Effects of dexamethasone and vitamin D3 on cartilage differentiation in a clonal chondrogenic cell population

We have investigated the regulation of chondroblast/chondrocyte differentiation using a unique clonal cell population, designated RCJ 3.1C5 (C5), which differentiates into discrete three-dimensional cartilage nodules when grown in the presence of 15% fetal calf serum. Histologically, the nodules resembled hyaline cartilage; they contained large rounded chondrocytes surrounded by a refractile matrix which stained intensely with Alcian blue, exhibited metachromasia after Toluidine blue staining, and stained with an antibody against type II collagen. The cartilage nodules that formed did not mineralize, despite the presence of organic phosphate in the culture medium. The synthetic glucocorticoid dexamethasone (DEX) increased the number of cartilage nodules formed in a dose-dependent manner (ED50, approximately 10(-9) M), with a maximal stimulatory dose of 10(-8) M. DEX had no effect on the population doubling time and saturation density. The effects of DEX on the number of cartilage nodules were similar whether it was added from the beginning of the culture period (starting during exponential growth) or at confluence. In contrast, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited cartilage nodule formation in a dose-dependent manner (IC50, approximately 5 x 10(-10) M), with maximum inhibition at 10(-7) M. In addition, 1,25-(OH)2D3 decreased cell proliferation and saturation density. Equimolar doses of the vitamin D3 metabolites 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 had no effect. C5 cells treated with 1,25-(OH)2D3 in the absence of DEX during the exponential growth phase exhibited a reduced capacity to form cartilage nodules upon subsequent exposure to DEX. At confluence, before cartilage nodules had formed, C5 cells responded to PTH and prostaglandin-E2 with increases in intracellular cAMP of about 10- and 95-fold respectively. After cartilage nodules were present, prostaglandin-E2 responsiveness decreased to about 25-fold, whereas there was no significant change in PTH responsiveness. DEX decreased the population alkaline phosphatase levels at all times measured, whereas 1,25-(OH)2D3 had a biphasic effect: an increase at 5 days in culture, followed by a decrease at later times in culture. These data indicate that the clonal cell line RCJ 3.1C5 is a useful model system in which to investigate cartilage differentiation.     

Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvaria cells in vitro

Isolated rat calvaria cells plated at low density in medium supplemented with ascorbic acid and organic phosphate form discrete three-dimensional mineralized nodules having the characteristics of bone. We have studied the effects of glucocorticoids on the formation of bone nodules by these cell populations. Cells isolated from 21-day-old fetal rat calvaria were maintained in vitro for up to 27 days. Dexamethasone (Dex) induced a dose-related increase in the number of nodules formed, with a peak at 10 nM and a half-maximal response at about 1 nM. Dex (10 nM) also significantly increased the size of bone nodules formed (P less than 0.002). High concentrations of Dex (1 microM) did not increase nodule number. In cells in primary culture maintained in medium containing 10 nM Dex, the increase in nodule number was 50-100% over the control value. The effect of Dex was much greater in first subculture cells, where the number of nodules was 600-800% higher than the control value. Dishes collected and quantitated from 12-27 days showed that nodule formation ceased between 15 and 18 days in cultures without Dex, whereas in the presence of Dex the number of nodules increased up to 27 days. Addition of 10 nM Dex only during specific periods resulted in significantly more nodules than in control cultures, but significantly fewer nodules than in cultures constantly exposed to Dex. Cell population doubling times during log phase growth were unaltered, but a significant increase in saturation density (P less than 0.001) was observed with 10 nM Dex. Hydrocortisone also caused an increase in the number of nodules formed, with a maximal effect of 50 nM and a half-maximal response at 8 nM. The results indicate that physiological levels of glucocorticoids stimulate bone nodule formation in long term cell culture by increasing the number of cells forming bone nodules and that maximization of the stimulatory effect of glucocorticoids on bone formation may require constant exposure to low levels of the hormone.     

Specificity and sensitivity of RHD genotyping methods by PCR-based DNA amplification

We have compared the sensitivity and specificity of four PCR methods of RHD gene detection using different sets of primers located in the regions of highest divergence between the RHD and RHCE genes, notably exon 10 (method I), exon 7 (method II), exon 4 (method III) and intron 4 (method IV). Methods I–III were the most sensitive and gave a detectable signal with D-pos/D-neg mixtures containing only 0.001% D-positive cells. Moreover, method II could detect the equivalent DNA amount present in only three nucleated cells in the assay without hybridization of PCR products, whereas the sensitivity of the other methods was 10–50 times less. Investigation of D variants indicated that false-negative results were obtained with method II (D^IVb variant), method III (D^VI and DFR variants) and method IV (D^VI variants), but not method I. Weak D (D^u) was correctly detected as D-positive by all methods, but most cases of Rh_null appeared as false-positives, as they carry normal RH genes that are not phenotypically expressed. Some false-positive results were obtained with method I in a few Caucasian DNA samples serotyped as RhD-neg but carrying a C - or E -allele, whereas a high incidence of false-positives was found among non-Caucasian Rh-negative samples by all methods. In the Caucasian population, however, we found a full correlation between the predicted genotype and observed phenotype at birth of 92 infants. Although we routinely use the four methods for RHD genotyping, a PCR strategy based on at least two methods is recommended.     

