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Biomedical subjects

S Migliaccio

Publications and source records attributed to S Migliaccio.

At least 19 recordsLinked to original sources
PubMed2005

Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment.

BACKGROUND: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). METHODS: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). RESULTS: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. CONCLUSIONS: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.

Adolescent↗
PubMed2004

Reduction of risk factors for cardiovascular diseases in morbid-obese patients following biliary-intestinal bypass: 3 years' follow-up.

BACKGROUND: Obese patients are often affected by hypertension, dyslipidaemia, impaired glucose metabolism, and suffer from cardiovascular disease (CVD), related to the characteristic metabolic alterations. AIM OF THE STUDY: To evaluate reduction of risk factors for CVDs in morbid-obese patients (body mass index (BMI)>40 kg/m2) after weight loss upon bariatric surgery intervention of biliary-intestinal bypass. SUBJECTS: 45 (17 men, 28 women) morbid-obese patients (age: 19-49 y, BMI>40 kg/m2). All patients were selected on the basis of medical history, physical and biochemical evaluation and of psychiatric tests, which were performed on all individuals admitted to our Day Hospital to verify the safety of surgical intervention. MEASUREMENTS: Body weight, body composition (by dual X-ray absorptiometry, DXA), blood pressure, lipid profile, fibrinogen and glucose metabolism were monitored at baseline and 1, 3, 6, 9, 12, 24 and 36 months after surgery. RESULTS: A significant and persistent weight loss was present in all patients at the end of the 3 y follow-up period (P<0.001), with a progressive reduction of total and trunk fat mass as evaluated by means of DXA. Additionally, a parallel significant reduction in systolic (P<0.001) and diastolic (P<0.001) blood pressure was observed. Total and LDL cholesterol were significantly reduced (P<0.001), while HDL showed no modifications; triglycerides declined progressively during the 3 y follow-up (P<0.001). Fibrinogen decreased from 364.5+/-82.4 to 266.4+/-45.7 mg/dl at the end of the period (P<0.001). Fasting glucose levels and glucose levels 120 min after an oral glucose tolerance test were reduced from 95.1+/-20.3 to 78.6+/-9.1 mg/dl (P<0.001) and from 116.9+/-34.7 to 77.6+/-15.5 mg/dl (P<0.001), respectively, at baseline and at the end of the study. Moreover, fasting insulin decreased from 30.0+/-20.4 to 8.6+/-2.9 microUI/ml (P<0.001) after 3 y, while insulin levels after (120 min) oral glucose load decreased from 105.5+/-61.5 to 12.0+/-6.0 microUI/ml (P<0.001). CONCLUSION: Our results show that biliary-intestinal bypass may represent a valid and alternative therapeutic approach in patients with morbid obesity since it induces a significant and stable reduction of body weight and obesity-related risk factors for CVD.

Adult↗
PubMed2003

Type II benign osteopetrosis (Albers-Schönberg disease) caused by a novel mutation in CLCN7 presenting with unusual clinical manifestations.

A 16-year-old male patient with type II autosomal dominant benign osteopetrosis (ADO) was genotyped and found to harbor a novel mutation in exon 25 of the gene encoding for the osteoclast-specific chloride channel, CLCN7, inherited from the father, who was asymptomatic. The patient had normal biochemical findings and acid-base balance, except for increased serum levels of creatine kinase, lactic dehydrogenase, and the bone formation markers bone alkaline phosphatase isoenzyme, osteocalcin and N-terminal type I collagen telopeptide/creatinine ratio. Unusual generalized osteosclerosis was observed together with a canonical increase in vertebral and pelvis bone mass. An affected first grade cousin presented with normal biochemical findings and a milder osteosclerotic pattern of the pelvis. At the cellular level, cultured osteoclasts from the patient showed increased motility, with lamellipodia, membrane ruffling and motile pattern of podosome distribution, all of which could have contributed to functional impairment of bone resorption. The present report documents a novel mutation of the CLCN7 gene causing osteopetrosis in a radiologically uncertain form of the diseases, with apparent incomplete penetrance.

