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Abstract Telmisartan, a selective inhibitor of angiotensin II receptor type 1 (AT1), demonstrates nonlinear pharmacokinetics (PK) when orally administered in ascending doses to healthy volunteers, but the underlying mechanisms remain unclear. This study presents a physiologically based pharmacokinetic model integrated with target‐mediated drug disposition (TMDD‐PBPK model) to explore the mechanism of its nonlinear PK. We employed the Cluster‐Gauss Newton method for top‐down analysis, estimating the in vivo Km,OATP1B3 (Michaelis–Menten constant for telmisartan hepatic uptake via Organic Anion Transporting Polypeptide 1B3) to be 2.0–5.7 nM. This range is significantly lower than the reported in vitro value of 810 nM, obtained in 0.3% human serum albumin (HSA) conditions. Further validation was achieved through in vitro assessment in plated human hepatocytes with 4.5% HSA, showing a Km of 4.5 nM. These results underscore the importance of albumin‐mediated uptake effect for the hepatic uptake of telmisartan. Our TMDD‐PBPK model, developed through a “middle‐out” approach, underwent sensitivity analysis to identify key factors in the nonlinear PK of telmisartan. We found that the nonlinearity in the area under the concentration–time curve (AUC) and/or maximum concentration (Cmax) of telmisartan is sensitive to Km,OATP1B3 across all dosages. Additionally, the dissociation constant (Kd) for telmisartan binding to the AT1 receptor, along with its receptor abundance, notably influences PK at lower doses (below 20 mg). In conclusion, the nonlinear PK of telmisartan appears primarily driven by hepatic uptake saturation across all dose ranges and by AT1‐receptor binding saturation, notably at lower doses.

Summary: Renal transporters play critical roles in predicting potential drug-drug interactions. However, current in vitro models often fail to adequately express these transporters, particularly solute carrier proteins, including organic anion transporters (OAT1/3), and organic cation transporter 2 (OCT2). Here, we developed a hiPSC-derived kidney organoids-based proximal tubule-on-chip (OPTC) model that emulates in vivo renal physiology to assess transporter function. Compared to chips based on immortalized cells, OPTC derived from the two most commonly used differentiation protocols exhibited significant improvement in expression level and polarity of OAT1/3 and OCT2. Hence, the OPTC demonstrates enhanced functionality in efflux and uptake assessments, and nephrotoxicity. Furthermore, these functionalities are diminished upon adding inhibitors during substrate-inhibitor interactions, which were closer to in vivo observations. Overall, these results support that OPTC can reliably assess the role of renal transporters in drug transport and nephrotoxicity, paving the way for personalized models to assess renal transport and disease modeling.

OAT10 (SLC22A13) is a transporter highly expressed in renal tubules and transporting organic anions including nicotinate, β-hydroxybutyrate, p-aminohippurate, and orotate. In transport assays using Xenopus oocytes and HEK293 cells, we found that apparent substrate selectivity of OAT10 was different between the expression systems, particularly less pronounced uptake of β-hydroxybutyrate in HEK293 cells. Because functional coupling between transporters may interfere with functional properties of the transporter, we searched for endogenous transporters in HEK293 cells that could affect OAT10. By means of comprehensive approach with co-immunoprecipitation followed by LC-MS/MS analysis, we identified monocarboxylate transporter MCT1 (SLC16A1) as physically coupled with OAT10. The knockdown of MCT1 in OAT10-expressing HEK293 cells increased the uptake of β-hydroxybutyrate and nicotinate, common substrates of OAT10 and MCT1, whereas the uptake of orotate, a substrate of only OAT10, was not affected. MCT1 is supposed to act as an escape route and mediate the efflux of nicotinate and β-hydroxybutyrate taken up by OAT10 localized nearby MCT1, as suggested by co-immunoprecipitation. This functional coupling would explain altered apparent substrate selectivity in HEK293 cells compared with Xenopus oocytes. The findings in this study warn in transporter studies that the expression system can interfere with assessing correct transport properties due to unexpected interactions with endogenous transporters.

Study Design Retrospective study. Purpose We aimed to use motor evoked potentials (MEPs) to examine the association of electrophysiological assessment of the trapezius muscle with neck pain. Overview of Literature Previous reports on the association of neck pain with the trapezius muscle have focused on surface electromyograms and muscle oxygenation; however, to our knowledge, none of these studies included detailed data on MEPs. Methods The study included 100 patients with cervical myelopathy who underwent surgery at the National Center for Geriatrics and Gerontology in Obu, Japan from June 2010 to March 2013. Before the surgery, neck pain was evaluated using a Visual Analog Scale (a score ≥50 indicated neck pain and a score

Background: A portable navigation system (PNS) was recently introduced. The PNS enables surgeons to place the acetabular component accurately. While the margin of the error for the cup abduction and anteversion was larger than the values obtained from a computed tomography-based navigation system. We hypothesized that the accuracy of the PNS might be affected by pelvic tilt. Material and Methods: A bone substitute model of the pelvis was used in this in vitro study. We set the acetabular component using PNS. We set the acetabular component angle after changing the sagittal, coronal, and axial pelvic tilt. We calculated the difference between the angle displayed on the PNS display and the actual angle of the acetabular component. The difference in inclination angle was defined as ΔRI, and the difference in the anteversion angle was defined as ΔRA. We evaluated the trends in this ΔRI and ΔRA due to the pelvic tilt. Results: In this in vitro study, the placement of the acetabular component was accurate in the neutral position; ΔRI was 0.5 ± 0.7° and ΔRA was 1.0 ± 0.7°. Sagittal pelvic tilt and axial pelvic tilt increased both the ΔRA and ΔRI (P = .017). Coronal tilt increased ΔRI but did not change ΔRA. Conclusions: While the PNS may enable surgeons to place accurate component placement in the neutral position, its accuracy decreased by pelvic tilt. The surgeons should use a solid pelvic lateral positioner for reducing discrepancies in pelvic tilt when using the PNS in the lateral decubitus position.

