Gut microbiota impacts bone via Bacteroides vulgatus-valeric acid-related pathways.

Although the gut microbiota has been reported to influence osteoporosis risk, the individual species involved, and underlying mechanisms, remain largely unknown. We performed integrative analyses in a Chinese cohort of peri-/post-menopausal women with metagenomics/targeted metabolomics/whole-genome sequencing to identify novel microbiome-related biomarkers for bone health. Bacteroides vulgatus was found to be negatively associated with bone mineral density (BMD), which was validated in US white people. Serum valeric acid (VA), a microbiota derived metabolite, was positively associated with BMD and causally downregulated by B. vulgatus. Ovariectomized mice fed B. vulgatus demonstrated increased bone resorption and poorer bone micro-structure, while those fed VA demonstrated reduced bone resorption and better bone micro-structure. VA suppressed RELA protein production (pro-inflammatory), and enhanced IL10 mRNA expression (anti-inflammatory), leading to suppressed maturation of osteoclast-like cells and enhanced maturation of osteoblasts in vitro. The findings suggest that B. vulgatus and VA may represent promising targets for osteoporosis prevention/treatment.

A Randomized Clinical Trial of Intravenous Methylprednisolone With 2 Protocols in Patients With Graves Orbitopathy.

CONTEXT: Intravenous glucocorticoid (IVGC) is an accessible and affordable treatment for Graves orbitopathy (GO); the 4.5-g protocol is well studied, but many details of treatment protocols need to be clarified. OBJECTIVE: To compare the efficacy and safety of weekly and monthly protocol of IVGC in GO. METHODS: A prospective, randomized, observer-masked, single-center clinical trial, followed up to week 24, at the third affiliated hospital of Southern Medical University; 58 patients with active and moderate to severe GO, aged 18-60 years old, who had not received relevant treatment were included. The intervention was weekly protocol or monthly protocol of IVGC; both received a cumulative dose of methylprednisolone 4.5 g and had a duration of 12 weeks. The overall effective rate, improvement of quality of life (QOL) and signal intensity ratio (SIR) were measured. RESULTS: There was no significant difference in the effective rate between the 2 groups at week 12 and week 24 (86.21% vs 72.41%, P = .195; 86.21% vs 82.61%, P = .441), there was no significant difference in the improvement of clinical activity score, exophthalmos, soft tissue involvement, diplopia, and QOL. At week 24, the mean SIR and maximum SIR of the 2 groups were lower than those before treatment, and there were no statistically significant difference between the 2 groups. There was no significant difference in the incidence of adverse events between the 2 groups (31.03% vs 27.59%, P = .773). CONCLUSION: The efficacy and safety of the 2 protocols are comparable; the monthly protocol could be used as an alternative to the weekly protocol.

Application of Multiparameter Quantitative Magnetic Resonance Imaging in the Evaluation of Graves' Ophthalmopathy.

BACKGROUND: Assessment of the activity of Graves' ophthalmopathy (GO) is difficult. Existing methods need improvement. PURPOSE: Investigate the application of multiparametric magnetic resonance imaging (MRI) in GO. STUDY TYPE: Retrospective. POPULATION: A total of 235 GO patients (age: 38.8 ± 13.4 years; 90 male; 96 active patients). FIELD STRENGTH/SEQUENCE: Short-tau inversion recovery (STIR) fast spin echo, multiecho spin echo T2 mapping and 3D T1-weighted fast field echo sequences at 3.0 T. ASSESSMENT: Two physicians assessed the mean and maximum signal intensity ratio of extraocular muscles to white matter (SIR), T2 relaxation time (T2RT), extraocular muscle area (EMA), fat fraction (FF), retrobulbar fat volume (RFV), and extraocular muscle volume (EMV). Clinical activity score (CAS) â‰§ 3 was in active stage. STATISTICAL TESTS: The optimal cut-off point of diagnostic efficacy was selected using receiver operating characteristic (ROC) curve analysis and evaluated using area under the curve (AUC), compared using Student's t test, analysis of variance or Kruskal-Wallis H test. The correlation used Pearson correlation analysis. The discriminant equation used a binary logistic regression analysis. P < 0.05 was considered statistically significant. RESULTS: The SIRmean, SIRmax, T2RTmean, T2RTmax, EMA, and EMV in active GO patients were significantly higher than those in inactive and were positively correlated with CAS (r = 0.276, 0.228, 0.438, 0.388, 0.502, and 0333, respectively). The FFmax of active patients was significantly lower than that of inactive patients and was negatively correlated with CAS (r = -0.44). Logistic regression analysis indicated that T2RTmean was independently associated with GO active periods and had good diagnostic performance (area under ROC curve = 0.736, sensitivity 70.7%, specificity 69.3%). T2RTmean â‰§ 74.295 could be a diagnostic cut-off for judging GO activity (sensitivity 55.3%). CONCLUSION: SIR, T2RT, EMV, and FF can quantitatively assess the activity and severity of GO and can potentially provide a basis for clinical judgment and selection of treatment options. EVIDENCE LEVEL: 4. TECHNICAL EFFICACY: Stage 2.

