Bone Microarchitecture Phenotypes Identified in Older Adults Are Associated With Different Levels of Osteoporotic Fracture Risk

Prevalence of osteoporosis is more than 50% in older adults, yet current clinical methods for diagnosis that rely on areal bone mineral density (aBMD) fail to detect most individuals who have a fragility fracture. Bone fragility can manifest in different forms, and a “one-size-fits-all” approach to diagnosis and management of osteoporosis may not be suitable. High-resolution peripheral quantitative computed tomography (HR-pQCT) provides additive information by capturing information about volumetric density and microarchitecture, but interpretation is challenging because of the complex interactions between the numerous properties measured. In this study, we propose that there are common combinations of bone properties, referred to as phenotypes, that are predisposed to different levels of fracture risk. Using HR-pQCT data from a multinational cohort (n = 5873, 71% female) between 40 and 96 years of age, we employed fuzzy c-means clustering, an unsupervised machine-learning method, to identify phenotypes of bone microarchitecture. Three clusters were identified, and using partial correlation analysis of HR-pQCT parameters, we characterized the clusters as low density, low volume, and healthy bone phenotypes. Most males were associated with the healthy bone phenotype, whereas females were more often associated with the low volume or low density bone phenotypes. Each phenotype had a significantly different cumulative hazard of major osteoporotic fracture (MOF) and of any incident osteoporotic fracture (p < 0.05). After adjustment for covariates (cohort, sex, and age), the low density followed by the low volume phenotype had the highest association with MOF (hazard ratio = 2.96 and 2.35, respectively), and significant associations were maintained when additionally adjusted for femoral neck aBMD (hazard ratio = 1.69 and 1.90, respectively). Further, within each phenotype, different imaging biomarkers of fracture were identified. These findings suggest that osteoporotic fracture risk is associated with bone phenotypes that capture key features of bone deterioration that are not distinguishable by aBMD. © 2021 American Society for Bone and Mineral Research (ASBMR).     

胃癌患者外周血来源树突状细胞诱导培养及表型分析

目的分析胃癌患者外周血单个核细胞，经诱导培养获取树突状细胞(DC)的形态学特点及表面分子表达的特点，为基于DC的胃癌生物学治疗提供实验基础。方法胃癌患者外周血单个核细胞经GM-CSF，IL-4及TNF-α诱导培养，获取成熟DC。利用光镜、电镜观察其形态特点。利用流式细胞术检测其表面分子表达特点。结果诱导培养7d即可获取成熟DC，细胞表面出现大量典型树枝状突起；细胞表面分子表达随细胞的成熟而逐渐升高，至培养7d时逐渐趋于稳定。结论胃癌患者外周血单个核细胞经GM-CSF，IL-4及TNF-α诱导培养，可获得大量成熟DC。所获取DC具备典型形态学特点及表面分子表达特点。该研究结果可作为胃癌生物学治疗进一步研究的基础。     

Osteocyte‐Secreted Wnt Signaling Inhibitor Sclerostin Contributes to Beige Adipogenesis in Peripheral Fat Depots

Cells of the osteoblast lineage are increasingly identified as participants in whole‐body metabolism by primarily targeting pancreatic insulin secretion or consuming energy. Osteocytes, the most abundant bone cells, secrete a Wnt‐signaling inhibitor called sclerostin. Here we examined three mouse models expressing high sclerostin levels, achieved through constitutive or inducible loss of the stimulatory subunit of G‐proteins (Gsα in mature osteoblasts and/or osteocytes). These mice showed progressive loss of white adipose tissue (WAT) with tendency toward increased energy expenditure but no changes in glucose or insulin metabolism. Interestingly beige adipocytes were increased extensively in both gonadal and inguinal WAT and had reduced canonical β‐catenin signaling. To determine if sclerostin directly contributes to the increased beige adipogenesis, we engineered an osteocytic cell line lacking Gsα which has high sclerostin secretion. Conditioned media from these cells significantly increased expression of UCP1 in primary adipocytes, and this effect was partially reduced after depletion of sclerostin from the conditioned media. Similarly, treatment of Gsα‐deficient animals with sclerostin‐neutralizing antibody partially reduced the increased UCP1 expression in WAT. Moreover, direct treatment of sclerostin to wild‐type mice significantly increased UCP1 expression in WAT. These results show that osteocytes and/or osteoblasts secrete factors regulating beige adipogenesis, at least in part, through the Wnt‐signaling inhibitor sclerostin. Further studies are needed to assess metabolic effects of sclerostin on adipocytes and other metabolic tissues. © 2016 American Society for Bone and Mineral Research.     

