Inactivation of estrogen receptor α in bone-forming cells induces bone loss in female mice.

The role of the estrogen receptor α (ERα) in bone-forming cells is incompletely understood at present. To examine the in vivo effects of ERα in these cells, we generated a mouse strain in which the ERα gene is inactivated in osteoblasts via osteocalcin promoter-regulated cyclic recombinase (Cre) activity (ERα(ΔOB/ΔOB)). This enabled micro-computed tomography- and histomorphometry-based analysis of ERα-mediated effects in bone-forming cells in mice, in which the systemic ERα-mediated effects are intact. In female ERα(ΔOB/ΔOB) mice, trabecular and cortical bone volumes were significantly reduced (31.5 and 11.4%, respectively) at 3.5 mo of age compared with control ERα(fl/fl) animals, and their response to ovariectomy was small compared with that of controls. In contrast with females, no differences could be detected in the bone phenotype of young males, whereas in 6-mo-old ERα(ΔOB/ΔOB) males, trabecular bone volume (Tb.BV) was decreased (27.5%). The ERα inactivation-related effects were compared with those of controls having a similar genetic background. Parental osteocalcin-Cre mice did not show Cre-related changes. Our results suggest that in female mice, Tb.BV and cortical bone volume are critically dependent on the ERα regulation of osteoblasts, whereas in male mice, osteoblastic ERα is not required for the regulation of bone formation during rapid skeletal growth, but it is involved in the maintenance of Tb.BV.

Neurofibromatosis 1-related osteopenia often progresses to osteoporosis in 12 years.

The current study is based on our earlier investigation carried out in 1999, where bone mineral density (BMD) of 35 neurofibromatosis type 1 (NF1) patients was measured and osteoporosis was shown to be common in NF1. The findings have been confirmed by a number of later publications. The purpose of the current longitudinal study was to assess the bone health of these 35 NF1 patients 12 years after the initial study. A total of 28 patients were reached, and BMD of 19 patients was subsequently remeasured. Fracture history of 28/35 NF1 patients who were reached was verified from the medical records. Six NF1 patients had osteoporosis in 1999, and three of them had an osteoporotic fracture between 1999 and 2011, showing an increased fracture risk compared to NF1 patients without osteoporosis. BMD of 19 patients was remeasured in 2011, and four patients who had osteopenia in 1999 had osteoporosis in 2011. The decrease in BMD was not explained by changes in smoking habits, physical activity, sunlight exposure, body mass index, or laboratory parameters, even though secondary hyperparathyroidism was common. Osteoporosis was found in 2011 in patients aged 37 years or older, both men and women. The results showed that NF1-related osteopenia often progresses to osteoporosis since BMD decreases with aging even in young patients. Even though our sample size was 19 patients, we recommend follow-up of NF1 patients with osteopenia and consideration of prophylactic measures to prevent osteoporosis and associated fracture risk.

Phenotypic characterization of transgenic mice harboring Nf1+/- or Nf1-/- osteoclasts in otherwise Nf1+/+ background.

Skeletal abnormalities in neurofibromatosis type 1 syndrome (NF1) are observed in ∼50% of patients. Here, we describe the phenotype of Nf1(Ocl) mouse model with Nf1-deficient osteoclasts. Nf1Ocl mice with Nf1+/- or Nf1-/- osteoclasts in otherwise Nf1+/+ background were successfully generated by mating parental Nf1flox/flox and TRAP-Cre mice. Contrary to our original hypothesis, osteoporotic or fragile bone phenotype was not observed. The µCT analysis revealed that tibial bone marrow cavity, trabecular tissue volume, and the perimeter of cortical bone were smaller in Nf1 Ocl-/- mice compared to Nf1 Ocl+/+ control mice. Nf1 Ocl-/- mice also a displayed narrowed growth plate in the proximal tibia. In vitro analysis showed increased bone resorption capacity and cytoskeletal changes including irregular cell shape and abnormal actin ring formation in Nf1-/- osteoclasts. Surprisingly, the size of spleen in Nf1 Ocl-/- mice was two times larger than in controls and histomorphometric analysis showed splenic megakaryocytosis. In summary, Nf1Ocl mouse model presented with a mild but specific bone phenotype. This study shows that NF1-deficiency in osteoclasts may have a role in the development of NF1-related skeletal abnormalities, but Nf1-deficiency in osteoclasts in Nf1+/+ background is not sufficient to induce skeletal abnormalities analogous to those observed in patients with NF1.

