Usha Bava

Effects of calcium supplementation on serum lipid concentrations in normal older women:: A randomized controlled trial

PURPOSE To determine the effect of supplementation with calcium citrate on circulating lipid concentrations in normal older women. SUBJECTS AND METHODS As part of a study of the effects of calcium supplementation on fractures, we randomly assigned 223 postmenopausal women (mean [+/- SD] age, 72 +/- 4 years), who were not receiving therapy for hyperlipidemia or osteoporosis, to receive calcium (1 g/d, n = 111) or placebo (n = 112) for 1 year. Fasting serum lipid concentrations, including high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol, were obtained at baseline, and at 2, 6, and 12 months. RESULTS After 12 months, HDL cholesterol levels and the HDL cholesterol to LDL cholesterol ratio had increased more in the calcium group than in the placebo group (mean between-group differences in change from baseline: for HDL cholesterol, 0.09 mmol/L (95% confidence interval [CI]: 0.02 to 0.17; P = 0.01); for HDL/LDL cholesterol ratio, 0.05 (95% CI: 0.02 to 0.08; P = 0.001). This was largely due to a 7% increase in HDL cholesterol levels in the calcium group, with a nonsignificant 6% decline in LDL cholesterol levels. There was no significant treatment effect on triglyceride level (P = 0.48). CONCLUSION Calcium citrate supplementation causes beneficial changes in circulating lipids in postmenopausal women. This suggests that a reappraisal of the indications for calcium supplementation is necessary, and that its cost effectiveness may have been underestimated.

Effects of calcitonin, amylin, and calcitonin gene-related peptide on osteoclast development

Amylin and calcitonin gene-related peptide (CGRP) are homologous 37 amino acid peptides that are found in the circulation. Both peptides belong to the calcitonin family. Similar to calcitonin, amylin and CGRP inhibit osteoclast activity, although they are much less potent than calcitonin. Calcitonin is known to act on the latter stages of osteoclast development, inhibiting the fusion of committed preosteoclasts to form mature multinucleated cells; however, whether or not calcitonin acts earlier in the formation of the precursor osteoclasts is controversial. The question of osteoclast development has never been examined with respect to amylin and CGRP. These issues are addressed in the present study. We studied the effects of calcitonin (salmon and rat), amylin (human and rat), and CGRP (human and rat) in mouse bone marrow cultures stimulated to generate osteoclasts using 1alpha,25-dihydroxyvitamin D3. Calcitonin dose-dependently decreased the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells as well as TRAP-positive mono-/binucleated cells at concentrations >10(-13) mol/L. Amylin and CGRP showed similar effects at concentrations >10(-9) mol/L. In addition, calcitonin substantially reduced the ratio of TRAP-positive multinucleated to mono-binucleated cells, indicating an effect on fusion of osteoclast precursors. The present data establish that this family of peptides not only acts on mature osteoclasts but also inhibits their development in bone marrow cultures. This activity is shared by amylin and CGRP. The much greater potency of calcitonin than amylin and CGRP is consistent with the action of these peptides being mediated by calcitonin receptors.

Differential Gene Expression in Cultured Osteoblasts and Bone Marrow Stromal Cells From Patients With Paget's Disease of Bone†

Pagets disease is a focal condition of bone. To study changes in cells within pagetic lesions, we cultured osteoblasts and stromal cells from 22 patients and compared gene expression in these cells to cells from healthy bone. We identified several differentially regulated genes, and we suggest that these changes could lead to the formation of the lesions.

Failure to Detect Measles Virus Ribonucleic Acid in Bone Cells from Patients with Paget’s Disease

BACKGROUND Pagets disease is a condition of focal accelerated bone turnover. Electron-microscopy investigations of osteoclasts from pagetic lesions have identified nuclear inclusion bodies that have a similar appearance to viral nucleocapsid particles. Subsequently, RNA from several paramyxoviruses has been detected in pagetic tissue, and it was suggested that these viruses, in particular measles, might play a role in the etiology of Pagets disease. We have tested for measles virus sequences in osteoblasts and bone marrow cells collected from pagetic lesions and healthy bone. METHODS Bone and bone marrow samples were taken from Pagets patients and control subjects, and cells were cultured from each of these tissues. RNA was extracted from 13 osteoblast cultures and 13 cultures of bone marrow cells derived from pagetic lesions, and from 26 and 23 control osteoblast and bone marrow cultures, respectively. These samples were sourced from 22 patients with Pagets disease and 31 controls. RT-PCR-nested PCR amplification was used for the detection of the genes for the measles nucleocapsid and matrix proteins. RESULTS Measles virus sequences were not detected in any of the pagetic or control samples. However, measles virus sequences were identified in samples of a measles virus culture isolate included as a positive control, and in a brain sample from a patient with subacute sclerosing panencephalitis, a condition associated with chronic measles infection. CONCLUSION The results of the study do not support the hypothesis that measles virus plays a role in the pathogenesis of Pagets disease.

