−45G>C and c.−27+18G>A in a putative binding domain for paired-box 2 transcription factor. As a whole, eight patients showed these variations: four patients carried the c.[−45G>C; −27+18G>A] complex allele, and the others had the c.−27+18G>A alone. A case-control study revealed that these two alleles were significantly associated with MSK. Five of the eight cases were found to be familial, and the allele variants cosegregated with the disease in a seemingly dominant pattern of inheritance. Patients revealed no mutations in the RET gene. Conclusions: This is the first report identifying GDNF gene sequence variations in patients with MSK and suggesting a role for this gene in the pathogenesis of some cases of the disease.

It is now widely accepted that insulin-like growth factor-I (IGF-I) has a local regulatory role in bone remodeling. IGF-I has also been demonstrated to regulate proliferation of bone-derived endothelial cells. Such studies suggest a role of IGF-I in skeletal angiogenesis. Using BBE cells, a bovine bone endothelial cell line, we characterized the kinetics and chemical properties of IGF-I receptors and examined the effect of IGF-I on bone endothelium migration. Two classes of binding sites with high affinity for IGF-I were detected by binding experiments on bone endothelial cells. Both competition analyses and cross-linking studies revealed the presence of type I IGF receptor in bone endothelial cells. Moreover, these cells produced and released authentic IGF-I into the medium, as evidenced by radioimmunoassay analyses of gel-filtered conditioned media. Both IGF-I binding capacity and release decreased either with increases in cell number or after treatment with 17 beta-estradiol (17 beta E2) and parathyroid hormone (PTH). Both hormones also inhibited chemotactic responses of bone endothelial cells to IGF-I. Taken together, these results strongly suggest that IGF-I, a growth factor that promotes the proliferation of various bone cell types, also induces growth and chemotactic responses in bone endothelium acting through the type I IGF receptor. This may be part of a generalized response of bone cells to IGF-I that facilitates cell migration.

Protective effects of ipriflavone, an isoflavone derivative, in osteoporosis are believed to be caused by the inhibitory action on bone resorption. A direct effect of ipriflavone on bone formation is as yet unknown. Ipriflavone and four of its metabolites (I, II, III, and V) were examined for their effects on parathyroid hormone response, collagen synthesis, alkaline phosphatase activity, and cell proliferation in a clonal cell population of rat osteoblastic cells. Pretreatment of osteoblasts with high concentrations of ipriflavone for 48 h significantly inhibited the cAMP response to parathyroid hormone, producing a shift in the dose-response curve; at lower concentrations metabolites II and III potentiated the cAMP accumulation induced by low doses of parathyroid hormone. The 48 h treatment with metabolite V at the 1 nM dose significantly stimulated collagen synthesis in osteoblastic cells. Ipriflavone and metabolite I showed a biphasic stimulatory action on the alkaline phosphatase activity of osteoblasts, with a maximal effect at the 0.1 and 1 nM doses, respectively. A similar biphasic response was observed with ipriflavone and metabolite I on osteoblastic cell growth, with a maximal effect at the 0.1 nM concentration. These results suggest a direct role of ipriflavone in modulating the synthetic and growth properties of osteoblast-like cells.

Development of tools to be used forin vivobone tissue regeneration focuses on cellular models and differentiation processes. In searching for all the optimal sources, adipose tissue-derived mesenchymal stem cells (hADSCs or preadipocytes) are able to differentiate into osteoblasts with analogous characteristics to bone marrow mesenchymal stem cells, producing alkaline phosphatase (ALP), collagen, osteocalcin, and calcified nodules, mainly composed of hydroxyapatite (HA). The possibility to influence bone differentiation of stem cells encompasses local and systemic methods, including the use of drugs administered systemically. Among the latter, strontium ranelate (SR) represents an interesting compound, acting as an uncoupling factor that stimulates bone formation and inhibits bone resorption. The aim of our study was to evaluate thein vitroeffects of a wide range of strontium (Sr2+) concentrations on proliferation, ALP activity, and mineralization of a novel finite clonal hADSCs cell line, named PA20-h5. Sr2+promoted PA20-h5 cell proliferation while inducing the increase of ALP activity and gene expression as well as HA production duringin vitroosteoinduction. These findings indicate a role for Sr2+in supporting bone regeneration during the process of skeletal repair in general, and, more specifically, when cell therapies are applied.

Bone tissue engineering is an emerging field, representing one of the most exciting challenges for scientists and clinicians. The possibility of combining mesenchymal stem cells and scaffolds to create engineered tissues has brought attention to a large variety of biomaterials in combination with osteoprogenitor cells able to promote and regenerate bone tissue. Human adipose tissue is officially recognized as an easily accessible source of mesenchymal stem cells (AMSCs), a significant factor for use in tissue regenerative medicine. In this study, we analyze the behavior of a clonal finite cell line derived from human adipose tissue seeded on poly(ε-caprolactone) (PCL) film, prepared by solvent casting. PCL polymer is chosen for its good biocompatibility, biodegradability, and mechanical properties. We observe that AMSCs are able to adhere to the biomaterial and remain viable for the entire experimental period. Moreover, we show that the proliferation process and osteogenic activity of AMSCs are maintained on the biofilm, demonstrating that the selected biomaterial ensures cell colonization and the development of an extracellular mineralized matrix. The results of this study highlight that AMSCs and PCL film can be used as a suitable model to support regeneration of new bone for future tissue engineering strategies.

One of the most promising genetic approaches to dissecting a multifactorial disease is represented by genetically isolated population studies. We studied a genetic marker in a cohort of women living on the Mediterranean island of Lampedusa, a geographically isolated population. Lampedusa, located between the African coast and Sicily, consists of a young genetic isolate (

The calcium-sensing receptor (CaSR), a key player in the maintenance of calcium homeostasis, can influence bone modeling and remodeling by directly acting on bone cells, as demonstrated by in vivo and in vitro evidence. The modulation of CaSR signaling can play a role in bone anabolism.The calcium-sensing receptor (CaSR) is a key player in the maintenance of calcium homeostasis through the regulation of PTH secretion and calcium homeostasis, thus indirectly influencing bone metabolism. In addition to this role, in vitro and in vivo evidence points to direct effects of CaSR in bone modeling and remodeling. In addition, the activation of the CaSR is one of the anabolic mechanisms implicated in the action of strontium ranelate, to reduce fracture risk.This review is based upon the acquisition of data from a PubMed enquiry using the terms "calcium sensing receptor," "CaSR" AND "bone remodeling," "bone modeling," "bone turnover," "osteoblast," "osteoclast," "osteocyte," "chondrocyte," "bone marrow," "calcilytics," "calcimimetics," "strontium," "osteoporosis," "skeletal homeostasis," and "bone metabolism."A fully functional CaSR is expressed in osteoblasts and osteoclasts, so that these cells are able to sense changes in the extracellular calcium and as a result modulate their behavior. CaSR agonists (calcimimetics) or antagonists (calcilytics) have the potential to indirectly influence skeletal homeostasis through the modulation of PTH secretion by the parathyroid glands. The bone anabolic effect of strontium ranelate, a divalent cation used as a treatment for postmenopausal and male osteoporosis, might be explained, at least in part, by the activation of CaSR in bone cells.Calcium released in the bone microenvironment during remodeling is a major factor in regulating bone cells. Osteoblast and osteoclast proliferation, differentiation, and apoptosis are influenced by local extracellular calcium concentration. Thus, the calcium-sensing properties of skeletal cells can be exploited in order to modulate bone turnover and can explain the bone anabolic effects of agents developed and employed to revert osteoporosis.