Functional Characterization of PHEX Gene Variants in Children With X-Linked Hypophosphatemic Rickets Shows No Evidence of Genotype–Phenotype Correlation

X-linked hypophosphatemia (XLHR) is caused by loss-of-function mutations in the phosphate regulating endopeptidase homolog X-linked (PHEX) gene. Considerable controversy exists regarding genotype–phenotype correlations in XLHR. The present study describes the clinical features and molecular genetic bases of 53 pediatric patients with XLHR. Overall, 47 different mutations were identified, of which 27 were not previously described in the literature or entered in the Human Gene Mutation Database (HGMD). A high prevalence (72.34%) of truncating variants was observed in XLHR patients. The clinical presentation and severity of XLHR did not show an evident correlation between the truncating and non-truncating mutation types in our cohort. To further delineate the characteristics of PHEX variants underlying this nonsignificant trend, we assessed the effects of 10 PHEX variants on protein expression, cellular trafficking, and endopeptidase activity. Our results showed that the nonsense mutations p.Arg567*, p.Gln714*, and p.Arg747* caused a reduction of protein molecular weight and a trafficking defect. Among seven non-truncating mutations, the p.Cys77Tyr, p.Cys85Ser, p.Ile281Lys, p.Ile333del, p.Ala514Pro, and p.Gly572Ser mutants were not secreted into the medium and remained trapped inside cells in an immature form, whereas the p.Gly553Glu mutant was terminally glycosylated and secreted into the medium. We further assessed the endopeptidase activity of the p.Gly553Glu mutant using a quenched fluorogenic peptide substrate and revealed that the activity of p.Gly553Glu significantly reduced to 13% compared with the wild type, which indicated disruption of catalytic function. These data not only support the clinical results showing no correlation between disease severity and the type of PHEX mutation but also provide helpful molecular insights into the pathogenesis of XLHR. © 2020 American Society for Bone and Mineral Research.     

Alpha globin gene numbers: an important modifier of HbE/β thalassemia

Abstract

HbE [β 26 Glu-Lys] is the second most common abnormal Hb in humans. HbE when associated with β thalassemia (HbE/β thalassemia) in compound heterozygous state results in a clinically severe condition. HbE/β thalassemia has a very variable clinical phenotype. One of the possible explanations for the observed variable clinical severity is variation of α gene number. The main aims of this study were to determine the frequency of α deletion/mutations and triplication in HbE/β thalassemia patients and to study the effect of α gene numbers on the phenotype of these patients. 240 HbE/β thalassemia patients who attended the Hematology Outpatient Department of the Institute were studied. Patients were divided into three groups mild (score=0–3·5), moderate (score=4–7) and severe (score=7·5–10). α deletion was found in 22 (7·5%), out of these 22 patients 16 (11 αα/-α^3·7, 4–α^3·7/-α^3·7 & 1 αα/--^SA) were from Gp I and 6 (αα/-α^3·7) were from Gp II. α triplication was found in 7 (2·9%) and out of these 7 patients 5 were (αα/ααα^anti-3·7) from Gp III and 2 were (αα/ααα^anti-3·7) from Gp II. The most common interaction was found to be HbE/β thalassemia and deletional α⁺-thalassemia (αα/-α^3·7) followed by others. α deletions and point mutations genotype were mostly observed in the individuals from Gp I while α triplication was most frequent in Gp III individuals. Interaction of HbE/β thal with α-thalassemia is common in the Indian population. Patients inheriting α deletions and point mutations behaved mildly with some exceptions, while patients with α triplication had a severe phenotype requiring frequent transfusions.     

Microsatellite Instability Is Absent in Liver and Biliary Mucosa of Patients with Primary Sclerosing Cholangitis

Microsatellite instability occurs in the colonicmucosa of patients with inflammatory bowel disease andmay predispose the mucosa to neoplastic transformation.It is unknown whether microsatellite instability also plays a role in the neoplastic riskassociated with primary sclerosing cholangitis. Weexamined 134 tissue samples from 21 patients withsclerosing cholangitis for microsatellite instability ateight loci. All tissues were also stainedimmunohistochemically using an antibody to theproliferation marker Ki-67. Microsatellite instabilitydid not occur in any samples from the intrahepatic orextrahepatic biliary system, although one patientdemonstrated instability in the colon. Ki-67 indicesranged from 0 to 2.5 in nondysplastic biliary epitheliumand from 1.5 to 29.4 in areas of dysplasia. The absence of microsatellite instability in sclerosingcholangitis suggests that the genetic basis ofneoplastic progression in chronic inflammatory diseaseof the bile ducts differs from that of intestinalcancers arising in the setting of chronic inflammatorybowel disease and may relate to differences in themicroenvironment in these two sites.     

