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.

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.

Platelet cyclic guanosine monophosphate production during menstrual cycle in healthy women.

UNLABELLED: The incidence of cardiovascular disease among women during their reproductive years is considerably less than in men and this difference decreases after menopause. Since in cultured endothelial cells and in platelets E2 increases nitric oxide (NO) production, it is possible that their cardioprotective effect may be mediated by NO. The aim of this study was to evaluate platelet cyclic guanosine monophosphate (cGMP), as a marker of NO production, during menstrual cycle. Fifteen women aged 26-40 yr were studied to evaluate: LH, FSH, E2, P and cGMP on the 5th follicular and 22nd luteal day of the cycle and during the ovulatory period. Platelet cGMP was evaluated in basal condition (3-isobuthyl 1-methylxanthine-IBMX) and with ionomycine (IONO) and sodium nitroprusside (SNP). RESULTS: LH, FSH, E2 and P demonstrated the typical patterns of ovulatory cycle. During follicular and luteal IBMX, SNP and IONO phase were homogeneous while they increased during the ovulatory period. A correlation between IBMX cGMP and E2 (p<0.002, rs=0.456) was found. In conclusion the data show an increase in platelet cGMP during the ovulatory period and a correlation between E2 and cGMP suggesting that E2 modulates NO production. The cardioprotective effect of E2 may be, at least in part, mediated by the increase in NO production.

Administration of glutathione in patients with type 2 diabetes mellitus increases the platelet constitutive nitric oxide synthase activity and reduces PAI-1.

Several studies suggest that nitric oxide (NO) production is impaired in diabetes mellitus. Reduced levels of NO could contribute to cardiovascular mortality. Furthermore, NO synthesis is impaired in glutathione (GSH)-depleted human umbilical vein endothelial cells and GSH is reduced in patients with type 2 diabetes mellitus (T2DM). We tested the hypothesis that treatment with GSH may improve platelet constitutive NO sinthase (cNOS) activity in patients with T2DM. Fifteen patients with T2DM underwent a treatment with GSH 600 mg/day i.m. for 10 days. With respect to the basal values on the 10th day of treatment, the red blood cell GSH concentration and platelets cNOS increased (1.4+/-0.1 vs 1.9+/-0.1 micromol/10(10) RBC, p<0.001 and 0.7+/-0.1 vs 2.9+/-0.2 fmol x min(-1) x 10(-9) PLTs, p<0.001, respectively) and the plasma PAI-1 levels diminished (81.4+/-3.7 vs 68.7+/-4.0 ng/ml, p<0.002). A negative correlation between the cNOS and the PAI-1 was found on the basal values. After a wash-out of 30 days the values of red blood cell GSH concentration, platelet cNOS activity and PAI-1 Ag returned to the basal levels. These data suggest that the administration of GSH, in patients with T2DM, is able to improve platelet cNOS activity together with a reduction of PAI-1.

Serum DHEAS levels correlate with platelet cGMP in normal women.

Several studies suggest that DHEAS is a protective factor against atherosclerosis and coronary artery disease in man, but the mechanism of its biological role is unclear. Recently, it has been suggested that DHEAS can retard atherosclerosis formation through an increase in nitric oxide (NO) production by increasing E2 synthesis. The aim of the study was to evaluate the platelet cGMP concentrations (i.e. a marker of NO production) and the serum levels of DHEAS and E2 in normal females. Blood samples were taken from 51 normal women (age 42.3+/-1.9 yr, range: 22-67 yr, BMI 23.0+/-0.6 kg/m2) to evaluate platelet cGMP concentrations and serum levels of DHEAS and E2. To determine the platelet cGMP content, platelet rich plasma (PRP) was incubated at 37 C (30 min) in the presence of IBMX. The amount of platelet cGMP was measured by a cGMP (3H) assay kit. In all subjects the mean of platelet cGMP was 536.2+/-45.3 fmol/10(6) platelets and the mean of serum DHEAS and E2 was 151.4+/-13.9 microg/dl and 34.7+/-6.1 pg/ml, respectively. In all subjects DHEAS positively correlates with cGMP (p<0.001, r=0.513) and with E2 (p<0.001, r=0.650); furthermore E2 positively correlates with cGMP (p<0.001, r=0.663). In conclusion our data support the hypothesis that DHEAS exerts its antiatherogenic effect by increasing the NO production directly and/or by increasing the E2 synthesis.