Several strands of evidence indicate that oestrogens exert a protective role against the development of colon cancer through indirect and direct effects on colonic epithelium. Oestrogen receptor β (ERβ), the predominant ER subtype in human colon, is significantly decreased in colonic tumours compared with normal mucosa suggesting a potential role in the regulation of colon tumour growth. To investigate this hypothesis we engineered human colon cancer ERα-negative HCT8 cells in order to obtain ERβ protein over-expression. Stably transfected cells were cloned and ERβ expression and functionality were monitored by RT-PCR, Western blotting and transactivation in an assay using oestrogen-responsive reporter constructs. Over-expression of ERβ inhibited cell proliferation and increased cell adhesion in a ligand-independent manner. Its constitutive activation is possibly due to cross-talk with intracellular signalling pathways, as epidermal growth factor and IGF-I were able to induce ERβ transactivation. A possible mechanism by which ERβ over-expression inhibits proliferation in HCT8 cells is by modulation of some key regulators of the cell cycle; there is a decrease in cyclin E and an increase in the cdk inhibitor p21CIP1. In fact, flow cytometry analysis provided evidence for blocking of the G1-S phase progression induced by ERβ over-expression. The magnitude of this effect was affected by the level of ERβ expression. These results provide the first direct evidence that ERβ plays an important role in colon cancer as a regulator of cell proliferation through the control of key cell cycle modulators and arrest in G1-S phase transition. These findings are compatible with the hypothesis that the loss of ERβ expression could be one of the events involved in the development or progression of colon cancer.

Goals The study was designed to determine whether high-calcium mineral water is an efficient additional source of dietary calcium, optimizing a method for calcium determination never used for mineral waters. Background It is generally agreed that an adequate calcium intake is necessary for the acquisition of an ideal peak bone mass and for the maintenance of the bone mineral density in adults, in postmenopausal women, and in the elderly. Mineral waters are calorie free, and some, with high calcium levels, might be significant sources of calcium. Study The availability of the calcium contained in a high-calcium mineral water was measured in 27 healthy subjects. In 8 subjects the calcium availability of the water was compared with the calcium availability ingested with milk at the same calcium load. Milk and water were labeled extrinsically with 30 mg Ca. Fractional absorption from the oral dose was determined from plasma samples using ICP-MS technique. Results At an ingested calcium load of 3.18 mmol, percentage of absorption for water averaged 22.53 +/- 2.53 (mean +/- SD) for men, 22.57 +/- 2.10 (mean +/- SD) for premenopausal women and 21.62 +/- 3.12 (mean +/- SD) for postmenopausal women. Percentage absorption from milk was 23.15 +/- 4.06 (mean +/- SD). Discussion The calcium from the mineral water is thus highly bioavailable, at least as bioavailable as milk calcium, and ICP-MS appears to represent a reliable and reproducible method for calcium absorption from alimentary sources.

Summary Background Sputum examination is being increasingly used as a non-invasive method for studying airway inflammation. However, the application of sputum still presents some methodological problems and the results of sputum analysis may be substantially flawed by salivary contamination, cell and mucus debris. In addition, much work is needed to deepen the possibility of extensive application of cell and molecular biology techniques to sputum analysis. Objective In an attempt to improve the technique of sputum processing, we investigated the effect of: (i) 20 and 11 µm filtration in addition to 40 µm on salivary contamination; (ii) Percoll density gradient centrifugation on sputum slides quality; (iii) a culture medium (Minimum Essential Medium containing HEPES 22 mm, pH 7.4: MEM) as washing and suspension solution compared to PBS on cell viability. Methods Induced sputum samples were obtained in 37 asthmatics. 21 samples were processed as selected sputum and 16 samples as entire expectorates. After dithiotreitol (DTT) homogenization, each specimen was aliquoted in two parts of equal volume. One portion was processed with the usual method, the other using a modified method: cell pellet was suspended in sterile MEM, filtered through 40, 20 and 11 µm net filters and separated from the residual debris by Percoll gradient centrifugation. Results As compared to the current sputum processing this method resulted in: (i) no selective bronchial cellular loss; (ii) a significant decrease of salivary contamination, particularly in entire expectorates in which squamous cells were reduced from 47 (36) to 15.5% (20) as median values and interquartile range; (iii) a higher proportion of good quality cytospins; (iv) maintenance of cell viability over the time (88% vs. 81% in MEM and PBS, respectively) 1 h after sample collection. Conclusion In the present study we demonstrated that the proposed method is feasible and makes it possible to overcome most of the technical limits met with the commonly used method, pointing to a potential extension of induced sputum application for more sophisticated techniques.

The complete repair of periodontal structures remains an exciting challenge that prompts researchers to develop new treatments to restore the periodontium. Recent research has suggested strontium ion to be an attractive candidate to improve osteogenic activity. In this study, we have isolated a clonal finite cell line derived from human periodontal ligament (PDL) in order to assess whether and in which way different doses of SrCl2 (from 0.5 to 500 μg/ml) can influence both the proliferation and the mineralization process, for future application in oral diseases. PDL cells were cloned by dilution plating technique and characterized by FACS. Cell proliferation analysis and mineralization were performed by [3H]-thymidine incorporation and spectrofluorometric assay. Results have evidenced that the higher SrCl2 concentrations tested, from 25 to 500 μg/ml, have increased the proliferation activity after only 24 h of treatment. Interestingly, the same higher concentrations have decreased the mineralization, which was conversely increased by the lower ones, from 0.5 to 10 μg/ml. Our findings suggest the possible use of SrCl2 in appropriate delivery systems that release, at different time points, the specific dose, depending on the biological response that we want to induce on periodontal ligament stem cells, providing a more efficient periodontal regeneration.

