Resveratrol Increases Osteoblast Differentiation In Vitro Independently of Inflammation.

Low-grade inflammation negatively affects bone. Resveratrol is a natural compound proven to possess both anti-inflammatory and bone protective properties. However, it is uncertain if the bone effects are mediated though anti-inflammatory effects. Firstly, we investigated if resveratrol affects proliferation and differentiation of human bone marrow-derived mesenchymal stem cells. Secondly, we investigated if inflammation negatively affects proliferation and differentiation, and if resveratrol counteracts this through anti-inflammatory effects. Mesenchymal stem cells were obtained from bone marrow aspiration in 13 healthy individuals and cultured towards the osteoblast cell lineage. The cells were stimulated with resveratrol, lipopolysaccharide (LPS), LPS + resveratrol, or vehicle (control) for 21 days. Compared to control, resveratrol decreased cell number by 35 % (p < 0.05) and induced differentiation (a 3-fold increase in alkaline phosphatase (p < 0.002), while P1NP and OPG showed similar trends). LPS induced inflammation with a 44-fold increase in interleukin-6 (p < 0.05) and an extremely prominent increase in interleukin-8 production (p < 0.05) relative to control. In addition, LPS increased cell count (p < 0.05) and decreased differentiation (a reduction in P1NP production (p < 0.02)). Co-stimulation with LPS + resveratrol did not reduce interleukin-6 or interleukin-8, but nonetheless, cell count was reduced (p < 0.05) and alkaline phosphatase, P1NP, and OPG increased (p < 0.05 for all). Thus, resveratrol stimulates osteoblast differentiation independently of inflammation.

Two novel HLA class I alleles, HLA-C*04:489 and -C*07:01:115, were identified by next-generation sequencing.

The novel HLA-C*04:489 and -C*07:01:115 alleles were detected during routine HLA typing by next-generation sequencing.

Identification of the novel HLA class I allele, HLA-A*31:215, identified by Next Generation Sequencing.

The novel HLA allele HLA-A*31:215 differs from HLA-A*31:01:02:01 by one nucleotide substitution c.G832A in exon 4.

Polymorphisms in the osteoprotegerin gene are associated with osteoporotic fractures.

Osteoprotegerin (OPG) is a soluble receptor for RANKL and therefore a competitive inhibitor of osteoclast differentiation and activity. With this key role in the control of resorptive activity, we found that OPG is a candidate gene for genetic control of bone mass. We examined the promoter and the five exons with surrounding intron sequences of the OPG gene for polymorphisms in 50 normal patients and 50 patients with osteoporosis. We found 12 polymorphisms. Two sets of four and five polymorphisms, respectively, were in complete linkage. Subsequently, we examined the effect of the informative polymorphisms A163-G (promoter), T245-G (promoter), T950-C (promoter), G1181-C (exon 1), and A6890-C (intron 4) on the prevalence of osteoporotic fractures, bone mass, and bone turnover in 268 osteoporotic patients and 327 normal controls. In A163-G the variant allele G was more common among fracture patients: 34.0% versus 26.3% in normal controls (p < 0.05) and the odds ratio (OR) for a vertebral fracture, if an individual has the G allele, was 1.44 (1.00-2.08). In T245-G the variant allele G was more common in osteoporotic patients: 12.4% versus 6.5% (p < 0.02) and the OR for vertebral fracture, if an individual has the G-allele, was 2.00 (1.10-3.62). G1181-C is located in the first exon and causes a shift in the third amino acid from lysine to asparagine. The CC genotype was less common among fracture patients: 26.3% versus 36.7% in the normal controls (p < 0.01). T950-C and A6890-C were not distributed differently among patients with osteoporosis and normal controls. None of the polymorphisms affected bone mineral density (BMD) or biochemical markers of bone turnover in the normal controls. In conclusion, we have examined the human OPG gene for polymorphisms and found 12. The rare alleles of the A163-G and T245-G were significantly more common among patients with vertebral fractures.

Polymorphisms in the transforming growth factor beta 1 gene and osteoporosis.