Study Design Single-center retrospective study in pediatric patients. Purpose The purpose of the study was to investigate the effects of the presence and severity of leg-length discrepancy (LLD) on scoliosis in children. Overview of Literature LLD is a common orthopedic problem that can result in standing imbalance, low back pain, and stress fractures. It may cause structural changes in the spine over time, but the exact effect of LLD on scoliosis is unknown. Methods The subjects were 23 children with LLD ≥20 mm (range, 27–65 mm) treated at Nagoya University Hospital between 2007 and 2017. Whole spine posteroanterior standing radiographs and whole lower limb radiographs in a supine position were recorded. Data were collected for demographics, LLD, Cobb angle, pelvic obliquity, and Nash/Moe index. Scoliosis was defined as a Cobb angle ≥10°. Leg length was measured from the top of the femoral head to the middle of the tibial plafond, and LLD was defined as the difference between the left and right leg lengths. Results The patients (nine males and 14 females) had a mean age of 14.0 years (range, 5–18 years). The average LLD was 44.3±17.2 mm, with LLDs of 20 to 39 mm, 40 to 59 mm, and ≥60 mm in 13, five, and five subjects, respectively. The average Cobb angle was 13.0°±7.0°, and 15 subjects (65%) had scoliosis. Convexity of the scoliosis was to the short leg side in all cases. The Cobb angle was significantly related to the severity of the LLD (R=0.736, p

Abstract This study aimed to evaluate the short-term effectiveness and safety profiles of baricitinib and explore factors associated with improved short-term effectiveness in patients with rheumatoid arthritis (RA) in clinical settings. A total of 113 consecutive RA patients who had been treated with baricitinib were registered in a Japanese multicenter registry and followed for at least 24 weeks. Mean age was 66.1 years, mean RA disease duration was 14.0 years, 71.1% had a history of use of biologics or JAK inhibitors (targeted DMARDs), and 48.3% and 40.0% were receiving concomitant methotrexate and oral prednisone, respectively. Mean DAS28-CRP significantly decreased from 3.55 at baseline to 2.32 at 24 weeks. At 24 weeks, 68.2% and 64.1% of patients achieved low disease activity (LDA) and moderate or good response, respectively. Multivariate logistic regression analysis revealed that no previous targeted DMARD use and lower DAS28-CRP score at baseline were independently associated with achievement of LDA at 24 weeks. While the effectiveness of baricitinib was similar regardless of whether patients had a history of only one or multiple targeted DMARDs use, patients with previous use of non-TNF inhibitors or JAK inhibitors showed lower rates of improvement in DAS28-CRP. The overall retention rate for baricitinib was 86.5% at 24 weeks, as estimated by Kaplan–Meier analysis. The discontinuation rate due to adverse events was 6.5% at 24 weeks. Baricitinib significantly improved RA disease activity in clinical practice. Baricitinib was significantly more effective when used as a first-line targeted DMARDs.

Study Design Retrospective study in a single center. Purpose To examine denosumab persistence in patients of different ages with severe osteoporosis in Japan. Overview of Literature Denosumab is an antibody drug used for the treatment of osteoporosis. It is mainly used in patients with severe osteoporosis who might have high motivation for treatment, and the need for only semi-annual subcutaneous injection might improve the continuation rate. However, no English-language articles have reported on denosumab persistence in the Japanese population, including young people, despite the importance of this issue in a super-aging society. Methods The subjects started treatment with subcutaneous denosumab in our department from July 2013 until December 2017. Persistence rates were calculated using Kaplan-Meier curves. Patients were defined as “persistent” or “non-persistent” according to the use of therapy after 60 months. Results The study included 101 patients (84 females) with a median follow-up period of 23.6±14.2 months. The persistence rate declined to 85.3%, 78.3%, 74.1%, 71.3%, and 69.3% at 12, 24, 36, 48, and 60 months, respectively. Age at the initiation of denosumab therapy differed significantly between non-persistent (n=31) and persistent (n=70) patients (81.3 vs. 72.8 years, p

Metformin is an important antidiabetic drug and often used as a probe for drug–drug interactions (DDIs) mediated by renal transporters. Despite evidence supporting the inhibition of multidrug and toxin extrusion proteins as the likely DDI mechanism, the previously reported physiologically‐based pharmacokinetic (PBPK) model required the substantial lowering of the inhibition constant values of cimetidine for multidrug and toxin extrusion proteins from those obtained in vitro to capture the clinical DDI data between metformin and cimetidine.1 We constructed new PBPK models in which the transporter‐mediated uptake of metformin is driven by a constant membrane potential. Our models successfully captured the clinical DDI data using in vitro inhibition constant values and supported the inhibition of multidrug and toxin extrusion proteins by cimetidine as the DDI mechanism upon sensitivity analysis and data fitting. Our refined PBPK models may facilitate prediction approaches for DDI involving metformin using in vitro inhibition constant values.

Introduction: Nonsteroidal anti-inflammatory drugs (NSAIDs) are the first-line treatment for acute lumbar radicular pain accompanying lumbar disc herniation (LDH), but their effects are minimal. The purpose of this study was to evaluate the efficacy and safety of pregabalin (PGB) as an alternative therapy for this condition. Methods: Patients with acute lumbar radicular pain accompanying LDH were randomly administered either NSAIDs plus PGB (30 patients) or NSAIDs alone (30 patients) for up to 4 weeks. The primary outcome was leg pain at 2 and 4 weeks. Secondary outcomes were reduction in sleep disturbances and patient global impressions of change (PGIC) at 2 and 4 weeks. Results: Four patients in the NSAIDs plus PGB group were deemed ineligible and excluded from the study. Fewer sleep disturbances were reported by patients administered NSAIDs plus PGB compared with the NSAID monotherapy group at both 2 and 4 weeks. Additionally, the NSAIDs plus PGB group showed greater improvement in pain than the NSAID monotherapy group at 4 weeks, although this difference was not significant. PGIC was also significantly better in the NSAIDs plus PGB group than in the NSAID monotherapy group at 4 weeks. The incidence of adverse events was significantly greater in the NSAIDs plus PGB group than in the NSAID monotherapy group. Conclusions: The combination of NSAIDs plus PGB is more effective against sleep disturbance than NSAIDs alone in patients with acute LDH, although the control of sciatic pain is minimal. Patients reported satisfactory recoveries could also be obtained, and thus, this combination therapy could be a good option for the conservative treatment of acute lumbar radicular pain, including LDH.

