The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-β2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP)α and C/EBPβ α at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor γ (PPARγ2) transcripts at 7 days. TGF-β2 administration in unloaded rats corrected the rise in C/EBPα and C/EBPβ transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPARγ2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBPα and C/EBPβ expression by TGF-β2 was associated with increased PPARγ serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPARγ transactivating activity. The sequential inhibitory effect of TGF-β2 on C/EBPα, C/EBPβ, and PPARγ2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-β2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBPα, C/EBPβ, and PPARγ expression and activity, which provides a sequential mechanism by which TGF-β2 regulates adipogenic differentiation of bone marrow stromal cells in vivo.

BackgroundSerum sickness-like reaction (SSLR) is an acute inflammatory condition affecting predominantly children. The pathophysiology remains unclear, but drugs are considered the main trigger.ObjectiveThe aim of this study was to describe the clinical and laboratory features, triggers, and treatment modalities in children diagnosed with SSLR.MethodsWe conducted a 10-year retrospective cohort study including all paediatric patients (0 to 18 years old) with query SSLR referred to the Adverse Drug Reactions Clinic at the Children's Hospital of Western Ontario. Diagnostic criteria included acute skin rash plus joint inflammation with or without fever.ResultsWe included 83 patients (47 females). Age ranged from 11 months to 12 years (mean 3.2 years). Amoxicillin was the trigger in 82.7% of patients. The mean time between the exposure to the triggering drug and the development of the symptoms was 8.5 days. Urticaria-like and Erythema multiforme-like lesions were present in 35% and 38.5% of the cases, respectively. Joint inflammation affecting hands/feet was present in 60%. Pruritus, lip/eye swelling, and fever were reported in 33, 31, and 45% of patients, respectively. The lymphocyte toxicity assay (LTA) showed incremental T-cell toxicity in 32 of 34 patients. Children that received treatment with antihistamines/nonsteroidal anti-inflammatory drugs (NSAIDs) plus oral steroids had a mean recovery time shorter than those treated only with antihistamines/NSAIDs (6 versus 8 days; P=0.09).ConclusionsIn our study, SSLR was mostly triggered by amoxicillin and had a mean time presentation of 8.5 days. Further prospective and well-conducted studies are needed.

During spaceflights in the shuttle, rats are provided specific food bars. To determine whether this diet allows normal body and skeletal growth, we used four groups of rats fed either standard pellet food or space food bars during a 2-wk unloading experiment. We recorded food intake, body weight, tibial bone mass, and mineral content by ash analyses, cancellous bone volume, and cell activities by histomorphometry. We found that food intake was not different when comparing the two types of food, but that suspended animals had a lower food intake than normal loaded animals. Body weight and bone mass were found lower in suspended animals than in normal loaded animals. Finally, longitudinal growth rate, cancellous bone volume, and bone formation rate were lower in suspended animals, irrespective of the type of food. These results show that space food bar feeding did not affect normal body and skeletal growth, and that body and bone changes due to unloading were not significantly different in animals fed space food bars and standard food.

Skeletal unloading induced by hindlimb suspension in rats reduces bone formation and induces osteopenia, but its effect on adipogenesis is unknown. We assessed the effects of unloading and transforming growth factor (TGF) beta2 on bone marrow stromal cell adipocyte differentiation in relation with osteoblast differentiation. Skeletal unloading rapidly (4-7 days) decreased osteoblast transcription factor Runx2, osteocalcin (OC), and type I collagen messenger RNA (mRNA) levels and reduced bone formation in the long bone metaphysis. Conversely, unloading increased expression of the adipocyte transcription factor peroxisome proliferator-activated receptor gamma2 (PPARgamma2) at 4 days and increased expression of the adipocyte differentiation genes lipoprotein lipase (LPL) and aP2 in the bone marrow stroma at 7 days. Consistently, unloading increased the number and volume of adipocytes in the bone marrow stroma. Continuous (0-7 days) and late (4-7 days) treatments with TGF-beta2 corrected the abnormal expression of Cbfa1/Runx2, OC, and type I collagen mRNAs and normalized bone formation in unloaded metaphyseal bone. Moreover, both TGF-beta2 treatments decreased PPARy2 and C/EBPalpha mRNA levels at 4 days and normalized aP2 and LPL expression and adipocyte number and volume at 7 days. These results show that skeletal unloading increases adipocyte differentiation concomitantly with inhibition of osteoblast differentiation. These abnormalities are prevented and reversed by TGF-beta2, suggesting a role for TGF-beta in the control of adipogenic differentiation in the bone marrow stroma.