Adolescent↗
PubMed2003

Isoflavones and skeletal health: are these molecules ready for clinical application?

A review of the recent literature on the effects of isoflavones was undertaken to determine whether molecules such as genistein and daidzein, aglycone derivatives of soybeans, might have benefit in the prevention and treatment of osteoporosis. The current standard for science-based medicine is the documentation of efficacy of an agent under controlled, randomized, prospective conditions. A few short clinical investigations have been undertaken using isoflavones (along with soy protein), but they had limitations in study design, and the numbers of women studied were small. Other evidence from animal models, in vitro experiments, and epidemiological reports suggest that the isoflavones have skeletal benefits in women with little or no ovarian estrogen production. A clear need exists for prospective human trials, using the required conditions of randomized clinical trials and designs, to satisfy objectively the needs for science-based medicine and for appropriate clinical applications.

Animals↗
PubMed2003

Pyrrolopyrimidine c-Src inhibitors reduce growth, adhesion, motility and invasion of prostate cancer cells in vitro.

Two bona fide c-Src inhibitors, denominated CGP77675 and CGP76030, reduced in a time- and concentration-dependent manner (i) the proliferation of the PC3 prostate carcinoma cell line, as assessed by the [3H]-thymidine incorporation test, (ii) the capacity of PC3 cells to adhere and spread on Matrigel substrate, as determined by crystal violet staining, (iii) the ability of PC3 cells to migrate through a gelatine boundary and invade a Matrigel substrate. The latter effect was not due to a decrease of urokinase-type plasminogen activator (uPA), nor of metalloproteinase-2 (MMP-2) activities. The MMP-9 activity, along with the expression of the Tissue Inhibitor of Metalloproteinases (TIMP)-1 and TIMP-2, were reduced by the two inhibitors, consistent with the ability of c-Src to enhance MMP-9 and TIMP expression levels. Collectively, these data demonstrate that the pyrrolopyrimidine-derived c-Src inhibitors significantly reduced PC3 cell activities associated with their malignant phenotype.

Antineoplastic Agents↗
PubMed2002

The role of the alphaVbeta3 integrin in the development of osteolytic bone metastases: a pharmacological target for alternative therapy?

Common cancers frequently develop bone metastases, which are often osteolytic in nature due to activation of osteoclast differentiation and bone resorption. This may result from direct stimulation of these cells by the metastasis, or may be due to indirect enhancement of osteoclast activity by osteoblasts. A further feature of the bone metastasis is an extensive medullary angiogenesis which supports tumor growth. The alphaVbeta3 integrin is highly expressed in bone metastatic cells, as well as in osteoclasts and in the activated endothelium, where it plays a major role in cell function. In contrast, this receptor is barely expressed in other cell types. Our hypothesis is that inhibition of this mechanism, which is not widespread in most tissues and at the same time is common to several steps of cancer-induced osteolysis (i.e., homing, growth, and survival of metastatic cells, osteoclast bone resorption, and angiogenesis), should represent a suitable target to block the development of bone spreading of metastatic tumors. We extend this hypothesis to downstream signalling molecules activated by ligation of the alphaVbeta3 integrin, some of which (i.e., Src, PYK2, and Shc) could have similar specific roles in tumor cells, activated endothelium and osteoclasts, but not in other cell types.

Antineoplastic Agents↗
PubMed2002

The selective estrogen receptor modulator raloxifene regulates osteoclast and osteoblast activity in vitro.

