Loss of equilibrative nucleoside transporter 1 in mice leads to progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis in humans

Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory spondyloarthropathy, characterized by ectopic calcification of spinal tissues. Symptoms include spine pain and stiffness, and in severe cases dysphagia and spinal cord compression. The etiology of DISH is unknown and there are no specific treatments. Recent studies have suggested a role for purine metabolism in the regulation of biomineralization. Equilibrative nucleoside transporter 1 (ENT1) transfers hydrophilic nucleosides, such as adenosine, across the plasma membrane. In mice lacking ENT1, we observed the development of calcified lesions resembling DISH. By 12 months of age, ENT1-/- mice exhibited signs of spine stiffness, hind limb dysfunction, and paralysis. Micro-computed tomography (μCT) revealed ectopic mineralization of paraspinal tissues in the cervical-thoracic region at 2 months of age, which extended to the lumbar and caudal regions with advancing age. Energy-dispersive X-ray microanalysis of lesions revealed a high content of calcium and phosphorus with a ratio similar to that of cortical bone. At 12 months of age, histological examination of ENT1-/- mice revealed large, irregular accumulations of eosinophilic material in paraspinal ligaments and entheses, intervertebral discs, and sternocostal articulations. There was no evidence of mineralization in appendicular joints or blood vessels, indicating specificity for the axial skeleton. Plasma adenosine levels were significantly greater in ENT1 -/- mice than in wild-type, consistent with loss of ENT1 - a primary adenosine uptake pathway. There was a significant reduction in the expression of Enpp1, Ank, and Alpl in intervertebral discs from ENT1-/- mice compared to wild-type mice. Elevated plasma levels of inorganic pyrophosphate in ENT1-/- mice indicated generalized disruption of pyrophosphate homeostasis. This is the first report of a role for ENT1 in regulating the calcification of soft tissues. Moreover, ENT1-/- mice may be a useful model for investigating pathogenesis and evaluating therapeutics for the prevention of mineralization in DISH and related disorders. © 2013 American Society for Bone and Mineral Research. Copyright © 2013 American Society for Bone and Mineral Research

Disruption of biomineralization pathways in spinal tissues of a mouse model of diffuse idiopathic skeletal hyperostosis

© 2015 Elsevier Inc. Equilibrative nucleoside transporter 1 (ENT1) mediates passage of adenosine across the plasma membrane. We reported previously that mice lacking ENT1 (ENT1-/-) exhibit progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. Here, we investigated mechanisms underlying aberrant mineralization in ENT1-/- mice. Micro-CT revealed ectopic mineralization of spinal tissues in both male and female ENT1-/- mice, involving the annulus fibrosus of the intervertebral discs (IVDs) of older mice. IVDs were isolated from wild-type and ENT1-/- mice at 2 months of age (prior to disc mineralization), 4, and 6 months of age (disc mineralization present) and processed for real-time PCR, cell isolation, or histology. Relative to the expression of ENTs in other tissues, ENT1 was the primary nucleoside transporter expressed in wild-type IVDs and mediated the functional uptake of [3H]2-chloroadenosine by annulus fibrosus cells. No differences in candidate gene expression were detected in IVDs from ENT1-/- and wild-type mice at 2 or 4 months of age. However, at 6 months of age, expression of genes that inhibit biomineralization Mgp, Enpp1, Ank, and Spp1 were reduced in IVDs from ENT1-/- mice. To assess whether changes detected in ENT1-/- mice were cell autonomous, annulus fibrosus cell cultures were established. Compared to wild-type cells, cells isolated from ENT1-/- IVDs at 2 or 6 months of age demonstrated greater activity of alkaline phosphatase, a promoter of biomineralization. Cells from 2-month-old ENT1-/- mice also showed greater mineralization than wild-type. Interestingly, altered localization of alkaline phosphatase activity was detected in the inner annulus fibrosus of ENT1-/- mice in vivo. Alkaline phosphatase activity, together with the marked reduction in mineralization inhibitors, is consistent with the mineralization of IVDs seen in ENT1-/- mice at older ages. These findings establish that both cell-autonomous and systemic mechanisms contribute to ectopic mineralization in ENT1-/- mice

Standardization of clinical protocols in oral malodor research

The objective of this study is to standardize protocols for clinical research into oral malodor caused by volatile sulfur compounds (VSCs). To detect VSCs, a gas chromatograph (GC) using a flame photometric detector equipped with a bandpass filter (at 393 nm) is the gold standard (sensitivity: 5 x 10 (11) gS s (1)). The baselines of VSC concentrations in mouth air varied considerably over a week. When the subjects refrained from eating, drinking and oral hygiene including mouth rinsing, the VSC concentrations remained constant until eating. Over a 6 h period after a meal, VSC concentrations decreased dramatically (p <0.01). These results point to optimal times and conditions for sampling subjects. Several portable devices were compared with the measurements by the GCs. Portable GCs demonstrated capabilities similar to those of the GCs. We also applied the recommended protocols described below to clinical research testing the efficacy of ZnCl2 products, and confirmed that using the recommended protocols in a randomized crossover design would provide very clear results. Proposed protocols include: (a) a short-term study rather than a long-term study is strongly recommended, since the VSC concentrations are constant in the short term; (b) a crossover study would be the best design to avoid the effects of individual specificities on each clinical intervention; (c) measurements of VSCs should preferably be carried out using either a GC or portable GCs