Long-term nocturnal calcium infusions can cure rickets and promote normal mineralization in hereditary resistance to 1,25-dihydroxyvitamin D.

We report the beneficial effects of calcium infusions in a child with hereditary resistance to 1,25(OH)2D and alopecia. This patient after transient responsiveness to vitamin D derivatives became unresponsive to all therapy despite serum 1,25(OH)2D concentrations maintained at levels approximately 100-fold normal. A 7-mo trial with calcium infusions led to correction of biochemical abnormalities and healing of rickets. Bone biopsies (n = 3) showed a normal mineralization and the disappearance of the osteomalacia. Cultures of bone-derived cells demonstrated a lack of activation of 25-hydroxyvitamin D 24-hydroxylase and osteocalcin synthesis by 1,25(OH)2D3 (10(-9) and 10(-6) M). These results demonstrate that even in the absence of a normal 1,25(OH)2D3 receptor-effector system in bone cells, normal mineralization can be achieved in humans if adequate serum calcium and phosphorus concentrations are maintained; and calcium infusions may be an efficient alternative for the management of patients with this condition who are unresponsive to large doses of vitamin D derivatives.

Cultured circulating mononuclear cells from osteopetrotic infants express the osteoclast-associated vitronectin receptor and form multinucleated cells in response to 1,25-dihydroxyvitamin D3.

Malignant osteopetrosis is characterized by impaired osteoclast activity. Osteoclasts derive from hematopoietic stem cells. In osteopetrosis, marrow cavities fail to develop, resulting in extramedullary hematopoiesis and the presence of stem cells in the bloodstream. Resistance to 1,25-(OH)2D3 may be involved in the pathogenesis of the disease. Sensitivity to 1,25-(OH)2D3, calcitonin sensitivity, and expression of the osteoclast-associated vitronectin receptor (VR) was examined in cultures of circulating mononuclear cells of seven osteopetrotic infants (1.5-6 months old). Since peripheral blood from age-matched children contains few stem cells, umbilical cord blood was used as control. Mononucleated cells were isolated by the Ficoll-Hypaque method and cultured (10(6) cells per ml) in alpha-MEM containing 20% horse serum in presence or absence of added 1,25-(OH)2D3. VR was identified by immunochemical staining with MAb 23C6. 1,25-(OH)2D3 at 10(-8) M significantly stimulated the formation of multinucleated cells (MNC) in cultures from all osteopetrotic patients and cord blood samples. Cells from three of five patients responded to 10(-9) M 1,25-(OH)2D3, the minimal stimulatory concentration for cord blood. Salmon calcitonin (100 ng/ml) partially inhibited the 10(-8) M 1,25-(OH)2D3-induced MNC formation in cultures from three of six patients and in cultures of all cord blood samples. In both types of cultures mononuclear cells and MNC cross-reacted with MAb 23C6, and 1,25-(OH)2D3 concentration did not influence the number and percentage of these cells. This study does not support the hypothesis of 1,25-(OH)2D3 resistance in osteopetrotic infants and shows that mononuclear cells expressing VR, possibly osteoclast progenitors, develop in cultures of circulating mononuclear cells from these infants. 1,25-(OH)2D3 may not be closely involved in VR expression.

Mineral metabolism in infants with malignant osteopetrosis: heterogeneity in plasma 1,25-dihydroxyvitamin D levels and bone histology.

A group of 16 infants, 2 weeks to 11 months old, with malignant osteopetrosis were investigated to examine their vitamin D metabolism and parathyroid function. Bone biopsies from 6 children were studied by light microscopic histomorphometry and by electron microscopy. Considerable heterogeneity existed among the patients with respect to the parameters reflecting mineral metabolism and with respect to the histological manifestations of the disease. The most constant findings were as follows. Immunoreactive parathyroid hormone (iPTH) was elevated in all children, except in 1 patient who had tubular acidosis, and plasma calcium was low or normal, suggesting skeletal resistance to PTH. Plasma 1,25-dihydroxyvitamin D [1,25-(OH)2D] was not constantly elevated and appeared to depend on plasma phosphorus, as both parameters were negatively correlated (r = 0.704, p less than 0.01). Osteoblast activity, as evaluated by circulating alkaline phosphatase and osteocalcin and osteoblast number, measured for 6 children by bone histology, were not increased, despite hyperparathyroidism, suggesting PTH resistance or defective osteoblasts. Osteoclasts could be detected in 5 of the 6 children who had a biopsy. Osteoclast number (5.7-13.3% of bone surface) was normal or mildly increased, and marrow spaces were relatively well developed in 4 patients, whereas 1 child had markedly increased osteoclast number (28.3% of bone surface) and reduced marrow cavities. These 5 children received transplants, and engraftment occurred in all, except in the "hyperosteoclastic" patient. Further studies are necessary to establish the prognostic significance of this histologic feature.