Excess dietary zinc drives a Cushing's-like syndrome in ovariectomized mice - Implications for postmenopausal obesity.

Postmenopausal women have an increased risk of obesity, but the underlying cause is not clear. We unexpectedly found that excess dietary zinc induced severe obesity and a Cushing's-like syndrome without increased food intake in ovariectomized (Ovx) but not in sham-operated mice. Zinc accumulated in the adrenal glands and inhibited adrenal 17,20-lyase activity and steroid synthesis. As adrenal steroids are the only source of estrogen in Ovx mice, estrogen deficiency induced adrenal hyperplasia, glucocorticoid overproduction, and consequent development of a Cushing's-like syndrome. Adrenal steroid supplementation prevented the effects of zinc. Plasma zinc was positively correlated with cortisol level and negatively correlated with the levels of adrenal steroids and estrogen in obese postmenopausal women. The finding of a link between dietary zinc, estrogen deficiency, and postmenopausal obesity, implies that postmenopausal obesity might be prevented by supplementation with a adrenal steroid and avoiding excess dietary zinc.

Identification and Functional Characterization of Metabolites for Bone Mass in Peri- and Postmenopausal Chinese Women.

CONTEXT: Although metabolic profiles appear to play an important role in menopausal bone loss, the functional mechanisms by which metabolites influence bone mineral density (BMD) during menopause are largely unknown. OBJECTIVE: We aimed to systematically identify metabolites associated with BMD variation and their potential functional mechanisms in peri- and postmenopausal women. DESIGN AND METHODS: We performed serum metabolomic profiling and whole-genome sequencing for 517 perimenopausal (16%) and early postmenopausal (84%) women aged 41 to 64 years in this cross-sectional study. Partial least squares regression and general linear regression analysis were applied to identify BMD-associated metabolites, and weighted gene co-expression network analysis was performed to construct co-functional metabolite modules. Furthermore, we performed Mendelian randomization analysis to identify causal relationships between BMD-associated metabolites and BMD variation. Finally, we explored the effects of a novel prominent BMD-associated metabolite on bone metabolism through both in vivo/in vitro experiments. RESULTS: Twenty metabolites and a co-functional metabolite module (consisting of fatty acids) were significantly associated with BMD variation. We found dodecanoic acid (DA), within the identified module causally decreased total hip BMD. Subsequently, the in vivo experiments might support that dietary supplementation with DA could promote bone loss, as well as increase the osteoblast and osteoclast numbers in normal/ovariectomized mice. Dodecanoic acid treatment differentially promoted osteoblast and osteoclast differentiation, especially for osteoclast differentiation at higher concentrations in vitro (eg,10, 100 µM). CONCLUSIONS: This study sheds light on metabolomic profiles associated with postmenopausal osteoporosis risk, highlighting the potential importance of fatty acids, as exemplified by DA, in regulating BMD.

