Gian Piero Pescarmona

Estrogen deficiency increases osteoclastogenesis up-regulating T cells activity: A key mechanism in osteoporosis

Compelling evidences suggest that increased production of osteoclastogenic cytokines by activated T cells plays a relevant role in the bone loss induced by estrogen deficiency in the mouse. However, little information is available on the role of T cells in post-menopausal bone loss in humans. To investigate this issue we have assessed the production of cytokines involved in osteoclastogenesis (RANKL, TNFalpha and OPG), in vitro osteoclast (OC) formation in pre and post-menopausal women, the latter with or without osteoporosis. We evaluated also OC precursors in peripheral blood and the ability of peripheral blood mononuclear cells to produce TNFalpha in both basal and stimulated condition by flow cytometry in these subjects. Our data demonstrate that estrogen deficiency enhances the production of the pro-osteoclastogenetic cytokines TNFalpha and RANKL and increases the number of circulating OC precursors. Furthermore, we show that T cells and monocytes from women with osteoporosis exhibit a higher production of TNFalpha than those from the other two groups. Our findings suggest that estrogen deficiency stimulates OC formation both by increasing the production of TNFalpha and RANKL and increasing the number of OC precursors. Women with post-menopausal osteoporosis have a higher T cell activity than healthy post-menopausal subjects; T cells thus contribute to the bone loss induced by estrogen deficiency in humans as they do in the mouse.

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. Copyright

Spontaneous osteoclast formation from peripheral blood mononuclear cells in postmenopausal osteoporosis

Osteoclasts are cells involved in bone reabsorbing and hence in postmenopausal bone loss. There is no evidence of increased in vitro spontaneous osteoclast formation in postmenopausal osteoporosis. The aim of our study was to evaluate spontaneous osteoclastogenesis in osteoporosis. Bone mineral density, markers of bone turnover, and cultures of peripheral blood mononuclear cells (PBMC) on dentine slices with or without the addition of 1,25‐OH vitamin D3 ([10−8 M]) were obtained from 18 osteoporotic women and 15 controls. To verify cytokine production by PBMC cultures, supernatants were collected on days 3 and 6 and tested for TNF‐α and RANKL. The data obtained were compared between patients and controls by one‐way ANOVA and correlated by Pearsons coefficient. We found a significant increase in osteoclast formation and bone reabsorbing activity in patients with respect to controls; in addition, the production of TNF‐α and RANKL is significantly higher in patients. Furthermore, osteoclast number is inversely correlated with bone mineral density and directly with RANKL in culture supernatants. Our data demonstrated an increased spontaneous osteoclastogenesis in women affected by postmenopausal osteoporosis: this increase may be explained by the higher production of TNF‐α and RANKL by PBMC cultures of osteoporotic patients.

Risedronate reduces osteoclast precursors and cytokine production in postmenopausal osteoporotic women

This paper studies the effect of oral risedronate on osteoclast precursors, osteoclast formation, and cytokine production in 25 osteoporotic women. Risedronate is effective in reducing the number of osteoclast precursors, their formation, vitality, and activity and the level of RANKL and TNF‐α in cultures.

Role of iron metabolism and oxidative damage in postmenopausal bone loss

It has been suggested that iron-deficient rats have lower bone mass than iron-replete animals, but a clear association between bone and iron repletion has not been demonstrated in humans. A growing body of evidences also suggests a relation between lipid oxidation and bone metabolism and between iron metabolism and LDL oxidation. Iron availability to cells also depends on haptoglobin (Hp) phenotypes. Hp has also important antioxidant properties according to its phenotype, hence we evaluate whether Hp phenotype could influence bone density, iron metabolism and lipid oxidation. This cross-sectional study enrolled 455 postmenopausal women affected by osteoporosis (260) or not (195). Bone mineral density, markers of bone and iron metabolism, levels of oxidized LDL (oxLDL) and Hp phenotype were measured in all the subjects. Hp 1.1 and 2.2 frequency was higher and Hp 2.1 was lower in the patients with fragility fractures (80) compared with the controls. We therefore evaluate different Hp phenotypes as risk or protective factors against fragility fracture: Hp 2.1 is a protective factor against fracture while 1.1 is an important and 2.2 a moderate risk factor for fragility fractures. Lower serum iron was associated with elevated transferrin in patients with Hp 1.1; moreover patients had relative iron deficiency compared with the controls and fractured patients had higher level of oxLDL. We found that both iron metabolism and oxLDL varies according to Hp phenotypes and are predictive of bone density. Our data indicate that Hp 2.1 is a protective factor for fragility fractures, depending on its role on iron metabolism and its antioxidant properties.