Objectives Sex steroids are important regulators of bone physiology and play an essential role in the maintenance of bone health throughout the life. Hormonal replacement therapy (HRT) is a treatment commonly used to relieve symptoms and some undesirable consequences of menopause such as osteoporosis. Osteoporosis, characterized by the loss of bone mass and deterioration of microarchitecture with a consequent higher risk of fragility fractures, is under genetic influence. A tetranucleotide (TTTA)n microsatellite repeat polymorphism, at intron 4 of the CYP19 (aromatase) gene, has been previously associated with higher lumbar spine bone mineral density (LS-BMD) and lower risk of spine fracture in postmenopausal women. Moreover, the ERα encoded by the ESR1 gene is another important candidate for the regulation of bone mass of menopause. Moreover prospective analysis from >18.000 subjects at the GENOMOS study indicated that XX homozygotes genotype had a reduced risk of fracture independently from BMD. In the present study, we investigated in postmenopausal Italian women, at baseline and after 1 year of HRT, whether ESR1 and CYP19 gene polymorphisms could affect BMD through different statistical models. Methods This study has been performed on 100 post-menopausal Italian women, from a larger group of 250. The study group was administred HRT and LS-BMD was measured at baseline and after 1 year of therapy. Genetic analysis evaluating ESR1 and CYP19 gene polymorphisms was performed. Results Generalized Linear Models (GLMs) test showed that women with normal LS-BMD at the baseline had a major statistically significant BMD increase of 0.1426 gr/cm(2) (p= 0.0001) with respect to the osteoporotic patients. In addition, subjects with genotype 1 and 2 of CYP19 gene had a lower modification in LS-BMD after 1 year of HRT (0.0837 gr/cm(2) and 0,076 g/cm(2); p=0.0470 and 0,0547 respectively) when compared to genotype 3. No influences of the aromatase genotypes were observed in the variable difference using both Anova and GLMs test. Regarding the ESR1 gene polymorphism, the LS-BMD after 1 year of HRT was influenced by the diagnosis at the baseline and height and ERα genotypes were able to influence difference with statistical significant results with both test. Conclusions In the present study, we have demonstrated that CYP19 gene polymorphism is able to influence the effect of 1 year HRT on LS-BMD with no influence on pre-/ and post-/HRT LS-BMD differences. Although ESR1 gene polymorphism is not able to influence the LS-BMD after 1 year HRT, it influences the observed modifications during the year of therapy. These data underlie the complexity of the genetics of the bone mass and its importance in influencing the response to HRT.

Bone mineral density (BMD), the major determinant of osteoporotic fracture risk, has a strong genetic component. The discovery that inactivation of estrogen receptor alpha (ERalpha) gene is associated with low BMD indicated ERalpha as a candidate gene for osteoporosis. We have investigated the role of three ERalpha gene polymorphisms [intron 1 PVU:II and XBA:I RFLPs and TA dinucleotide repeat polymorphism 5' upstream of exon 1] in 610 postmenopausal women. There was a strong linkage disequilibrium between intron 1 polymorphic sites and also between these sites and the microsatellite (TA)(n) dinucleotide polymorphism, with a high degree of coincidence of the short TA alleles and the presence of PVU:II and XBA:I restriction sites. No significant relationship between intron 1 RFLPs and BMD was observed. A statistically significant correlation between (TA)(n) repeat allelic variants and lumbar BMD was observed (P = 0.04, ANCOVA), with subjects with a low number of repeats (TA15) showing the lowest BMD values. We observed a statistically significant difference in the mean +/- SD number of TA repeats between analyzed women with a vertebral fracture (n = 73) and the non-fracture group, equivalent to 2.9 (95% CI 1.56-5.72) increased fracture risk in women with a low number of repeats (TA15). We conclude that in this large population sample the (TA)(n) dinucleotide repeat polymorphism at the 5' end of the ERalpha gene accounts for part of the heritable component of BMD and might prove useful in the prediction of vertebral fracture risk in postmenopausal osteoporosis.

Recognition of a major genetic component in bone mass determination represented the basis for studies aiming to the identification of underlying major and minor genes. Bone mineral density (BMD) represents the continuous trait to be quantified in order to evaluate segregation of candidate genes with risk of osteoporosis. Polymorphisms at the vitamin D receptor (VDR), estrogen receptor, (ER), collagen type I, and interleukin 6 (IL6) gene loci have been correlated to BMD. However, in a polygenic disorder, such as osteoporosis, the number of genes expected to influence BMD is very large. In the present study we examined the presence of restriction fragment length polymorphisms (RFLPs) for the calcitonin receptor (CTR) gene in postmenopausal women. We identified a polymorphic (Tt) site at the CTR gene locus using the Taq I restriction fragment enzyme. Three genotypes were observed, whose Tt was the most frequent in our population (49.7%). In addition, Ancova analysis and Tukey's test showed that women with tt genotype had significantly lower lumbar BMD in comparison with Tt genotype (Tukey's test: p = 0.005). In conclusion, evidence of RFLPs at the CTR gene locus in Caucasian postmenopausal women of Italian origin made it possible to identify the involvement of another gene, the CTR gene, in the determination of bone mass.

There is much evidence that eosinophils play an important role in bronchial epithelial damage in asthma by releasing cationic proteins. However, the extent to which eosinophil inflammation relates to indices of asthma severity in chronic stable asthma is still a matter of debate. We studied 46 clinically stable patients with mild to severe chronic asthma (forced expiratory volume in one second (FEV1) 50-126% of predicted value). The clinical severity of asthma was graded from 1 to 4 according to the Aas scoring system. Twelve normal subjects were also studied as controls. Induction of sputum was performed by hypertonic saline to determine differential cell count, and eosinophil cationic protein (ECP) by the so-called "plug technique". The concentration of ECP was measured by a fluoroimmunoassay. Bronchial hyperresponsiveness was recorded by inhaling progressive concentrations of histamine, and the concentration that caused a 20% decrease in FEV1 (PC20) was calculated. Sputum eosinophils (range 0-61%), sputum ECP (range 24-10,800 microg x L[-1]) and serum ECP (range 4-61 microg x L[-1]) were significantly greater in asthmatics than in normal subjects, and distinguished the most severe group with the highest Aas score from the others. Sputum eosinophils and sputum ECP were strongly related to each other. The relationships between sputum or serum ECP and PC20 (range 0.016-7.5 mg x mL[-1]), and between sputum ECP and FEV1 were found to be weak. In conclusion, sputum outcomes of eosinophil activation and serum eosinophilic cationic protein appear to be useful indicators of disease. They do not accurately reflect current clinical or functional indices of asthma severity in chronic stable patients, and might therefore provide complementary data disease monitoring.

