Teriparatide increases the maturation of circulating osteoblast precursors.

UNLABELLED: This study shows that teriparatide promotes the circulating osteoblast (OB) precursor degree of maturation in patients affected by postmenopausal osteoporosis. INTRODUCTION: Anabolic treatment with teriparatide has proven effective for the therapy of postmenopausal osteoporosis and significantly reduces the risk of non-vertebral fragility fractures. The aim of this study was to investigate the effect of teriparatide on circulating OB precursors. METHODS: We evaluated by flow cytometry and real-time PCR the expression of OBs typical markers in peripheral blood mononuclear cells during treatment with teriparatide plus calcium and vitamin D, raloxifene plus calcium and vitamin D or calcium and vitamin D alone at various time points. Serum bone alkaline phosphatase and osteocalcin (OC) were measured as markers of bone turnover. RESULTS: Our results show that circulating OB precursors are more numerous and more immature in patients affected by fragility fractures than in osteoporotic patients without fractures. We also show that teriparatide treatment increases the expression of alkaline phosphatase and of OC in OB precursors; thus, it increases their degree of maturation. CONCLUSIONS: We suggest that teriparatide acts as anabolic agents also by promoting the maturation of OB precursors.

Bone and bone marrow pro-osteoclastogenic cytokines are up-regulated in osteoporosis fragility fractures.

UNLABELLED: This study evaluates cytokines production in bone and bone marrow of patients with an osteoporotic fracture or with osteoarthritis by real time PCR, Western blot and immunohistochemistry. We demonstrate that the cytokine pattern is shifted towards osteoclast activation and osteoblast inhibition in patients with osteoporotic fractures. INTRODUCTION: Fragility fractures are the resultant of low bone mass and poor bone architecture typical of osteoporosis. Cytokines involved in the control of bone cell maturation and function are produced by both bone itself and bone marrow cells, but the roles of these two sources in its control and the amounts they produce are not clear. This study compares their production in patients with an osteoporotic fracture and those with osteoarthritis. METHODS: We evaluated 52 femoral heads from women subjected to hip-joint replacement surgery for femoral neck fractures due to low-energy trauma (37), or for osteoarthritis (15). Total RNA was extracted from both bone and bone marrow, and quantitative PCR was used to identify the receptor activator of nuclear factor kB Ligand (RANKL), osteoprotegerin (OPG), macrophage colony stimulating factor (M-CSF), transforming growth factor ß (TGFß), Dickoppf-1 (DKK-1) and sclerostin (SOST) expression. Immunohistochemistry and Western blot were performed in order to quantify and localize in bone and bone marrow the cytokines. RESULTS: We found an increase of RANKL/OPG ratio, M-CSF, SOST and DKK-1 in fractured patients, whereas TGFß was increased in osteoarthritic bone. Bone marrow produced greater amounts of RANKL, M-CSF and TGFß compared to bone, whereas the production of DKK-1 and SOST was higher in bone. CONCLUSIONS: We show that bone marrow cells produced the greater amount of pro-osteoclastogenic cytokines, whereas bone cells produced higher amount of osteoblast inhibitors in patients with fragility fracture, thus the cytokine pattern is shifted towards osteoclast activation and osteoblast inhibition in these patients.

Alendronate reduces osteoclast precursors in osteoporosis.

UNLABELLED: This study evaluates the effect of alendronate on osteoclastogenesis, cytokine production, and bone resorption in postmenopausal women. We suggest that it acts on mature bone resorbing osteoclasts after 3 months of treatment, whereas, after 1 year, it diminishes their formation by reducing their precursors and serum RANKL. INTRODUCTION: Osteoclasts are the target cells of bisphosphonates, though the most drug-sensitive steps of their formation and activity have not been determined. The present study evaluates the effect of alendronate on osteoclastogenesis, cytokine production, and bone resorption in postmenopausal women. METHODS: The study was conducted on 35 osteoporotic women; 15 were pretreated with alendronate 70 mg/week, whereas, 20 were treated with calcium 1 g/day and vitamin D 800 IU/day. After 3 months, 30 received alendonate 70/mg, vitamin D 2800 IU/week, and calcium 1 g/day for 12 months (combined therapy), whereas, the other five patients remained on calcium 1 g/day and vitamin D 800 IU/day. The following parameters were assessed before and after therapy: changes in bone resorption markers, circulating osteoclast precursors, formation of osteoclasts in peripheral blood mononuclear cell cultures, their viability, and variations in cytokines production. RESULTS: After 3 months of alendronate, there was no significant reduction in the number of osteoclast precursors, osteoclast formation and viability, and cytokine levels, whereas, there was a significant reduction of bone resorption markers. One year of the combined therapy, on the other hand, reduced osteoclast precursors, osteoclast formation, and serum RANKL, whereas, calcium plus vitamin D alone had no effect. CONCLUSIONS: We suggest that alendronate mainly acts on mature bone resorbing osteoclasts in the short term, whereas, its long-term administration diminishes their formation by reducing their precursors and serum RANKL.