Objectives To clarify which rheumatoid arthritis (RA) patients benefit most from the anti-receptor activator of nuclear factor-κB ligand antibody denosumab to reduce the progression of joint destruction.Methods We pooled patient data from the 12-month, double-blind, placebo-controlled DRIVE (phase II) and DESIRABLE (phase III) studies. In DRIVE, concomitant treatment was limited to methotrexate, salazosulfapyridine and bucillamine. In DESIRABLE, patients could receive any disease-modifying antirheumatic drug. RA patients were randomised to denosumab 60 mg every 6 months (Q6M), every 3 months (Q3M) or placebo. Efficacy was assessed by van der Heijde-modified total Sharp score (mTSS), bone erosion score (ES) and joint space narrowing score (JSNS). Change in mTSS was assessed in subgroups stratified by risk factors for radiographic damage if the interaction factor was significant.Results The pooled analysis included 909 patients. Denosumab reduced worsening of mTSS (mean (SD)) at 12 months in the Q6M (0.88 (3.30), p=0.0024) and Q3M (0.66 (2.16), p=0.0002) groups versus placebo (1.50 (3.73)). This reduction in mTSS progression was due to the change in ES (Q6M, 0.44 (1.89), p=0.0006; Q3M, 0.20 (0.86), p

Abstract Background Achondroplasia (ACH), the most common form of short‐limbed skeletal dysplasia, is caused by gain‐of‐function mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. More than 97% of patients result from a heterozygous p.G380R mutation in the FGFR3 gene. We present here a child who had two de novo variants in the FGFR3 on the same allele, a common p.G380R mutation and a novel p.S378N variant. Methods A 3‐year‐old Japanese girl born from non‐consanguineous healthy parents showed more severe clinical and radiological phenotypes than classic ACH, including severe short‐limbed short stature with marked ossification defects in the metaphysis and epiphysis, hydrocephalus and cervicomedullary compression due to foramen magnum stenosis, prolonged pulmonary hypoplasia, and significant delay in the gross motor development. Genomic DNA was extracted from the proband and whole‐exome sequencing was performed. The variants were subsequently confirmed by Sanger sequencing. Results Mutation analysis demonstrated that the proband had p.S378N (c.1133G>A) and p.G380R (c.1138G>A) variants in the FGFR3 gene. Both variants were not detected in her parents and therefore considered de novo. An allele‐specific PCR was developed in order to determine whether these mutations were on the same allele (cis) or on different alleles (trans). The c.1138G>A mutation was found in the PCR product generated with the primer for the mutant 1133A, but it was not detected in the product with the wild‐type 1133G, confirming that p.S378N and p.G380R variants were located on the same allele (cis). Conclusion This is the second case who had two FGFR3 variants in the transmembrane domain on the same allele. The p.S378N variant may provide an additive effect on the activating receptor with the p.G380R mutation and alter the protein function, which could be responsible for the severe phenotype of the present case.

Aims: This study investigated effects of narrow-range ultraviolet irradiation (UVR) by a new UV–LED device on vitamin D supply and changes of bone in senescence-accelerated mouse P6 (SAMP6) with vitamin D deficiency. Main methods: We used female SAMP6 mice as a senile osteoporotic model. We set a total of 3 groups (n = 4 per group); D-UVR+ group (vitamin D deficient–dietary and UVR), D- (vitamin D deficient–dietary), and D+ groups (vitamin D contained–dietary). Mice in the D-UVR + group were UV–irradiated (305nm) with 1 kJ/m2 twice a week for 12 weeks from 20 to 32 weeks of age. Serum 25(OH)D, 1,25(OH)2D, and micro–computed tomography (CT) were assessed over time. Mechanical test, and histological assay were performed for femurs removed at 32 weeks of age. Key findings: UVR increased both serum 25(OH)D and 1,25(OH)2D levels at 4 and 8 weeks–UVR in the D-UVR+ group compared with that in the D- group (P < 0.05, respectively). Relative levels of trabecular bone mineral density in micro–CT were higher in the D-UVR+ group than in the D- group at 8 weeks–UVR (P = 0.048). The ultimate load was significantly higher in the D-UVR+ group than in the D- group (P = 0.036). In histological assay, fewer osteoclasts and less immature bone (/mature bone) could be observed in the D-UVR+ group than in the D- group, significantly. Significance: UVR may have possibility to improve bone metabolism associated with vitamin D deficiency in SAMP6 mice.

Achondroplasia (ACH) is the most common short-limbed skeletal dysplasia caused by activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. We identified that meclizine hydrochloride inhibited FGFR3 signaling in various chondrocytic cells and promoted longitudinal bone growth in mouse model of ACH. Meclizine has safely been used for more than 50 years, but it lacks the safety data for repeated administration and pharmacokinetics (PK) when administered to children. We performed a phase Ia study to evaluate the PK and safety of meclizine administered orally to ACH children. Twelve ACH children aged from 5 to younger than 11 years were recruited, and the first 6 subjects received once a day of meclizine in the fasted condition, subsequent 6 subjects received twice a day of meclizine in the fed condition. Meclizine was well tolerated in ACH children with no serious adverse events. The mean Cmax, Tmax, AUC0-24h, t1/2 during 24 hours in the fasted condition were 130 ng/mL, 1.7 hours, 761 ng·h/mL, and 8.5 hours respectively. The simulation of repeated administration of meclizine for 14 days demonstrated that plasma concentration apparently reached steady state around 10 days after the first dose both at once a day and twice a day administration. The AUC0-10h of the fasting and fed condition were 504 ng·h/mL and 813 ng·h/mL, respectively, indicating exposure of meclizine increased with the diet. Although higher drug exposure was confirmed in ACH children compared to adults, a single administration of meclizine seemed to be well tolerated.

Neurotrophic factors have been regarded having promising potentials for neuronal protection and regeneration, and thus promoting beneficial effects of kinesiological functions. They can be suspected to play important roles in cell/tissue grafting for various neural diseases. The clinical applications of such trophic factors to the central nervous system (CNS), however, have caused problematic side effects on account of the distinctive bioactive properties. In the course of developing synthetic compounds reflecting beneficial properties of basic fibroblast growth factor (bFGF), we conducted screening candidates that stimulate to trigger the intracellular tyrosine phosphorylation of FGF receptor and lead to the subsequent intracellular signaling in neurons. A small synthetic molecule SUN13837 was characterized by mimicking the beneficial properties of bFGF, which have been known as its specific activities when applied to CNS. What is more remarkable is that SUN13837 is eliminated the bioactivity to induce cell proliferation of non-neuronal somatic cells. On the bases of studies of pharmacology, behavior, physiology and histology, the present study reports that SUN13837 is characterized as a promising synthetic compound for treatment of devastating damages onto the rat spinal cord.