Establishment of magnetic resonance imaging 3D reconstruction technology of orbital soft tissue and its preliminary application in patients with thyroid-associated ophthalmopathy.

OBJECTIVE: Effective management of thyroid-associated ophthalmopathy (TAO) requires precise identification of the disease activity period as it is responsive to immunosuppressive treatment. Quantitative evaluations of orbital soft tissue are useful for analysing disease stages. We aimed to establish a method for orbital soft tissue volume calculation based on magnetic resonance imaging (MRI) data using 3D reconstruction technology. Furthermore, we validated the accuracy and precision of this method and investigated volume differences between patients with TAO and healthy individuals. MATERIALS AND METHODS: Using Mimics software for 3D reconstruction based on orbital MRI data, we quantitatively measured orbital fat volume (FV) and extraocular muscle volume (MV) using a manual phantom, and in patients with TAO and healthy volunteers (n = 10 each). The phantom was made using a combination of butter and chicken muscle and 2 observers measured its volume. Volume calculations were compared to a previously established standard volume. One observer measured a typical TAO case 10 times to calculate intra-observer variability while 3 observers independently measured 10 patients with TAO each to calculate interobserver variability. Orbital soft tissue volumes between 10 patients with TAO and 10 healthy individuals were compared. RESULTS: The precision of calculations for the phantom between the 2 observers varied from -4.60% to -2.78% for FV and between -4.13% to 0.71% for MV. Mean differences among repetitive calculations were lower than 4%, except during measurement of MV, which was 5.84%. The intraclass correlation coefficient varied from 0.976 to 0.996. FV was 15.53 ± 3.06 mL in patients with TAO and 11.32 ± 1.68 mL(P = .001)in healthy individuals, while MV was 3.19 ± 0.82 mL in patients with TAO and 2.45 ± 0.57 mL(P = .030)in healthy individuals. CONCLUSIONS: This method of calculating orbital soft tissue volumes based on MRI data and 3D reconstruction is both reliable and accurate as it yielded significant differences in tissue volume between patients with TAO and healthy individuals.

Identifying potentially common genes between dyslipidemia and osteoporosis using novel analytical approaches.

Dyslipidemia (DL) is closely related to osteoporosis (OP), while the exact common genetic mechanisms are still largely unknown. We proposed to use novel genetic analysis methods with pleiotropic information to identify potentially novel and/or common genes for the potential shared pathogenesis associated with OP and/or DL. We assessed the pleiotropy between plasma lipid (PL) and femoral neck bone mineral density (FNK BMD). We jointly applied the conditional false discovery rate (cFDR) method and the genetic analysis incorporating pleiotropy and annotation (GPA) method to the summary statistics provided by genome-wide association studies (GWASs) of FNK BMD (n = 49,988) and PL (n = 188,577) to identify potentially novel and/or common genes for BMD/PL. We found strong pleiotropic enrichment between PL and FNK BMD. Two hundred and forty-five PL SNPs were identified as potentially novel SNPs by cFDR and GPA. The corresponding genes were enriched in gene ontology (GO) terms "phospholipid homeostasis" and "chylomicron remnant clearance". Three SNPs (rs2178950, rs9939318, and rs9368716) might be the pleiotropic ones and the corresponding genes NLRC5 (rs2178950) and TRPS1 (rs9939318) were involved in NF-κB signaling pathway and Wnt signaling pathway as well as inflammation and innate immune processes. Our study validated the pleiotropy between PL and FNK BMD, and corroborated the reliability and high-efficiency of cFDR and GPA methods in further analyses of existing GWASs with summary statistics. We identified potentially common and/or novel genes for PL and/or FNK BMD, which may provide new insight and direction for further research.

[A novel method for quantitative measurement of orbital fat volume based on magnetic resonance images].

