Immune Suppressive and Bone Inhibitory Effects of Prednisolone in Growing and Regenerating Zebrafish Tissues.

Glucocorticoids are widely used as therapeutic agents to treat immune-mediated diseases in humans because of their anti-inflammatory and immunosuppressive effects. However, glucocorticoids have various adverse effects, in particular rapid and pronounced bone loss associated with fractures in glucocorticoid-induced osteoporosis, a common form of secondary osteoporosis. In zebrafish, which are increasingly used to study processes of bone regeneration and disease, glucocorticoids show detrimental effects on bone tissue; however, the underlying cellular mechanisms are incompletely understood. Here, we show that treatment with the glucocorticoid prednisolone impacts on the number, activity and differentiation of osteoblasts, osteoclasts, and immune cells during ontogenetic growth, homeostasis, and regeneration of zebrafish bone. Macrophage numbers are reduced in both larval and adult tissues, correlating with decreased generation of myelomonocytes and enhanced apoptosis of these cells. In contrast, osteoblasts fail to proliferate, show decreased activity, and undergo incomplete differentiation. In addition, prednisolone treatment mitigates the number and recruitment of osteoclasts to sites of bone regeneration in adult fish. In combination, these effects delay bone growth and impair bone regeneration. Our study demonstrates the many-faceted effects of glucocorticoids in non-mammalian vertebrates and helps to further establish the zebrafish as a model to study glucocorticoid-induced osteoporosis. © 2017 American Society for Bone and Mineral Research.

CT Texture Analysis of Ex Vivo Renal Stones Predicts Ease of Fragmentation with Shockwave Lithotripsy.

INTRODUCTION: Understanding the factors affecting success of extracorporeal shockwave lithotripsy (SWL) would improve informed decision-making on the most appropriate treatment modality for an individual patient. Although stone size and skin-to-stone distance do correlate with fragmentation efficacy, it has been shown that stone composition and architecture, as reflected by structural heterogeneity on CT, are also important factors. This study aims to determine if CT texture analysis (CTTA), a novel, nondestructive, and objective tool that generates statistical metrics reflecting stone heterogeneity, could have utility in predicting likelihood of SWL success. MATERIALS AND METHODS: Seven spontaneously passed, intact renal tract stones, were scanned ex vivo using standard CT KUB and micro-CT. The stones were then fragmented in vitro using a clinical lithotripter, after which, chemical composition analysis was performed. CTTA was used to generate a number of metrics that were correlated to the number of shocks needed to fragment the stone. RESULTS: CTTA metrics reflected stone characteristics and composition, and predicted ease of SWL fragmentation. The strongest correlation with number of shocks required to fragment the stone was mean Hounsfield unit (HU) density (r = 0.806, p = 0.028) and a CTTA metric measuring the entropy of the pixel distribution of the stone image (r = 0.804, p = 0.039). Using multiple linear regression analysis, the best model showed that CTTA metrics of entropy and kurtosis could predict 92% of the outcome of number of shocks needed to fragment the stone. This was superior to using stone volume or density. CONCLUSIONS: CTTA metrics entropy and kurtosis have been shown in this experimental ex vivo setting to strongly predict fragmentation by SWL. This warrants further investigation in a larger clinical study for the contribution of CT textural metrics as a measure of stone heterogeneity, along with other known clinical factors, to predict likelihood of SWL success.

Gα11 mutation in mice causes hypocalcemia rectifiable by calcilytic therapy.

Heterozygous germline gain-of-function mutations of G-protein subunit α11 (Gα11), a signaling partner for the calcium-sensing receptor (CaSR), result in autosomal dominant hypocalcemia type 2 (ADH2). ADH2 may cause symptomatic hypocalcemia with low circulating parathyroid hormone (PTH) concentrations. Effective therapies for ADH2 are currently not available, and a mouse model for ADH2 would help in assessment of potential therapies. We hypothesized that a previously reported dark skin mouse mutant (Dsk7) - which has a germline hypermorphic Gα11 mutation, Ile62Val - may be a model for ADH2 and allow evaluation of calcilytics, which are CaSR negative allosteric modulators, as a targeted therapy for this disorder. Mutant Dsk7/+ and Dsk7/Dsk7 mice were shown to have hypocalcemia and reduced plasma PTH concentrations, similar to ADH2 patients. In vitro studies showed the mutant Val62 Gα11 to upregulate CaSR-mediated intracellular calcium and MAPK signaling, consistent with a gain of function. Treatment with NPS-2143, a calcilytic compound, normalized these signaling responses. In vivo, NPS-2143 induced a rapid and marked rise in plasma PTH and calcium concentrations in Dsk7/Dsk7 and Dsk7/+ mice, which became normocalcemic. Thus, these studies have established Dsk7 mice, which harbor a germline gain-of-function Gα11 mutation, as a model for ADH2 and have demonstrated calcilytics as a potential targeted therapy.

