Polymorphisms in genes involved in the NF-κB signalling pathway are associated with bone mineral density, geometry and turnover in men

Introduction

In this study, we aimed to investigate the association between single nucleotide polymorphisms (SNPs) within two genes involved in the NF-κB cascade (GPR177 and MAP3K14) and bone mineral density (BMD) assessed at different skeletal sites, radial geometric parameters and bone turnover.

Methods

Ten GPR177 SNPs previously associated with BMD with genome-wide significance and twelve tag SNPs (r²≥0.8) within MAP3K14 (±10 kb) were genotyped in 2359 men aged 40–79 years recruited from 8 centres for participation in the European Male Aging Study (EMAS). Measurement of bone turnover markers (PINP and CTX-I) in the serum and quantitative ultrasound (QUS) at the calcaneus were performed in all centres. Dual energy X-ray absorptiometry (DXA), at the lumbar spine and hip, and peripheral quantitative computed tomography (pQCT), at the distal and midshaft radius, were performed in a subsample (2 centres). Linear regression was used to test for association between the SNPs and bone measures under an additive genetic model adjusting for study centre.

Results

We validated the associations between SNPs in GPR177 and BMD_a previously reported and also observed evidence of pleiotrophic effects on density and geometry. Rs2772300 in GPR177 was associated with increased total hip and LS BMD_a, increased total and cortical vBMD at the radius and increased cortical area, thickness and stress strain index. We also found evidence of association with BMD_a, vBMD, geometric parameters and CTX-I for SNPs in MAP3K14. None of the GPR177 and MAP3K14 SNPs were associated with calcaneal estimated BMD measured by QUS.

Conclusion

Our findings suggest that SNPs in GPR177 and MAP3K14 involved in the NF-κB signalling pathway influence bone mineral density, geometry and turnover in a population-based cohort of middle aged and elderly men. This adds to the understanding of the role of genetic variation in this pathway in determining bone health.     

The ESR1 (6q25) locus is associated with calcaneal ultrasound parameters and radial volumetric bone mineral density in European men

Purpose

Genome-wide association studies (GWAS) have identified 6q25, which incorporates the oestrogen receptor α gene (ESR1), as a quantitative trait locus for areal bone mineral density (BMD_a) of the hip and lumbar spine. The aim of this study was to determine the influence of this locus on other bone health outcomes; calcaneal ultrasound (QUS) parameters, radial peripheral quantitative computed tomography (pQCT) parameters and markers of bone turnover in a population sample of European men.

Methods

Eight single nucleotide polymorphisms (SNP) in the 6q25 locus were genotyped in men aged 40–79 years from 7 European countries, participating in the European Male Ageing Study (EMAS). The associations between SNPs and measured bone parameters were tested under an additive genetic model adjusting for centre using linear regression.

Results

2468 men, mean (SD) aged 59.9 (11.1) years had QUS measurements performed and bone turnover marker levels measured. A subset of 628 men had DXA and pQCT measurements. Multiple independent SNPs showed significant associations with BMD using all three measurement techniques. Most notably, rs1999805 was associated with a 0.10 SD (95%CI 0.05, 0.16; p = 0.0001) lower estimated BMD at the calcaneus, a 0.14 SD (95%CI 0.05, 0.24; p = 0.004) lower total hip BMD_a, a 0.12 SD (95%CI 0.02, 0.23; p = 0.026) lower lumbar spine BMD_a and a 0.18 SD (95%CI 0.06, 0.29; p = 0.003) lower trabecular BMD at the distal radius for each copy of the minor allele. There was no association with serum levels of bone turnover markers and a single SNP which was associated with cortical density was also associated with cortical BMC and thickness.

Conclusions

Our data replicate previous associations found between SNPs in the 6q25 locus and BMD_a at the hip and extend these data to include associations with calcaneal ultrasound parameters and radial volumetric BMD.     

The regulatory framework for similar biotherapeutic products in Cuba

Biopharmaceuticals make up a significant proportion of medicinal products used for the treatment of diseases such as cancer, arthritis, cardiac dysfunctions and AIDS. Access to therapies based on the use of these products has been limited as a result of the high marketing costs. Cuba has a biopharmaceutical industry with great potential for innovation, capable of developing new products and to produce others, like the biosimilars destined to fulfill the needs of its National Health System. The Center for State Control on the Quality of Drugs (CECMED) the Cuban NRA, is facing the challenge of regulating the approval of biosimilar products manufactured locally. Consequently, CECMED has issued a position paper establishing the basic principles for regulation of these products and a specific guideline on this was elaborated.     

