Osteogenic Mechanisms of Basal Ganglia Calcification and its ex vivo Model in the Hypoparathyroid Milieu.

CONTEXT: Basal-ganglia calcification (BGC) is common (70%) in patients with chronic hypoparathyroidism. Interestingly, cortical gray matter is spared from calcification. The mechanism of BGC, role of hyperphosphatemia, and modulation of osteogenic molecules by parathyroid hormone (PTH) in its pathogenesis is not clear. OBJECTIVE: We assessed the expression of a large repertoire of molecules with proosteogenic or antiosteogenic effects, including neuroprogenitor cells in caudate, dentate, and cortical gray matter from normal autopsy tissues. The effect of high phosphate and PTH was assessed in an ex vivo model of BGC using striatum tissue culture of the Sprague-Dawley rat. METHODS: The messenger RNA and protein expression of 39 molecules involved in multiple osteogenic pathways were assessed in 25 autopsy tissues using reverse-transcriptase polymerase chain reaction, Western blot, and immunofluorescence. The striatal culture was maintained in a hypoparathyroid milieu for 24 days with and without (a) high phosphate (10-mm ß-glycerophosphate) and (b) PTH(1-34) (50 ng/mL Dulbecco's modified Eagle's medium-F12 media) for their effect on striatal calcification and osteogenic molecules. RESULTS: Procalcification molecules (osteonectin, ß-catenin, klotho, FZD4, NT5E, LRP5, WNT3A, collagen-1α, and SOX2-positive neuroprogenitor stem cells) had significantly higher expression in the caudate than gray matter. Caudate nuclei also had higher expression of antiosteogenic molecules (osteopontin, carbonic anhydrase-II [CA-II], MGP, sclerostin, ISG15, ENPP1, and USP18). In an ex vivo model, striatum culture showed an increased propensity for calcified nodules with mineral deposition similar to that of bone tissue on Fourier-transformed infrared spectroscopy, alizarin, and von Kossa stain. Mineralization in striatal culture was enhanced by high phosphate and decreased by exogenous PTH through increased expression of CA-II. CONCLUSION: This study provides a conceptual advance on the molecular mechanisms of BGC and the possibility of PTH therapy to prevent this complication in a hypoparathyroid milieu.

Central Immune Tolerance of T and B Cells in Patients With Idiopathic Hypoparathyroidism, T1D, and Autoimmune Thyroiditis.

CONTEXT: Pathogenesis of idiopathic hypoparathyroidism (IH) is under investigation. Abnormalities in central immune tolerance have yet not been investigated in this condition. T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs), formed during receptor gene rearrangements, are tools to assess central T- and B-cell output. OBJECTIVE: We assessed the number of circulating TRECs and KRECs in patients with IH, autoimmune type 1 diabetes (T1D), and autoimmune thyroiditis (ATs) and healthy controls (HCs). DESIGN: Comparative case-control at tertiary care center. SUBJECTS AND METHODS: Absolute and relative TRECs and KRECs were measured in DNA extracted from whole blood of patients with IH (n = 181, 22 of whom were reassessed after a decade of follow-up) and T1D (n = 133), AT (n = 53), and HC (n = 135) using a quantitative real-time PCR/TaqMan® probe technique. RESULTS: Absolute and relative means of TRECs and KRECs in IH were comparable to HCs, and no differences were found between IH with and without calcium-sensing receptor antibodies or class I HLA-A*26:01 association. TRECs and KRECs did not change after a decade of follow-up. T1D had significantly higher absolute TRECs than IH, AT, and HCs, whereas AT patients showed lower TRECs and the highest KRECs; these levels showed no noteworthy correlation with thyroid dysfunctions. CONCLUSION: Patients with IH showed TRECs and KRECs comparable to HCs, indicating an intact mechanism of T- and B-cell central immune tolerance. Interestingly, absolute TRECs were significantly higher in T1D than HCs, suggesting impaired central immune tolerance in T1D.