OBJECTIVE: To establish a new method for rapid and quantitative measurement of orbital fat volume based on magnetic resonance imaging (MRI) data. METHODS: We collected MRI data from normalized mold and patients with the diagnosis of thyroid-associated ophthalmopathy (TAO). The cross-sectional areas of the orbital fat on each MR image slice were measured to calculate the fat volume on each slice and then the total orbital fat volume. We recorded the time for completing the measurement and assessed the precision, reliability, repeatability and interoperator variations of the results. RESULTS: This MRI data-based method allowed precise measurement of the orbital fat volumes with an absolute value of the mean percentage difference <1%. This method was fast and the results showed a good repeatability (with CVs <1%), a high reliability (ICC=0.996, 95%CI: 0.985-0.999) and a high interoperator concordance (95%CI of the Bland-Altman: -0.54-0.90). CONCLUSION: The novel method we established for orbital fat volume measurement is rapid, accurate, reliable and reproducible with a low learning cost for clinical use.

[Calculation of orbital fat volumes for determining treatment timing for thyroid- associated ophthalmopathy].

OBJECTIVE: To analyze the relationship between orbital fat volume and the progression and prognosis of thyroid- associated ophthalmopathy (TAO) and determine the optimal treatment timing for TAO. METHODS: The clinical data were collected from 35 patients (70 orbits) with a definite diagnosis of TAO between January, 2016 and December, 2016. The correlation between orbital fat volume and the clinical parameters was evaluated. We also analyzed the correlation of the signal intensity ratio (SIR) of the extraocular muscles with the clinical parameters. The orbital fat volume was compared between patients with TAO and 12 control subjects. RESULTS: The orbital fat volume was significantly correlated with the duration of TAO (r=0.480, P<0.01), but showed no significant difference between patients with a disease course within 6 months and those with a disease course of 6 to 12 months (P=0.084). The patients with a disease course beyond 12 months had a significantly greater orbital fat volume than those with a disease course of 6 months (P<0.01) or 6 to 12 months (P<0.05). The orbital fat volume was correlated with the degree of proptosis (r=0.622, P<0.01), and an increase of exophthalmos by 1 mm was associated with a total orbital volume increment of 0.88 mL. The clinical activity score was correlated with the SIR of the extraorbital muscles (r=0.536, P<0.01) and levels of anti-thyroid-stimulating hormone receptor antibody (r=0.416,P<0.01). The orbital fat volume was significantly greater in TAO patients than in the healthy individuals (P<0.01). CONCLUSION: In patients with TAO, the peak increase of orbital fat volume occurs one year after the disease onset. Measurement of the orbital fat volume combined with SIR of the extraorbital muscles can serve as an indicator for determining the optimal timing for intervention of TAO and helps in the evaluation of prognosis of the patients.

[Comparison of endothelial differentiation capacity of adipose-derived stem cells and bone marrow mesenchymal stem cells from rats].

OBJECTIVE: To compared the differentiation capacity of rat adipose-derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs) into endothelial cells. METHODS: Rat BMSCs and ASCs were isolated, cultured and identified for cell surface markers using flow cytometry. The cell growth curves were drawn by CCK-8 assay, and the cells in active growth were induced for endothelial differentiation following standard protocols. On day 21 of induction, the cells were examined for mRNA expressions of endothelial cell specific markers CD31, KDR, and vWF using qPCR. Immunostaining was performed to observe the expression of CD31 on the cells. The induced cells were also tested for Dil-labeled acetylated low-density lipoprotein (ac-LDL) uptake ability. The tube-forming ability of the induced cells was verified on Matrigel. RESULTS: We successfully isolated rat ASCs and BMSCs. Morphologically, ASCs were similar with BMSCs, both having long spindle-shaped and fibroblast-like morphology. Flow cytometry showed that both BMSCs and ASCs had high expressions of mesenchymal markers CD29 and CD90 and a low expression of hematopoietic cell surface markers CD45. CCK-8 assay showed that ASCs proliferated more quickly than BMSCs. The cells with induced endothelial differentiation exhibited increased levels of CD31, KDR, and vWF mRNA expressions and immunofluorescent staining identified CD31 antigen expression on the cell membrane. Fluorescence microscopy revealed red fluorescence in the induced cells suggesting uptake of Dil-Ac-LDL by the cells. The induced cells were capable of forming tube on Matrigel, confirming their identity of endothelial cells. CONCLUSION: Both rat BMSCs and ASCs can be induced to differentiate into endothelial cells, but ASCs differentiate more quickly into endothelial cells and possess a stronger proliferation ability, suggesting its greater potential than BMSCs in future applications.