Nanoarchaeal 16S rRNA gene sequences are widely dispersed in hyperthermophilic and mesophilic halophilic environments

The Nanoarchaeota, proposed as the fourth sub-division of the Archaea in 2002, are known from a single isolate, Nanoarchaeum equitans, which exists in a symbiotic association with the hyperthermophilic Crenarchaeote, Ignicoccus. N. equitans fails to amplify with standard archaeal 16S PCR primers and can only be amplified using specifically designed primers. We have designed a new set of universal archaeal primers that amplify the 16S rRNA gene of all four archaeal sub-divisions, and present two new sets of Nanoarchaeota-specific primers based on all known nanoarchaeal 16S rRNA gene sequences. These primers can be used to detect N. equitans and have generated nanoarchaeal amplicons from community DNA extracted from Chinese, New Zealand, Chilean and Tibetan hydrothermal sites. Sequence analysis indicates that these environments harbour novel nanoarchaeal phylotypes, which, however, do not cluster into clear phylogeographical clades. Mesophilic hypersaline environments from Inner Mongolia and South Africa were analysed using the nanoarchaeal-specific primers and found to contain a number of nanoarchaeal phylotypes. These results suggest that nanoarchaeotes are not strictly hyperthermophilic organisms, are not restricted to hyperthermophilic hosts and may be found in a large range of environmental conditions.     

Effect of Food Restriction on Ghrelin in Normal‐Cycling Female Rats and in Pregnancy

Objective: Ghrelin is a 28‐amino‐acid acylated peptide that was recently identified as the endogenous ligand for the growth hormone secretagogue receptor. Previous studies have shown that ghrelin potently increases growth hormone release and food intake. The aim of this study was to clarify the physiological implications of ghrelin in the regulation of energy balance, by assessing the effect of undernutrition throughout 21 days in normal‐cycling and pregnant rats on ghrelin. Research Methods and Procedures: We have determined ghrelin levels by radioimmunoassay and gastric ghrelin mRNA expression by Northern blot analysis during 21 days of chronic food restriction (30% of ad libitum available diet) in normal‐cycling female rats and in pregnancy. Results: Our results show that chronic food restriction led to an increase in plasmatic ghrelin levels in normal‐cycling female rats. In pregnancy, ghrelin plasmatic levels were enhanced particularly during the latter part of gestation (19 and 21 days) compared with pregnant rats with free access to food. Gastric ghrelin mRNA expression showed a similar expression pattern, being higher in the food‐restricted group than in the group fed ad libitum, in normal‐cycling as well as in pregnant rats. Discussion: These observations indicate that ghrelin plasmatic levels and ghrelin gastric mRNA are up‐modulated during undernutrition in normal‐cycling rats and in pregnancy. These findings suggest that increased ghrelin levels may have a role in mediating the physiological responses to undernutrition and could represent an adaptative response to prevent long‐lasting alterations in energy balance and body weight homeostasis.     

Lipid factor (bVLF) from bovine vitreous body evokes in EGFR-T17 cells a Ca2+-dependent K+ current associated with inositol 1,4,5-trisphosphate-independent Ca2+ mobilization

Bovine vitreous lipid factor (bVLF) is a complex phospholipid isolated from bovine vitreous body with strong Ca(2+)-mobilizing activity. In this study, the effects of bVLF on membrane potential were investigated in EGFR-T17 fibroblasts with the whole-cell patch clamp technique on monolayer cells, as well as with the fluorescent dye bis-oxonol as membrane potential-sensitive probe on monolayer and suspension cells. bVLF induced a transient hyperpolarization characterized by an initial peak and subsequent return to resting membrane potential levels within 1-2 min. The increase of [Ca(2+)](i) was concomitant with an outward current responsible for the hyperpolarizing response. Results with: (a) high [K(+)](o) media; (b) the monovalent cation ionophore gramicidin; and (c) substitution of K(+) with Cs(+) in the intracellular solution were consistent with the involvement of K(+) channels. The bVLF-induced hyperpolarization was blocked by the K(+) channel blockers, quinine and tetraethylamonium chloride, and partially affected by 4-aminopyridine. The calcium ionophore ionomycin caused a similar hyperpolarization as bVLF. When intracellular calcium was buffered by adding BAPTA to the pipette solution, bVLF-activated outward current was prevented. Moreover, the hyperpolarization response was strongly reduced at low doses (3 nM) of specific Ca(2+)-activated K(+) channel blockers, charybdotoxin and iberiotoxin. Based on these observations we conclude that bVLF hyperpolarizes the cells via the activation of a Ca(2+)-dependent K(+) current. In addition, it was observed that bVLF did not have a significant effect on intercellular communication measured by a single patch-electrode technique. Thus, membrane potential changes appeared to belong to the earliest cellular responses triggered by bVLF, and are closely associated with phosphatidic acid-dependent [Ca(2+)](i) mobilization.     