Bone has two fundamental roles, i.e. reservoir of inorganic elements (calcium, phosphorus, magnesium), and mechanical support for soft tissues. Together with the combined action of muscles, tendons, and ligaments, this support permits motion and locomotion. These two functions rely on a sophisticated bone tissue architecture, and on the adaptability of this structure, with modeling and remodeling processes, themselves associated with the Coupled activity of specialized bone cell populations. Microgravity associated with spaceflight has been shown in numerous experiments to be associated with marked skeletal changes. However, thirty years after the first changes in phospho-calcium metabolism were observed in astronauts after spaceflight, current knowledge does not provide a full understanding of this phenomenon. These changes have been extensively studied in rats and in monkeys flown for one to three weeks onboard Russian Biosatellite and American Shuttle missions. Changes ranged from decrease in bone volume to alterations in biomechanical properties, associated with reduction in both cortical and trabecular bone formation. Although skeletal unloading is at least one of the most promising ways of explanation of this alteration, the mechanisms by which bone senses loads and adjusts to them are not clearly known. The involvement of endocrine-type systemic phenomena in this process has not been formally proven. Moreover, the fact that weight-bearing bones are most affected is suggestive of a predominantly local or loco-regional phenomenon and local growth factors represent good candidates for at least one part of the explanation of these phenomenons. However, the transmission of mechanical constraints within bone tissue as cell messages, and the nature of these messages remains so far poorly known, and needs further investigations.

Using analyses of iliac crest cell and tissue, back-scattered electron imaging, and biochemical techniques, we characterized the effects of a 14-day spaceflight (Bion 11) on bone structure and bone formation in two 3- to 4-yr-old male rhesus monkeys compared with eight age-matched Earth-control monkeys. We found that postflight bone volume was 35% lower than preflight values in flight monkeys. This was associated with reduced osteoid (-40%) and mineralizing (-32%) surfaces and decreased bone formation rate (-53%). Moreover, flight monkeys exhibited trends to lower values of mineralization profile in iliac bone (back-scattered electron imaging) and to decreased osteocalcin serum levels (P = 0.08). The initial number of trabecular bone cells yielded in cultures did not differ in flight and control animals before or after the flight. However, osteoblastic cell proliferation was markedly lower in postflight vs. preflight at 9 and 14 days of culture in one flight monkey. This study suggests that a 14-day spaceflight reduces iliac bone formation, osteoblastic activity, and/or recruitment in young rhesus monkeys, resulting in decreased trabecular bone volume.

Bone formation and structure have been shown repeatedly to be altered after spaceflight. However, it is not known whether these changes are related to a stress-related altered status of the corticosteroid axis. We investigated the role of corticosteroids on spaceflight-induced effects in rat pelvis and thoracic vertebrae. Thirty-six male Sprague-Dawley rats were assigned to a flight, flight control, or vivarium group (n = 12/group). Bilateral adrenalectomy was performed in six rats per group, the additional six rats undergoing sham surgery. Adrenalectomized (ADX) rats were implanted with corticosteroid pellets. On recovery from spaceflight, thoracic vertebrae and the whole pelvis were removed and processed for biochemistry, histomorphometry, or bone cell culture studies. The 17-day spaceflight resulted in decreased bone volume (BV) in the cotyle area of pelvic bones (-12%; p < 0.05) associated with approximately 50% inhibition of bone formation in the cancellous area of pelvic metaphyses and in thoracic vertebral bodies. The latter effect was associated with a decreased number of endosteal bone cells isolated from the bone surface (BS) in these samples (-42%; p < 0.05). This also was associated with a decreased number of alkaline phosphatase positive (ALP+) endosteal bone cells at 2 days and 4 days of culture, indicating decreased osteoblast precursor cell recruitment. Maintaining basal serum corticosterone levels in flight-ADX rats did not counteract the impaired bone formation in vertebral or pelvic bones. Moreover, the decreased ex vivo number of total and ALP+ endosteal bone cells induced by spaceflight occurred independent of endogenous corticosteroid hormone levels. These results indicate that the microgravity-induced inhibition of bone formation and resulting decreased trabecular bone mass in specific areas of weight-bearing skeleton in growing rats occur independently of endogenous glucocorticoid secretion.

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 hone 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 OD vs. controls) and osteoclastic (+31%, p < 0.1 OD vs. controls) surfaces. This was not paralleled by increased hone 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 OD vs. controls) acid 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 OD 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, (C) 2000 by Elsevier Science Inc. All rights reserved.

