The complement inhibitor low molecular weight dextran sulfate prevents TLR4-induced phenotypic and functional maturation of human dendritic cells.

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

White matter maturation reshapes structural connectivity in the late developing human brain.

From toddler to late teenager, the macroscopic pattern of axonal projections in the human brain remains largely unchanged while undergoing dramatic functional modifications that lead to network refinement. These functional modifications are mediated by increasing myelination and changes in axonal diameter and synaptic density, as well as changes in neurochemical mediators. Here we explore the contribution of white matter maturation to the development of connectivity between ages 2 and 18 y using high b-value diffusion MRI tractography and connectivity analysis. We measured changes in connection efficacy as the inverse of the average diffusivity along a fiber tract. We observed significant refinement in specific metrics of network topology, including a significant increase in node strength and efficiency along with a decrease in clustering. Major structural modules and hubs were in place by 2 y of age, and they continued to strengthen their profile during subsequent development. Recording resting-state functional MRI from a subset of subjects, we confirmed a positive correlation between structural and functional connectivity, and in addition observed that this relationship strengthened with age. Continuously increasing integration and decreasing segregation of structural connectivity with age suggests that network refinement mediated by white matter maturation promotes increased global efficiency. In addition, the strengthening of the correlation between structural and functional connectivity with age suggests that white matter connectivity in combination with other factors, such as differential modulation of axonal diameter and myelin thickness, that are partially captured by inverse average diffusivity, play an increasingly important role in creating brain-wide coherence and synchrony.

Neonatal dexamethasone induces premature microvascular maturation of the alveolar capillary network.

Postnatal glucocorticoid treatment of preterm infants was mimicked by treating newborn rats with dexamethasone (0.1-0.01 microg/g, days 1-4). This regimen has been shown to cause delayed alveolarization. Knowing that microvascular maturation (transformation of double- to single-layered capillary networks in alveolar septa) and septal thinning prevent further alveolarization, we measured septal maturation on electron photomicrographs in treated and control animals. In treated rats and before day 10, we observed a premature nonreversing microvascular maturation and a transient septal thinning, which both appeared focally. In vascular casts of both groups, we observed contacts between the two capillary layers of immature alveolar septa, which were predictive for capillary fusions. Studying serial electron microscopic sections of human lungs, we were able to confirm the postulated fusion process for the first time. We conclude that alveolar microvascular maturation indeed occurs by capillary fusion and that the dexamethasone-induced impairment of alveolarization is associated with focal premature capillary fusion.

Trabecular Bone Score Helps Classifying Women At Riskof Fracture Prospectively In The Ofely Study

Objectives: Trabecular Bone Score (TBS, Med-Imaps, France) is an index of bone microarchitecture calculated from antero-posterior spine DXA scan and reported to be associated with fracture in prior case-control studies and in a large prospective study with the Prodigy DXA device. Our aim was to assess the ability of TBS to predict incident fracture and improve the classification of fracture prospectively in the OFELY study.Materials/Methods: TBS was assessed in 564 postmenopausal women (66±8 years old) from the OFELY cohort, who had a spine DXA scan (QDR 4500A, Hologic, USA) between year 2000 and 2001. During a mean follow up of 7.8±1.3 years, 94 women sustained a fragility fracture.Results: At the time of baseline DXA scan, women with incident fracture were significantly older (70±9 vs. 65± 8 years), had a lower spine BMD (T-score: −1.9±1.2 vs. −1.3±1.3, p<0.001) and spine TBS (−3.1%, p<0.001) than women without incident fracture. After adjustment for age, BMI and the presence of prevalent fracture, the magnitude of fracture prediction was similar for spine BMD (OR=1.42 [1.11;1.82] per SD decrease [95% CI]) and TBS (OR=1.34 [1.04;1.74]) but the combination of TBS and spine BMD did not improve fracture prediction. Spine BMD and TBS were both correlated with age (respectively r=−0.17 and −0.49, p<0.001) and correlated together with 39% of TBS explained by spine BMD (r=0.63, p<0.001). When using the WHO classification, 38% of the fractures occurred in osteoporotic (fracture rate=29%), 47% in osteopenic (fracture rate=16%) and 15% in women with T-score >−1 (fracture rate=9%). By classifying our population in tertiles of TBS, we found that 47% of the fractures occurred in the lowest tertile of TBS (fracture rate=23%) and 39% of the fracture that occurred in osteopenic women were in the lowest tertile of TBS.Conclusions: Spine BMD and TBS predicted fractures equally well. The addition of TBS to spine BMD added only limited information on fracture risk prediction in our cohort when considering the all range of BMD. Nevertheless combining the osteopenic T-score and the lowest TBS helped defining a subset of osteopenic women at higher risk of fracture.Disclosure of Interest: None declared.