Rac-GTPase, osteoclast cytoskeleton and bone resorption.

The members of the Rho-GTPase subfamily, Rac1 and Rac2, are intimately involved in the organization of the cytoskeleton, and the p21-activated kinases or PAKs are targets of these proteins. Rac1 and Rac2 are also essential components of NADPH oxidase, the enzyme responsible for generating free radicals. The cytoskeleton modulates the adhesion of osteoclasts to bone and its subsequent resorption. These cells contain NADPH diaphorase activity, and free radicals influence bone resorption. The influence of Rac1, Rac2 and PAK1 on the cytoskeleton, resorbing activity and NADPH diaphorase activity of disaggregated rat osteoclasts was investigated by permeabilisation with saponin and introducing specific anti-Rac1, anti-Rac2 or anti-PAK1 antibodies. Rhodamine-phalloidin stain was used to identify actin in osteoclasts cultured on plastic slides, and the bone-slice method was used to measure resorption. Saponin permeabilisation did not affect the cytoskeletal organization or bone resorption. Anti-Rac antibodies caused dose- and time-dependent cytoskeletal changes. The osteoclasts rounded up and developed retraction fibers; actin rings were disrupted and large actin dots were seen at the periphery of the cells. Osteoclast resorptive activity was depressed after incubation with the antibodies. The total area resorbed by treated cells and the mean pit area were smaller than those of controls. Anti-PAK1 antibody caused similar changes. None of the antibodies altered the NADPH diaphorase activity. Thus, Rac-GTPases are present in rat osteoclasts and are involved in the organization of the actin cytoskeleton and in resorptive activity. These effects may be mediated by PAK1 kinase, but do not influence osteoclast NADPH diaphorase activity.

In vivo bone metabolism and ex vivo bone marrow osteoprogenitors in vitamin D-deprived pigs.

Vitamin D insufficiency is still a concern in countries where there is no routine food supplementation, such as France. A low vitamin D status is clearly associated with an increased risk of fracture in the elderly, but the long-term consequences of latent vitamin D insufficiency in young people and adults are not known. We fed 26 growing pigs a high calcium diet (1.1%) with a 1000 IU cholecalciferol/kg diet (controls), or without vitamin D (0D) for 4 months. We then analyzed the overall impact of low vitamin D status on osteotropic hormones (calcitriol and immunoreactive parathyroid hormone), plasma markers of bone remodeling (alkaline phosphatase [ALP] activity, carboxyterminal propeptide of type I procollagen [PICP], osteocalcin, hydroxyproline), whole bone parameters (ash content, bending moment), histomorphometry, and the populations of marrow osteoblastic and osteoclastic precursors by ex vivo cultures. The fall in plasma 25-dihydroxyvitamin [25(OH)D] in the 0D pigs indicated severe depletion of their vitamin D stores. However, they remained normocalcemic, were mildly hyperparathyroid after 2 months of vitamin D deprivation, and showed only a slight decrease in plasma calcitriol. The bone mineral content and bending moment of metatarsals decreased and they had increased osteoblastic (+59%, p < 0.05 0D vs. controls) and osteoclastic (+31%, p < 0.1 0D vs. controls) surfaces. This was not paralleled by increased bone turnover, because plasma hydroxyproline and ALP were unchanged and PICP and osteocalcin were decreased. The adherent fraction of bone marrow cells showed a great increase in the number of total stromal colony-forming units (CFU-F; +93%, p < 0.05 0D vs. controls) and in the percent of ALP(+) CFU-F (+58%, p < 0.01 0D vs. controls) in cultures from 0D pigs. More tartrate-resistant acid phosphatase-positive (TRAP(+)) multinucleated cells were generated in cultures of nonadherent marrow cells from 0D pigs, and the area of resorption was 345% greater than in controls. Thus, vitamin D deprivation caused only moderate hormonal changes in growing pigs fed a high-calcium diet, but affected their bone characteristics and greatly enhanced the pool of osteoblasts and osteoclasts by stimulating the commitment of their precursors in bone marrow.

PP60c-src expression in osteoclasts from osteopetrotic children and in giant tumor cells.