Attempt to treat congenital pseudarthrosis of the tibia with mesenchymal stromal cell transplantation.

BACKGROUND AIMS: Congenital pseudarthrosis of the tibia (CPT) caused by neurofibromatosis type 1 (NF1) is a refractory disease occurring in childhood. We present two cases that had failed all earlier treatment attempts and, as a last treatment attempt, the patients were chosen to receive mesenchymal stromal cell (MSC) transplantation prior to amputation. METHODS: The MSC from bone marrow (BM) were harvested from the iliac crest and cultured in osteoinductive medium for 3 weeks. The cultured MSC were injected in solution into BM canals of the tibia and around the resection line or bone defect in a 3-dimensional collagen sponge scaffold. After the MSC transplantation, the patients were monitored during a 10-month follow-up period. In both cases, bone formation at the pseudarthrosis site was observed and two of three treated bone defects healed. For clinical reasons not related to cell transplantation, such as new infection and pseudarthrosis and severe shortening of the leg, both extremities were finally amputated and bone samples were analyzed to evaluate MSC therapy effect and safety. RESULTS: MSC transplantation normalized bone remodeling, promoted bone resorption and improved the overall structure of bone. The number of osteoclasts in the cortical bone was 2-fold higher compared with the monitored situation before MSC transfer. In addition, the mineral content of the bone improved after transplantation. We could see no sign of aberrant bone formation or malignant transformation. CONCLUSIONS: Our data suggest that MSC transplantation is a possibility for treatment of CPT caused by NF1 in less severe cases without adjunct defects.

Interaction of spin-labeled inhibitors of the vacuolar H+-ATPase with the transmembrane Vo-sector.

The osteoclast variant of the vacuolar H(+)-ATPase (V-ATPase) is a potential therapeutic target for combating the excessive bone resorption that is involved in osteoporosis. The most potent in a series of synthetic inhibitors based on 5-(5,6-dichloro-2-indolyl)-2-methoxy-2,4-pentadienamide (INDOL0) has demonstrated specificity for the osteoclast enzyme, over other V-ATPases. Interaction of two nitroxide spin-labeled derivatives (INDOL6 and INDOL5) with the V-ATPase is studied here by using the transport-active 16-kDa proteolipid analog of subunit c from the hepatopancreas of Nephrops norvegicus, in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Analogous experiments are also performed with vacuolar membranes from Saccharomyces cerevisiae, in which subunit c of the V-ATPase is replaced functionally by the Nephrops 16-kDa proteolipid. The INDOL5 derivative is designed to optimize detection of interaction with the V-ATPase by EPR. In membranous preparations of the Nephrops 16-kDa proteolipid, the EPR spectra of INDOL5 contain a motionally restricted component that arises from direct association of the indolyl inhibitor with the transmembrane domain of the proteolipid subunit c. A similar, but considerably smaller, motionally restricted population is detected in the EPR spectra of the INDOL6 derivative in vacuolar membranes, in addition to the larger population from INDOL6 in the fluid bilayer regions of the membrane. The potent classical V-ATPase inhibitor concanamycin A at high concentrations induces motional restriction of INDOL5, which masks the spectral effects of displacement at lower concentrations of concanamycin A. The INDOL6 derivative, which is closest to the parent INDOL0 inhibitor, displays limited subtype specificity for the osteoclast V-ATPase, with an IC(50) in the 10-nanomolar range.