The effects of nutrition, puberty and dancing on bone density in adolescent ballet dancers.

Ballet dancers have on average a low bone mineral content (BMC), with elevated fracture-risk, low body mass index (BMI) for age (body mass index, kg/m2), low energy intake, and delayed puberty. This study aims at a better understanding of the interactions of these factors, especially with regard to nutrition. During a competition for pre-professional dancers we examined 127 female participants (60 Asians, 67 Caucasians). They averaged 16.7 years of age, started dancing at 5.8 years, and danced 22 hours/week. Assessments were made for BMI, BMC (DXA), and bone mineral apparent density (BMAD) at the lumbar spine and femoral neck, pubertal stage (Tanner score), and nutritional status (EAT-40 questionnaire and a qualitative three-day dietary record). BMI for age was found to be normal in only 42.5% of the dancers, while 15.7% had a more or less severe degree of thinness (12.6% Grade2 and 3.1% Grade 3 thinness). Menarche was late (13.9 years, range 11 to 16.8 years). Food intake, evaluated by number of consumed food portions, was below the recommendations for a normally active population in all food groups except animal proteins, where the intake was more than twice the recommended amount. In this population, with low BMI and intense exercise, BMC was low and associated with nutritional factors; dairy products had a positive and non-dairy proteins a negative influence. A positive correlation between BMAD and years since menarche confirmed the importance of exposure to estrogens and the negative impact of delayed puberty. Because of this and the probable negative influence of a high intake of non-dairy proteins, such as meat, fish, and eggs, and the positive association with a high dairy intake, ballet schools should promote balanced diets and normal weight and should recognize and help dancers avoid eating disorders and delayed puberty caused by extensive dancing and inadequate nutrition.

Bone microarchitecture assessed by TBS predicts osteoporotic fractures independent of bone density: The manitoba study.

The measurement of BMD by dual-energy X-ray absorptiometry (DXA) is the "gold standard" for diagnosing osteoporosis but does not directly reflect deterioration in bone microarchitecture. The trabecular bone score (TBS), a novel gray-level texture measurement that can be extracted from DXA images, correlates with 3D parameters of bone microarchitecture. Our aim was to evaluate the ability of lumbar spine TBS to predict future clinical osteoporotic fractures. A total of 29,407 women 50 years of age or older at the time of baseline hip and spine DXA were identified from a database containing all clinical results for the Province of Manitoba, Canada. Health service records were assessed for the incidence of nontraumatic osteoporotic fracture codes subsequent to BMD testing (mean follow-up 4.7 years). Lumbar spine TBS was derived for each spine DXA examination blinded to clinical parameters and outcomes. Osteoporotic fractures were identified in 1668 (5.7%) women, including 439 (1.5%) spine and 293 (1.0%) hip fractures. Significantly lower spine TBS and BMD were identified in women with major osteoporotic, spine, and hip fractures (all p < 0.0001). Spine TBS and BMD predicted fractures equally well, and the combination was superior to either measurement alone (p < 0.001). Spine TBS predicts osteoporotic fractures and provides information that is independent of spine and hip BMD. Combining the TBS trabecular texture index with BMD incrementally improves fracture prediction in postmenopausal women. © 2011 American Society for Bone and Mineral Research.

Proteomics fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake.