INTRODUCTION:Physical exercise is known to augment bone mass, but the specific mechanisms by which physical activity influences skeletal metabolism is still not thoroughly understood. So far, time related events of bone tissue adaptation to physical training have not been investigated. We, therefore, studied the time-course effects of daily spontaneous exercise training on bone metabolism in the rat by biochemical and tissue analyses.METHODS:Forty-five 4-wk-old female Dark Agouti rats were used, randomly assigned to voluntary exercising groups of five animals for 1, 2, 3, and 4 wk, or to age-matched sedentary controls. At sacrifice, blood was sampled for determination of parameters of bone and calcium metabolism (osteocalcin, alkaline phosphatases, total and ionized calcium, phosphorus, parathyroid hormone). Right and left tibiae were removed for bone mass (dry and ash mass, mineral content) and histomorphometric analyses.RESULTS:We found that the animals performed large amounts of exercise (reaching 50 +/- 8 km x wk(-1)), and that this voluntary physical training induced significant changes in bone metabolism. An increase (approximately 32%) in serum alkaline phosphatase activity was already measurable at the end of the first week of exercising and persisted throughout the experiment, accompanied by a concomitant increase in osteoblastic bone formation (approximately 27%), as evaluated in bone tissue. These changes were associated with consecutive increases in tibial bone mass (approximately 7.6%), mineral content (approximately 7.4%) and metaphyseal bone volume (approximately 17%) measurable 4 wk after the onset of exercise, with interesting positive correlations between tibial bone mass parameters and soleus muscle mass. Conversely, no change was evidenced in biochemical parameters of calcium metabolism, except for an increase in phosphorus serum levels in trained animals.CONCLUSION:Daily spontaneous exercise training induced an increase in bone mass and bone volume in less than 4 wk. Further studies are needed to fully investigate these changes during the very first days of exercising.

(2000). Role of Growth Factors in Osteoblast Alterations Induced by Skeletal Unloading in Rats. Growth Factors: Vol. 18, No. 1, pp. 1-10.

In normal life on earth, the locomotor system is exposed to two types of stimulation: gravity (passive stimulation) and motion (active stimulation). Both permanently combine, and the interactions between locomotion and gravity induce an overall recruitment which is repeated daily and maintains the bone tissue structure within the range of constraints to which it is adapted. This range is one of the basic hypotheses underlying the mechanical concepts of bone structure control, and it has been considered as logical to assume that weightlessness of spaceflight should produce bone loss since astronauts are outside of the terrestrial gravitational field of forces, no longer relying on muscular work to change positions or move. But, thirty years after the first changes in phospho-calcium metabolism were observed in astronauts after spaceflight, current knowledge does not provide a full understanding of this pathogeny, and prove the G-factor is now considered as an essential component of the experimental tools available to study bone physiology. The study of the physiology of bone tissue usually consists in the investigation of its two fundamental roles, i.e. reservoir of inorganic elements (calcium, phosphorus, magnesium) and mechanical support for soft tissues. Together with the combined action of muscles, tendons, and ligaments, this support permits motion and locomotion. These two functions rely on a sophisticated bone tissue architecture, and on the adaptability of this structure, with modeling and remodeling processes, themselves associated with the coupled activity of specialized bone cell populations.

Studies on calcium nutrition in appropriate large animal models can be directly relevant to humans. We have examined the effect of dietary Ca deficiency on various bone and bone-related variables, including plasma markers, histomorphometry, mineral content and breaking strength in pigs. Three groups of eight 38-d-old female pigs were fed adequate (0.9%; control), low (0.4%; LCa) or very low (0.1%; VLCa) Ca diets for 32 d. Plasma Ca significantly decreased over time only in the VLCa-deficient pigs. The concentrations of the parathyroid hormones (PTH) and calcitriol increased as Ca deficiency developed, and the plasma PTH and calcitriol levels varied inversely with dietary Ca. The total bone ash contents, bending moments, trabecular bone volume and the mineral apposition rate all decreased as the calcium intake decreased. The osteoclast surface areas were greater than those of controls in both Ca-deficient groups, whereas the osteoblast surface areas were greater only in the VLCa group. The plasma osteoblast-related markers (alkaline phosphatase, carboxy-terminal propeptide of type I procollagen and osteocalcin) were either greater or unaffected in the Ca-deficient pigs. The results indicate that deficient bone mineralization combined with an increased bone resorption led to bone loss and fragility. The differences in the changes in bone cells (number and activity) between LCa and VLCa groups might be due to differences (time and extent) of circulating PTH and calcitriol. The defective mineralization in both Ca-depleted groups resulted mainly from the lack of Ca because their osteoblast activity was either maintained or stimulated. The results also underline the progressive sensitivity of pigs to Ca supply and the usefulness of this model.