Acute Molecular Changes in Synovial Fluid Following Human Knee Injury: Association With Early Clinical Outcomes.

OBJECTIVE: To investigate whether molecules found to be up-regulated within hours of surgical joint destabilization in the mouse are also elevated in the analogous human setting of acute knee injury, how this molecular response varies between individuals, and whether it is related to patient-reported outcomes in the 3 months after injury. METHODS: Seven candidate molecules were analyzed in blood and synovial fluid (SF) from 150 participants with recent structural knee injury at baseline (<8 weeks from injury) and in blood at 14 days and 3 months following baseline. Knee Injury and Osteoarthritis Outcome Score 4 (KOOS4 ) was obtained at baseline and 3 months. Patient and control samples were compared using Meso Scale Discovery platform assays or enzyme-linked immunosorbent assay. RESULTS: Six of the 7 molecules were significantly elevated in human SF immediately after injury: interleukin-6 (IL-6), monocyte chemotactic protein 1, matrix metalloproteinase 3 (MMP-3), tissue inhibitor of metalloproteinases 1 (TIMP-1), activin A, and tumor necrosis factor-stimulated gene 6 (TSG-6). There was low-to-moderate correlation with blood measurements. Three of the 6 molecules were significantly associated with baseline KOOS4 (those with higher SF IL-6, TIMP-1, or TSG-6 had lower KOOS4 ). These 3 molecules, MMP-3, and activin A were all significantly associated with greater improvement in KOOS4 over 3 months, after adjustment for other relevant factors. Of these, IL-6 alone significantly accounted for the molecular contribution to baseline KOOS4 and change in KOOS4 over 3 months. CONCLUSION: Our findings validate relevant human biomarkers of tissue injury identified in a mouse model. Analysis of SF rather than blood more accurately reflects this response. The response is associated with patient-reported outcomes over this early period, with SF IL-6 acting as a single representative marker. Longitudinal outcomes will determine if these molecules are biomarkers of subsequent disease risk.

Glycosphingolipid synthesis inhibition limits osteoclast activation and myeloma bone disease.

Glycosphingolipids (GSLs) are essential constituents of cell membranes and lipid rafts and can modulate signal transduction events. The contribution of GSLs in osteoclast (OC) activation and osteolytic bone diseases in malignancies such as the plasma cell dyscrasia multiple myeloma (MM) is not known. Here, we tested the hypothesis that pathological activation of OCs in MM requires de novo GSL synthesis and is further enhanced by myeloma cell-derived GSLs. Glucosylceramide synthase (GCS) inhibitors, including the clinically approved agent N-butyl-deoxynojirimycin (NB-DNJ), prevented OC development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of transcriptional activator NFATc1. GM3 was the prevailing GSL produced by patient-derived myeloma cells and MM cell lines, and exogenous addition of GM3 synergistically enhanced the ability of the pro-osteoclastogenic factors RANKL and insulin-like growth factor 1 (IGF-1) to induce osteoclastogenesis in precursors. In WT mice, administration of GM3 increased OC numbers and activity, an effect that was reversed by treatment with NB-DNJ. In a murine MM model, treatment with NB-DNJ markedly improved osteolytic bone disease symptoms. Together, these data demonstrate that both tumor-derived and de novo synthesized GSLs influence osteoclastogenesis and suggest that NB-DNJ may reduce pathological OC activation and bone destruction associated with MM.

Low-dose TNF augments fracture healing in normal and osteoporotic bone by up-regulating the innate immune response.

The mechanism by which trauma initiates healing remains unclear. Precise understanding of these events may define interventions for accelerating healing that could be translated to the clinical arena. We previously reported that addition of low-dose recombinant human TNF (rhTNF) at the fracture site augmented fracture repair in a murine tibial fracture model. Here, we show that local rhTNF treatment is only effective when administered within 24 h of injury, when neutrophils are the major inflammatory cell infiltrate. Systemic administration of anti-TNF impaired fracture healing. Addition of rhTNF enhanced neutrophil recruitment and promoted recruitment of monocytes through CCL2 production. Conversely, depletion of neutrophils or inhibition of the chemokine receptor CCR2 resulted in significantly impaired fracture healing. Fragility, or osteoporotic, fractures represent a major medical problem as they are associated with permanent disability and premature death. Using a murine model of fragility fractures, we found that local rhTNF treatment improved fracture healing during the early phase of repair. If translated clinically, this promotion of fracture healing would reduce the morbidity and mortality associated with delayed patient mobilization.