Alendronate decreases orthotopic PC-3 prostate tumor growth and metastasis to prostate-draining lymph nodes in nude mice.

BACKGROUND: Metastatic prostate cancer is associated with a high morbidity and mortality but the spreading mechanisms are still poorly understood. The aminobisphosphonate alendronate, used to reduce bone loss, has also been shown to inhibit the invasion and migration of prostate cancer cells in vitro. We used a modified orthotopic PC-3 nude mouse tumor model of human prostate cancer to study whether alendronate affects prostate tumor growth and metastasis. METHODS: PC-3 cells (5 x 10(5)) were implanted in the prostates of nude mice and the mice were treated with alendronate (0.5 mg/kg/day in PBS, s.c.) or vehicle for 4 weeks. After sacrifice, the sizes of tumor-bearing prostates were measured and the tumors and prostate-draining regional iliac and sacral lymph nodes were excised for studies on markers of proliferation, apoptosis, angiogenesis and lymphangiogenesis, using histomorphometry and immunohistochemistry. RESULTS: Tumor occurrence in the prostate was 73% in the alendronate-treated group and 81% in the control group. Mean tumor size (218 mm3, range: 96-485 mm3, n = 11) in the alendronate-treated mice was 41% of that in the control mice (513 mm3, range: 209-1350 mm3, n = 13) (p < 0.05). In the iliac and sacral lymph nodes of alendronate-treated mice, the proportion of metastatic area was only about 10% of that in control mice (p < 0.001). Immunohistochemical staining of tumor sections showed that alendronate treatment caused a marked decrease in the number of CD34-positive endothelial cells in tumors (p < 0.001) and an increase in that of ISEL positive apoptotic cells in tumors as well as in lymph node metastases (p < 0.05) compared with those in the vehicle-treated mice. The density of m-LYVE-1-stained lymphatic capillaries was not changed. CONCLUSION: Our results demonstrate that alendronate treatment opposes growth of orthotopic PC-3 tumors and decreases tumor metastasis to prostate-draining lymph nodes. This effect could be at least partly explained by decreased angiogenesis and increased apoptosis. The results suggest that bisphosphonates have anti-tumoral and anti-invasive effects on primary prostate cancer.

Urinary osteocalcin and other markers of bone metabolism: the effect of risedronate therapy.

OBJECTIVE: Serum osteocalcin (S-OC) is widely used as an index of bone formation. However, there is evidence that some urinary fragments of OC reflect resorption and might be useful in monitoring antiresorptive therapy. Here, we report 6-month changes in urinary midfragments of osteocalcin (U-MidOC) and other bone turnover markers in response to risedronate treatment. MATERIAL AND METHODS: The study group comprised 19 patients with postmenopausal osteoporosis, aged 49-66 years, and receiving risedronate therapy. Fifty-four premenopausal women served as controls. Osteoporosis was diagnosed by lumbal bone mineral density (BMD). Urinary osteocalcin was measured by the U-MidOC assay for midfragments. Bone formation was assessed by S-PINP and S-OC, and resorption by S-CTx-I. RESULTS: At baseline, U-MidOC was significantly correlated only with S-OC. After the 1st month of therapy, a similar decrease was observed in the values of U-MidOC and S-CTx-I, but in formation markers S-P1NP and S-OC only after three months. The rapid decrease in U-MidOC, analogous to S-CTX-I, and the different kinetics for urinary and serum OC suggest that urinary OC midfragments are more associated with resorption than S-OC. An association was also observed between the 1-month change in U-MidOC and 12-month gain in lumbar BMD. The response in U-MidOC after only the 1st month of therapy makes it a potential marker for monitoring the effect of risedronate, presumably reflecting different aspects of bone resorption than S-CTx-I does.

Estrogen receptor α contributes to T cell-mediated autoimmune inflammation by promoting T cell activation and proliferation.