Besides functional estrogen receptors, the presence of signalling cell surface binding sites for 17beta-estradiol (17betaE2) has been reported in osteoblast- and osteoclast-like cells, suggesting that 17betaE2 may influence bone remodelling by a dual mechanism of action: to affect gene expression mediated by the nuclear activity of the steroid-receptor complex, and to initiate rapid responses triggered by a signal-generating receptor on the cell surface. Recently, we demonstrated that the human pre-osteoclastic cell line FLG 29.1 bears functional estrogen receptors. In this study we examined FLG 29.1 cells for the presence of cell surface binding sites for 17betaE2, and whether 17betaE2 could elicit cell signalling. Using a cell-impermeant and fluorescent estrogen conjugate, 17beta-estradiol-6-carboxymethyloxime-bovine serum albumin-fluorescein isothiocyanate, we demonstrated the presence of specific plasma membrane binding sites for 17betaE2. Stimulation of FLG 29.1 cells with low (1 nM) and high (1 microM) doses of 17betaE2 induced a prompt and significant (P0.05) increase of cellular pH, as measured in single cells using an image analysis system. In addition, both cAMP and cGMP were significantly increased by 17betaE2 with a dose-dependent response. Finally, a rapid increase of intracellular calcium ion concentration [Ca2+] was also induced by 1 nM 17betaE2, as measured in single cells using an image analysis system. Our findings strongly suggest a non-genomic action of 17betaE2 on osteoclast precursors.

Using a clonal line of bovine parathyroid endothelial cells (BPE-1) we defined the presence on these cells of a histamine H2 receptor and characterized its pharmacological properties. Interaction of histamine with its receptor induced an increase of cAMP accumulation in a dose- and time-dependent fashion. This effect appears unique for parathyroid endothelial cells, in fact, clonal parathyroid epithelial cells did not exhibit a similar response. No effect of histamine was observed on BPE-1 cell proliferation.

The primary biological effect of the estrogen estradiol-17 beta (17 beta E2) on bone is to decrease bone resorption. However, whether 17 beta E2 affects osteoclast differentiation or function directly or through its action on osteoblasts is unclear. To investigate this question we examined the human preosteoclastic cell line FLG 29.1 for evidence of functional estrogen receptors (ERs). Southern blotting of reverse transcription-PCR amplification products with a 32P-labeled cDNA probe for the human ER mRNA demonstrated that FLG 29.1 cells express ER mRNA. Binding of [3H]17 beta E2 to nuclear ERs was steroid specific with approximately 400 saturable, high affinity (Kd approximately 1 nM) binding sites per cell nucleus. Nuclear ERs covalently labeled with [3H]tamoxifen aziridine showed an apparent molecular weight of 65,000 by SDS/PAGE and Western blotting with the D75 monoclonal antibody to human ER. Pretreatment of cells with 0.1, 1.0, or 10 nM 17 beta E2 induced a dose- and time-dependent specific binding of progesterone to FGL 29.1 cells, and stimulation of the cells with 10 nM and 100 nM 17 beta E2 significantly (P < 0.05) reduced cell proliferation. Transcriptional activity of the ER gene was detected by transient transfection of cells with the pERE-BLCAT plasmid containing the estrogen response element for the vitellogenin A2 gene and the bacterial chloramphenicol acetyltransferase reporter gene. Treatment of FLG 29.1 cells with 10 nM 17 beta E2 increased chloroamphenicol acetyltransferase expression from 5- to 29-fold compared to controls. These observations suggest a potential role for estrogen in osteoclastogenesis.

This study comprised a comprehensive analysis of the glutathione (GSH) redox system during osteogenic differentiation in human osteoblast-like SaOS-2 cells. For the first time, a clear relationship between expression of specific factors involved in bone remodeling and the changes in the GSH/oxidized GSH (GSSG) redox couple induced during the early phases of the differentiation and mineralization process is shown. The findings show that the time course of differentiation is characterized by a decrease in the GSH/GSSG ratio, and this behavior is also related to the expression of osteoclastogenic markers. Maintenance of a high GSH/GSSG ratio due to GSH exposure in the early phase of this process increases mRNA levels of osteogenic differentiation markers and mineralization. Conversely, these events are decreased by a low GSH/GSSG ratio in a reversible manner. Redox regulation of runt-related transcription factor-2 (RUNX-2) activation through phosphorylation is shown. An inverse relationship between RUNX-2 activation and extracellular signal-regulated kinases related to GSH redox potential is observed. The GSH/GSSG redox couple also affects osteoclastogenesis, mainly through osteoprotegerin down-regulation with an increase in the ratio of receptor activator of NF-κB ligand to osteoprotegerin and vice versa. No redox regulation of receptor activator of NF-κB ligand expression was found. These results indicate that the GSH/GSSG redox couple may have a pivotal role in bone remodeling and bone redox-dysregulated diseases. They suggest therapeutic use of compounds that are able to modulate not just the GSH level but the intracellular redox system through the GSH/GSSG redox couple.

Background. Clodronate is used in high bone resorption diseases. Its action was defined as "cytotoxic" based on the induced cellular ATP loss, without any experimental verification of reversibility. In the present report the reversibility of clodronate action was tested on cultured human osteoclastic cell cultures. As "in vitro" bioeffects of clodronate are reversible, this compound should not be defined as "cytotoxic".Introduction. Bisphosphonates are pyrophosphate analogs able to inhibit osteoclast-mediated bone resorption widely used in the treatment of diseases with high bone turnover. Several evidences have shown that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R(2)side chain. The nitrogen-containing bisphosphonates act on osteoclasts by preventing protein prenylation, while non-nitrogen-containing bisphosphonates, like clodronate, are metabolized intracellularly to a β-γ-methylene analog of ATP that induces inhibition of the ADP/ATP translocase.Materials and Methods. In order to evaluate clodronate effects on osteoclastic cells and the bioreversibility of its action, we have used a human preosteoclastic (FLG 29.1) cell line and primary cultures of human osteoclast-like (HOC) cells. Functional and differentiative modifications were evaluated with immunocytochemical tartrate-resistant acid phosphatase activity (TRAcP) assay and with rapid quantitative detection of the complex "matrix metalloproteinase 9/tissue inhibitor of metalloproteinase" (MMP9/TIMP1) by RT-PCR analysis based on "TaqMan" technology. The apoptosis phenomenon were detected by DNA ladder analysis and quantified by counting apoptotic cells with Transmission Electron Microscopy (TEM) analysis. Adenosine-5'-[ β - γ -dichloromethylene] triphosphate (AppCCl(2)p) was detected and identified in cell extract by HPLC-ESI-MS-MS Mass Spectrometry. Intracellular ATP modulation in the presence of clodronate was evaluated by luciferin-luciferase assay. The Mann-Whitney "U" test was conducted for statistical analysis.Results. We found that clodronate inhibited both proliferation and differentiative features of cells of the osteoclastic lineage. Furthermore, treatment of both cell types with clodronate caused apoptosis, generation of measurable levels of AppCCl(2)p, and reduction of intracellular ATP levels. Addition of ATP to the culture medium caused an inhibition of the biological actions of clodronate on the human osteoclastic cell lineage.Conclusions. These data indicate that intracellular accumulation of the metabolite AppCCl(2)p is the likely route by which clodronate inhibits osteoclastic function and this effect is reversed by ATP.

