The Role of MKP-1 in the Anti-Proliferative Effects of Glucocorticoids in Primary Rat Pre-Osteoblasts.

Glucocorticoid (GC)-induced osteoporosis has been attributed to a GC-induced suppression of pre-osteoblast proliferation. Our previous work identified a critical role for mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in mediating the anti-proliferative effects of GCs in immortalized pre-osteoblasts, but we subsequently found that MKP-1 null mice were not protected against the pathological effects of GCs on bone. In order to reconcile this discrepancy, we have assessed the effects of GCs on proliferation, activation of the MAPK ERK1/2 and MKP-1 expression in primary adipose-derived stromal cells (ADSCs) and ADSC-derived pre-osteoblasts (ADSC-OBs). ADSCs were isolated by means of collagenase digestion from adipose tissue biopsies harvested from adult male Wistar rats. ADSC-OBs were prepared by treating ADSCs with osteoblast differentiation media for 7 days. The effects of increasing concentrations of the GC dexamethasone on basal and mitogen-stimulated cell proliferation were quantified by tritiated thymidine incorporation. ERK1/2 activity was measured by Western blotting, while MKP-1 expression was quantified on both RNA and protein levels, using semi-quantitative real-time PCR and Western blotting, respectively. GCs were strongly anti-proliferative in both naïve ADSCs and ADSC-OBs, but had very little effect on mitogen-induced ERK1/2 activation and did not upregulate MKP-1 protein expression. These findings suggest that the anti-proliferative effects of GCs in primary ADSCs and ADSC-OBs in vitro do not require the inhibition of ERK1/2 activation by MKP-1, which is consistent with our in vivo findings in MKP-1 null mice.

Vertebral fracture prevalence in black and white South African women.

UNLABELLED: Black women are generally regarded as being less prone to the development of osteoporosis. This study reports a similar prevalence of morphometric vertebral fractures in black (9.1 %) and white (5.0 %) South African women. Clinical risk factors and bone strength parameters contributed differently to fracture risk in the two ethnic cohorts. PURPOSE: Vertebral fracture represents one of the most common osteoporotic fractures and is a significant cause of morbidity and mortality. Little is known regarding the prevalence of vertebral fractures on the African continent. We therefore prospectively examined the prevalence of vertebral fracture on radiographs of the thoraco-lumbar spine in otherwise healthy community-dwelling older black and white South African women. METHODS: Radiographs of the spine (T4-L5) were obtained randomly in 189 women (47 % black), aged 40 years or older, for the analysis of vertebral fracture. Radiographs were evaluated by a single radiologist, blinded to clinical data, using Genant's semi-quantitative method. Clinical risk factors for osteoporosis, risk factors for falls (fall history, quadriceps strength, lateral sway and reaction time), areal and volumetric bone mineral density of the spine and hip, calcaneal ultrasonography (QUS) and vertebral macro-geometry were assessed in the two ethnic groups and the association with prevalent vertebral fractures examined. RESULTS: Vertebral fracture prevalence in older South African black and white women was similar (9.1 % in black and 5.0 % in white women). In black women, lower body weight and lower areal and volumetric bone mineral density (BMD) at all sites could serve as markers of increased fracture risk. Older age, physical inactivity, lower muscle strength and lower femoral BMD were associated with vertebral fracture risk in whites. CONCLUSION: Our findings are noteworthy and the first attempt to compare vertebral fracture risk in women of different ethnicities on the African continent. A similar vertebral fracture risk between black and white women in South Africa must be considered at present to ensure appropriate evaluation in all subjects who present with clinical risk factors for osteoporosis, regardless of ethnicity.

Validating and extending the three process model of alertness in airline operations.

Sleepiness and fatigue are important risk factors in the transport sector and bio-mathematical sleepiness, sleep and fatigue modeling is increasingly becoming a valuable tool for assessing safety of work schedules and rosters in Fatigue Risk Management Systems (FRMS). The present study sought to validate the inner workings of one such model, Three Process Model (TPM), on aircrews and extend the model with functions to model jetlag and to directly assess the risk of any sleepiness level in any shift schedule or roster with and without knowledge of sleep timings. We collected sleep and sleepiness data from 136 aircrews in a real life situation by means of an application running on a handheld touch screen computer device (iPhone, iPod or iPad) and used the TPM to predict sleepiness with varying level of complexity of model equations and data. The results based on multilevel linear and non-linear mixed effects models showed that the TPM predictions correlated with observed ratings of sleepiness, but explorative analyses suggest that the default model can be improved and reduced to include only two-processes (S+C), with adjusted phases of the circadian process based on a single question of circadian type. We also extended the model with a function to model jetlag acclimatization and with estimates of individual differences including reference limits accounting for 50%, 75% and 90% of the population as well as functions for predicting the probability of any level of sleepiness for ecological assessment of absolute and relative risk of sleepiness in shift systems for safety applications.