It has long been appreciated that most autoimmune disorders are characterized by increased prevalence in females, suggesting a potential role for sex hormones in the etiology of autoimmunity. To study how estrogen receptor α (ERα) contributes to autoimmune diseases, we generated mice in which ERα was deleted specifically in T lymphocytes. We found that ERα deletion in T cells reduced their pathogenic potential in a mouse model of colitis and correlated with transcriptomic changes that affected T cell activation. ERα deletion in T cells contributed to multiple aspects of T cell function, including reducing T cell activation and proliferation and increasing the expression of Foxp3, which encodes a critical transcription factor for the differentiation and function of regulatory T cells. Thus, these data demonstrate that ERα in T cells plays an important role in inflammation and suggest that ERα-targeted immunotherapies could be used to treat autoimmune disorders.

Pathophysiology, risk factors and management of bisphosphonate-associated osteonecrosis of the jaw: Is there a diverse relationship of amino- and non-aminobisphosphonates?

Reports of osteonecrosis of the jaw (ONJ) in patients receiving long-term bisphosphonate therapy have appeared in the literature since 2003. This condition involves avascular necrotic bone in the area of maxilla or mandibula and there may be a secondary infection. Most cases of ONJ have been reported in cancer patients receiving the intravenous aminobisphosphonates zoledronic acid and pamidronate monthly or q 3 week; of note these are also the two most commonly used agents of this class. Risk factors for ONJ include a history of trauma, dental surgery or dental infection and intravenous bisphosphonate administration; in addition, the extent and duration of exposure to bisphosphonates also seem to correlate with the risk. Although a direct causal relationship with bisphosphonates cannot be assumed, these agents may possibly contribute to the development of ONJ by suppression of bone remodeling in the jaw which leads to increased rates of bone mineralisation and accumulation of microfractures. Clodronate, a non-aminobisphosphonate, appears to have a different mechanism of suppressing bone remodeling compared with aminobisphosphonates, and this may explain why few cases of ONJ have been reported with clodronate despite extensive use over the past 20 years; however, the potential of clodronate to reduce the risk of ONJ while providing equivalent clinical benefit to the aminobisphosphonates needs to be substantiated in controlled clinical trials. Use of bisphosphonate therapy should be carefully planned in patients with metastatic bone disease who have risk factors for ONJ, and appropriate preventive measures taken to avoid the development of this condition.

Periapical cemental dysplasia is common in women with NF1.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a genetic disorder with skeletal involvement. Periapical cemental dysplasia is a rare finding in the normal population. METHOD: A total of 55 patients with NF1, 29 female and 26 male patients, were evaluated with orthopantomograms, supplemented with periapical radiographs if necessary. The vitality of the teeth was measured by two different testing methods. RESULTS: A novel finding was the occurrence of cemental dysplasia affecting the periapical area of vital mandibular teeth in 8 adult women with NF1. Thus, cemental dysplasia was detected in 34.8% of the adult female NF1 patients, while cemental dysplasia was not present in men or children with NF1. CONCLUSION: Periradicular cemental dysplasia is indeed a new NF1 related bone lesion type. Our finding suggests that this is the first reported sexual dimorphism in the manifestations of NF1. Cemental dysplasia of NF1 patients should not be confused with periapical findings caused by endodontic pathoses. The former do not require active therapy whereas in the latter root canal treatment is necessary.