Epidemiological studies suggest that consumption of isoflavones rich diets can improve several postmenopausal complications. The aim of this study was to investigate the absorption and the efficacy of isoflavonic supplementation in the treatment of menopausal symptoms.36 postmenopausal women received 75 mg/day of isoflavones in the form of tablets, for six months. 21 subjects concluded the treatment. Plasmatic and urinary samples were collected before and after the treatment, along with a dietary interview. Isoflavones were determined in biological samples and in commercial administered supplements by a HPLC/DAD system.Results showed the presence of genistein (from 0.043 to 1.820 micromol/L) in plasma samples, and of genistein (from 2.486 to 20.363 micromol/24h) and daidzein (from 11.106 to 98.091 micromol/24h) in the urines of the treated women. In the 21 completers the Greene Climateric scale value for hot flushes changed from 3 to 1 or 0. No changes of the endometrial thickness and of the breast tissue were detected. The analysis of the supplement content in the tablets was in agreement with what declared by the producer.Administration of isoflavone supplements produced a decrease of symptoms in this cohort of postmenopausal women monitored for isoflavone absorption.

Objective: Familial hypocalciuric hypercalcemia (FHH) syndrome is a rare benign condition, inherited as an autosomal dominant trait, in which inactivating mutations of the calcium-sensing receptor (CASR) gene affects the body’s ability to regulate calcium homeostasis. Its outcome is featured by increased levels of serum calcium, moderate hypophosphatemia, and inadequately normal or elevated circulating parathyroid hormone levels. Affected patients are mostly asymptomatic and do not benefit from surgical resection of their mildly enlarged parathyroids. Design: We evaluated for hypercalcemia an Italian family that was identified via a young adult male proband referred to our center for parathyroidectomy. Methods: The patients and the family members were evaluated both biochemically and genetically as suspected FHH subjects. An in vitro functional study was performed by site-directed mutagenesis, and CASR activity was monitored by measuring intracellular calcium ([Ca 2C ]i). Results: The patient had a novel germline heterozygous CASR mutation (c.361_364GATT; p.D121del/fsX122). The mutation caused a premature stop codon at codon 122, exiting a truncated protein. The biochemical phenotype of all family members carrying the heterozygous deletion was concordant with classic FHH syndrome. Conclusions: Our findings confirm the role of CASR gene mutational analysis to offer a valuable addition for the recognition of FHH in hypercalcemic patients not yet characterized for a positive familial history of hypercalcemia, the only condition that identifies CASR gene mutations in hypercalcemia.

In recent years, there has been an increasing interest in interactive application principles of biology and engineering for the development of valid biological systems for tissue regeneration, such as for the treatment of bone fractures or skeletal defects. The application of stem cells together with biomaterials releasing bioactive factors promotes the formation of bone tissue by inducing proliferation and/or cell differentiation. In this study, we used a clonal cell line from human adipose tissue-derived mesenchymal stem cells (hADSCs or preadipocytes), named PA2-E12, to evaluate the effects of strontium (Sr2+) released in the culture medium from an amidated carboxymethylcellulose (CMCA) hydrogel enriched with different Sr2+concentrations on osteodifferentiation. The osteoinductive effect was evaluated through both the expression of alkaline phophatase (ALP) activity and the hydroxyapatite (HA) production during 42 days of induction. Present data have shown that Sr2+released from CMCA promotes the osteodifferentiation induced by an osteogenic medium as shown by the increase of ALP activity at 7 and 14 days and of HA production at 14 days. In conclusion, the use of biomaterials able to releasein situosteoinductive agents, like Sr2+, could represent a new strategy for future applications in bone tissue engineering.

Increasing evidence suggests that transforming growth factor-beta (TGF-beta) is involved in bone formation during remodeling. Using a recently cloned human leukemic cell line (FLG 29.1 cells) we demonstrate that these cells synthesize and secrete TGF-beta 1 and that exogenous or autocrine TGF-beta 1 can induce the same features of osteoclastic-like cells, exerting its effects through the binding to TGF-beta specific receptors. Scatchard analysis of 125I-labeled TGF-beta 1 to FLG 29.1 cells revealed the presence of a single high affinity binding site with a Kd value of approximately 25 pM and a binding capacity of approximately 900 sites/cell. Affinity labeling experiments showed that FLG 29.1 cells express type I and type II TGF-beta receptors. Stimulation of FLG 29.1 cells with low TGF-beta 1 doses reduced cell proliferation and increased cell adhesion and tartrate resistant acid phosphatase (TRAcP) activity. Pretreatment of FLG 29.1 cells with TGF-beta 1 caused a significant and dose-dependent response to calcitonin. Northern blot of total mRNA and analysis of the conditioned media (CM) showed that TGF-beta 1 was synthesized by FLG 29.1 cells. TPA treatment, which induces partial differentiation of these cells, markedly increased TGF-beta 1 mRNA expression and growth factor release. The majority of TGF-beta 1 secreted by TPA-treated cells was in its latent form. However, anti-TGF-beta antibodies inhibited TGF-beta 1 and TPA-induced growth inhibition, calcitonin responsiveness, and TRAcP activity, suggesting that the TPA effect is mediated in part by autocrine TGF-beta 1 and indicating that the cells can activate and respond to the TGF-beta that they secrete. These findings support a potential autocrine role for TGF-beta 1 in osteoclast differentiation.

In the present study, we investigated the effects of estrogens on bone endothelial cell metabolism and the presence of estrogen binding sites in the same cells. For these studies, we have used a continuous cell line of clonal bovine bone endothelial cells for evidence of a direct response to estrogens in vitro. Receptor analysis to intact viable cells was steroid specific and saturable, with an apparent dissociation constant of 17.2 nM and a Bmax of 3.2 x 10(4) sites/cell. Northern blot analysis revealed a 6.5-kilobase mRNA that hybridized with a cDNA to human estrogen receptor. The 6.5-kilobase size is in close agreement with the reported size of the human estrogen receptor mRNA. In vitro estrogen responses of bone endothelial cells included a stimulation of cell proliferation as well as an inhibition of parathyroid hormone responsiveness. These findings clearly demonstrate the presence of functional estrogen receptors in bone endothelial cells in vitro, suggesting a role of estrogens in bone angiogenesis and in the entire process of bone remodeling.