Bone density in black and white South African women: contribution of ethnicity, body weight and lifestyle.

UNLABELLED: Ethnic differences in bone mineral density (BMD) between healthy adult black and white South African women were studied. Higher BMD was only noted at the femoral sites in black women. Body weight significantly impacted these findings. A lower fracture risk at all skeletal sites cannot be assumed in black South African (SA) women. PURPOSE: Bone mineral density (BMD) varies amongst women of different ethnicities. African-Americans have higher BMD at all skeletal sites compared with whites. On the African continent, bone density studies suggest site-specific ethnic differences in BMD. To examine the contribution of body weight and lifestyle characteristics to ethnic differences in BMD between adult black and white South African women, we assessed lumbar spine (SBMD), femoral neck (FNBMD) and total femoral BMD (FTBMD) by dual-energy X-ray absorptiometry (DXA) in 184 black and 143 white women aged between 23 and 82 years. METHODS: BMDs were compared amongst pre- and postmenopausal blacks and whites before and after adjustment for covariates with significant univariate association with BMD. Volumetric bone mineral apparent density (BMAD) of the spine and femoral neck was also calculated to account for ethnic differences in bone size. RESULTS: Before adjustment, SBMD was lower (p < 0.05), FTBMD similar and FNBMD (p < 0.01) higher in premenopausal black women. Similar SBMD, but significantly higher BMD at the femoral sites (p < 0.01), was noted in postmenopausal blacks compared with whites. Amongst anthropometric measures and lifestyle factors, only adjustment for weight significantly altered these observed ethnic differences in bone density. After adjustment for weight, SBMD remained lower in premenopausal blacks and became lower in young postmenopausal blacks. Weight adjustment eliminated all ethnic differences in proximal femoral BMD measurements, with the exception of FNBMD that remained higher in younger postmenopausal blacks. Before adjustment, calculated SBMAD was similar and FNBMAD consistently higher in blacks in all the menstrual groups. Adjustment for weight did not alter these findings. CONCLUSION: Most of the observed ethnic difference in BMD was explained by differences in body weight between black and white SA women. The higher femoral BMD in older blacks may explain, the lower hip fracture prevalence in black South African women. The lower SBMD in pre- and postmenopausal black women in this study suggests that factors other than BMD should be considered to explain a lower vertebral fracture prevalence in blacks, if a lower fracture prevalence does indeed exist.

Screening for hypothalamic-pituitary-adrenal axis suppression in asthmatic children remains problematic: a cross-sectional study.

OBJECTIVE: To determine which parameter is the most useful screening test for hypothalamic-pituitary-adrenal suppression in asthmatic children. DESIGN: Cross-sectional study. SETTING: Paediatric allergy clinics in Cape Town, South Africa. PARTICIPANTS: 143 asthmatic children of mostly mixed ancestry, aged 5-12 years. OUTCOME MEASURES: Primary outcome measures included Spearman correlation coefficients (r) calculated between the postmetyrapone (PMTP) serum adrenocorticotropic hormone (ACTH), 11-deoxycortisol (11DOC), 11DOC+ cortisol (C) and height, weight, height velocity, weight velocity, change in systolic blood pressure from supine to standing, early morning urinary free cortisol (UFC), morning C, ACTH and dehydroepiandrosterone sulfate (DHEAS). Secondary outcome measures were the receiver operating characteristics (ROC) curve and the diagnostic statistics for the most promising test. RESULTS: All screening variables were weakly correlated with the three PMTP outcomes. Only DHEAS and UFC (nmol/m(2)) were statistically significant-DHEAS for PMTP ACTH and 11DOC (r=0.20, p=0.025 and r=0.21, p=0.017); UFC (nmol/m(2)) for PMTP 11DOC and 11DOC+C (r=0.19, p=0.033 and r=0.20, p=0.022). The area under ROC curve for DHEAS in the 5-year to 9-year age group was 0.69 (95% CI 0.47 to 0.92). At DHEAS cut-off of 0.2 µmol/L: sensitivity=0.88 (CI 0.47 to 1.00), specificity=0.61 (CI 0.42 to 0.78), positive predictive value=0.37 (CI 0.16 to 0.62), negative predictive value=0.95 (CI 0.75 to 1.00), accuracy=0.67 (CI 0.50 to 0.81), positive likelihood ratio=2.26 (CI 1.35 to 3.78), negative likelihood ratio=0.20 (CI 0.03 to 1.30). CONCLUSIONS: No parameter is useful as a universal screening test. DHEAS may be suitable to exclude HPAS before adrenarche. Further research is needed to confirm these findings and identify factors, for example, genetic that may predict or protect against HPAS.