Inhibition of cyclooxygenase-2 down-regulates osteoclast and osteoblast differentiation and favours adipocyte formation in vitro.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenases (COX) and are widely used for post-trauma musculoskeletal analgesia. In animal models, NSAIDs have been reported to delay fracture healing and cause non-union, possibly due to the drug-induced inhibition of osteoblast recruitment and differentiation. To further investigate the cellular effects of these drugs in the context of bone healing, we examined the effects of COX-1 inhibitor indomethacin and COX-2 inhibitors, parecoxib and NS398 on osteoclast and osteoblast differentiation and activity in vitro. We discovered that all tested COX-inhibitors significantly inhibited osteoclast differentiation, by 93%, 94% and 74% of control for 100 microM indomethacin, 100 microM parecoxib and 3 microM NS398, respectively. Furthermore, inhibition of COX-2 reduced also the resorption activity of mature osteoclasts. All tested COX-inhibitors also significantly inhibited osteoblast differentiation from human mesenchymal stem cells. Simultaneously, the number of adipocytes was significantly increased. The adipocyte covered areas in the cultures with 1 microM indomethacin, 1 microM parecoxib and 3 microM NS398 were 9%, 29% and 24%, respectively, as compared with 6% in the control group. This data suggests that COX-2 inhibition disturbs bone remodelling by inhibiting osteoclast differentiation and diverting stem cell differentiation towards adipocyte lineage instead of osteoblast lineage. In conclusion, our results further suggest cautious use of COX-2 inhibitors after osseous trauma.

Effects of bioactive peptide, valyl-prolyl-proline (VPP), and lactobacillus helveticus fermented milk containing VPP on bone loss in ovariectomized rats.

BACKGROUND: Valyl-prolyl-proline (VPP), a bioactive peptide formed during the fermentation with Lactobacillus helveticus LBK-16H (L. helveticus), has been shown to increase bone formation in vitro. The aim of the study was to determine whether VPP and L. helveticus fermented milk prevent bone loss in ovariectomized (OVX) rats. METHODS: During the 12-week intervention study, the OVX rats received VPP in water or L. helveticus fermented milk, containing VPP. Sham-operated rats receiving water acted as controls. The trabecular and cortical bone mineral density were determined by peripheral quantitative computed tomography before the operation and at 4 and 12 weeks. The mechanical testing and ash weight analysis as well as the static and dynamic histomorphometrical parameters were assessed at the end of the intervention. RESULTS: VPP given in water showed no clear effect on bone loss. L. helveticus fermented milk prevented bone loss by decreasing bone turnover and increasing the bone mineral density. Ovariectomy caused a 57% loss in the trabecular bone, which was attenuated by 16% in the L. helveticus group. CONCLUSIONS: VPP peptide did not prevent ovariectomy-induced bone loss, which could be due to the poor bioavailability of VPP from water solution. L. helveticus fermented milk prevented bone loss, whether this is due to the VPP peptide cannot be concluded.

Effect of exercise on extraskeletal risk factors for hip fractures in elderly women with low BMD: a population-based randomized controlled trial.

UNLABELLED: We conducted a 30-month population-based, randomized, controlled trial in 160 elderly women at risk for fractures on the basis of a low baseline BMD. Mainly home-based weight-bearing exercise was effective in improving strength, balance, and gait. INTRODUCTION: Evidence on the effect of exercise on extraskeletal risk factors for hip fractures comes mainly from studies in voluntary low-risk women, and no population-based, long-term interventions have been performed in elderly women with low bone mass. The aim of this study was to determine the effect of long-term weight-bearing exercise on balance, muscle strength, and gait in elderly women at risk for fractures on the basis of a low baseline BMD. MATERIALS AND METHODS: A birth cohort of 1690 women 70-73 years of age were invited to the radius and hip BMD measurements; 96 women were excluded because of medical reasons; 160 women with radius and hip BMD values of >2 SD below the reference value were included in the trial. The participants were randomly assigned to 30 months of impact, balancing, and strengthening exercises or to no intervention. Main outcome measures were body sway length and leg strength at month 30. Secondary endpoints included gait speed, endurance, and grip strength. Outcomes were assessed at 0, 12, 24, and 30 months using blinded operators. Repeated-measures ANOVA was used to determine statistical significance. The analyses were performed on an intention-to-treat basis. RESULTS: Body sway increased more in the control group than in the exercise group over time (time-group interaction, p < 0.001). Leg strength improved in the exercise group and decreased in the control group (interaction, p < 0.001). A significant time-group interaction (p < 0.001) in favor of the exercise group was found on the following secondary endpoints: the timed up and go test score, walking speed, and distance walked in 2 minutes. CONCLUSIONS: Weight-bearing exercise is an effective way of modifying extraskeletal risk factors for fractures in elderly women.