Raloxifene is a selective estrogen receptor modulator (SERM) that prevents bone loss. Although it is largely used for the treatment of osteoporosis, the mechanisms by which this compound modulates the activity of bone cells are still poorly understood. In this study we investigate whether raloxifene affects osteoclast and osteoblast activity in vitro. Bone marrow cultures were established from neonatal mice and treated with 1,25(OH)(2) vitamin D(3) (VitD(3), 10(-8) mol/L) to induce osteoclast generation. Similar to 17beta-estradiol, raloxifene significantly reduced the number of osteoclasts in a concentration-dependent manner, with maximal inhibition at 10(-11) mol/L (-48%). However, as for 17beta-estradiol, at a high concentration (10(-7) mol/L), the inhibitory effect of raloxifene was abolished. In a pit assay, raloxifene inhibited bone resorption. A maximal effect was observed at 10(-9) mol/L, and maintained at a high concentration, indicating that inhibition of osteoclast formation and inhibition of bone resorption may be due to activation of, at least in part, different pathways. Osteoblasts from neonatal mice calvariae were also exposed to raloxifene. In these cells, this compound induced a concentration-dependent increase of proliferation, which was blocked by the estrogen-receptor antagonist ICI 164,384. Raloxifene also increased the osteoblast-specific transcription factor Cbfa1/Runx2 and alpha2 procollagen type I chain mRNAs, with a pattern that only partially coincided with that of 17beta-estradiol. Consistent with decreased osteoclastogenesis, raloxifene inhibited the mRNA expression of interleukin (IL)-1beta and IL-6 at a low concentration, but not at a high concentration, whereas 17beta-estradiol had similar effects on IL-6 and inhibited IL-1beta at both concentrations. Furthermore, both compounds were able to inhibit tumor necrosis factor (TNF)-alpha-induced IL-1beta, but not IL-6, increase. In conclusion, these data show that raloxifene negatively modulates osteoclasts, and positively affects osteoblasts, suggesting not only an antiresorptive role, but also an osteoblast stimulatory role.

Animals↗
PubMed2001

Melanoma cells stimulate osteoclastogenesis, c-Src expression and osteoblast cytokines.

Malignant melanomas metastasise to the bone and enhance osteoclast bone resorption. We demonstrated that a 48-h-B16 melanoma cell conditioned media (B16CM) induced osteoclastogenesis in mouse bone marrow cultures, without the requirement of B16 cell-bone marrow cell co-culture. B16 cells transcriptionally expressed detectable levels of TGFbeta1, IL-6, M-CSF, GM-CSF and TNFalpha mRNAs, albeit to a lower extent compared with levels in osteoblasts, and failed to express PTHrP, OPGL, OPG and IL-1beta. Interestingly, B16CM greatly upregulated IL-1beta, IL-6 and GM-CSF, and modestly enhanced TNFalpha and OPGL mRNA expression in osteoblasts, suggesting a potential indirect stimulation of osteoclastogenesis via the osteogenic lineage. B16CM barely upregulated c-Fos, but strongly and time-dependently enhanced c-Src expression in the total bone marrow cultures during osteoclast differentiation. Moreover, c-Src expression was enhanced in differentiated and purified osteoclast preparations to higher levels than in stromal cells. In conclusion, melanoma induces osteoclast generation with a paracrine mechanism independent of cell-cell contact, specifically upregulating c-Src in osteoclasts and cytokine expression in osteoblasts.

Animals↗
PubMed2001

Reduced growth and skeletal changes in zinc-deficient growing rats are due to impaired growth plate activity and inanition.

We investigated the effects of dietary zinc deficiency on skeletal metabolism in an animal model. Thirty 21-d-old male Sprague-Dawley rats were fed for 28 d either a zinc-deficient (ZD) diet (1 mg zinc/kg) or a normal diet ad libitum (AL, 50 mg zinc/kg) or in the same quantity as the ZD (pair-fed, PF). Only in the ZD group were general physical signs of zinc deficiency observed. Compared with the AL and PF rats, ZD rats showed significantly lower mean values in ponderal growth rate, femur weight and length, circulating levels of insulin-like growth factor-I, bone mechanical properties and concentration of zinc and, on histomorphometry, a decrease in the thicknesses of the overall growth plate and hypertrophic cartilage. In contrast, although bone volume was significantly lower in the ZD and PF rats than in the AL rats, no difference was observed between the ZD and PF rats. Osteoclast surface/bone surface and osteoclast number/bone surface ratios were significantly greater in PF rats than in the other two groups and not different in ZD and AL rats. Collectively, these data indicate that zinc deficiency has profound effects on the skeletal system of growing rats. In particular, the effects of zinc deficiency on bone growth and mass are the result of the reduced activity of the growth plate, likely mediated by impairment in the insulin-like growth factor-I system. We did not demonstrate an effect on bone mass via increased bone resorption.