High prevalence of lipoatrophy in pre-pubertal South African children on antiretroviral therapy: a cross-sectional study.

BACKGROUND: Despite changes in WHO guidelines, stavudine is still used extensively for treatment of pediatric HIV in the developing world. Lipoatrophy in sub-Saharan African children can be stigmatizing and have far-reaching consequences. The severity and extent of lipoatrophy in pre-pubertal children living in sub-Saharan Africa is unknown. METHODS: In this cross-sectional study, children who were 3-12 years old, on antiretroviral therapy and pre-pubertal were recruited from a Family HIV Clinic in South Africa. Lipoatrophy was identified and graded by consensus between two HIV pediatricians using a standardized grading scale. A professional dietician performed formal dietary assessment and anthropometric measurements of trunk and limb fat. Previous antiretroviral exposures were recorded. In a Dual-Energy X-ray Absorbtiometry (DXA) substudy body composition was determined in 42 participants. RESULTS: Among 100 recruits, the prevalence of visually obvious lipoatrophy was 36% (95% CI: 27%-45%). Anthropometry and DXA measurements corroborated the clinical diagnosis of lipoatrophy: Both confirmed significant, substantial extremity fat loss in children with visually obvious lipoatrophy, when adjusted for age and sex. Adjusted odds ratio for developing lipoatrophy was 1.9 (95% CI: 1.3 - 2.9) for each additional year of accumulated exposure to standard dose stavudine. Cumulative time on standard dose stavudine was significantly associated with reductions in biceps and triceps skin-fold thickness (p=0.008). CONCLUSIONS: The prevalence of visually obvious lipoatrophy in pre-pubertal South African children on antiretroviral therapy is high. The amount of stavudine that children are exposed to needs review. Resources are needed to enable low-and-middle-income countries to provide suitable pediatric-formulated alternatives to stavudine-based pediatric regimens. The standard stavudine dose for children may need to be reduced. Diagnosis of lipoatrophy at an early stage is important to allow timeous antiretroviral switching to arrest progression and avoid stigmatization. Diagnosis using visual grading requires training and experience, and DXA and comprehensive anthropometry are not commonly available. A simple objective screening tool is needed to identify early lipoatrophy in resource-limited settings where specialized skills and equipment are not available.

Hypothalamic-pituitary-adrenal axis suppression in asthmatic children on inhaled and nasal corticosteroids--more common than expected?

BACKGROUND: Hypothalamic pituitary adrenal axis suppression (HPAS) when treating asthmatic children with inhaled corticosteroids (ICS) is thought to be rare. OBJECTIVE: To determine the prevalence of HPAS in asthmatic children treated with ICS and nasal steroids (NS). METHODS: Twenty-six asthmatic children were recruited. Clinical features of HPAS, height, weight, height and weight velocity, steroid dose, adherence, symptom control and lung functions were documented. Metyrapone test was performed if the serum cortisol was > 83 nmol/L (> 3 microg/dL). RESULTS: No child had a serum cortisol < 83 nmol/L (< 3 microg/dL). Prevalence of HPAS was 35 (CI = 17%-56%). Daily NS dose/ m2 and cumulative NS dose/m2 were significantly (p = 0.03) inversely correlated with the post-metyrapone ACTH (r = -0.42 for both). Current ICS dose was not associated with the post-metyrapone ACTH (r = 0). There was a weak correlation with the daily ICS dose/m2 (r = -0.12) and cumulative ICS dose/m2 (r = -0.26). CONCLUSIONS: A third of asthmatic children on ICS and NS develop HPAS. Contributing factors are the use of NS, body size and cumulative dose of ICS.

Hypothalamic-pituitary-adrenal axis suppression in asthmatic children on inhaled and nasal corticosteroids: is the early-morning serum adrenocorticotropic hormone (ACTH) a useful screening test?