Fam3c modulates osteogenic cell differentiation and affects bone volume and cortical bone mineral density.

Fam3c, a cytokine-like growth factor, has been suggested to have a role in epithelial-to-mesenchymal transition (EMT), tumor growth and metastasis. A single-nucleotide polymorphism affecting bone mineral density has been found in the first intron of the Fam3c gene in a study analyzing an Asian population cohort. Other independent studies on different population cohorts have found the fam3c locus to be associated with bone mineral density and fractures. In order to investigate the role of Fam3c in bone biology, we have generated a Fam3c knock-out (KO) mouse strain. The Fam3c KO mice were found to have normal appearance, behavior and fertility, but small changes in bone morphology and content were also observed. Micro-CT analysis of tibiae of the female mice revealed decreased number of trabeculae. In male mice the changes in the bone phenotype were smaller, but hematological changes were observed. Furthermore, there was a negative correlation between body weight and tibial trabecular and cortical bone volume in the male KO mice. There was a small increase in cortical bone mineral density, but in the lateral direction of tibiae the breaking strength was reduced. Fam3c KO bone marrow cells showed accelerated osteogenic differentiation and mineralization in vitro. The reduced number of bone trabeculae in Fam3c KO mice and the stimulated osteogenic differentiation indicate a role for Fam3c in osteoblast differentiation and bone homeostasis.

Mechanism of osteoclast mediated bone resorption--rationale for the design of new therapeutics.

Bone resorption is an important cellular function in the development and physiology of the skeleton. Pathophysiology of several skeletal diseases includes either increased (for instance osteoporosis, metastatic bone disease and Paget's disease of bone) or decreased (various syndromes of osteopetrosis) bone resorption rate. Thus there is a genuine need to regulate, especially inhibit, bone resorption rate in several diseases. Bone resorption can be inhibited by several strategies. One can prevent osteoclast formation, inhibit their action or induce premature cell death. All these strategies have been used in pharmacology to inhibit bone resorption and there are also physiological regulators for each of these three different phases of in osteoclast life. Many present resorption inhibitors inhibit osteoclast formation via osteoblastic cells since they are producing a number of factors that are essential for osteoclast differentiation. Best characterized of these factors are macrophage colony stimulating factor (M-CSF) and receptor activator of NFêB ligand (RANKL). For instance sex steroids, parathyroid hormone and some interleukins are known to exert their positive or negative effects on osteoclast differentiation via the RANK/RANKL/osteoprotegrin pathway. It is not yet clear enough how specific intervention to osteoclast formation is since also other cell lineages derived from hematopoetic precursors use similar signalling pathways. An effective inhibition of bone resorption can also be achieved by inhibiting osteoclast activity to resorb bone. Examples of this category of physiological and pharmacological inhibitors are calcitonin and aminobisphosphonates, respectively. Finally one can reduce bone resorption by shortening osteoclast lifespan with substances that induce apoptosis in osteoclasts. A good example of these substances is a first generation bisphosphonate, clodronate. Several new potential molecular targets have been revealed during recent years since many individual molecules in osteoclast differentiation, function and apoptosis have been identified and their physiological functions revealed. Thus we are expecting several new bone inhibitors to be developed in following years both for experimental studies and finally also for clinical use.

Effects of calcium, dairy product, and vitamin D supplementation on bone mass accrual and body composition in 10-12-y-old girls: a 2-y randomized trial.