[125I]Atrial natriuretic peptide (ANP) was used to identify ANP receptors on a clonal line of bovine bone endothelial (BBE) cells. Specific binding of [125I]ANP was saturable and of high affinity. Computer analysis of the equilibrium binding data indicated that the Scatchard plots are best fit by a straight line (Kd = 69.3 +/- 20.9 pM; binding capacity = 37.9 fmol/10(6) cells). The order of potency for competing with [125I]ANP binding was human ANP (hANP)rat atriopeptin-1 (rAP-1)porcine brain natriuretic peptide (pBNP)porcine C-type natriuretic peptide. Affinity cross-linking studies indicated the presence of two major 130- and 70-kilodalton bands that specifically bound to hANP, rAP-1, pBNP, and porcine C-type natriuretic peptide. The binding of natriuretic peptides to BBE cells resulted in an increase in cGMP production and a significant decrease in Na+/K+/Cl- cotransport, without effects on cAMP intracellular accumulation. hANP, rAP-1, and pBNP at 100-nM concentrations, significantly inhibited PTH-induced cAMP production. Treatment with natriuretic hormones was also associated with an increase in 6-keto-prostaglandin F1 alpha levels in the culture medium of BBE cells and a higher cell growth rate. These studies demonstrate that bone endothelial cells bear receptors for natriuretic hormones associated with changes in PTH-induced cAMP production, prostaglandin production, and cell proliferation.

BACKGROUND: Calcium-sensing receptor gene (CaSR) is a candidate to explain susceptibility to calcium kidney stones. Thus, we studied CaSR gene single-nucleotide polymorphisms (SNPs) and haplotypes associated with stones. METHODS: Four hundred and sixty-three calcium stone formers and 213 healthy controls were genotyped for 21 SNPs mapping the whole CaSR gene. CaSR gene structure was studied. SNPs and haplotypes were analysed for association with stones. RESULTS: Three haplotype blocks were identified in the CaSR gene. The first block was characterized by six SNPs and included gene promoters. The rs7652589 and rs1501899 SNPs and the CATTCA haplotype of the first block were significantly more frequent in normocitraturic calcium kidney stone formers than controls. The risk of stones was increased in normocitraturic homozygous patients and heterozygotes for the CATTCA haplotype. The rate of stones was higher in stone formers with the CATTCA haplotype. In a three-generation family, calcium stones were associated with the CATTCA haplotype. The bioinformatic analysis identified a new site for the octamer-binding transcription factor 1 in the presence of the variant alleles at the rs7652589 and rs1501899 SNPs. This transcription factor may downregulate the transcription of vitamin D-dependent genes and the CaSR expression. Conclusion. SNPs and CATTCA haplotype of the CaSR gene first block is associated with kidney stones in normocitraturic patients.

Studies on human osteoclast formation have been hampered by lack of a defined isolated progenitor cell population. We describe here the establishment of a human leukemic cell line (designated FLG 29.1) from bone marrow of a patient with acute monoblastic leukemia. The cultured cells are predominantly undifferentiated leukemic blasts, but addition of 12-o-tetradecanoylphorbol 13-acetate (TPA; 0.1 microM) induces irreversible differentiation into adherent, non-dividing, multinucleated cells. TPA-treated cells bear surface antigens typical of fetal osteoclasts, degrade 45Ca-labeled devitalized bone particles, display tartrate-resistant acid phosphatase in both mononuclear and multinuclear cells and receptors for calcitonin. Calcitonin increases intracellular cAMP accumulation in TPA-treated cells. TPA-treated cells show some ultrastructural features of osteoclasts as evidenced by transmission EM. These results indicate that FLG 29.1 cells may represent an osteoclast committed cell population, which upon induction with TPA acquire some morphological, phenotypical, and functional features of differentiated osteoclasts.

Objective To evaluate estrogen receptor (ER) expression in human melanoma tissues and in the adjacent healthy skin with the aim of explaining whether the ERα : ERβ expression ratio has a role in neoplastic progression. Design Prospective study. Setting Department of Dermatology, University of Florence, Florence, Italy. Patients Fourteen patients, 12 with cutaneous melanoma (6 women and 6 men) and 2 with melanocytic nevi (1 woman and 1 man). Main Outcome Measures Using quantitative reverse transcriptase–polymerase chain reaction and immunohistochemical analysis, we analyzed ERα and ERβ messenger RNA (mRNA) and ERβ protein expression in cutaneous melanoma and in the healthy skin surrounding the lesions. Results All melanocytic lesions expressed detectable levels of ERα and ERβ mRNA as well as ERβ protein. Dividing melanoma cases into 2 groups according to Breslow thickness, we found lower ERα and ERβ mRNA levels and lower ERβ protein levels in thicker, more invasive tumors. Conclusions These observations suggest a role for ERs in the metastatic process of melanoma cells, pointing at the possibility of using ERβ expression as a prognostic indicator of melanoma. The possibility of distinguishing proliferative melanomas, which are associated with dismal prognosis, from the so-called dormant melanomas opens up novel avenues in tailoring individual treatments, as already happens for other tumors.

Background: Tumoral calcinosis is a rare disease characterized by hyperphosphatemia due to hypophosphaturia and by ectopic calcifications. Phosphatonins are important hormones that regulate phosphorus homeostasis. Tumoral calcinosis is a rare congenital disorder in which the differential diagnosis from other syndromes associated with extraskeletal calcifications may be difficult. Mutations in the UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-3 ( GALNT3 ) and fibroblast growth factor-23 ( FGF23 ) genes have been described. Mutational analysis is important for the early recognition of the disorder, for prevention of its complications, and for family screening strategies. We examined two unrelated white patients affected by tumoral calcinosis. Methods: The first patient was a woman with a history of an ectopic calcification in the left shoulder. The second patient was a man with a history of an ectopic calcification in the right buttock. Routine biochemistry and FGF-23 assays were performed on serum samples. Genomic DNA was extracted from peripheral blood. The FGF23 and GALNT3 genes were analyzed by direct sequencing. Results: A new homozygous H41Q codon 41, C→A transversion at position 123 (c.123C>A) in exon 1 of the FGF23 gene was evidenced in both patients. No mutation of the GALNT3 gene was detected in these patients. As determined by an ELISA assay, intact FGF-23 circulating protein was low in both patients. Conclusions: This is the fourth mutation of the FGF23 gene described in subjects with tumoral calcinosis. Clinical Relevance: The biochemical and clinical profile of the two unrelated patients bearing the mutation reinforces the relevant role of FGF-23 in bone metabolism and in the pathogenesis of tumoral calcinosis.