Dual effect of insulin on in vitro leptin secretion by adipose tissue

Although it is widely accepted that insulin stimulates leptin secretion, a dual action was observed using a validated in vitro system, i.e., an early (less than 48 h) inhibitory action, followed later (48-96 h) by a clear-cut stimulation. While the inhibitory phase was observed at every glucose concentration tested (from 1 to 25 mM), the stimulatory phase required the presence of physiological or supraphysiological glucose concentrations. In fact, leptin secretion was virtually eliminated in the presence of glucose uptake inhibitors. This dual effect of insulin was not due to modifications of the ob mRNA levels, suggesting that it depends entirely on posttranslational mechanisms. In conclusion, insulin appears to induce an early inhibition of leptin secretion by the adipose cell, followed later by a stimulatory effect secondary to the metabolic changes triggered by the insulin-induced increase in glucose uptake.     

Short-term fasting abolishes the sex-related difference in GH and leptin secretion in humans

We studied growth hormone (GH) and leptin secretion in eight male (age 29.3 +/- 1.2 yr, body mass index 22.2 +/- 0.5 kg/m(2)) and seven female normal subjects (28.0 +/- 0.8 yr, 20.1 +/- 0.7 kg/m(2)) before and after 36 h of fasting. In the fed state, 8-h mean GH and leptin concentrations were higher in females (P < 0.05 and P < 0. 0001, respectively). Fasting increased GH and decreased leptin in both sexes. There was significant interaction between gender and fasting (P < 0.05 for GH and P < 0.005 for leptin). Females showed a slighter increase in GH but a more marked decrease in leptin, so that there was no significant gender-related difference in GH and leptin after fasting. Fasting did not modify insulin-like growth factor (IGF) I, IGF binding protein (IGFBP)-3, acid-labile subunit, or GH binding protein; increased IGFBP-1 and free fatty acids (P < 0.0001) but decreased glucose (P < 0.001) and insulin levels (P < 0.05). In males, insulin levels were higher (P < 0.05) in the fed state and underwent deeper reduction after fasting (interaction P < 0.03). In conclusion, GH and leptin secretions are higher in women than in men in the fed but not in the fasting condition, which abolishes these gender-related differences in humans.     

Chronic exposure of diesel exhaust particles induces alveolar enlargement in mice

Background

Diesel exhaust particles (DEPs) are deposited into the respiratory tract and are thought to be a risk factor for the development of diseases of the respiratory system. In healthy individuals, the timing and mechanisms of respiratory tract injuries caused by chronic exposure to air pollution remain to be clarified.

Methods

We evaluated the effects of chronic exposure to DEP at doses below those found in a typical bus corridor in Sao Paulo (150 μg/m³). Male BALB/c mice were divided into mice receiving a nasal instillation: saline (saline; n = 30) and 30 μg/10 μL of DEP (DEP; n = 30). Nasal instillations were performed five days a week, over a period of 90 days. Bronchoalveolar lavage (BAL) was performed, and the concentrations of interleukin (IL)-4, IL-10, IL-13 and interferon-gamma (INF-γ) were determined by ELISA-immunoassay. Assessment of respiratory mechanics was performed. The gene expression of Muc5ac in lung was evaluated by RT-PCR. The presence of IL-13, MAC2+ macrophages, CD3+, CD4+, CD8+ T cells and CD20+ B cells in tissues was analysed by immunohistochemistry. Bronchial thickness and the collagen/elastic fibers density were evaluated by morphometry. We measured the mean linear intercept (Lm), a measure of alveolar distension, and the mean airspace diameter (D0) and statistical distribution (D2).

Results

DEP decreased IFN-γ levels in BAL (p = 0.03), but did not significantly alter IL-4, IL-10 and IL-13 levels. MAC2+ macrophage, CD4+ T cell and CD20+ B cell numbers were not altered; however, numbers of CD3+ T cells (p ≤ 0.001) and CD8+ T cells (p ≤ 0.001) increased in the parenchyma. Although IL-13 (p = 0.008) expression decreased in the bronchiolar epithelium, Muc5ac gene expression was not altered in the lung of DEP-exposed animals. Although respiratory mechanics, elastic and collagen density were not modified, the mean linear intercept (Lm) was increased in the DEP-exposed animals (p ≤ 0.001), and the index D2 was statistically different (p = 0.038) from the control animals.

Conclusion

Our data suggest that nasal instillation of low doses of DEP over a period of 90 days results in alveolar enlargement in the pulmonary parenchyma of healthy mice.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0172-z) contains supplementary material, which is available to authorized users.