BACKGROUND: Hypothalamic-pituitary-adrenal axis suppression (HPAS) in asthmatic children treated with inhaled corticosteroids (ICS), with or without nasal steroids (NS), may be more common than previously thought. Only dynamic testing will identify children at risk of adrenal crisis. It is impractical to test all asthmatic children for HPAS with a gold standard adrenal function test, i.e. the metyrapone or insulin tolerance test. OBJECTIVE: To determine which clinical or biochemical parameter is the most useful screening test for HPAS in asthmatic children. METHODS: Twenty-six asthmatic children, 5-18 yr old, on ICS ± NS, not treated with oral or topical steroids in the preceding year were recruited. Height, weight, height velocity, weight velocity and a change in systolic blood pressure from the recumbent to the standing position (ΔSBP) were recorded. Early-morning urine for urinary free cortisol (UFC) and urinary cortisol metabolites (UCM) was collected. UFC was analysed by both a chemiluminescent assay and gas chromatography/mass spectrometry (GC-MS). Morning serum cortisol and adrenocorticotropic hormone (ACTH) levels were measured. The overnight metyrapone test was performed if the fasting morning serum cortisol was >83 nmol/l. HPAS was diagnosed if the ACTH failed to rise >100 pg/ml after metyrapone. Spearman correlation coefficients (r) were calculated between the post-metyrapone ACTH and each variable. A receiver-operating characteristics (ROC) curve was drawn for the most promising test, and the diagnostic performance was calculated. RESULTS: All clinical and biochemical parameters investigated were weakly and non-significantly correlated with the post-metyrapone ACTH, except for the morning serum ACTH (r = 0.68; p <0.001). The best discrimination between those who have and those who do not have HPAS is a morning serum ACTH level of 11.7 pg/ml. This corresponds to a sensitivity of 0.89 (0.57-0.98), a specificity of 0.77 (0.53-0.90), a positive predictive value of 0.67 (0.39-0.87), a negative predictive value of 0.93 (0.69-0.99), an accuracy of 0.81 (0.61-0.94), a positive likelihood ratio of 3.78 (1.68-9.49) and a negative likelihood ratio of 0.15 (0.03-0.60). CONCLUSIONS: The morning serum ACTH level was found to be the most useful screening test to detect HPAS in this sample of children receiving ICS ± NS. A larger study should be undertaken to refine the diagnostic precision of the morning serum ACTH level.

MKP-1 knockout does not prevent glucocorticoid-induced bone disease in mice.

Glucocorticoid-induced osteoporosis (GCOP) is predominantly caused by inhibition of bone formation, resulting from a decrease in osteoblast numbers. Employing mouse (MBA-15.4) and human (MG-63) osteoblast cell lines, we previously found that the glucocorticoid (GC) dexamethasone (Dex) inhibits cellular proliferation as well as activation of the MAPK/ERK signaling pathway, essential for mitogenesis in these cells, and that both these effects could be reversed by the protein tyrosine phosphatase (PTP) inhibitor vanadate. In a rat model of GCOP, the GC-induced changes in bone formation, mass, and strength could be prevented by vanadate cotreatment, suggesting that the GC effects on bone were mediated by one or more PTPs. Employing phosphatase inhibitors, qRT-PCR, Western blotting, and overexpression/knockdown experiments, we concluded that MKP-1 was upregulated by Dex, that this correlated with the dephosphorylation of ERK, and that it largely mediated the in vitro effects of GCs on bone. To confirm the pivotal role of MKP-1 in vivo, we investigated the effects of the GC methylprednisolone on the quantitative bone histology of wild-type (WT) and MKP-1 homozygous knockout (MKP-1(-/-)) mice. In WT mice, static bone histology revealed that GC administration for 28 days decreased osteoid surfaces, volumes, and osteoblast numbers. Dynamic histology, following time-spaced tetracycline labeling, confirmed a significant GC-induced reduction in osteoblast appositional rate and bone formation rate. However, identical results were obtained in MKP-1 knockout mice, suggesting that in these animals upregulation of MKP-1 by GCs cannot be regarded as the sole mediator of the GC effects on bone.

Effect of rapamycin on hepatic osteodystrophy in rats with portasystemic shunting.

AIM: To study if T-cell activation related to portasystemic shunting causes osteoclast-mediated bone loss through RANKL-dependent pathways. We also investigated if T-cell inhibition using rapamycin would protect against bone loss in rats. METHODS: Portasystemic shunting was performed in male Sprague-Dawley rats and rapamycin 0.1 mg/kg was administered for 15 wk by gavage. Rats received powderized chow and supplemental feeds to prevent the effects of malnutrition on bone composition. Weight gain and growth was restored after surgery in shunted animals. At termination, biochemical parameters of bone turnover and quantitative bone histology were assessed. Markers of T-cell activation, inflammatory cytokine production, and RANKL-dependent pathways were measured. In addition, the roles of IGF-1 and hypogonadism were investigated. RESULTS: Portasystemic shunting caused low turnover osteoporosis that was RANKL independent. Bone resorbing cytokine levels, including IL-1, IL-6 and TNFalpha, were not increased in serum and TNFalpha and RANKL expression were not upregulated in PBMC. Portasystemic shunting increased the circulating CD8+ T-cell population. Rapamycin decreased the circulating CD8+ T-cell population, increased CD8+ CD25+ T-regulatory cell population and improved all parameters of bone turnover. CONCLUSION: Osteoporosis caused by portasystemic shunting may be partially ameliorated by rapamycin in the rat model of hepatic osteodystrophy.