Phagocytosis, whether of food particles in protozoa or bacteria and cell remnants in the metazoan immune system, is a conserved process. The particles are taken up into phagosomes, which then undergo complex remodeling of their components, called maturation. By using two-dimensional gel electrophoresis and mass spectrometry combined with genomic data, we identified 179 phagosomal proteins in the amoeba Dictyostelium, including components of signal transduction, membrane traffic, and the cytoskeleton. By carrying out this proteomics analysis over the course of maturation, we obtained time profiles for 1,388 spots and thus generated a dynamic record of phagosomal protein composition. Clustering of the time profiles revealed five clusters and 24 functional groups that were mapped onto a flow chart of maturation. Two heterotrimeric G protein subunits, Galpha4 and Gbeta, appeared at the earliest times. We showed that mutations in the genes encoding these two proteins produce a phagocytic uptake defect in Dictyostelium. This analysis of phagosome protein dynamics provides a reference point for future genetic and functional investigations.

Treatment of large bone defects with the Ilizarov technique.

Between 1985 and 1990 we treated 11 large segmental bone defects (average 6.7 cm) in ten patients with the Ilizarov technique. Open fractures, type III according to Gustilo, represented the largest group (8 of 11 cases). The average delay before the Ilizarov technique was initiated was 8.9 months. The external fixator was usually maintained for 1 year. Bone regeneration was obtained in every case. Consolidation was not fulfilled with this technique in three cases. The complications observed were one refracture, four leg-length discrepancies (average 1.5 cm), and five axial deformities exceeding 5 degrees. No pin-track infection was observed. In our limited series of four type IIIC open fractures treated by the Ilizarov technique, no patients required amputation. The Ilizarov technique is particularly useful in the treatment of large bone defects, without major complications, especially if there is an adequate initial debridement.

Maturation-dependent neurotoxicity of lead acetate in vitro: implication of glial reactions.

Despite a wealth of data on the neurotoxic effects of lead at the cellular and molecular levels, the reasons for its development-dependent neurotoxicity are still unclear. Here, the maturation-dependent effects of lead acetate were analyzed in immature and differentiated brain cells cultured in aggregates. Markers of general cytotoxicity as well as cell-type-specific markers of glial and neuronal cells showed that immature brain cells were more sensitive to lead than the differentiated counterparts, demonstrating that the development-dependent neurotoxicity of lead can be reproduced in aggregating brain cell cultures. After 10 days of treatment, astrocytes were found to be more affected by lead acetate than neurons in immature cultures, and microglial cells were strongly activated. Eleven days after cessation of the treatment, lead acetate caused a partial loss of astrocytes and an intense reactivity of the remaining ones. Furthermore, microglial cells expressed a macrophagic phenotype, and the loss of activity of neuron-specific enzymes was aggravated. In differentiated cultures, no reactive gliosis was found. It is hypothetized that the intense glial reactions (microgliosis and astrogliosis) observed in immature cultures contribute to the development-dependent neurotoxicity of lead.

Bone anabolic potential of soy isoflavones and plant extracts and genomic changes during differentiation of hPOB-tert osteoblast-like cells

Summary For the nutritional management of bone health and the prevention of osteoporosis it is important to identify nutrients that positively influence the bone remodeling process at the cellular level. Soy isoflavones show promising osteoprotective effects in animals and humans but their mechanism of action in bone cells is yet poorly understood. Firstly, soy tissue cultures were characterized as a new and optimized source of isoflavones. A large variability in the isoflavone content was observed and high-producing strains (46.3 mg/g dry wt isoflavones) were identified. In the Ishikawa cells bioassay, the estrogenicity of isoflavones was confirmed to be 1000 to 10000 less than 17Mestradiol and that of the malonyl forms was shown for the first time (EC50 of 350 nM and 1880 nM for malonylgenistin and malonyldaidzin, respectively). The estrogenic activity of soya tissue culture extracts correlated to their isoflavone content. Secondly, the effects of phytonutrients on BMP-2 gene expression and on the mevalonate synthesis pathway, as key mediators of bone formation, were investigated. Dietary achievable concentrations of genistein and daidzein (10vM), and statins (4xM) but not 17M estradiol (10nM), induced BMP-2 gene expression (by up to 3-fold) and inhibited the cholesterol biosynthetic pathway (by up to 50%) in the human osteoblastic cell line hP0B¬tert. In addition, several plant extracts (Cyperus rotundus, Lindera benzoin and Cnidium monnieri) induced BMP-2 gene expression but this induction was not restricted to the inhibition of the cholesterol synthesis pathway neither to the estrogenicity. Finally, the gene expression profiles during hP0B-tert differentiation induced by vitamin D and dexamethasone were analyzed with the Affymetrix human GeneChip. 1665 different genes and 98 ESTs were significantly regulated. The expression profiles of bone-related genes was largely in agreement with previously documented patterns, supporting the physiological relevance of the genomic results and the hP0B-tert cell line as a valid model of human osteoblast differentiation. The expression of alternative differentiation markers during the osteogenic treatment of hP0B-tert cells indicated that the adipocyte and myoblast differentiation pathways were repressed, confirming that these culture conditions allowed only osteoblast differentiation. The gene ontology analysis identified further sub-groups of genes that may be involved in the bone formation process. Aims of the thesis In order to define new strategies for the nutritional management of bone health and for the prevention of osteoporosis the major goal of the present work was to investigate the potential of phytonutrients to positively modulate the bone formation process at the cellular level and, in particular: 1.To select and optimise alternative plant sources containing high levels of isoflavones with estrogenic activity (Chapter 3). 2.To compare the effects of statins and phytonutrients on BMP-2 gene expression and on the mevalonate synthesis pathway and to select new plant extracts with a bone anabolic potential (Chapter 4). 3.To further characterize the new human periosteal cell line, hP0B-tert, as a bone- formation model, by elucidating its gene expression profile during differentiation induced by vitamin D and dexamethasone (Chapter 5).