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

SummaryThis 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.IntroductionFragility 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.MethodsWe 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 β (TGFβ), 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.ResultsWe found an increase of RANKL/OPG ratio, M-CSF, SOST and DKK-1 in fractured patients, whereas TGFβ was increased in osteoarthritic bone. Bone marrow produced greater amounts of RANKL, M-CSF and TGFβ compared to bone, whereas the production of DKK-1 and SOST was higher in bone.ConclusionsWe 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.

Microparticles in physiological and in pathological conditions.

Chronic diseases pose a severe burden to modern National Health Systems. Individuals nowadays have a far more extended lifespan than in the past, but healthy living was only scantily extended. As much as longer life is desirable, it is saddened by chronic diseases and organ malfunctions. One contributor to these problems was recognized to be represented by microparticles (MPs). Our purpose is to better understand MPs, to contrast their ominous threat and possible clinical importance. For this intent we correlated MPs with thrombotic pathologies, hemophilia, malaria, diabetes, cardiovascular diseases, endothelial dysfunctions, pulmonary hypertension, ischemic stroke, pre‐eclampsia, rheumatologic diseases—rheumatoid arthritis, polymyositis—dermatomyositis, angiogenesis and tumor progression—cancer; we listed the possibilities of using them to improve transfusion methods, as a marker for acute allograft rejection, in stem cell transplantation, as neuronal biomarkers, to understand gender‐specific susceptibility for diseases and to improve vaccination methods and we presented some methods for the detection of MPs. Copyright

Long-term exposure to dehydroepiandrosterone affects the transcriptional activity of the glucocorticoid receptor

Although the antiglucocorticoid effects of dehydroepiandrosterone (DHEA) have been demonstrated in vivo in many systems, controversial results have been reported by in vitro studies. In order to elucidate the long-term antiglucocorticoid effects of DHEA in vitro in a context more physiological than what proposed by previous works, we set up a system consisting of a carcinoma cell line relying on endogenously produced glucocorticoid receptor (GR) and stably expressing a reporter gene ErbB-2 under the control of a GR-dependent MMTV promoter. These cells grown in presence of low levels of serum glucocorticoids (GC) showed a basal translocation and activity of endogenous GR. The cells reacted to high concentrations of dexamethasone increasing GR nuclear import, although down-regulating receptor expression, and enhancing GR-dependent transcriptional activity, as shown by EMSA assay and expression of the reporter gene ErbB-2. The response to GC was also functional since the increase of ErbB-2 boosted cellular growth. On the contrary, 72h of incubation with DHEA diminished basal GR-dependent reporter expression and abated cellular proliferation. Analysing molecular mechanisms responsible for this failed transcriptional activity, upon prolonged treatment with DHEA we observed a slow nuclear import of GR not followed by its recruitment to DNA. These data add novel information about the long-term effects of DHEA in vitro.

Synergistic effect of retinoic acid and dehydroepiandrosterone on differentiation of human neuroblastoma cells

Retinoic acid (RA) affects many cell types by either promoting their survival or inducing their differentiation. Dehydroepiandrosterone (DHEA), a precursor for both androgenic and estrogenic steroids and abundantly produced by brain, is known as an inhibitor of cell proliferation. Differentiation of a human neuroblastoma cell line (SK‐N‐BE) was evaluated measuring growth rate, motility, neurite extension and GAP‐43 expression. We report that DHEA enhances the differentiating effect of RA on neuroblastoma cells via a signalling that is not RA receptor‐mediated. Instead, we show a differential expression of matrix metalloproteinases: RA enhances the activity of MMP‐2, whereas MMP‐9 expression is up‐regulated by DHEA. The concerted modulation of these proteinases may support the neurite outgrowth observed after co‐treatment with the two drugs.

Pluripotent plasticity of stem cells and liver repopulation

Different types of stem cells have a role in liver regeneration or fibrous repair during and after several liver diseases. Otherwise, the origin of hepatic and/or extra‐hepatic stem cells in reactive liver repopulation is under controversy. The ability of the human body to self‐repair and replace the cells and tissues of some organs is often evident. It has been estimated that complete renewal of liver tissue takes place in about a year. Replacement of lost liver tissues is accomplished by proliferation of mature hepatocytes, hepatic oval stem cells differentiation, and sinusoidal cells as support. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types of epithelial cells found in the liver, hepatocytes, and bile ductular cells. In gastroenterology and hepatology, the first attempts to translate stem cell basic research into novel therapeutic strategies have been made for the treatment of several disorders, such as inflammatory bowel diseases, diabetes mellitus, celiachy, and acute or chronic hepatopaties. In the future, pluripotent plasticity of stem cells will open a variety of clinical application strategies for the treatment of tissue injuries, degenerated organs. The promise of liver stem cells lie in their potential to provide a continuous and readily available source of liver cells that can be used for gene therapy, cell transplant, bio‐artificial liver‐assisted devices, drug toxicology testing, and use as an in vitro model to understand the developmental biology of the liver. Copyright