Study Design Retrospective study. Purpose To examine the relationship between postoperative bowel bladder disorder (BBD) and the efficacy of needle electrodes for the external anal sphincter (EAS) in intraoperative spinal cord monitoring with transcranial muscle action potentials (Tc-MsEP). Overview of Literature Spinal surgery for spina bifida, spinal cord tumor, and spinal tethered cord syndrome has a high rate of postoperative BBD. Monitoring of the EAS with Tc-MsEP is frequently performed during spinal surgery. We initially used plug-surface electrodes for this purpose, but have more recently switched to needle electrodes for the monitoring of the EAS. To date, there has been no comparison between the utility of these electrodes. Methods Waveform derivation, exacerbation of postoperative BBD, and sensitivity and specificity for prediction of BBD by 70% amplitude reduction of EAS activity using needle and plug-surface electrodes were investigated in 239 spine surgeries. The cut-off for the % drop in amplitude for BBD prediction was determined for EAS monitoring using a needle electrode. Results The overall rate of postoperative BBD aggravation was 7.1% (17/239 cases), with the individual rates using needle and plug-surface electrodes being 6.9% (8/116) and 7.3% (9/123), respectively. The waveform derivation rate was significantly higher using needle electrodes (91.3% [106/116] vs. 76.4% [94/123], p

Study Design Single-center retrospective study. Purpose To optimize the perioperative management of patients with hemophilia who are undergoing spinal surgery. Overview of Literature Hemophilia is a rare disease in which there is a tendency of bleeding because of a congenital deficiency in blood coagulation factor activity. There has been no previous report on spinal surgery in patients with hemophilia. Methods The subjects were five patients (all males) with hemophilia who underwent spinal surgery at Nagoya University Hospital. Two patients had hemophilia A (deficiency of factor VIII) and three had hemophilia B (deficiency of factor IX). The mean age at the time of surgery was 63 years (range, 46–73 years). The following surgeries were performed: posterior lumbar interbody fusion (PLIF) in two patients, and lumbar fenestration, cervical laminoplasty and lumbar fenestration, and cervical laminoplasty and PLIF in one patient each. Results Coagulation factor at a mean dose of 4.8 ×103 U (range, 3–6 ×103 U) was intravenously injected before surgery, and a mean dose of 5.2 ×103 U (rang, 4–6 ×103 U) was continuously administered for 24 hours after surgery. Factor activity was maintained at ≥80% until postoperative day 14 and at ≥50% thereafter. The average duration of surgery was 178 minutes (range, 133–233 minutes), the estimated blood loss was 661 mL (range, 272–1,344 mL), and a drain tube was left subfascially in place for 2 days in all patients. Reoperation due to postoperative surgical site infection was required in one patient, but there were no complications due to hemorrhagic diathesis. The total dose of coagulation factor administered during hospitalization was 102 ×103 U (range, 46–198 ×103 U). Conclusions Coordination with a hematologist and dose adjustment of the coagulation factor preparation to maintain a target level of coagulation factor activity facilitated a smooth postoperative course with perioperative control of bleeding during spinal surgery for patients with hemophilia.

Abstract Background Thyroid metastasis of soft tissue sarcoma is very rare, and the diagnosis is especially difficult when only a single lesion is present. Case presentation A 50-year-old man was diagnosed with myxoid liposarcoma of the right thigh and treated with wide resection. Two and a half years after the surgery, a growing low-density area was incidentally observed in the right lobe of his thyroid gland on follow-up chest computed tomography. Fine needle aspiration biopsy was performed twice, and the thyroid mass was suspected of being a sarcoma metastasis. He was treated by hemithyroidectomy, and the lesion was pathologically confirmed as a metastasis of myxoid liposarcoma. Conclusion We experienced single thyroid gland metastasis in patients with myxoid liposarcoma in whom a growing mass is observed in the thyroid gland after radical surgery of the primary site.

Introduction: An occipital-cervical surgery for children is challenging for surgeons because of the immature bone quality, extensive anatomical variability, and small osseous structures. Furthermore, occipital-C2 fusion in children results in great stress on the C2 screws. We report a technique that uses both C2 pedicle and bilateral lateral mass screws (C2 hybrid screws) in children with an upper cervical disorder to preserve motion segment and secure strength in those who require occipital-cervical fusion. Case Report: Case 1 was that of a 5-year-old girl with Down syndrome who had atlantoaxial dislocation and os odontoideum. Owing to the C1 hypoplasia, the posterior arch was fractured by the C1 lateral mass screw. Therefore, O-C2 fusion was performed. C2 bilateral lamina screws were added along with the C2 bilateral pedicle screws for reinforcement. Case 2 was that of an 8-year-old boy who presented with torticollis and neck pain. The patient was diagnosed as having atlantoaxial rotatory fixation. The right vertebral artery was obstructed, and the left vertebral artery was dominant. The C1 posterior arch was bifid and assimilated with the occipital bone. C2 bilateral lamina screws were added with the right C2 pedicle screw for reinforcement. Both cases attained bone union after O-C2 fusion surgery using hybrid screws. Conclusions: The use of C2 hybrid screws with both C2 pedicle and bilateral lateral mass screws can preserve mobile segments in the fusion area in young children who require occipital-cervical fixation.

Abstract Background We aimed to determine whether synovial fluid (SF) biomarkers can predict the progression of articular cartilage damage as determined by arthroscopic evaluation during and after anterior cruciate ligament (ACL) reconstruction. Methods Arthroscopic assessment of articular cartilage damage was performed twice in 62 patients, first during ACL reconstruction and then approximately 2 years later during implant removal for ligament fixation. SF levels of the collagenase-generated cleavage neoepitope of type II collagen (C2C) and proteoglycan glycosaminoglycans keratan sulfate (KS), chondroitin-4-sulfate (Δdi-C4S), and chondroitin-6-sulfate (Δdi-C6S) were measured at ACL reconstruction. Associations between baseline biomarker levels and subsequent progression of cartilage damage were determined using receiver operating characteristic analysis and multivariable logistic regression analysis. Results No radiographic changes were observed in any of the patients. Progression of high-grade cartilage damage, observed arthroscopically, was negatively correlated with levels of Δdi-C6S and KS, as well as the ratio of Δdi-C6S to Δdi-C4S (C6S/C4S). Logistic regression analysis revealed significant associations of Δdi-C6S (cut-off: 55.7 nmol/ml, odds ratio (OR) 0.231, 95% confidence interval (CI) 0.061–0.879), KS (cut-off: 10.6 μg/ml, OR 0.114, 95% CI 0.024–0.529), and C6S/C4S ratio (cut-off: 4.6, OR 0.060, 95% CI 0.005–0.737) with the progression of high-grade cartilage damage after adjusting for age, the duration from injury to first surgery, sex, and the number of high-grade lesions (grades III and IV) at baseline. Conclusions The progression of high-grade cartilage damage was significantly associated with baseline levels of proteoglycan glycosaminoglycan biomarkers; namely, Δdi-C6S, KS, and C6S/C4S ratio.