Polymorphisms of genes encoding enzymes of the mevalonate pathway could modulate the response to amino-bisphosphonate treatment in postmenopausal osteoporosis.A characterisation of 234 Danish osteoporotic postmenopausal women (as part of the Prospective Epidemiological Risk Factors study (PERF)), treated for at least 2 years with amino-bisphosphonates, with respect to the adenosine/cytosine (A/C) rs2297480 farnesyl pyrophosphate synthase (FDPS) gene polymorphism, was carried out by PCR-based enzymatic digestion and quantitative PCR allelic discrimination on genomic DNA extracted from blood leukocytes. The association between these polymorphism genotypes and the response of spine and femur bone mineral density (BMD) and of biochemical bone biomarkers to treatment with amino-bisphosphonates was statistically examined.FDPS polymorphism did not show any relationship to baseline spinal and femoral BMD in Danish postmenopausal women. BMD response to treatment with amino-bisphosphonates was similar in the AA and the AC genotypes, while the CC genotype showed a lower BMD response to 2-year-treatment with amino-bisphosphonates at all examined skeletal sites (p=0.60 at the spine and p=0.59 at the femur). Interestingly, after 2 years of treatment the response of urinary Cross-laps to amino-bisphosphonates treatment was significantly (p0.05) lower in the CC genotype when compared to both the AC and AA genotypes. Even the response of serum osteocalcin was lower in the CC genotype, but without reaching a statistical significance (p=0.65).Danish postmenopausal women with osteoporosis bearing the homozygous CC genotype for rs2297480 FDPS polymorphism showed a decreased response of bone turnover markers to amino-bisphosphonate therapy, when compared to the heterozygous AC and to the homozygous AA genotypes. Further investigation on larger and different populations, together with polymorphism functional studies are required to confirm these data.

Long-term stability of arthroplasty prosthesis depends on the integration between the bone tissue and the implanted biomaterials, which requires the contribution of osteoblastic precursors and their continuous differentiation into the osteoblastic phenotype. Classically, these interactions are tested in vitro using mesenchymal stem cells (MSCs) isolated and ex vivo expanded from bone marrow aspirates. Human adipose tissue-derived stromal cells (AMSCs) may be a more convenient source of MSCs, according to their abundance and accessibility, but no data are available on their in vitro interactions with hard biomaterials. The aim of this work is to compare the osteogenic potential of human AMSCs and bone marrow-derived MSCs (BMMSCs) and to evaluate their response to Ti6Al4V alloy in terms of adhesion, proliferation and differentiation features, using the human osteosarcoma cell line SaOS-2 for comparison. The overall results showed that AMSCs have the same ability to produce bone matrix as BMMSCs and that Ti6Al4V surfaces exhibit an osteoinductive action on AMSCs, promoting their differentiation into functional osteoblasts and increasing bone formation. In conclusion, adipose tissue is a promising autologous source of osteoblastic cells with important clinical implications for bone tissue engineering.

Bone mineral density (BMD) contributes to bone strength, and methods for clinical assessment of bone quality characteristics beyond what can be gathered by BMD are awaited. Peripheral quantitative computed tomography (pQCT) allows for separate assessments of cortical and trabecular bone, providing information on bone geometry. Previous studies examining the relationship between estrogen receptor alpha (ERalpha) gene polymorphisms and BMD have been performed in large populations. However, only limited information is available on the possible segregation of ERalpha gene polymorphisms with bone structural properties. The aim of our study was to evaluate the association of XbaI and PvuII ERalpha gene polymorphisms with QCT parameters. We studied 900 subjects (541 women, 449 men) participating to the InCHIANTI study. By tibial pQCT we evaluated trabecular volumetric BMD, cortical volumetric BMD, cortical bone area, and cortical thickness (CtTh). Subjects were genotyped for ERalpha gene PvuII and XbaI polymorphisms. Analysis of variance was used for statistical analysis. Male subjects with PP and XX genotypes had higher geometric parameters, and female subjects with XX and PP genotypes showed higher densitometric parameters than other genotypes; however, the differences did not reach statistical significance. After adjustment for potential confounders, we found a significant (P = 0.002) CtTh difference across PvuII polymorphism in male subjects, with higher CtTh values in PP genotypes with respect to Pp and pp genotypes. These results show a relationship between the presence of the P allele and higher values of CtTh in male subjects, indicating for ERalpha a role in the control of tibial bone geometry.

The soy isoflavone genistein targets adipose tissue and elicits physiological effects that may vary based on dietary intake. We hypothesized that the adipose effects of genistein are dose and gender dependent. Four-week-old C57BL/6 male and female mice received daily oral doses of genistein (50-200,000 microg/kg.d) or 17beta-estradiol (E2) (5 microg/kg.d) for 15 d or a diet containing 800 ppm genistein. Genistein increased epididymal and renal fat pad and adipocyte size at doses up to 50,000 microg/kg.d or at 800 ppm in the diet in males but not in females. The alteration in adipocity correlated with changes in peripheral insulin resistance. These treatments increased genistein serum concentrations from 35+/-6 to 103+/-26 nM 12 h after treatment and lowered plasma triglycerides and cholesterol levels. The 200,000 microg/kg.d genistein dose decreased adipose tissue weight similarly to E2. This genistein dose down-regulated estrogen receptor (beta more than alpha) and progesterone receptor expression and induced estrogen-dependent adipose differentiation factors; it did not change expression of the minimal consensus estrogen-responsive element in ERE-tK-LUC mice, which was positively modulated in other tissues (e.g. the lung). E2 down-regulated almost all examined adipogenic factors. Gene microarray analysis identified factors in fat metabolism and obesity-related phenotypes differentially regulated by low and high doses of genistein, uncovering its adipogenic and antiadipogenic actions. The lower dose induced the phospholipase A2 group 7 and the phospholipid transfer protein genes; the 200,000 microg/kg.d dose inhibited them. The antiadipogenic action of genistein and down-regulation of adipogenic genes required the expression of ERbeta. In conclusion, nutritional doses of genistein are adipogenic in a gender-specific manner, whereas pharmacological doses inhibited adipose deposition.

Current evidence suggests that estrogen plays a dominant role in determining bone mineral density (BMD) in men, and inactivating mutations in the aromatase CYP19 gene have been associated with low bone mass in young males. We previously reported an association between a TTTA repeat polymorphism in intron 4 of the CYP19 gene and osteoporotic risk in postmenopausal females. Here we explore the role of this polymorphism as a genetic determinant of BMD in a sample of elderly males who were recruited by direct mailing and followed longitudinally for 2 (n = 300) and 4 (n = 200) yr. Six different allelic variants, containing seven, eight, nine, 10, 11, and 12 TTTA repeats, were detected. There was a bimodal distribution of alleles, with two major peaks at seven and 11 repeats and a very low distribution of the nine-repeat allele. Men with a high-repeat genotype (>nine repeats) showed higher lumbar BMD values, lower bone turnover markers, higher estradiol levels, and a lower rate of BMD change than men with a low-repeat genotype (25), suggesting that the effect of CYP19 genotypes on bone may be masked by the increase in fat mass. Moreover, the high-repeat genotype was less represented, although not significantly, in the vertebral fracture group with respect to the nonvertebral fracture group. Functional in vitro analysis after incubation with [3H]-androstenedione showed a higher aromatase activity in fibroblasts from subjects with a high-repeat genotype than in fibroblasts from subjects with a low-repeat genotype. In conclusion, differences in estrogen levels due to polymorphism at the aromatase CYP19 gene may predispose men to increased age-related bone loss and fracture risk.