Animals↗
PubMed2001

Apparent cure of a newborn with malignant osteopetrosis using prednisone therapy.

A newborn girl with hemorrhagic purpura, suspected neonatal sepsis, and pale and dry skin was lethargic with remarkable hepatosplenomegaly, convergent strabismus, severe anemia, and elevated alkaline phosphatase activity. Radiographs showed a generalized increase in bone density, small medullary cavities, sclerosis of the skull and vertebrae, transverse wavy stripes of sclerotic bone in the metaphyses, and bone-in-bone appearance in phalanges of hands and feet. On this basis, she was diagnosed with malignant infantile osteopetrosis. On the first day of life, the infant was given a blood transfusion and vitamin K (1 mg intravenously [iv]). Corticosteroid therapy was started with prednisone (2 mg/kg per day). She showed marked improvement of symptoms. On the 26th day and 42nd day of life, she received additional blood transfusions. On the 49th day, the patient was discharged and corticosteroid therapy was continued at a regimen of 5 mg/day. Subsequent blood sample analyses revealed normal values for age. At 1 year of life, a bone marrow sample showed normal white and red cell lineages. X-ray confirmed attenuation of the bone sclerosis; therefore, bone marrow transplantation (BMT) was not implemented. At the age of 1.5 years, prednisone therapy was discontinued gradually and withdrawn before the age of 2 years. Subsequent follow-up showed normalization of all radiological and hematologic parameters. At present, the patient is 3 years old and appears healthy with apparently complete regression of the disease.

Ankle↗
PubMed2000

Decreased c-Src expression enhances osteoblast differentiation and bone formation.

c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src(-/-) relative to Src(+/+) mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by approximately 60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src(-/-) osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.

Alkaline Phosphatase↗
PubMed2000

Qualitative assessment of natural apatite in vitro and in vivo.

Among the natural and synthetic materials investigated as bone graft substitutes, much interest has been focused on natural apatite obtained from low temperature heat-deproteinated compact bone. Previous research demonstrates that, when treated at a temperature below 500 degrees C, this material maintains its characteristic ultrastructural features, with a high surface/volume ratio, while as an implant material, it offers the host tissue a large surface of interaction. In vitro and in vivo tests showed that natural apatite is well tolerated and is a good osteoconducing material. The present in vivo study in rabbits was carried out to first investigate the behavior and capacity of natural apatite implants to stimulate bone ingrowth, and then to analyze the cells located at the bone/material interface. Synthetic hydroxyapatite was used as a control material. In a parallel in vitro study, we investigated the activity of differentiated osteoblasts and periosteal cells obtained from rats and new-born rabbits, incubated with natural apatite and synthetic hydroxyapatite. The in vivo study showed that natural apatite allows osteoblasts to form new bone tissue, adhering to the implant with ingrowth into the implant structure. In the presence of synthetic hydroxyapatite, a less pronounced osteoblastic activity was observed. In agreement with these observations, the in vitro study showed that natural apatite is more effective in attracting cells, favoring their proliferation and stimulating alkaline phosphatase activity. These findings suggest that natural apatite is more suitable for bone filling or bone regeneration than synthetic hydroxyapatite.

Animals↗
PubMed2000

Transient estrogen exposure of female mice during early development permanently affects osteoclastogenesis in adulthood.