Abstract The catabolism of hyaluronan in articular cartilage remains unclear. The aims of this study were to investigate the effects of hyaluronidase 2 (Hyal2) knockdown in articular cartilage on the development of osteoarthritis (OA) using genetic manipulated mice. Destabilization of the medial meniscus (DMM) model of Col2a promoter specific conditional Hyal2 knockout (Hyal −/− ) mice was established and examined. Age related and DMM induced alterations of articular cartilage of knee joint were evaluated with modified Mankin score and immunohistochemical staining of MMP-13, ADAMTS-5, KIAA11199, and biotinylated- hyaluronan binding protein staining in addition to histomorphometrical analyses. Effects of Hyal2 suppression were also analyzed using explant culture of an IL-1α induced articular cartilage degradation model. The amount and size of hyaluronan in articular cartilage were higher in Hyal2 −/− mice. Hyal2 −/− mice exhibited aggravated cartilage degradation in age-related and DMM induced mice. MMP-13 and ADAMTS-5 positive chondrocytes were significantly higher in Hyal2 −/− mice. Articular cartilage was more degraded in explant cultures obtained from Hyal2 −/− mice. Knockdown of Hyal2 in articular cartilage induced OA development and progression possibly mediated by an imbalance of HA metabolism. This suggests that Hyal2 knockdown exhibits mucopolysaccharidosis-like OA change in articular cartilage similar to Hyal1 knockdown.

Abstract Achondroplasia (ACH) is the most common short-limbed skeletal dysplasia caused by gain-of-function mutations in the fibroblast growth factor receptor 3 (FGFR3). No effective FGFR3-targeted therapies for ACH are currently available. By drug repositioning strategies, we identified that meclozine, which has been used as an anti-motion-sickness, suppressed FGFR3 signaling in chondrocytes and rescued short-limbed phenotype in ACH mouse model. Here, we conducted various pharmacological tests for future clinical application in ACH. Pharmacokinetic analyses demonstrated that peak drug concentration (Cmax) and area under the concentration-time curve (AUC) of 2 mg/kg of meclozine to mice was lower than that of 25 mg/body to human, which is a clinical usage for anti-motion-sickness. Pharmacokinetic simulation studies showed that repeated dose of 2 mg/kg of meclozine showed no accumulation effects. Short stature phenotype in the transgenic mice was significantly rescued by twice-daily oral administration of 2 mg/kg/day of meclozine. In addition to stimulation of longitudinal bone growth, bone volume and metaphyseal trabecular bone quality were improved by meclozine treatment. We confirmed a preclinical proof of concept for applying meclozine for the treatment of short stature in ACH, although toxicity and adverse events associated with long-term administration of this drug should be examined.

Abstract Background Immunohistochemical staining with conventional anti-β-catenin antibody has been applied as a diagnostic tool for desmoid-type fibromatosis (DF). This study aimed to evaluate the diagnostic and prognostic value of immunohistochemical staining with anti-non-phospho β-catenin antibody, which might more accurately reflect the aggressiveness of DF, in comparison to the conventional anti-β-catenin antibody. Methods Between 2003 and 2015, 40 patients with extra-peritoneal sporadic DF were prospectively treated with meloxicam or celecoxib, a COX-2 inhibitor, therapy. The efficacy of this treatment was evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST). Immunohistochemical staining was performed on formalin-fixed material to evaluate the expression of β-catenin and non-phospho β-catenin, and the positivity was grouped as negative, weak, moderate, and strong. DNA was isolated from frozen tissue or formalin-fixed materials, and the CTNNB1 mutation status was determined by direct sequencing. Results Of the 40 patients receiving COX-2 inhibitor treatment, there was one with complete remission, 12 with partial remission, 7 with stable disease, and 20 with progressive disease. The mutation sites in CTNNB1 were detected in 22 (55%) of the 40 cases: T41A (17 cases), S45F (3 cases), and T41I and S45P (1 each). The positive nuclear expression of non-phospho β-catenin showed a significant correlation with positive CTNNB1 mutation status detected by Sanger method (p = 0.025), and poor outcome in COX-2 inhibitor therapy (p = 0.022). In contrast, nuclear expression of β-catenin did not show a significant correlation with either CTNNB1 mutation status (p = 0.43) or outcome of COX-2 inhibitor therapy (p = 0.38). Conclusions Nuclear expression of non-phospho β-catenin might more appropriately reflect the biological behavior of DF, and immunohistochemical staining with non-phospho β-catenin could serve as a more useful diagnostic and prognostic tool of COX-2 inhibitor therapy for patients with DF.

We managed perioperative hemostasis for a 72-year-old man with hemophilia A and low inhibitor titers (3 BU/mL), who underwent osteosynthesis for supracondylar fracture of the left humerus. He was treated perioperatively using the combination of high doses of factor VIII (FVIII) with recombinant human Factor VIII Fc fusion protein (rFVIIIFc), followed by emicizumab. On the day of surgery (day 0), he was administered bolus infusion of 150 IU/kg rFVIIIFc, followed by continuous infusion at a dose of 4 IU/kg/h. Emicizumab, 3 mg/kg, was injected subcutaneously once a week, on days 5, 12, 19, and 26. Inhibitors were detected on day 6 at a titer of 4 BU/mL and FVIII:C decreased to below assay sensitivity limits on day 10. The rate of increase in inhibitor titers was high, with inhibitors increasing to 343.4 BU/mL on day 14. The transition of thrombin production by thrombin generation assay (TGA) showed temporary decrease in thrombin production on day 7, although it was restored by day 10, i.e., five days after commencement of emicizumab therapy. Rotational thromboelastometry displayed consistent results with TGA, showing that clotting time was prolonged and the alpha angle decreased to less than measurable levels on day 6, although they were improved by day 10. There were no bleeding-related events or other adverse events throughout the perioperative period. In conclusion, emicizumab was effective for the management of perioperative hemostasis after development of an anamnestic response in a patient with hemophilia A with inhibitors. Combination therapy with high doses of FVIII followed by emicizumab could be a workable alternative for patients with hemophilia A with inhibitors.