Bone-bound bisphosphonate inhibits growth of adjacent non-bone cells.

The conventional view of the mode of action of bisphosphonates is that they are taken up by bone surfaces and then ingested by bone-resorbing osteoclasts, the activity of which they inhibit through their actions on the enzyme, farnesyl pyrophosphate (FPP) synthase. This model suggests that these compounds should only have effects on osteoclasts, and does not provide an explanation for their other actions, such as the epithelial abnormalities seen in osteonecrosis of the jaw, and their possible prolongation of disease-free survival in some malignancies. The present studies set out to determine whether cells other than osteoclasts are affected by bone-bound bisphosphonates. Bone slices were incubated overnight in PBS or in solutions of bisphosphonates (100 μM), washed, then transferred to 96-well plates (1 slice/well). Cells from 2 cell lines were seeded onto the bone slices: Caco-2 human colorectal adenocarcinoma epithelial cells and Chinese hamster ovary (CHO) cells. Cell proliferation (cell numbers and thymidine incorporation) was assessed at 4-72 h. Cell adhesion at 4 h was normal on bone slices pre-treated with bisphosphonates, but there were progressive reductions in cell numbers from 48 h and even greater reductions in thymidine incorporation from 24 h (>90% with zoledronate at 72 h). Growth inhibition was related to the clinical potency of the bisphosphonate used. There was no evidence of increased apoptosis in cells grown on bisphosphonate-coated bone, but levels of unprenylated Rap1A were increased, indicating inhibition of FPP synthase. Similar growth inhibition was observed in primary cultures of rat osteoblasts on bone, indicating that this was not specific to transformed cells. It is concluded that bisphosphonates bound to a bone surface can act on adjacent non-bone cells and inhibit their growth. This greatly widens the range of potential target cells for these drugs.

Absence of Somatic SQSTM1 Mutations in Paget’s Disease of Bone

BACKGROUND Pagets disease is a common focal bone disorder that appears to be caused by a combination of genetic and environmental factors. Mutations in the SQSTM1 gene are found in about one third of families with Pagets disease and 8% of sporadic cases. Other potential loci linked to the disease have also been identified, and a number of environmental factors have been suggested to be involved in the disease. However, the focal nature of Pagets is still unexplained. Therefore, we examined the possibility that somatic mutations in the SQSTM1 gene are present in the local lesions, using RNA collected from primary osteoblast and bone marrow cell cultures of patients with this condition. METHODS SQSTM1 was sequenced, and allelic discrimination for the common P392L mutation was performed in cDNA samples from 14 osteoblast cultures and from 14 cultures of bone marrow cells. RESULTS In these 28 samples drawn from 23 patients, the wild-type sequence of SQSTM1 was found in all but one marrow sample, which was heterozygous for the P392L mutation. DNA from peripheral blood in this subject had an identical sequence of SQSTM1, indicating that this was a germline mutation. CONCLUSION We conclude that somatic mutations for SQSTM1 are not commonly present in Pagets disease.

Alteration of bone cell function by RANKL and OPG in different in vitro models

Background  Receptor activator of nuclear factor‐κB ligand (RANKL) and osteoprotegerin (OPG) are well‐documented potent regulators of osteoclast development. However, their effects in mature bone cells and in organ cultures have not been well studied. It is uncertain whether their activities in different experimental models are comparable.

Tyrosine Kinase Inhibitors Regulate OPG through Inhibition of PDGFRβ

Nilotinib and imatinib are tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). In vitro, imatinib and nilotinib inhibit osteoclastogenesis, and in patients they reduce levels of bone resorption. One of the mechanisms that might underlie these effects is an increase in the production of osteoprotegerin (OPG). In the current work we report that platelet-derived growth factor receptor beta (PDGFRβ) signaling regulates OPG production in vitro. In addition, we have shown that TKIs have effects on RANKL signaling through inhibition of the PDGFRβ and other target receptors. These findings have implications for our understanding of the mechanisms by which TKIs affect osteoclastogenesis, and the role of PDGFRβ signaling in regulating osteoclastogenesis. Further studies are indicated to confirm the clinical effects of PDGFRβ-inhibitors and to elaborate the intracellular pathways that underpin these effects.