Estrogens modulate bone tissue turnover in both experimental animal models and postmenopausal women. Our previous studies have shown that exposure to diethylstilbestrol (DES) during the perinatal period increases peak bone mass in female mice in adulthood. We investigated whether developmental DES exposure can influence bone mass by affecting osteoclastogenesis. Female mice were injected with 100 microg/kg body weight DES from days 9-16 of gestation or, alternatively, pups received neonatal injections of 2 microg of DES from days 1-5 of life. Animals were weaned at 21 days of age and effects of estrogen on bone cells were evaluated in adulthood. A significant increase in bone mass in female mice was already observed at 2 months, with a maximal effect in older animals. Bone sections from DES-treated animals showed a significant decrease in osteoclast number and tartrate-resistant acid phosphatase (TRAP) enzymatic activity as compared with controls. To verify the importance of the estrogen surge at puberty in this event, a group of control and DES-treated mice were ovariectomized at 17 days to prevent puberty, and potential effect on osteoclastic cells was evaluated in adulthood. As expected, ovariectomy induced an increase of TRAP-positive cells. DES treatment blunted the ovariectomized-dependent increase of the total number of osteoclastic cells, suggesting a role of developmental DES exposure in the process of bone-cell imprinting. Our data indicate, for the first time, that transient changes in estrogen levels during development modulate bone turnover and osteoclastogenesis likely participating in bone-cell imprinting during early phases of bone development, and that this effect could be induced by direct alteration of bone microenvironment.

Animals↗
PubMed1999

Decreased proliferation and altered differentiation in osteoblasts from genetically and clinically distinct craniosynostotic disorders.

Craniosynostoses are a heterogeneous group of disorders characterized by premature fusion of cranial sutures. Mutations in fibroblast growth factor receptors (FGFRs) have been associated with a number of such conditions. Nevertheless, the cellular mechanism(s) involved remain unknown. We analyzed cell proliferation and differentiation in osteoblasts obtained from patients with three genetically and clinically distinct craniosynostoses: Pfeiffer syndrome carrying the FGFR2 C342R substitution, Apert syndrome with FGFR2 P253R change, and a nonsyndromic craniosynostosis without FGFR canonic mutations, as compared with control osteoblasts. Osteoblasts from craniosynostotic patients exhibited a lower proliferation rate than control osteoblasts. P253R and nonsyndromic craniosynostosis osteoblasts showed a marked differentiated phenotype, characterized by high alkaline phosphatase activity, increased mineralization and expression of noncollagenous matrix proteins, associated with high expression and activation of protein kinase Calpha and protein kinase Cepsilon isoenzymes. By contrast, the low proliferation rate of C342R osteoblasts was not associated with a differentiated phenotype. Although they showed higher alkaline phosphatase activity than control, C342R osteoblasts failed to mineralize and expressed low levels of osteopontin and osteonectin and high protein kinase Czeta levels. Stimulation of proliferation and inhibition of differentiation were observed in all cultures on FGF2 treatment. Our results suggest that an anticipated proliferative/differentiative switch, associated with alterations of the FGFR transduction pathways, could be the causative common feature in craniosynostosis and that mutations in distinct FGFR2 domains are associated with an in vitro heterogeneous differentiative phenotype.

Acrocephalosyndactylia↗
PubMed1999

Mechanisms of osteoclast dysfunction in human osteopetrosis: abnormal osteoclastogenesis and lack of osteoclast-specific adhesion structures.

Osteoclasts from a patient affected by osteopetrosis were examined in vivo and in vitro. Iliac crest biopsy revealed an osteosclerotic pattern, with prominent numbers of osteoclasts noted for hypernuclearity and incomplete adherence to the bone surface. A population comprising tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated and mononuclear cells, and alkaline phosphatase-positive stromal fibroblasts was obtained in vitro from bone marrow. Mononuclear TRAP-positive precursors spontaneously fused in culture to form giant osteoclast-like cells. These cells expressed the osteoclast marker MMP-9 and calcitonin receptor, and lacked the macrophage marker, Fc receptor. Expression and distribution of c-src, c-fms, and CD68, and response to steroid hormones relevant to osteoclast differentiation and function were apparently normal, whereas cell retraction in response to calcitonin was impaired. TRAP-positive multinucleated cells did not form osteoclast-specific adhesion structures (clear zone, podosomes, or actin rings). Bone resorption rate was severely reduced in vitro. Focal adhesions and stress fibers were observed en lieu of podosomes and actin rings. Adhesion structures contained low levels of immunoreactive vitronectin receptor, most of this integrin being retained in cytoplasmic vesicles. These data provide the first characterization of abnormal differentiation and function of human osteopetrotic osteoclast-like cells.

Acid Phosphatase↗