Bone mineral density (BMD), micro-architecture estimation (TBS) and vertebral fracture assessment (VFA) extracted from a single DXA device in combination with clinical risk factors improve significantly the identification of women at high risk of fracture

Introduction: Osteoporosis (OP) is a systemic skeletal disease characterized by a low bone mineral density (BMD) and a micro-architectural (MA) deterioration. Clinical risk factors (CRF) are often used as a MA approximation. MA is yet evaluable in daily practice by the Trabecular Bone Score (TBS) measure. TBS is a novel grey-level texture measurement reflecting bone micro-architecture based on the use of experimental variograms of 2D projection images. TBS is very simple to obtain, by reanalyzing a lumbar DXA-scan. TBS has proven to have diagnosis and prognosis value, partially independent of CRF and BMD. The aim of the OsteoLaus cohort is to combine in daily practice the CRF and the information given by DXA (BMD, TBS and vertebral fracture assessment (VFA)) to better identify women at high fracture risk. Method: The OsteoLaus cohort (1400 women 50 to 80 years living in Lausanne, Switzerland) started in 2010. This study is derived from the cohort COLAUS who started in Lausanne in 2003. The main goals of COLAUS is to obtain information on the epidemiology and genetic determinants of cardiovascular risk in 6700 men and women. CRF for OP, bone ultrasound of the heel, lumbar spine and hip BMD, VFA by DXA and MA evaluation by TBS are recorded in OsteoLaus. Preliminary results are reported. Results: We included 631 women: mean age 67.4±6.7 y, BMI 26.1±4.6, mean lumbar spine BMD 0.943±0.168 (T-score -1.4 SD), TBS 1.271±0.103. As expected, correlation between BMD and site matched TBS is low (r2=0.16). Prevalence of VFx grade 2/3, major OP Fx and all OP Fx is 8.4%, 17.0% and 26.0% respectively. Age- and BMI-adjusted ORs (per SD decrease) are 1.8 (1.2- 2.5), 1.6 (1.2-2.1), 1.3 (1.1-1.6) for BMD for the different categories of fractures and 2.0 (1.4-3.0), 1.9 (1.4-2.5), 1.4 (1.1-1.7) for TBS respectively. Only 32 to 37% of women with OP Fx have a BMD < -2.5 SD or a TBS < 1.200. If we combine a BMD < -2.5 SD or a TBS < 1.200, 54 to 60% of women with an osteoporotic Fx are identified. Conclusion: As in the already published studies, these preliminary results confirm the partial independence between BMD and TBS. More importantly, a combination of TBS subsequent to BMD increases significantly the identification of women with prevalent OP Fx which would have been miss-classified by BMD alone. For the first time we are able to have complementary information about fracture (VFA), density (BMD), micro- and macro architecture (TBS & HAS) from a simple, low ionizing radiation and cheap device: DXA. Such complementary information is very useful for the patient in the daily practice and moreover will likely have an impact on cost effectiveness analysis.