Malignant infantile osteopetrosis is a severe congenital disease characterized by impaired osteoclast activity. Among the multiple factors that influence bone resorption, the c-src protooncogene product pp60c-src plays an essential role, since mice which lack pp60c-src develop osteopetrosis. To gain insight into the possible role of pp60c-csrc in the pathogenesis of infantile osteopetrosis, we examined the osteoclasts of three children displaying the typical features of the disease, aged respectively one, four and seven months. pp60c-csrc expression and localization, together with the expression of a 80/85-kilodalton pp60c-src substrate, cortactin, were examined by immunoelectron microscopy. Osteoclasts from two giant cell tumors were used as controls. Bone and tumor samples were fixed in 4% paraformaldehyde, included in LR-White resin at -30 degrees C and the sections processed with mAb 327 or mAb anti p80/85 by an immunogold technique. pp60c-src was expressed in the cytoplasm, in nuclear membranes and in nuclei of the osteoclasts of the three osteopetrotic children. The subcellular localization of the kinase was not different from the localization in giant tumor cells. In both cases cortactin was abundant. In conclusion, in three children with malignant osteopetrosis, pp60c-src expression in osteoclasts does not appear to be involved in the pathogenesis of the disease. The presence of this protein, however, does not necessarily reflect normal c-src tyrosine kinase activity, nor normal c-src-dependent intracellular signaling pathways. Moreover the presence of the protein in nuclear membranes, and especially around nuclear pores supports the hypothesis that in osteoclasts, c-src may participate in the regulation of RNA processing.

Calcium distribution in high-pressure frozen bone cells by electron energy loss spectroscopy and electron spectroscopic imaging.

Subcellular localization of total calcium requires tissue processing that preserves the chemical composition of the samples and a highly sensitive microanalytical technique. In this study rat fetal bone samples were submitted to high-pressure freezing and freeze substitution. Ultrastructural preservation was good in the superficial sections: osteoblasts near the bone mineral had clearly defined plasma and nuclear membranes, dense mitochondria, and numerous ribosomes. Electron energy loss spectroscopy allowed high-resolution calcium-sensitive images to be obtained using ionization edge loss electrons. In biological samples, the Ca-L2,3 signal is superimposed on the carbon edge and artifacts may result from thickness and scattering effects. Therefore the relative thickness was established for each area analyzed (t/lambda <0.5). Background was subtracted using the three-images method, allowing high resolution calcium-sensitive images of intramitochondrial granules and of intracellular compartments, and semiquantitative data from the granules to be obtained. Calcium maps were confirmed by spectra collected on defined areas of the images and the shape of the net Ca-L2,3 edges was compared to the characteristic Ca-L2,3 edge of bone crystals. These procedures will provide new information about total calcium localization in bone cells and the possibility of examining the distribution of other elements.

Rat serum osteocalcin concentration is decreased by restriction of energy intake.

We studied the effects of energy restriction on serum osteocalcin concentration and bone formation rate in rats. The experiment was designed to achieve energy restriction by reducing the carbohydrate intake while providing identical quantities of protein, fat, vitamins and minerals. Energy intakes of three groups of post-weaning male rats were restricted by 20, 40 and 60% for 4 wk. Serum calcium, phosphorus, transthyretin, triiodothyronine (T3), thyroxine (T4), 1,25-dihydroxycholecalciferol, 25-hydroxycholecalciferol and immunoreactive parathyroid hormone (iPTH) concentrations were determined. Energy restriction (20, 40 and 60%) produced a significant and gradual drop of serum osteocalcin concentrations, although the serum concentrations of its key regulators, i.e., 1,25-dihydroxycholecalciferol and iPTH, were not significantly affected. On the contrary, serum concentrations of calcium, phosphorus, transthyretin, T3 and T4 were significantly lower in the energy-restricted groups. However, our results do not support their implication in the regulation of serum osteocalcin synthesis by energy intake. Serum osteocalcin concentration was positively correlated with bone mineral apposition (r = 0.50, P < 0.05) and bone mineralization (r = 0.50, P < 0.05) rates suggesting that its decrease resulted from a reduction of bone formation, and not from abnormal mineralization, because osteoid seam thickness was not modified. Energy intake seems to be an important determinant of serum osteocalcin concentration and bone formation; however, the exact mechanism underlying this regulation remains to be determined.

Bioactive parathyroid hormone in pregnant rats and fetuses.