4-Methyl-3-(arylsulfonyl)furoxans: a new class of potent inhibitors of platelet aggregation.

A series of 4-methyl-3-(arylthio)furoxans were synthesized by oxidation of 1-(arylthio)-2-methylglyoxymes with dinitrogen tetroxide. Reduction with trimethyl phosphite of the furoxan derivatives afforded the corresponding furazans, while oxidation with an equimolar amount of 30% hydrogen peroxide in acetic acid or with an excess of 81% hydrogen peroxide in trifluoroacetic acid afforded the corresponding arylsulfinyl and arylsulfonyl analogues, respectively. All the furoxan and furazan derivatives showed activity as inhibitors of platelet aggregation. 4-Methyl-3-(arylsulfonyl)furoxans were the most potent derivatives of the series. 4-Methyl-3-(phenylsulfonyl)furoxan (10a), one of the most active derivatives, inhibits the AA-induced increase of cytosolic free Ca2+ and production of malondialdehyde. A primary action of the compound on cyclooxygenase is excluded, as a stable epoxymethano analogue of prostaglandin H2 does not reverse the inhibitory effect of 10a. This compound produces a significant increase in cGMP which is likely to cause inhibition at an early stage of the platelet activation pathway.

Calcium blood level modulates endogenous nitric oxide action: effects of parathroidectomy in patients with hyperparathyroidism.

UNLABELLED: Platelet cyclic guanosine monophosphate (cGMP) is produced by soluble guanylate cyclase (sGC), the activity of which is modulated by the activity of nitric oxide (NO) constitutive synthase (cNOS) which, in turn, is activated by a calcium/calmodulin complex. In primary hyperparathyroidism (H-PTH) an increase in platelet free calcium levels is present. In this study we evaluate the platelet cGMP levels, as an expression of NO production, in the presence of 3-isobutyl-1-methylxanthine (IBMX) alone (IBMXcGMP) and after stimulation by ionomycine (IONO; IONOcGMP) and sodium nitroprusside (SNP; SNPcGMP), in eight subjects affected by H-PTH before and after removal of adenoma. Platelet cGMP levels were also measured in seven normal subjects. IBMXcGMP and IONOcGMP were elevated in H-PTH patients compared with normal subjects (1.9 +/- 0.3 vs 0.8 +/- 0.2 fmol/10(6) platelets and 2.7 +/- 0.4 vs 1.4 +/- 0.3; P < 0.02 and P < 0.05 respectively) but SNPcGMP was unaffected (3.9 +/- 0.6 vs 2.5 +/- 0.5). After parathyroidectomy, blood levels of intact parathyroid hormone (i-PTH), total calcium (t-Ca), IBMXcGMP and IONOcGMP all decreased (177.5 +/- 23.9 vs 45.0 +/- 8.8 pg/ml, P < 0.005; 6.5 +/- 0.5 vs 4.6 +/- 0.1 mEq/1, P < 0.005; 1.9 +/- 0.3 vs 0.8 +/- 0.2, P < 0.005; 2.7 +/- 0.4 vs 1.8 +/ 0.3, P < 0.05 respectively), while SNPcGMP was not modified (3.9 +/- 0.6 vs 4.3 +/- 0.9). t-Ca and i-PTH were directly correlated with IBMXcGMP (P < 0.02, rs = 0.613; P < 0.02, rs = 0.576 respectively) and i-PTH was also correlated with t-Ca (P < 0.001), rs = 0.840). IN CONCLUSION: (1) levels of IBMXcGMP and IONOcGMP are high in subjects with H-PTH; (2) after surgery both IBMXcGMP and IONOcGMP decrease to normal values. As IBMXcGMP expresses basal cGMP and IONOcGMP expresses the cGMP after cNOS stimulation, it can be speculated that the increase in NO production could be a mechanism to downregulate the vasoconstriction which may be caused by the high calcium levels in smooth muscle cells. After surgery, together with the normalization of calcium levels, NO production also returned to normal values.