Vitamin D and calcium supplementation, skeletal muscle strength and serum testosterone in young healthy adult males: Randomized control trial.

BACKGROUND: Cholecalciferol and/or calcium supplementation might increase skeletal muscle strength and serum testosterone in young adult males. OBJECTIVE: We performed a randomized control trial assessing the effect of cholecalciferol/calcium on skeletal muscle strength and serum testosterone in vitamin D deficient young males. DESIGN: Two-by-two factorial RCT. SUBJECT AND INTERVENTION: Two-hundred and twenty-eight young males were block-randomized to (i) double-placebo, (ii) calcium/placebo, (iii) cholecalciferol/placebo and (iv) cholecalciferol/calcium. Doses for cholecalciferol were 60 000 IU/wk for 8 weeks followed by 60 000 IU/fortnightly, and doses for elemental calcium were 500 mg/twice daily for 6 months. A total of 180 subjects completed the study protocol. Their  ean age, body mass index and baseline 25(OH)D were 20.2 ± 2.2 years, 23.0 ± 3.6 kg/m2 and 21.5 ± 9.5 nmol/L, respectively. MEASUREMENTS: Handgrip (primary outcome), pinch-grip strength, distance walked in 6 minutes, dyspnoea-score, quality of life by Short Form 36, serum 25(OH)D, 1,25(OH)2 D, iPTH, total testosterone and free androgen index (FAI). RESULTS: After intervention, mean serum 25(OH)D was >75.0 nmol/L in cholecalciferol groups. However, the handgrip strength (29.7 ± 4.4, 29.3 ± 4.6, 30.6 ± 5.0 and 28.8 ± 4.3 kg, P = .28) was comparable in the 4 groups. Subgroups analysis among subjects with baseline serum 25OH)D < 25.0 and <12.0 nmol/L showed similar results. The mean serum testosterone decreased significantly at 6 months; however, delta change was similar in 4 groups. Change in handgrip strength and other outcomes was similar in 4 groups with and without adjustment for delta testosterone and FAI. CONCLUSIONS: Six months of cholecalciferol/calcium supplementation had no significant effect on skeletal muscle strength and serum testosterone in young adult males.

Presence of Autoreactive, MHC Class I-Restricted, Calcium-Sensing Receptor (CaSR)-Specific CD8+ T Cells in Idiopathic Hypoparathyroidism.

Context: Major histocompatibility complex class I allele HLA-A*26:01 and human leukocyte antigen (HLA) supertype A01 (STA01) are increased in idiopathic hypoparathyroidism (IH). However, cell-mediated autoimmune responses directed against the calcium-sensing receptor (CaSR) have not been demonstrated. Objective: To study CaSR-specific cytotoxic T-cell responses in peripheral blood mononuclear cells of IH patients. Design: Twenty-four peptides of CaSR (RH1 to RH24) were evaluated for their ex vivo potential to stimulate PBMCs from IH patients and controls in interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assays. Setting: Tertiary patient care center and National Institute of Immunology, New Delhi, India. Patients and Other Participants: Forty-five patients with IH attending the endocrine clinic of the All India Institute of Medical Sciences and 22 healthy controls. Main Outcome Measures: Major histocompatibility complex class-I restricted, CaSR-specific cytotoxic CD8+ T-cell responses evaluated by IFN-γ ELISPOT assay. Results: Of IH patients, 82.2% showed IFN-γ-secreting cells when stimulated ex-vivo with CaSR peptides. Peptides RH7, RH9, and RH16 elicited HLA supertype A01-restricted responses in IH. RH8, RH14, RH15, RH20, and RH21 peptides induced significantly higher responses in STA01+ IH patients compared with healthy controls irrespective of their supertype A01 status. Conclusions: Our ex vivo IFN-γ ELISPOT assays demonstrate the presence of CaSR-specific memory CD8+ T cells in the peripheral circulation of patients with IH, suggesting the role of cell-mediated autoimmune responses in the etiopathogenesis of IH.