Absence of bone sialoprotein (BSP) impairs primary bone formation and resorption: the marrow ablation model under PTH challenge

Bone sialoprotein (BSP) is highly expressed in early bone deposition and may play a part in primary bone mineralization. We previously showed that while BSP?/? mice have a mild secondary bone phenotype and are responsive to mechanical (unloading) and hormonal (ovariectomy, parathyroid hormone (PTH)) challenges, repair of a cortical bone defect, which involves primary bone deposition is significantly delayed in these mice. In the present study, we investigated the role of BSP in a pure model of primary bone modeling. Bone marrow was ablated by trans-epiphysis aspiration in the femora of BSP+/+ and BSP?/? mice, and 7 days post surgery μCT analysis showed vigorous new bone formation in the shaft of BSP+/+ animals but much less in BSP?/? mice. After 14 days, the volume of medullary bone was significantly decreased as expected in BSP+/+ mice, while it remained stable in the BSP?/?. Osteoid thickness and surface were higher in BSP?/? at day 7, suggesting delayed mineralization, while osteoclast surface and number were significantly lower at day 14, a stage of high medullary bone resorption. At day 7, mRNA expression of early osteoblast marker genes (RUNX2, osterix, alkaline phosphatase, osteopontin) did not differ between the two genotypes, while markers of terminal differentiation (MEPE, DMP1, osteocalcin) as well as receptor activator of NF-kappaB ligand (RANKL) and tartrate-resistant acid phosphatase (TRAP) were significantly lower in BSP?/? than in BSP+/+ mice. PTH treatment maintained the volume of medullary bone up to 12 days after ablation in BSP+/+ mice, but failed to do so in BSP?/? mice. PTH significantly increased bone formation rate in both genotype, while it reduced osteoclast number and surface in BSP+/+, but not in BSP?/? medullary bone. In summary, medullary bone formation after marrow ablation is blunted in BSP?/? mice, with delayed resorption and impaired response to PTH. These findings confirm the hypothesis of a crucial role for BSP in primary ossification, which has long been suspected for mineralization, but here extends to bone deposition and turnover.     

Absence of Minocycline in Sebum?

The authors studied the bioavailability of minocycline in sebum and serum. Blood and sebum samples were collected weekly for 6 weeks from ten healthy volunteers taking 200 mg of minocycline every day for 4 weeks. Sebum was collected by direct extraction with petroleum ether from the forehead. After evaporation, sebum was weighed on a scale accurate to 10 micrograms. Determination of minocycline in serum and sebum was performed using a high performance liquid chromatography technique (HPLC), with a better detection limit at 352 nm than at 400 nm (20 ng/ml and 0.324 microgram/ml respectively). Our results contrast with other studies since no minocycline was detected in the sebum samples of treated subjects and microbiological assays of minocycline in sebum were also negative. In our opinion, the current hypothesis claiming that the effectiveness of minocycline in the treatment of acne vulgaris is based on sebaceous secretion should be reconsidered.     

Wilson's disease. X-ray CT and MRI aspects of the brain in a case before treatment

Wilson disease is an autosomal recessive disorder of copper metabolism. The case of a young girl is reported here, with CT and MRI data before any treatment. The unusual MR presentation on T2 weighted sequences is studied, and compared with those described in patients under treatment.     

Intra and interobserver variability of preoperative planning for surgical instrumentation in adolescent idiopathic scoliosis

Surgical instrumentation planning for the correction of scoliosis involves many difficult decisions, especially with the introduction of multi-segmental and other instrumentation technologies. A preliminary study has shown a high variability in planning among a small group of surgeons. The purpose of this paper was to evaluate and analyze the selection of fusion levels and instrumentation choices among a more extended group of scoliosis surgeons. Thirty-two experienced spinal deformity surgeons were asked to provide their preferred posterior instrumentation planning for five patients with adolescent idiopathic scoliosis (AIS) using a graphical worksheet and the usual preoperative X-rays. Overall, the number of implants used ranged from 8 to 30 per patient (mean 16; SD 6): 71% of these were mono-axial screws, 20% multi-axial screws, and 9% hooks. The selected superior and inferior instrumented vertebrae varied up to six levels. The following significant groups of strategies were identified: A- “All Pedicle Screws Constructs” [N _A = 103; 66%]; B- “All Hooks constructs” [N _B = 5; 3%]; C- “Hybrid Constructs” [N _C = 48; 31%]. A top-to-bottom attachment sequence was selected in 49% of all cases, a bottom-up in 46%, and an alternate order in 4%. A large variability in preoperative instrumentation strategy exists in AIS within an experienced group of orthopedic spine surgeons. The impact of such choices on the resulting correction is questioned and will need to be determined with adequate clinical, biomechanical, and computer simulation prospective studies.     

Post-transplant focal segmental glomerulosclerosis refractory to plasmapheresis and rituximab therapy

Sir, Primary focal segmental glomerulosclerosis (FSGS) recurs in20 to 50% of kidney transplants [1]. Recently, there have beenreports demonstrating the successful use of rituximab for recurrentpost-transplant FSGS [2–4]. We report a case of recurrentFSGS that was refractory to both