Parathyroid function at the end of gestation (day 21) was investigated by measuring plasma calcium (PCa), immunoreactive parathyroid hormone (iPTH), bioactive parathyroid hormone (bioPTH; cytochemical bioassay), and bone histology in intact and thyroparathyroidectomized (TPTX; day 12, ether anesthesia) rats and their fetuses. In pregnant intact rats, PCa was significantly lower, and iPTH, bioPTH, and osteoclast number were higher than in nonpregnant rats. In fetuses, PCa was higher than maternal PCa and correlated with fetal bioPTH. TPTX suppressed maternal bioPTH and decreased iPTH and osteoclast number, whereas fetal iPTH and bioPTH were decreased with no change in osteoclast number. Fetal PCa was near normal and was correlated with maternal PCa but not with fetal bioPTH. The fetomaternal calcium gradient was maintained and even increased. This study shows that there is maternal physiological hyperparathyroidism and functional fetal parathyroid glands at the end of gestation in the rat. Parathyroid hormone does not seem to be responsible for maintaining the high fetomaternal calcium gradient in TPTX animals.

PTH mRNA transcription analysis in infantile tumors associated with hypercalcemia.

The ability of infantile hypercalcemic tumors (three rhabdoid renal tumors, one cellular mesoblastic nephroma, and one hepatoblastoma) to produce parathyroid hormone (PTH) was tested using RNA-DNA hybridization. Results were compared with those obtained in one lung epidermoid carcinoma and one parathyroid adenoma from adult patients. Elevated plasma immunoreactive PTH (iPTH) concentrations were observed in three of five children. The only tumor in which PTH-RNA hybridization could be detected was the parathyroid adenoma. The integrity of the RNA preparations was further confirmed by positive hybridization obtained with a glucagon DNA probe in both normal pancreas and the rhabdoid tumors. Quantitative bone histomorphometry of tumor-bearing nude mice showed a reduction in bone formation and increased bone resorption, the opposite of what occurs in hyperparathyroidism. The PTH-like protein, which was detected by radioimmunoassays (RIA) in the sera of three patients, could not be correlated with tumor PTH mRNA transcription within the limits of our assays. In order to explain this discrepancy, we suggest that the tumors produce a factor (not PTH) which, in turn, elicits the excess iPTH which we detected by RIA.

Iron distribution in thalassemic bone by energy-loss spectroscopy and electron spectroscopic imaging.

Iron overload occurs frequently in thalassemia as a consequence of regular blood transfusions, and iron has been found to accumulate in bone, but skeletal toxicity of iron is not clearly established. In this study, bone biopsies of thalassemic patients were investigated by light (n = 6) and electron microscopy (n = 8) in order to analyze iron distribution and possible iron-associated cellular lesions. Sections (5 microns thick) were used for histomorphometry and iron histochemistry. Ultrathin sections were examined with an energy filtering transmission electron microscope. Iron was identified by electron energy loss spectroscopy (EELS), and iron distribution was visualized by electron spectroscopic imaging (ESI) associated with computer-assisted treatment (two-window method). This study shows that EELS allows the detection of 4500-9000 iron atoms, and that computer-assisted image processing is essential to eliminate background and to obtain the net distribution of an element by ESI. This study shows also that stainable iron was present along trabecular surfaces, mineralizing surfaces, and on cement lines in the biopsies of all patients. Moreover, iron was detected by EELS in small granules (diffusely distributed or condensed in large clusters), in osteoid tissue, and in the cytoplasm of bone cells, but not in the mineralized matrix. The shape and size (9-13 nm) of these granules were similar to those reported for ferritin. As for iron toxicity, all patients had osteoid volume and thickness and osteoblast surface in the normal range. Stainable iron surfaces did not correlate with osteoblast surfaces, plasma ferritin concentrations, or the duration of transfusion therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of iron oxide nanoparticles in rat lymph nodes studied using electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI).

Superparamagnetic iron nanoparticles have been developed as contrast agents for magnetic resonance lymphography. The kinetics of uptake of these particles has not yet been accurately determined. We have therefore monitored the distribution of individual iron particles (ferumoxtran, AMI-227, Sinerem) in rat lymph nodes 1.5, 3, 6, 12, and 24 hours after i.v. injection (two rats per time point). The ultrastructural distribution of the iron was determined by energy-filtered transmission electron microscopy (EFTEM). This method allows the identification of elements using element-specific energy-loss electrons. Iron was identified by the Fe-L(2,3) edge (EELS), and iron maps were obtained using iron-specific electrons for imaging (ESI). The background was calculated by simplex optimization (EELS) and by the two-window method (ESI). Ferumoxtran particles were regularly observed at the periphery of the lymph nodes but not in their centers. Isolated iron particles were seen extracellularly within lymph vessels and, 3 hours after injection, as small dots in phagocytic cells. Numerous dense clusters appeared within the cells at later times (6 and 12 hours after injection). These results suggest that the contrast agent moves rapidly across the capillary wall to the lymph and is then taken up by phagocytic cells. J. Magn. Reson. Imaging 2000;12:505-509.