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan essential for the homeostasis of cartilage-related tissues. Intracellular adhesion molecule-1 (ICAM-1) and CD44 have been identified as receptors for HA. Recently, transient receptor potential vanilloid 4 (TRPV4) has emerged as a potential research target in several areas of physiology. TRPV4 is a Ca2+-permeable, non-selective cation channel that appears to have mechanosensory or osmosensory roles in several musculoskeletal tissues. HA and TRPV4 play key roles in chondrogenesis; however, it has remained unclear whether they have interactive effects on chondrogenesis and, if so, how do they interact with each other? This study investigated the relationship between HA, its receptors ICAM-1 and CD44, and TRPV4 in the chondrogenic pathway using the ATDC5 cell line. It was found that the presence of HA is required for TRPV4-induced chondrogenesis. Loss of HA suppressed TRPV4-induced expression of the chondrogenic markers, SOX9 and Aggrecan. Moreover, HA affects TRPV4-induced chondrogenic development via each of ICAM-1 and CD44 partially. In conclusion, for the first time, the existence of an interaction between HA, its receptor ICAM-1 and CD44, and TRPV4-activity in chondrogenesis in the ATDC5 cell line was reported. TRPV4 is known to function as a mechanosensory channel in several musculoskeletal tissues. Therefore, findings of this study may suggest the existence of a molecular mechanism that underlies the interactive effects of HA and mechanical loading on joint chondrogenesis.

AimArteriosclerotic disease is increasing due to aging of the population, and is associated with diabetes, hypertension, hyperlipidemia, obesity, and smoking. This disease may result in fatal cerebrovascular disease, and especially cardiogenic cerebral embolism caused by artery plaque-based atherothrombotic cerebral infarction. The study was performed to examine the relationship of abdominal aortic calcification (AAC) on lumbar radiographs with carotid intima-media complex thickness (IMT), factors associated with carotid artery plaque, and cutoff values in middle-aged and elderly people.Patients and methodsThe subjects were 309 healthy volunteers (average age 63 years) who attended a health checkup supported by a local government in 2015. The AAC-24 score was determined on lumbar lateral standing radiographs and was categorized as 0 (54% of subjects),1-4 (31%), and ≥5 (severe, 15%). Carotid ultrasonography was used to evaluate IMT of the common carotid artery. Carotid artery plaque was defined as IMT >1.1 mm. Body mass index (BMI), hypertension, diabetes mellitus (DM), dyslipidemia, smoking, alcohol intake, and osteoporosis were examined.ResultsOf 309 cases, 142 (46%) had AAC and 104 (34%) had carotid artery plaque. Thus, 15% (n = 45) had severe AAC. Age, prevalence of DM and carotid artery plaque increased with severity of AAC. In patients with carotid artery plaque (n = 104), age (67.8±7.5 vs. 61.0±10.1 years), % male (56% vs. 39%), BMI (22.9±2.8 vs. 23.7±3.5), AAC rate (58% vs. 40%) and AAC-24 score (3 (0, 8) vs. 0 (0, 2)) were all significantly higher than in those (n = 205) without carotid artery plaque. In multivariate analysis, age (OR 1.172), male gender (OR 1.654), AAC (OR 1.352), and AAC-24 ≥5 (OR 4.191) were significantly associated with carotid artery plaque. Combining AAC-24 with age significantly increased the AUC from 0.632 to 0.834 (pConclusionThere was a significant relationship between AAC on lumbar radiographs and carotid IMT.

Study DesignRetrospective study.PurposeTo identify the prevalence of severe headache occurring after cervical posterior surgical fixation (PSF) and to evaluate the clinical and radiological findings associated with severe headache after surgery.Overview of LiteratureSeveral studies have reported on the axial pain after cervical surgery. However, to our knowledge, the incidence of severe headache after cervical PSF has not been elucidated.MethodsThe medical records and radiological assessment of patients who underwent surgical treatment from August 2002 to May 2012 were reviewed to identify the prevalence and risk factors for severe headaches occurring following PSF from C2 distally. Neck disability index scores (NDI) (the item for neck pain), the type of C2 screw, number of cervical fused levels (1–6), and smoking habit were calculated preoperatively and postoperatively. In addition, radiological parameters (T1 slope angle, C1/2 angle, C2–7 Cobb angle, C2–7 sagittal vertical axis and C1-implant distance) were assessed for all patients. Severe headache was defined as a high NDI headache score (>4 out of 5).ResultsEighty-two patients met the inclusion criteria. The mean age was 59.2 years (range, 21–78 years), and the mean number of fused levels was 5.1. The mean follow-up period was 2.9 years (range, 1–10.9 years). While only one severe headache occurred de novo postoperatively in a patient in the C3 or C4 distally group (total 30 patients, average age of 50.2 years), 11 patients in the C2 distally group (p=0.04) had severe headache occur postoperatively. The radiological parameters were not significantly different between the postoperative milder headache and severe headache (SH) groups. The SH group had a significantly higher preoperative NDI score (neck pain) (p

We describe successful vertebrectomy from a posterior approach using a computed tomography (CT)-based navigation system (O-arm) in a 53-year-old man with adenocarcinoma of the posterior apex of the right lung with invasion of the adjacent rib, thoracic wall, and T2 and T3 vertebral bodies. En bloc partial vertebrectomy for lung cancer adjacent to the thoracic spine was planned using O-arm. First, laminectomy was performed from right T2 to T3, and pedicles and transverse processes of T2 to T3 were resected. O-arm was used to confirm the location of the cutting edge in the T2 to 3 right vertebral internal body, and osteotomy to the anterior cortex was performed with a chisel. Next, the patient was placed in a left decubitus position. The surgical specimen was extracted en bloc. This case shows that O-arm can be used reliably and easily in vertebrectomy from a posterior approach and can facilitate en bloc resection.