High density lipoproteins (HDL) in women with postmenopausal osteoporosis: a preliminary study.

OBJECTIVE: Since a previous study showed an inverse correlation between high density lipoproteins (HDL) and bone mineral density (BMD), we searched for a possible relationship between HDL level and the presence of postmenopausal osteoporosis. DESIGN: We measured HDL levels in 37 women with postmenopausal osteoporosis, and compared them with a control group of 43 healthy postmenopausal women. The HDL levels were compared between the two groups using Student's t test and were correlated with BMD by Pearson's coefficient. To avoid possible selection bias, we compared patients and controls for body mass index by chi 2 test. The sensitivity and specificity of HDL level higher than 65 mg% (positive test) or lower than 45 mg% (negative test) was compared with double emission x-ray absorptiometry (considered the gold standard in the measurement of BMD). RESULTS: The level of HDL was significantly higher in the osteoporotic patients than in the controls (67.7 +/- 15.5 mg% vs 58.3 +/- 11.6 mg%, p = 0.0039). HDL was inversely correlated with BMD (r = -0.29, p = 0.0083). HDL higher than 65 mg% has a high specificity (77%) for patients with osteoporosis, while HDL lower than 45 mg% has a high sensitivity (97%) in detecting subject without osteoporosis. CONCLUSIONS: Our preliminary data suggest an interesting, as yet unexplained association between HDL and bone mineral density in postmenopausal women.

Effect of GSH depletion by 1-chloro-2,4-dinitrobenzene on human platelet aggregation, arachidonic acid oxidative metabolism and cytoskeletal proteins.

Platelet reduced glutathione (GSH) is completely depleted by 1-chloro-2,4-dinitrobenzene (CDNB), which is a substrate for GSH-S-transferase. GSH-depleted platelets: a) aggregate normally at high inducer concentration; b) respond with increased (after arachidonic acid) or depressed (after collagen) aggregability at low inducer concentration; c) show almost no arachidonic acid-induced stimulation of the hexose monophosphate shunt; d) are sensitized to oxidant agents such as diamide, which elicits a faster cytoskeletal protein oxidative polymerization and reversible aggregation. Our results suggest that GSH acts as a reducing cofactor and/or free radical scavenger in the PG-hydroperoxidase step of the cyclooxygenase pathway; moreover, GSH protects membrane and cytoskeletal protein -SH groups from oxidation.

Investigation of the potential role of membrane CD38 in protection against cell death induced by HIV-1.

The aim of the present study was to assess the possibility that mCD38, a bifunctional ectoenzyme with NAD+ hydrolase and ADP ribose cyclase activities, exerts a protective role in the development of acute, non-syncytial cell death from HIV-1. This hypothesis was tested in a panel of human T-cell lines with defined membrane CD4 (mCD4) expression. A negative correlation was found between the levels of mCD38 expression and the rate of acute cell death from HIV-1 observed 96 h after infection. The negligible rate of cell death from HIV-1 detected in some cell lines (H9 and Supt-1) is apparently unrelated to the level of mCD4 expression, whereas the association with high levels of mCD38 is confirmed. In H9 and Supt-1 cells, the fraction of cells positive to HIV-1 p24 is lower than in the mCD38low cell lines (MT-4, MT-2, C8166). This suggests that high CD38 expression is correlated to resistance to HIV-1 infection, resulting in a lower rate of cell death. A further finding supporting the work hypothesis is that the addition of nicotinamide, a reaction product of CD38, confers to MT-4 cells (mCD38low) partial protection against acute cell death from HIV-1. This indicates that nicotinamide may be at least partially responsible for the correlation observed between high levels of mCD38 and negligible rates of acute cell death from HIV-1.

Local interaction simulation approach for the response of the vascular system to metabolic changes of cell behavior.

The self-regulatory interactions between cells and the vascular system are mediated by signals propagating at a finite speed. In order to build up a physical model of these processes, several features, such as storing of internal energy, nonclassical nonlinear behavior, and delay and threshold effects, have to be taken into account. Considering cells as particles in different metabolic states according to their internal energy, we have developed a model based on the local interaction simulation approach. Several numerical results, in qualitative agreement with biological observations, illustrate the applicability of the model and the method to implement it.