Analysis of a "phase transition" from tumor growth to latency.

A mathematical model, based on the local interaction simulation approach, is developed in order to allow simulations of the spatiotemporal evolution of neoplasies. The model consists of a set of rules, which govern the interaction of cancerous cells among themselves and in competition with other cell populations for the acquisition of essential nutrients. As a result of small variations in the basic parameters, it leads to four different outcomes: indefinite growth, metastasis, latency, and complete regression. In the present contribution a detailed analysis of the dormant phase is carried on and the critical parameters for the transition to other phases are computed. Interesting chaotic behaviors can also be observed, with different attractors in the parameters space. Interest in the latency phase has been aroused by therapeutical strategies aiming to reduce a growing tumor to dormancy. The effect of such strategies may be simulated with our approach.

A physical-based model for the simulation of neoplastic growth and metastasis.

BACKGROUND AND OBJECTIVES: It is possible to formulate models capable of reproducing the main details of the physical processes involved in the evolution of biological systems. The complexity of the problem requires to begin with a simple and universal model for the description of the cellular growth, to be adapted successively to the local conditions found in clinically observed neoplastic growths. METHODS: A model based on the Local Interaction Simulation Approach (LISA) has been formulated for the simulation of growth, diffusion, and metastasis of neoplasms. The vascularization is described by a blood vessel located on one edge of the specimen in which a constant and homogeneous flow is assumed. A nutrient density is defined to mimic the blood flow within the tissue. RESULTS: Photograms taken at proper times may identify the main characteristics of the tumor evolution and describe its volume variations in a transversal section. Furthermore, it is possible to monitor constantly the volume of the neoplasm and of the necrotic tissue as a function of time, as well as the portion of cells that have migrated in the blood vessel. CONCLUSIONS: In spite of strong simplifying assumptions, the model presents good qualitative agreement with clinical data, which may be further improved by more detailed information about cancer cells properties or local vascular system patterns.

Iron metabolism and HIV infection: reciprocal interactions with potentially harmful consequences?

Humans with advanced human immunodeficiency virus (HIV) infection present some evidence suggestive of iron accumulation. Ferritin concentrations increase with HIV disease progression, and iron accumulates in several tissues. Iron excess may exert negative effects in individuals with HIV. Indeed, iron accumulation seems to be associated with shorter survival, and a number of investigations point to an iron-mediated oxidative stress in subjects with HIV infection. The observations on humans infected with HIV are in part supported by in-vitro findings. Indeed, in-vitro HIV infection is associated with changes in iron metabolism, and an iron-mediated oxidative stress is likely to contribute to viral cytopathogenicity. Furthermore, it is interesting to point out that the interaction between iron and HIV may be reciprocal, since viruses with a life-cycle involving a DNA phase require chelatable iron for optimum replication. This combined evidence suggests that iron metabolism is an important area for virus/host interaction. These observations may be relevant to both laboratory monitoring and clinical treatment of individuals with HIV.

Modulation of surface transferrin receptors in lymphoid cells de novo infected with human immunodeficiency virus type-1.

To investigate whether transferrin receptor (CD71) expression is affected by acute HIV-1 infection, three different lymphoid cell lines (MT-4, SUPT-1, H9) were infected with HIV-1 and tested for surface CD71 expression after different incubation periods depending on cell survival after infection. We found that expression of surface CD71 was lower in cells infected with HIV-1 than in uninfected controls: the timing and extent of this down-modulation depended apparently on the different susceptibility of the cell lines to HIV-1 infection and cytopathogenicity. Citrate, a molecule capable of chelating iron, dose-dependently prevented down-modulation of surface CD71 in HIV-1 infected cells as well as viral cytopathic effects. We conclude that (i) expression of surface transferrin receptors is down-modulated by acute HIV-1 infection in T lymphoid cells, that (ii) this cell phenotypic modulation is associated with the cytopathic effects of the virus, and that (iii) these phenomena are modulated by iron chelation. These results support the view that iron metabolism may be an important area for interaction between HIV-1 and human cells.

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.

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.

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.

Nitric oxide synthesis is impaired in glutathione-depleted human umbilical vein endothelial cells.