Night-time immobilization of the distal interphalangeal joint reduces pain and extension deformity in hand osteoarthritis.

OBJECTIVE: DIP joint OA is common but has few cost-effective, evidence-based interventions. Pain and deformity [radial or ulnar deviation of the joint or loss of full extension (extension lag)] frequently lead to functional and cosmetic issues. We investigated whether splinting the DIP joint would improve pain, function and deformity. METHODS: A prospective, radiologist-blinded, non-randomized, internally controlled trial of custom splinting of the DIP joint was carried out. Twenty-six subjects with painful, deforming DIP joint hand OA gave written, informed consent. One intervention joint and one control joint were nominated. A custom gutter splint was worn nightly for 3 months on the intervention joint, with clinical and radiological assessment at baseline, 3 and 6 months. Differences in the change were compared by the Wilcoxon signed rank test. RESULTS: The median average pain at baseline was similar in the intervention (6/10) and control joints (5/10). Average pain (primary outcome measure) and worst pain in the intervention joint were significantly lower at 3 months compared with baseline (P = 0.002, P = 0.02). Differences between intervention and control joint average pain reached significance at 6 months (P = 0.049). Extension lag deformity was significantly improved in intervention joints at 3 months and in splinted joints compared with matched contralateral joints (P = 0.016). CONCLUSION: Short-term night-time DIP joint splinting is a safe, simple treatment modality that reduces DIP joint pain and improves extension of the digit, and does not appear to give rise to non-compliance, increased stiffness or joint restriction. TRIAL REGISTRATION: clinical trials.gov, http://clinicaltrials.gov, NCT01249391.

Ultrasonographic measures of synovitis in an early phase clinical trial: a double-blind, randomised, placebo and comparator controlled phase IIa trial of GW274150 (a selective inducible nitric oxide synthase inhibitor) in rheumatoid arthritis.

OBJECTIVES: To test the sensitivity to change of ultrasonographic endpoints in early phase clinical trials in subjects with active rheumatoid arthritis (RA). METHODS: A double-blind, placebo and comparator controlled, randomised, two-centre study investigated the effect on synovial thickness and vascularity of 28 days repeat daily oral dosing of 60 mg of the inducible nitric oxide synthase inhibitor GW274150 or 7.5 mg prednisolone in RA. Fifty patients with DAS28 scores ≥4.0 were assigned to 3 treatment arms of 17, 19 and 14 (on placebo, GW274150 and prednisolone respectively). Synovial thickness and vascularity of all 10 metacarpophalangeal joints were assessed by ultrasonography using a semi-quantitative scale at baseline (Day 1), Day 15 and Day 28. Vascularity was also measured quantitatively by power Doppler area. RESULTS: At Day 28, the GW274150 group showed a trend towards reduction in synovial thickness compared with placebo, with an adjusted mean decrease of 33% (p=0.072); the prednisolone group decreased significantly by 44% (p=0.011). Similarly, there was a trend to reduced synovial vascularity with GW274150 by 42% compared with placebo (p=0.075); prednisolone resulted in a statistically significant decrease of 55% (p=0.012). There was a 55% decrease in power Doppler area for GW274150, compared with placebo although the result was not statistically significant (p=0.375). Prednisolone 7.5 mg resulted in a highly statistically significant decrease of 95% (p=0.003). CONCLUSIONS: This study advocates the use of ultrasonographic measures of metacarpophalangeal joint synovitis as an endpoint for clinical studies assessing therapeutic potential of new compounds in small patient cohorts over 28 days.

Dual-specificity phosphatase 1-null mice exhibit spontaneous osteolytic disease and enhanced inflammatory osteolysis in experimental arthritis.