Telomerase activity has been correlated to parathyroid carcinoma. Because its role in acquisition of a malignant phenotype by parathyroid cells is unclear, we treated telomerase-positive cultured human parathyroid cancer cells with the telomerase inhibitor AZT, evaluating cell telomerase activity, cytotoxic effects, growth, and morphological changes. In vitro exposure of these cells to AZT correlated with inhibition of cell proliferation. Introduction: Parathyroid carcinoma represents an uncommon cause of primary hyperparathyroidism, whose spectrum of clinical presentation, degree of malignancy, and prognosis are difficult to be properly identified. Neck surgery, specifically an en bloc resection of primary tumor, is the only curative treatment. Alternatively, affected patients could undergo repetitive palliative surgical exeresis of metastatic nodules. It has been previously shown that telomerase activity is specifically present in parathyroid carcinoma cells, being absent in hyperplastic and adenomatous tissues. Thus, determination of telomerase activity could represent either a useful diagnostic molecular marker for human parathyroid carcinoma or a potential target for pharmacological intervention in a malignant neoplasia usually resistant to chemo- and radiotherapeutic interventions. Materials and Methods: To further investigate the role of telomerase activity in acquisition of a malignant phenotype by parathyroid cells, we treated telomeric repeat amplification protocol-positive cultured human parathyroid cells with the telomerase inhibitor zidovudine, 3′-azido-3′deoxythymidine (AZT), evaluating cell telomerase activity, growth characteristics, potential cytotoxic effects, and morphological changes. Results: Our findings indicate that in vitro exposure of human parathyroid cancer cells to AZT resulted in intracellular accumulation of AZT-monophosphate (AZT-MP) and inhibition of telomerase, which correlate with inhibition of human parathyroid cancer cell proliferation. Moreover, we also found that AZT induced an apoptotic rather than a necrotic type of cellular death. None of these effects were observed in human adenomatous parathyroid cells in culture. Conclusions: Altogether these results indicate that AZT may be a highly effective agent against cancer parathyroid cells proliferation, which is an extremely important observation for a neoplasia which shows lack of response to classical pharmacological and physical antiblastic treatments.

Leiomyomas are the most common tumors in women, found in up to 30% of women in active reproductive life. These tumors are estrogen- and progestin-responsive. In fact, they do not occur before menarche, undergoing rapid increase in size during pregnancy with consequent fetal wastage. Conversely, they can regress or even calcify after menopause or castration. Given the central role of sex steroids in uterus trophism, estrogen and progesterone receptors are important mediators of hormonal bioeffects, and the genes encoding these receptors become easy candidates for uterine proliferative disorders.We examined the distribution of common polymorphisms of ERalpha, ERbeta and PR genes in 413 Caucasian females: 225 post-menopausal healthy controls and 188 premenopausal women affected by uterine leiomyomas. The polymorphisms were evaluated both in constitutive and tissue DNA by PCR amplification, specific endonuclease digestion, and then detection with agarose or polyacrylamide gel electrophoresis.The observed allele frequencies did not deviate from the expected Hardy-Weinberg distribution in our population. In case-control analysis, no variant frequency of the studied genes differed significantly between control subjects and patients.Polymorphisms in the genes encoding for ERalpha, ERbeta and PR did not correlate with the occurrence of uterine leiomyomas in our Caucasian population.

Calcium-sensing receptor (CaSR) is a plasma membrane protein that regulates tubular reabsorption of Ca. To establish its role in idiopathic hypercalciuria, the association of urinary Ca excretion with the polymorphisms of CASR gene has been studied in healthy subjects and in hypercalciuric and normocalciuric Ca stone formers. CASR exon 7 single nucleotide polymorphisms (SNP), G/T at codon 986, G/A at codon 990, and C/G at codon 1011, were evaluated by PCR amplification and direct sequencing in 97 normocalciuric stone formers, 134 hypercalciuric stone formers, and 101 normocalciuric healthy controls. Four haplotypes were defined on the basis of CASR gene SNP: haplotype 1 was characterized by the most frequent sequence; haplotypes 2, 3, or 4 by the presence of a single polymorphic variant at codon 986, 990, or 1011, respectively. The relative risk of hypercalciuria was calculated with multinomial logistic regression and was significantly increased only in individuals carrying haplotype 3 (Odds ratio, 13.0 [95% confidence interval, 1.7 to 99.4]). Accordingly, Ca excretion was higher in subjects bearing haplotype 3, whereas those bearing haplotype 2 showed a slight increase of plasma Ca concentration. Multiple regression analysis showed that haplotype 3 explained 4.1% of the total variance of Ca excretion and 12.6% of the variance explained by the variables considered in the study. In conclusion, CASR gene could be a component of the complex genetic background regulating Ca excretion. Arg990Gly polymorphism could facilitate activation of CaSR and increase Ca excretion and susceptibility to idiopathic hypercalciuria.

AIM: To investigate the effects of quercetin and genistein on colon cancer cell proliferation and their estrogen receptor β (ERβ) expression. METHODS: Colon cancer cells were stably transfected with a mammalian expression vector to overexpress ERβ (HCT8-β8-expressing cells) or a control vector (HCT8-pSV2neo-expressing cells). The proliferation of these cells was examined after treatment with quercetin or genistein (5-100 μmol/L), or 10 nmol/L 17β-estradiol (17β-E2). Cell viability was examined by acridine orange staining following treatments for 48 or 144 h. Effects of quercetin and genistein on ERβ transcriptional transactivation were examined by luciferase activity in HCT8-β8-expressing cells transiently transfected with a pEREtkLUC reporter vector. In addition, the regulation of ERβ transcription by phytoestrogens and 17β-E2 was examined by quantitative polymerase chain reaction. RESULTS: Proliferation of HCT8-β8-expressing cells was not reduced low doses (5 μmol/L) of quercetin and genistein, while it was reduced at 25-50 μmol/L with an effect similar to 10 nmol/L 17β-E2. Treatment with doses of phytoestrogens ≥ 75 μmol/L completely blocked cell growth and reduced overall cell counts, however no effects at any dose were observed in HCT8-pSV2neo-expressing cells. These results were supported by viability staining that revealed acridine orange-stained lysosomes with high doses or extended treatment periods. Genistein and quercetin (50 μmol/L) significantly increased ER-responsive luciferase activity similar to 10 nmol/L 17β-E2 (P < 0.05). Furthermore, genistein and quercetin (50 μmol/L), as well as 10 nmol/L 17β-E2 significantly increased ERβ mRNA levels in HCT8-β8-expressing cells (P < 0.05). In addition, treatment of HCT8-pSV2neo-expressing cells with 50 µmol/L quercetin or 10 nmol/L 17β-E2 significantly increased ERβ mRNA levels compared to untreated controls (P < 0.05), though the absolute levels were much lower than in HCT8-β8-expressing cells. CONCLUSION: The antitumorigenic effects of the phytoestrogenic compounds quercetin and genistein on colon cancers cells occur through ERβ activity and expression.