Gsα activity is reduced in erythrocyte membranes of patients with psedohypoparathyroidism due to epigenetic alterations at the GNAS locus

In pseudohypoparathyroidism (PHP), PTH resistance results from impairment of signal transduction of G protein–coupled receptors caused by a deficiency of the Gsα‐cAMP signaling cascade due to diminished Gsα activity in maternally imprinted tissues. In PHP‐Ia, inactivating mutations of the GNAS gene lead to haploinsufficiency in some tissues with biallelic expression, so in addition to PHP, Albright's hereditary osteodystrophy (AHO) is also present. In PHP‐Ib, caused by methylation defects at the GNAS locus, diminished Gsα activity was thought to be limited to maternally imprinted tissues, such as the renal proximal tubule and the thyroid, leading to a lack of AHO. Recently, we demonstrated methylation defects in patients with AHO signs, indicating a connection between epigenetic changes and AHO. Our objective was to determine Gsα activity in erythrocyte membranes in patients with epigenetic defects at the GNAS locus compared to normal controls and patients with inactivating GNAS mutations. Gsα activity and expression, mutation of the GNAS locus, and methylation status were studied in patients with PHP and mild signs of AHO (PHP‐Ia: 12; PHP‐Ib: 17, of which 8 had some features of AHO). Then, we statistically compared the Gsα activity of the different PHP subtypes. Patients with methylation defects at the GNAS locus show a significant decrease in erythrocyte Gsα activity compared to normal controls (PHP‐Ib versus controls, p < .001). This was significantly lower in patients with AHO signs (PHP‐Ib + mild‐AHO versus PHP‐Ib, p < .05). Our research shows that PHP‐Ia and PHP‐Ib classification is not only overlapped genetically, as reported, but also in terms of Gsα activity. Reduced expression of GNAS due to methylation defects could downregulate Gsα activity in other tissues beyond those described and could also be causative of AHO. © 2011 American Society for Bone and Mineral Research     

Immune Phenotype of Chronic Liver Disease

Immune disorders in chronic liver disease mayreflect common host propensities or diseasespecificfactors. Our aim was to determine the principal basesfor these expressions. Four hundred fifty-one patients with various chronic liver diseases wereassessed prospectively for concurrent immune disorders.Individuals with immune diseases were more frequentlywomen (73% vs 60%, P = 0.02) and they had HLA DR4 more often than counterparts with other HLA(46% vs 23%, P = 0.000008). The association between HLADR4 and immune disease was apparent within individualliver diseases and within different categories of liver disease. Women with HLA DR4 had ahigher frequency of immune disease than women withoutHLA DR4 (52% vs 22%, P 0.000001), and they also hadimmune diseases more commonly than DR4-positive men (52% vs 31%, P = 0.03). DR4-positive men,however, had higher frequencies of immune disease thanDR4-negative men, especially in the nonimmune types ofliver disease (26% vs 4%, P = 0.002). We conclude that HLA DR4 and female gender constitute animmune phenotype that is an important basis forautoimmune expression in chronic liverdisease.     

The effect of radio-frequency glow discharge treatment of polystyrene on the behavior of porcine chondrocytes in vitro

The aim of this study was to determine the effects of physicochemical surface properties of tissue-culture substrata on chondrocyte behavior. Polystyrene was modified by radio-frequency glow discharge (RFGD) plasma treatment with various monomers. The changes in surface properties of the modified polystyrene were verified by ESCA and water contact angle measurements. Porcine chondrocytes were seeded on these surfaces and cultured for 5 days. After 5 days of culture, the number of chondrocytes was highest on the N₂ plasma-treated surface, followed by the CH₂/N₂ plasma-treated surface, untreated polystyrene and CF₄ plasma-treated surface. The number of chondrocytes decreased with increasing water contact angle. The surface chemical properties influenced the morphology and gene expression of cultured chondrocytes. The cells cultured on the CF₄ plasma-treated surface retained a round morphology characteristic of chondrocytes after day 1, while most of the cells grown on the N₂ plasma-treated surface or the untreated polystyrene showed a flattened morphology. Using RT-PCR, expression of type-I collagen could not be detected in the chondrocytes cultured on the CF₄ plasma-treated surface and the CH₂/N₂ plasma-treated surface. In contrast, the chondrocytes grown on the N₂ plasma-treated surface or the untreated polystyrene surface expressed type-I collagen mRNA. This study shows that modification by RFGD treatment could modulate chondrocyte culture and gene expression.