OBJECTIVE: Bone formation and destruction are usually tightly linked; however, in disorders such as rheumatoid arthritis, periodontal disease, and osteoporosis, elevated osteoclast activity leads to bone destruction. Osteoclast formation and activation are controlled by many signaling pathways, including p38 MAPK. Dual-specificity phosphatase 1 (DUSP-1) is a factor involved in the negative regulation of p38 MAPK. The purpose of this study was to examine the effect of Dusp1 deficiency on bone destruction. METHODS: Penetrance, onset, and severity of collagen-induced arthritis were recorded in DUSP-1+/+ and DUSP-1-/- mice. Bone destruction was assessed by histologic and micro-computed tomographic examination of the joints. The in vitro formation and activation of osteoclasts from DUSP-1+/+ and DUSP-1-/- precursors were assessed in the absence or presence of tumor necrosis factor (TNF). RESULTS: The formation and activation of osteoclasts in vitro in the presence of TNF were enhanced by Dusp1 gene disruption. DUSP-1-/- mice exhibited higher penetrance, earlier onset, and increased severity of experimental arthritis, accompanied by greater numbers of osteoclasts in inflamed joints and more extensive loss of bone. A DUSP-1-/- mouse colony of mixed genetic background also demonstrated striking spontaneous osteolytic destruction of distal phalanges. CONCLUSION: DUSP-1 is a critical regulator of osteoclast activity and limits bone destruction in an experimental model of rheumatoid arthritis. Defects in the expression or activity of DUSP1 in humans may correlate with a propensity to develop osteolytic lesions in arthritis.

TNF-alpha promotes fracture repair by augmenting the recruitment and differentiation of muscle-derived stromal cells.

With an aging population, skeletal fractures are increasing in incidence, including the typical closed and the less common open fractures in normal bone, as well as fragility fractures in patients with osteoporosis. For the older age group, there is an urgent unmet need to induce predictable bone formation as well as improve implant fixation in situations such as hip joint replacement. Using a murine model of slow-healing fractures, we have previously shown that coverage of the fracture with muscle accelerated fracture healing and increased union strength. Here, we show that cells from muscle harvested after 3 d of exposure to an adjacent fracture differentiate into osteoblasts and form bone nodules in vitro. The osteogenic potential of these cells exceeds that of adipose and skin-derived stromal cells and is equivalent to bone marrow stromal cells. Supernatants from human fractured tibial bone fragments promote osteogenesis and migration of muscle-derived stromal cells (MDSC) in vitro. The main factor responsible for this is TNF-α, which promotes first MDSC migration, then osteogenic differentiation at low concentrations. However, TNF-α is inhibitory at high concentrations. In our murine model, addition of TNF-α at 1 ng/mL at the fracture site accelerated healing. These data indicate that manipulating the local inflammatory environment to recruit, then differentiate adjacent MDSC, may be a simple yet effective way to enhance bone formation and accelerate fracture repair. Our findings are based on a combination of human specimens and an in vivo murine model and may, therefore, translate to clinical care.

Effects of extensor synovectomy and excision of the distal ulna in rheumatoid arthritis on long-term function.

PURPOSE: Objective outcomes data after excision of the distal ulna in rheumatoid arthritis are lacking. The aim of this study was to evaluate the functional results of this surgery in the long term. METHODS: We prospectively collected data on range of motion (22 wrists), visual analog pain scores (14 wrists), and grip strength measured using a Jamar dynamometer (20 hands) in a group of 23 patients (26 wrists) preoperatively and at 3 months, 12 months, and a minimum of 5 years postoperatively (range, 5.3-10.4 y). The Jebsen-Taylor hand function test was administered to 9 patients at the same time points. A subgroup of patients also underwent extensor carpi radialis longus to extensor carpi ulnaris tendon transfer (11 wrists). RESULTS: At one year, there were improvements in wrist pronation and supination, which were maintained at final follow-up. Active radial deviation decreased significantly at 3 months (p = .01) and one year (p = .02); this remained reduced at final follow-up (not significant). Wrist extension and active ulnar deviation showed slight improvements by one year, but reduced to levels below that measured preoperatively by final follow-up. Wrist flexion was significantly reduced at all time points postoperatively. Grip strength showed improvement from 10.0 kg (standard deviation [SD] 4.1 kg) preoperatively to 12.5 kg (SD 4.6 kg) 1 year after surgery and returned to preoperative levels (9.5 kg, SD 5.6 kg) by final follow-up. Wrist pain was significantly reduced from a mean score of 5 (SD 4) preoperatively to 2 (SD 2) postoperatively (p = .01). The Jebsen-Taylor hand function test showed improvements in writing and card turning. CONCLUSIONS: In the long term, excision of the distal ulna in rheumatoid patients results in an improvement in some aspects of hand function. There is a significant (p = .01) reduction in wrist pain but a reduction of wrist flexion. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.