Non-linear model of cancer growth and metastasis: a limiting nutrient as a major determinant of tumor shape and diffusion.

A new approach for modelling the spatio-temporal evolution of tumors is presented. To test its validity, a very basic model is considered, which, in spite of its simplicity, is capable of generating a multiplicity of morphologies and growth and migration rates. From an in-vivo scenario of basic life processes, cancer cell proliferation is described as a competition for basic nutrients. The chosen mathematical treatment and simulation techniques permit a direct implementation of the local nonlinear couplings existing between the various cell populations and the free and bound nutrient concentration. A discussion of the results and proposed improvements and applications of the model is also presented.

Platelet vitamin D receptor is reduced in osteoporotic patients.

AIM: It is well known that vitamin D plays an important role in maintaining bone homeostasis and in regulating calcium absorption. The active form of vitamin D interacts with its receptor the VDR that is expressed in multiple tissues and it is involved in platelets (PLTs) function. In the present study we evaluate PLTs' VDR expression in osteoporotic as opposed to healthy subjects. METHODS: We enrolled in the study 77 women with postmenopausal osteoporosis, 33 healthy women of childbearing age, 49 healthy men, and 11 healthy women matched with patients for age and postmenopausal period. Thirty-nine patients had had one femoral fracture occurred after the age of fifty and attributable to primary osteoporosis. Bone mineral density, markers of bone metabolism and VDR levels were measured in all the subjects. RESULTS: Our data show that VDR level is lower in patients as respect to controls and is positively correlated with bone density, but not with markers of bone metabolism. We also found a decrease in the phosphorus levels in patients without differences in vitamin D levels and in the dietary calcium intake. CONCLUSION: The lower VDR expression in osteoporotic could indicate a lower ability to respond to vitamin D, and could be the explanation of the increase in the PTH and decrease in the phosphorus levels in patients with respect to controls.

Glutathione levels in patients with erectile dysfunction, with or without diabetes mellitus.

The reduced form of glutathione (GSH) is the most important cell antioxidant and is also an essential cofactor for nitric oxide (NO) synthase that synthesizes NO from l-arginine. Reduced levels of GSH, due both to a hyperglycaemia-induced increase of free radical production and to a decrease of NADPH levels [like in diabetes mellitus (DM)], can hamper the endothelial cell functions. This condition may play an important role in the aetiology of some clinical signs, like erectile dysfunction (ED). The aim of this study was to test the hypothesis that GSH concentration is reduced in patients with ED and type 2 diabetes mellitus. We studied 111 male patients with ED: 64 with diabetes (ED/DM) and 47 without diabetes (ED/wDM); 20 patients with diabetes but without ED (DM) and 26 male normal subjects as a control group (C). The GSH red blood cell concentration was significantly lower in ED than in C (X +/- SD; 1782.12 +/- 518.02 vs. 2269.20 +/- 231.56 mumol/L, p < 0.001). In particular, GSH was significantly reduced in ED/DM vs. ED/wDM (1670.74 +/- 437.68 vs. 1930.63 +/- 581.01 micromol/L, p < 0.01). In DM, GSH was significantly lower than in C and significantly higher than in ED/DM (2084.20 +/- 118.14 vs. 2269.20 +/- 231.56 and vs. 1670.74 +/- 437.68 micromol/L, p < 0.002 and p < 0.001 respectively). GSH showed a negative correlation with fasting glucose concentrations (r = -0.34, p < 0.01) and with the duration of DM (r = -0.25, p < 0.05). A GSH depletion can lead to a reduction of NO synthesis, thus impairing vasodilation in the corpora cavernosa.

[Aspects of the decrease of some enzyme activities in erythrocytes of subjects with beta-thalassemia]. / Aspetti del decadimento di albune attivita' enzimatiche in eritrociti di soggetti beta-talassemici.

G6PD, GSH-Px and 6PGD activity was found to be elevated in beta-thalassemia heterozygous erythrocytes. Estimates of the activities of the three enzymes in red cell fractions of differing mean age separated by centrifugation through a density gradient of Ficoll-Triosil layers, showed that the rate of in vivo decline was normal for G6PD and GSH-Px, and decreased for 6PGD. The increased enzyme activity results from increased rate of synthesis for G6PD and GSH-Px, and from higher in vivo stability for 6PGD.