Study DesignAnimal study.PurposeTo review the present warning point criteria of the compound muscle action potential (CMAP) and investigate new criteria for spinal surgery safety using an animal model.Overview of LiteratureLittle is known about correlation palesis and amplitude of spinal cord monitoring.MethodsAfter laminectomy of the tenth thoracic spinal lamina, 2-140 g force was delivered to the spinal cord with a tension gage to create a bilateral contusion injury. The study morphology change of the CMAP wave and locomotor scale were evaluated for one month.ResultsFour different types of wave morphology changes were observed: no change, amplitude decrease only, morphology change only, and amplitude and morphology change. Amplitude and morphology changed simultaneously and significantly as the injury force increased (p

Study DesignProspective comparative study.PurposeTo compare the incidence and severity of adverse reactions associated with myelography performed in outpatients vs. in inpatients and report the safety and usefulness of outpatient myelography in Japanese patients.Overview of LiteratureMyelography is normally performed as an inpatient procedure in most hospitals in Japan. No studies have reported the usefulness and adverse effects of outpatient myelography in Japanese patients.MethodsWe performed 221 myelography procedures. Eighty-five of the 221 patients underwent outpatient myelography using our new protocol. The incidence and severity of adverse reactions were compared with the other 136 patients, who underwent conventional inpatient myelography. We further compared the cost of outpatient and inpatient myelography.ResultsThe overall rate of adverse effects was 9.4% in outpatients, as compared with 7.4% in inpatients. Overall, 1.2% of outpatients and 0.74% inpatients experienced "severe" adverse effects (requiring hospitalization). There were no significant differences between the 2 groups in either the overall rate of adverse effects or the rate of "severe" adverse effects. Moreover, the average outpatient procedure cost was only one-third to one-half that of the inpatient procedure.ConclusionsThis was the first study to address the safety and usefulness of outpatient myelography in Japanese patients. If selected according to proper inclusion criteria for outpatient procedure, no significant differences were observed in the adverse effects between inpatients and outpatients. The outpatient procedure is more economical and has the added benefit of being more convenient and time-efficient for the patient.

The transcription factor, runt-related transcription factor 2 (Runx2), plays a pivotal role in the differentiation of the mesenchymal stem cells to the osteochondroblast lineages. We found by the drug repositioning strategy that a proton pump inhibitor, lansoprazole, enhances nuclear accumulation of Runx2 and induces osteoblastogenesis of human mesenchymal stromal cells. Systemic administration of lansoprazole to a rat femoral fracture model increased osteoblastogenesis. Dissection of signaling pathways revealed that lansoprazole activates a noncanonical bone morphogenic protein (BMP)-transforming growth factor-beta (TGF-β) activated kinase-1 (TAK1)–p38 mitogen-activated protein kinase (MAPK) pathway. We found by in cellulo ubiquitination studies that lansoprazole enhances polyubiquitination of the TNF receptor-associated factor 6 (TRAF6) and by in vitro ubiquitination studies that the enhanced polyubiquitination of TRAF6 is attributed to the blocking of a deubiquitination enzyme, cylindromatosis (CYLD). Structural modeling and site-directed mutagenesis of CYLD demonstrated that lansoprazole tightly fits in a pocket of CYLD where the C-terminal tail of ubiquitin lies. Lansoprazole is a potential therapeutic agent for enhancing osteoblastic differentiation.

Purpose: Oxidative stress is closely associated with the pathogenesis of nontraumatic osteonecrosis of the femoral head (ONFH). This study aimed to determine whether the serum levels of antioxidant nutrients were decreased in patients with ONFH. Methods: We analyzed the serum levels of antioxidant nutrients in 39 patients with ONFH (ONFH group) and 78 age- and gender-matched healthy people (control group) who voluntarily participated in the Yakumo study, which is a comprehensive health examination program. We measured and compared the serum levels of α-tocopherol (vitamin E) and total carotenoids, including zeaxanthin/lutein, β-cryptoxanthin, lycopene, α-carotene, and β-carotene, in the ONFH and control groups using high-performance liquid chromatography. Results: The mean serum levels of total carotenoids were significantly lower in the ONFH group than in the control group (2.36 ± 1.26 and 3.79 ± 2.36 µmol/l, respectively, p < 0.001). However, no significant difference was found in α-tocopherol between the two groups (26.37 ± 6.90 µmol/l in the ONFH group and 26.24 ± 6.28 µmol/l in the control group, p = 0.920). Among each carotenoid, the serum levels of zeaxanthin/lutein, lycopene, and β-carotene were significantly lower in the ONFH group than in the control group ( p < 0.001). Conclusions: The serum levels of carotenoids were lower in patients with ONFH than in healthy, community-living people. This result suggests that carotenoids may be related to the pathogenesis of ONFH.

Localized scleroderma, also known as morphea, is a rare condition characterized by progressive sclerosis of the skin and associated atrophy of the underlying tissues. The linear type of localized scleroderma is the most frequent form in childhood, usually affecting unilateral extremities. Fibrosclerosis of the fasciae and muscles can spread across joints and impair the range of motion of the joint. Dysplastic and/or atrophic bones of the affected lower extremity can lead to clinically significant leg length discrepancy (LLD). Limb reconstruction surgery has rarely been indicated for LLD in patients with linear morphea. We report on a case of extensive bone lengthening for appreciable LLD in a pediatric patient with linear morphea. A Japanese girl with linear morphea underwent staged simultaneous lengthening of the femur and tibia twice at seven and eleven years of age using a unilateral external fixator. A healing index exceeded 100 days/cm except for the first femoral lengthening that was complicated by regenerate fracture. At the final follow-up, LLD of 38 mm remained, but she could walk independently without a brace or a crutch. Due to soft tissue tightness and poor regenerative ability in the affected limb, cautions should be taken to prevent regenerate fracture and/or malalignment of the limb.