Transforming growth factor (TGF)-beta1 is the most abundant growth factor in human bone. It is produced by osteoblasts and inhibits osteoclast proliferation and activity and stimulates proliferation and differentiation of preosteoblasts. Several polymorphisms have been described in the TGF-beta1 gene. Previously, we and others have found associations between some of these polymorphisms and bone mass. We therefore wanted to examine if these polymorphisms are also predictors of osteoporotic fractures. The polymorphisms G(-1639)-A, C(-1348)-T, C(-765)insC, T(29)-C, G(74)-C, 713-8delC, C(788)-T, and T(816-20)-C were examined using RFLP and sequencing in 296 osteoporotic patients with vertebral fractures and 330 normal individuals. Bone mineral density (BMD) was examined at the lumbar spine and at the femoral neck by DXA. Genotype distributions were in H-W equilibrium. Linkage disequilibrium was found between the polymorphisms. The T(816-20)-C genotypes were distributed differently among osteoporotic patients and normal controls. The TT genotype was less common in individuals with osteoporotic fractures (chi(2) = 6.02, P < 0.05). BMD was higher in individuals with the TT-genotype (T(816-20)-C) at the lumbar spine, 0.960 +/- 0.173 g/cm(2) compared with individuals with the TC or CC genotypes: 0.849 +/- 0.181 g/cm(2) and 0.876 +/- 0.179 g/cm(2), respectively (P < 0.001, ANOVA). Similar differences between genotypes were found at the different hip regions as well as at the total hip. Individuals with the TT-genotype (C(-1348)-T) had higher bone mass at the femoral neck: 0.743 +/- 0.134 g/cm(2) compared with 0.703 +/- 0.119 g/cm(2) in individuals with TC or CC genotypes (P < 0.05). Individuals with the CC-genotype (T(29)-C) had higher bone mass at the femoral neck, 0.735 +/- 0.128 g/cm(2) compared with 0.703 +/- 0.120 g/cm(2) in individuals with TC or TT genotypes (P < 0.05) and at the total hip: 0.852 +/- 0.166 g/cm(2) vs. 0.818 +/- 0.149 g/cm(2), respectively (P < 0.05). None of the other polymorphisms were distributed differently in patients and controls and did not affect BMD. In conclusion, The TT genotype of the T(816-20)-C polymorphism is less common in patients with osteoporotic fractures and is associated with higher bone mass both at the lumbar spine and at the hip. The C(-1348)-T and T(29)-C polymorphisms were distributed similarly in osteoporotic patients and normal controls, however, the rare genotypes were associated with higher bone mass at the hip.

Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is associated with osteoporotic vertebral fractures, but is a weak predictor of BMD.

Osteoporosis is a common disease with a strong genetic component. Linkage studies have suggested linkage between BMD and loci on chromosome 1. The MTHFR gene is located on chromosome 1. MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate, which is used for homocysteine methylation to methionine. The rare genotype (TT) of the C677T polymorphism has previously been demonstrated to be associated with increased plasma homocysteine levels in individuals with inadequate plasma folate levels. Recently, the TT genotype has been found to be associated with reduced bone mass. We therefore examined if the C677T polymorphism in the MTHFR gene is associated with changes in bone mass and risk of osteoporotic fractures in 388 osteoporotic patients and 336 normal individuals. The distributions of the genotypes CC, CT and TT in women with osteoporotic vertebral fractures and normal controls were 43.5%, 42.2% and 14.3% and 52.0%, 42.0% and 8.0%, respectively, chi2 = 5.62, P = 0.06. Since studies of the functionality of this polymorphism have revealed that only the TT genotype is associated with biochemical changes, we also compared the prevalence of the TT genotype versus the CT- and CC genotypes in patients and controls and found that the TT genotype is significantly more common in women with vertebral fractures (14.3%) compared with normal controls (8.0%), chi2 = 4.31, P < 0.05. Logistic regression analysis demonstrated that vertebral fractures were significantly associated with BMD (lumbar spine) and height but only marginally with the MTHFR genotype (P = 0.06). Multiple linear regression analysis revealed that weight, age and the MTHFR polymorphism were predictors of lumbar spine BMD in women. However, age- and gender-corrected BMD of the lumbar spine and the hip was not significantly different between MTHFR genotypes. Furthermore, individuals with the TT genotype did not have BMD significantly lower than the combined group of individuals with the CT- or CC genotypes. In conclusion, we have demonstrated that the rare TT genotype of the C677T polymorphism in the MTHFR gene is associated with increased risk of osteoporotic fractures in women and a weak predictor of lumbar spine BMD.