BACKGROUND: Little is known about the relative effectiveness of calcium supplementation from food or pills with or without vitamin D supplementation for bone mass accrual during the rapid growth period. OBJECTIVE: The purpose was to examine the effects of both food-based and pill supplements of calcium and vitamin D on bone mass and body composition in girls aged 10-12 y. DESIGN: This placebo-controlled intervention trial randomly assigned 195 healthy girls at Tanner stage I-II, aged 10-12 y, with dietary calcium intakes <900 mg/d to 1 of 4 groups: calcium (1000 mg) + vitamin D3 (200 IU), calcium (1000 mg), cheese (1000 mg calcium), and placebo. Primary outcomes were bone indexes of the hip, spine, and whole body by dual-energy X-ray absorptiometry and of the radius and tibia by peripheral quantitative computed tomography. RESULTS: With the use of intention-to-treat or efficacy analysis, calcium supplementation with cheese resulted in a higher percentage change in cortical thickness of the tibia than did placebo, calcium, or calcium + vitamin D treatment (P = 0.01, 0.038, and 0.004, respectively) and in higher whole-body bone mineral density than did placebo treatment (P = 0.044) when compliance was >50%. With the use of a hierarchical linear model with random effects to control for growth velocity, these differences disappeared. CONCLUSIONS: Increasing calcium intake by consuming cheese appears to be more beneficial for cortical bone mass accrual than the consumption of tablets containing a similar amount of calcium. Diverse patterns of growth velocity may mask the efficacy of supplementation in a short-term trial of children transiting through puberty.

Association of low 25-hydroxyvitamin D concentrations with elevated parathyroid hormone concentrations and low cortical bone density in early pubertal and prepubertal Finnish girls.

BACKGROUND: Very few studies have evaluated both parathyroid hormone (PTH) and 25-hydroxyvitamin D [25(OH)D] and their effects on bone mass in children. OBJECTIVE: We studied the associations of serum 25(OH)D and intact PTH (iPTH) with bone mineral content (BMC) and bone mineral density (BMD) at different bone sites and the relation between serum 25(OH)D and iPTH in early pubertal and prepubertal Finnish girls. DESIGN: The subjects were 10-12-y-old girls (n = 193) at Tanner stage 1 or 2, who reported a mean (+/- SD) dietary calcium intake of 733 +/- 288 mg/d. 25(OH)D, iPTH, tartrate-resistant acid phosphatase 5b (TRAP 5b), urinary calcium excretion, BMC, areal BMD, and volumetric BMD were assessed by using different methods. RESULTS: Thirty-two percent of the girls were vitamin D deficient [serum 25(OH)D < or = 25 nmol/L], and 46% of the girls had an insufficient concentration (26-40 nmol/L). iPTH and TRAP 5b concentrations were significantly higher in the deficient group than in the insufficient and sufficient groups [iPTH: 43.9 +/- 15.7 compared with 38.6 +/- 11.2 pg/L (P = 0.049) and 32.7 +/- 12.1 pg/L (P < 0.001), respectively; TRAP 5b: 12.2 +/- 2.9 compared with 11.0 +/- 2.8 U/L (P = 0.009) and 10.9 +/- 1.9 U/L (P = 0.006), respectively]. The girls in the deficient group also had significantly lower cortical volumetric BMD of the distal radius (P < 0.001) and tibia shaft (P = 0.002). High iPTH concentrations were also associated with low total-body apparent mineral density and urinary calcium excretion (P < 0.007). CONCLUSIONS: Vitamin D-deficient girls have low cortical BMD and high iPTH concentrations, which are consistent with secondary hyperparathyroidism. A low vitamin D concentration accompanied by high bone resorption (TRAP 5b) may limit the accretion of bone mass in young girls.

Effects of Lactobacillus helveticus fermented milk on bone cells in vitro.