Human endothelial cells cultured from umbilical vein (HUVEC) were tested for their ability to synthesize nitric oxide (NO), which has been identified as an endothelium-derived relaxing factor. The synthesis of this free radical (detected as citrulline, which is produced stoichiometrically with NO from arginine) in HUVEC is Ca2+ dependent, is increased sevenfold by the calcium ionophore ionomycin, and accounts for most basal and ionomycin-induced guanosine 3',5'-cyclic monophosphate (cGMP) production. Loading of cells with reduced glutathione (GSH), but not with N-(2-mercaptopropionyl)- glycine (MPG), led to increased citrulline production, both basally and after ionomycin stimulation. When the cells were depleted of GSH by incubation with 1-chloro-2,4-dinitrobenzene (CDNB), citrulline synthesis and cGMP production were inhibited in a concentration-dependent way. CDNB was not cytotoxic and did not inhibit cGMP increase elicited by sodium nitroprusside; cell loading with GSH (but not with MPG) relieved the block of citrulline synthesis. These results suggest that GSH is necessary in HUVEC for NO synthesis rather than for the NO effect on guanylate cyclase.

Proliferative and migratory responses of murine microvascular endothelial cells to granulocyte-colony-stimulating factor.

Microvascular murine endothelial cells lines transformed by middle T oncogene of polyoma virus maintain the biological characteristics of nontransformed microvascular endothelial cells (EC). By using cell lines originated from different anatomical districts (thymus, brain, heart, and skin), we demonstrated that murine granulocyte-colony-stimulating factor (G-CSF) induces proliferation of murine microvascular endothelial cells at nanomolar concentrations without any cooperation with fetal calf serum. The proliferative effect on murine cells is less than that elicited by epidermal growth factor (EGF), used as standard for this function. G-CSF also promotes the migration of tEnd.1 endothelial cell line assayed by Boyden chamber technique. The analysis of transcript for G-CSF receptor (G-CSFR) by Northern blot hybridization and by reverse-transcriptase polymerase chain reaction (RT-PCR) shows that these cell lines have specific mRNA, with the size of that present in myeloid cells. These results indicate that G-CSF operates in the microvascular endothelial cells by a mechanism related to the presence of a specific receptor.

Pyrimidine 5'-nucleotidase and oxidative damage in red blood cells transfused to beta-thalassemic children.

Pyrimidine 5' nucleotidase (P5'N) acquired deficiency has been found in several hematologic disorders including beta-thalassemia. Our previous studies suggested that the aldehydes produced during membrane lipid peroxidation could play a role in P5'N inactivation in thalassemia. To evaluate the effects of the thalassemic "environment" on transfused red blood cells, we tested P5'N, pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G-6PD) activity, creatine content, reduced glutathione (GSH) levels and the hexose monophosphate shunt (HMS) in the red cells of homozygous transfusion-dependent thalassemic children, immediately following and again one month after transfusion. In red cells aged in thalassemic plasma, P5'N activity, creatine level, GSH stability and stimulated HMS flux were significantly decreased. These results fit in with the presence in thalassemic plasma of molecules interfering with antioxidant red cell defenses. Normal red cells incubated in thalassemic plasma display a significant stimulation of the basal HMS (p less than 0.01). Transfused red cell metabolic alterations could be explained by the plasma pro-oxidant activity and may contribute to reducing red cell survival in the host plasma.

Pyrimidine 5'-nucleotidase acquired deficiency in beta-thalassemia: involvement of enzyme-SH groups in the inactivation process.

Pyrimidine 5'-nucleotidase (P5'N) partial deficiency has been described in several hematological disorders and also in the beta-thalassemic trait. To check if the P5'N deficiency in thalassemia was acquired we used thalassemic red cells (from either homo- or heterozygous subjects), whose P5'N activity was lower than in control cells. After separation on a density gradient, activity in lighter cells was similar to controls and less than 50% in denser cells. The most probable mechanism for this faster inactivation involves enzyme -SH groups modification by oxidation and reaction with monofunctional aldehydes produced by membrane lipid peroxidation. In vitro challenge of thiol enzymes as pyruvate kinase (PK), adenylate kinase (AK) and P5'N with increasing concentrations of GSSG, hexanal (HEX) and 4-hydroxynonenal (HNE), showed that HNE is the most powerful among the enzyme inhibitors tested and that P5'N activity is a more sensitive index of -SH groups damage, when compared to PK and AK.