Osteoclast adhesion and activity on synthetic hydroxyapatite, carbonated hydroxyapatite, and natural calcium carbonate: relationship to surface energies.

This study investigates the adhesion, cytoskeletal changes, and resorptive activity of disaggregated rat osteoclasts cultured on polished slices of three biomaterials: crystalline synthetic hydroxyapatite (HA), carbonated hydroxyapatite (C-HA), and natural calcium carbonate (C). The surface chemistry of each substrate was defined by X-ray diffraction and IR spectroscopy, surface wettability by the dispersive, and the polar components of the surface energies. Osteoclast adhesion was modulated by the polar component of the surface energy: fewer (p < 0.01) osteoclasts adhered to C-HA (97 +/- 20/slice, surface energy 9 +/- 5 mJ/m2) than to HA (234 +/- 16/slice, surface energy 44 +/- 2 mJ/m2) or to C (268 +/- 37/slice, surface energy 58 +/- 0.5 mJ/m2). Actin rings, which are the cytoskeletal structure essential for resorption, developed on all three materials. The area of the actin ring, which is resorbed by local acidification, and the osteoclast area, which reflects osteoclast spreading, were both greater in osteoclasts cultured on HA and C-HA than in those cultured on C. C was resorbed, but HA and C-HA were not. Thus, the surface energy plays an essential role in osteoclast adhesion, whereas osteoclast spreading may depend on the surface chemistry, especially on protein adsorption and/or on newly formed apatite layers. Resorption may be limited to the solubility of the biomaterial.

The protein tyrosine kinase p60c-Src is not implicated in the pathogenesis of the human autosomal recessive form of osteopetrosis: a study of 13 children.

Osteopetrosis has been described in mice generated by homozygous gene disruption of c-src gene encoding for the p60c-Src protein tyrosine kinase (Src-/- mice). The similarities of bone histologic findings in this murine model to those observed in some patients first seen with autosomal recessive osteopetrosis, "malignant" osteopetrosis, led us to investigate the potential role of p60c-Src in the pathogenesis of malignant osteopetrosis in 13 children. In 4 patients a c-src mutation was ruled out by an intragenic microsatellite segregation study. In the other 9 we analyzed p60c-Src expression and function, as well as c-src sequence. The expression was normal in all of the patients tested. In addition, the tyrosine phosphorylation and kinase activity of p60c-Src were also normal in all of the patients. Moreover, in these patients, sequences of the coding region of c-src were identical to the published sequence of the human c-src complementary DNA. These results exclude a role for c-src in the pathogenesis of human malignant osteopetrosis in the 13 patients analyzed.

Plasma vitamin D metabolites in a patient with sporadic hypophosphataemic osteomalacia (adult-onset type).

Sporadic hypophosphataemic osteomalacia (adult-onset type) was demonstrated in a 40-year-old man on the basis of severe osteomalacia, hypophosphataemia, hyperphosphaturia and glycinuria. Plasma immunoreactive parathyroid hormone (iPTH) concentration was 9.3 ng prot./ml (normal range: 4-8 ng prot./ml). Plasma 25-hydroxy-vitamin D and 24,25-dihydroxy-vitamin D concentrations were 11 and 2.4 ng/ml respectively. Basal 1 alpha,25-dihydroxy-vitamin D concentrations were slightly elevated (116 and 96 pg/ml) and increased to 240 pg/ml after 3 days on a low-phosphorus diet. The patient was put on oral treatment with 25-hydroxycholecalciferol (100 microgram per day) and phosphorus (1500 mg per day). On the 4th month on treatment, a clinical improvement was apparent. Plasma 25(OH)D was 44 ng/ml, plasma 1,25(OH)2D was 256 pg/ml. However, plasma phosphorus remained low (0.77 mmol/l). On the 9th month of treatment a radiological improvement was evident despite a persistent hypophosphataemia (0.68 mmol/l). These facts suggest in our patient the existence of a vitamin D-independent renal phosphorus leak.