Milk fermented with Lactobacillus helveticus (L. helveticus) contains small peptides such as isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP), which inhibit the angiotensin converting enzyme (ACE). We investigated the effects of L. helveticus fermented milk whey (Lh-whey) and its components, sour milk whey, calcium and IPP and VPP peptides, on bone cells in vitro. An osteoblast assay was performed by determining the amount of deposited calcium as an index of bone formation in cultures of mouse osteoblasts formed from bone marrow-derived osteoblast precursor cells. An osteoclast assay was performed by determining the activity of tartrate-resistant acid phosphatase released into the culture medium in cultures of mouse osteoclasts formed from bone marrow-derived osteoclast precursor cells. The Lh-whey increased bone formation 1.3-1.4 times with the 1 x 10(-5), 1 x 10(-4) and 1 x 10(-3) solutions. The IPP and VPP peptides also demonstrated a significant 5-fold activation of bone formation in in vitro osteoblast cultures, whereas the sour milk whey and calcium had no effect. No significant effects were observed on osteoclasts in vitro with any of the study products. L. helveticus fermented milk whey contains bioactive components that increase osteoblastic bone formation in vitro. The effect may be due to the ACE-inhibitory IPP and VPP peptides, which showed a similar effect to that of the L. helveticus fermented milk whey.

Serum myoglobin/carbonic anhydrase III ratio as a marker of reperfusion after myocardial infarction.

BACKGROUND: Coronary patency is important for short- and long-term outcome after myocardial infarction. Serum myoglobin concentration is a sensitive marker of myocardial damage and its specificity can be improved by simultaneous measurement of carbonic anhydrase III, a skeletal muscle marker. In the present study we evaluated the role of myoglobin/carbonic anhydrase III ratio as a non-invasive marker of reperfusion. METHODS: We measured myoglobin, carbonic anhydrase III and creatine kinase MB-fraction release serially in 29 patients with acute myocardial infarction treated with thrombolysis and in 28 patients treated with primary coronary angioplasty. RESULTS: Thrombolytic therapy was followed by a 9.1+/-2.2-fold increase in myoglobin and 10.8+/-3.3-fold increase in creatine kinase MB-fraction during the first hour of treatment, while carbonic anhydrase III remained unchanged. The peak value of myoglobin/carbonic anhydrase III ratio was found at 2 h and that of creatine kinase MB-fraction at 8 h after thrombolysis. Knowledge of the reperfusion time point during primary angioplasty and follow-up of cardiac markers revealed that cut-off points of 3 and 10 h for the peak values of myoglobin/carbonic anhydrase III ratio and creatine kinase MB-fraction can be used as indicators for reperfusion, respectively. Myoglobin/carbonic anhydrase III ratio measured before treatment and at 2 and 4 h after the onset of treatment screened 23 of those 25 patients with probable reperfusion after thrombolysis. CONCLUSIONS: We conclude that measuring myoglobin/carbonic anhydrase III ratio during the first hours after initiation of thrombolysis may be useful in evaluating the success of reperfusion after acute myocardial infarction.

Novel interaction of Rab13 and Rab8 with endospanins.

Rab GTPases regulate vesicular traffic in eukaryotic cells by cycling between the active GTP-bound and inactive GDP-bound states. Their functions are modulated by the diverse selection of effector proteins that bind to specific Rabs in their activated state. We previously described the expression of Rab13 in bone cells. To search for novel Rab13 interaction partners, we screened a newborn rat bone marrow cDNA library for Rab13 effectors with a bacterial two-hybrid system. We found that Rab13 binds to the C-terminus of Endospanin-2, a small transmembrane protein. In addition to Rab13 also Rab8 bound to Endospanin-2, while no binding of Rab7, Rab10, Rab11 or Rab32 was observed. Rab13 and Rab8 also interacted with Endospanin-1, a close homolog of Endospanin-2. Rab13 and Endospanin-2 colocalised in perinuclear vesicular structures in Cos1 cells suggesting direct binding also in vivo. Endospanin-2 is implicated in the regulation of the cell surface growth hormone receptor (GHR), but the inhibition of Rab13 expression did not affect GHR cell surface expression. This suggests that the Rab13-Endospanin-2 interaction may have functions other than GHR regulation. In conclusion, we have identified a novel interaction for Rab13 and Rab8 with Endospanin-2 and Endospanin-1. The role of this interaction in cell physiology, however, remains to be elucidated.