Th1, Th2, and Th17 Cytokine Involvement in Thyroid Associated Ophthalmopathy.

To determine serum cytokine profiles in Graves' disease (GD) patients with or without active and inactive thyroid associated ophthalmopathy (TAO), we recruited 65 subjects: 10 GD only (without TAO), 25 GD + active TAO, 20 GD + TAO, and 10 healthy controls. Liquid chip assay was used to measure serum Th1/Th2/Th17 cytokines including IFN-γ (interferon-gamma), TNF-α (tumor necrosis factor-alpha), IL-1α (interleukin-1 alpha), IL-1Ra (IL-1 receptor antagonist), IL-2, IL-4, IL-6, and IL-17 and two chemokines: RANTES (regulated upon activation, normal T cell expressed and secreted) and IP-10 (IFN-γ-induced protein 10). Serum levels of TSH (thyroid stimulating hormone) receptor autoantibodies (TRAb) were measured using an enzyme linked immunosorbent assay. Compared with healthy controls, TAO patients showed significantly elevated serum levels of IFN-γ, TNF-α, IL-1α, IL-4, IL-6, IL-17, and IP-10. Comparing active and inactive TAO, serum Th1 cytokines IFN-γ and TNF-α were elevated in active TAO, while serum Th2 cytokine IL-4 was elevated in inactive TAO. Serum Th17 cytokine IL-17 was elevated in GD but reduced in both active and inactive TAO. A positive correlation was found between TRAb and IFN-γ, TNF-α, IL-1α, IL-2, IL-4, and IL-6. Taken together, serum Th1/Th2/Th17 cytokines and chemokines reflect TAO disease activity and may be implicated in TAO pathogenesis.

[Role of Treg/Th17 cells and related cytokines in Graves' ophthalmopathy].

OBJECTIVE: To explore the role of CD4⁺ CD25⁺ Foxp3⁺ Treg/CD4⁺ IL-17A(+)Th17 cells and the related cytokines in Graves' ophthalmopathy. METHODS: Based on clinical activity scores (CAS), we divided patients with untreated Graves' ophthal- mopathy into active group (AGO group with CAS ≥ 3 (15 cases) and non-active group (NGO group) with CAS<3 (15 cases), with another 15 patients with untreated Graves' disease free of eye symptoms (GD group) and 15 normal subjects as controls. Peripheral venous blood Treg/Th17 cell ratio was determined using flow cytometry. RT-PCR was used to detect the mRNA expression levels of Treg-specific transcription factor Foxp3 and Th17-specific transcription factor RORγt. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of Th17 cell-related cytokines (IL-17A, IL-23, and IL-6) and Treg-related cytokines (TGF-ß, IL-10, and IL-35). RESULTS: Compared with the normal subjects, the patients in GD, NGO, AGO groups all showed significantly increased Th17 cell count (P<0.05), which was the highest in AGO group. RT-PCR results revealed significantly increased RORγt in GD, NGO, and AGO groups, also the highest in AGO group. Serum IL-17A, IL-23, and IL-6 levels all showed significant increments in GD, NGO, and AGO groups (P<0.05), especially in AGO group. Among the Treg-related cytokines, TGF-ß and IL-35 levels decreased (P<0.05) but IL-10 increased significantly (P<0.05) in GD, NGO, AGO groups. CONCLUSION: Decreased immunosuppressive capacity of Treg cells can be an important factor in the pathogenesis of Graves' ophthalmopathy. Th17 cells may also participate in the occurrence and progression of Graves' ophthalmopathy and can serve along with related cytokines as novel indicators of the disease activity. Impaired Treg/Th17 balance may importantly contribute to the occurrence of Graves' ophthalmopathy.