Spinal fusion and bone defect after injuries, removal of bone tumors, and infections need to be repaired by implantation. In an aging society, recovery from these procedures is often difficult. In this study, we found that injection of SPG-178 leads to expression of several bone marker genes and mineralization in vitro, and revealed a significantly higher degree of newly formed bone matrix with use of SPG-178 in vivo. MC3T3-E1 cells were used to evaluate osteoblast differentiation promoted by SPG-178. To analyze gene expression, total RNA was isolated from MC3T3-E1 cells cultured for 7 and 14 days with control medium or SPG-178 medium. Among the several bone marker genes examined, SPG-178 significantly increased the mRNA levels for ALP, BMP-2 and Osteocalcin, OPN, BSP and for the Osterix. Ten-week-old female Wistar rats were used for all transplantation procedures. A PEEK cage was implanted into a bony defect (5 mm) within the left femoral mid-shaft, and stability was maintained by an external fixator. The PEEK cages were filled with either a SPG-178 hydrogel plus allogeneic bone chips (n = 4) or only allogeneic bone chips (n = 4). The rats were then kept for 56 days. Newly formed bone matrix was revealed inside the PEEK cage and there was an increased bone volume per total volume with the cage filled with SPG-178, compared to the control group. SPG-178 has potential in clinical applications because it has several benefits. These include its favorable bone conduction properties its ability to act as a support for various different cells and growth factors, its lack of infection risk compared with materials of animal origin such as ECM, and the ease with which it can be used to fill defects with complex shapes and combined with a wide range of other materials.

Struma ovarii is a rare tumor that is defined as an ovarian teratoma with a thyroid tissue component exceeding 50%. Most of these tumors are benign, with malignant struma ovarii occurring in

Thoracic ossification of the ligamentum flavum (T-OLF) is a relatively rare spinal disorder that generally requires surgical intervention, due to its progressive nature and the poor response to conservative therapy. The prevalence of OLF has been reported at 3.8%-26%, which is similar to that of cervical ossification of the posterior longitudinal ligament (OPLL). The progression of OPLL after cervical laminoplasty for the treatment of OPLL is often shown in long-term follow-up. However, there have been no reports on the progression of OLF following surgery. We report a case of thoracic myelopathy secondary to the progressive relapse of OLF following laminectomy.

After tendon injuries, biomechanical properties of the injured tendon are not fully recovered in most cases. Modulation of signaling pathways, which are involved in tendon development and tendon repair, is one of attractive modalities to facilitate proper regeneration of the injured tendon. The roles of TGF-β signaling in tendon homeostasis and tendon development have been elucidated. In contrast, the roles of Wnt/β-catenin signaling in tendon remain mostly elusive. We found that the number of β-catenin-positive cells was increased at the injured site, suggesting involvement of Wnt/β-catenin signaling in tendon healing. Activation of Wnt/β-catenin signaling suppressed expressions of tenogenic genes of Scx, Mkx, and Tnmd in rat tendon-derived cells (TDCs) isolated from the Achilles tendons of 6-week old rats. Additionally, activation of Wnt/β-catenin reduced the amounts of Smad2 and Smad3, which are intracellular mediators for TGF-β signaling, and antagonized upregulation of Scx induced by TGF-β signaling in TDCs. We found that Wnt/β-catenin decreased Mkx and Tnmd expressions without suppressing Scx expression in Scx-programmed tendon progenitors. Our studies suggest that Wnt/β-catenin signaling is a repressor for tenogenic gene expressions.

A recent study reported that heat stress stimulates osteogenesis in an in vivo rat model using alginate gel and magnetite cationic liposomes. However, for clinical use, the efficacy for promoting osteogenesis needs to be investigated using clinically approved materials, and preferably with animals larger than rats. The aim of this study was to evaluate multiple heat stimuli-triggered osteogenesis in rat tibial defect models using already clinically applicable materials (Resovist® and REGENOS®) and determine the efficacy also in the rabbit. Fifty-eight rats and 10 rabbits were divided into two groups, respectively, with or without hyperthermia treatment at 45°C for 15 min. (hyperthermia; 20 rats once a week, 8 rats three times a week, 5 rabbits once a week, control; 30 rats and 5 rabbits). Micro-CT assessment at 4 weeks revealed that a significantly stimulated osteogenesis was observed in the once a week group of both rats and rabbits as compared to the control group (p = 0.018 and 0.036, respectively). In contrast, the three times a week group did not show enhanced osteogenesis. Histological examination and image analysis showed consistent results in which the area of mineralized bone formation in the once a week hyperthermia group was significantly increased compared with that in the control group at four weeks (rat; p = 0.026, rabbit; p = 0.031). Newly formed bone was observed in the grafted materials from the periphery toward the center, and more osteoclasts were found in the once a week group. Heat stress also induced enhanced alkaline phosphatase expression in cultured osteoblastic cells, MC3T3, in vitro (p = 0.03). On the other hand, heat stress had no obvious effects on chondrogenic differentiation using ATDC5 cells. Our study demonstrates that heat-stimuli with clinically applicable novel heating materials can promote significant osteogenesis, and may thus be a promising treatment option for diseases associated with bone defects.

Abnormal activation of the Wnt/β-catenin signaling is implicated in the osteoarthritis (OA) pathology. We searched for a pre-approved drug that suppresses abnormally activated Wnt/β-catenin signaling and has a potency to reduce joint pathology in OA. We introduced the TOPFlash reporter plasmid into HCS-2/8 human chondrosarcoma cells to estimate the Wnt/β-catenin activity in the presence of 10 μM each compound in a panel of pre-approved drugs. We found that fluoxetine, an antidepressant in the class of selective serotonin reuptake inhibitors (SSRI), down-regulated Wnt/β-catenin signaling in human chondrosarcoma cells. Fluoxetine inhibited both Wnt3A- and LiCl-induced loss of proteoglycans in chondrogenically differentiated ATDC5 cells. Fluoxetine increased expression of Sox9 (the chondrogenic master regulator), and decreased expressions of Axin2 (a marker for Wnt/β-catenin signaling) and Mmp13 (matrix metalloproteinase 13). Fluoxetine suppressed a LiCl-induced increase of total β-catenin and a LiCl-induced decrease of phosphorylated β-catenin in a dose-dependent manner. An in vitro protein-binding assay showed that fluoxetine enhanced binding of β-catenin with Axin1, which is a scaffold protein forming the degradation complex for β-catenin. Fluoxetine suppressed LiCl-induced β-catenin accumulation in human OA chondrocytes. Intraarticular injection of fluoxetine in a rat OA model ameliorated OA progression and suppressed β-catenin accumulation.

To explore the effect of hyaluronan oligosaccharides (HAoligos) on interactions between HA and its principal receptor, CD44, in rheumatoid synovial fibroblasts (RSFs) and matrix metalloproteinase (MMP) production.RSFs were isolated from rheumatoid synovial tissue. HA distribution was visualized by immunocytochemistry. MMP-1 and MMP-3 induction was analyzed by real-time RT-PCR and immunoblotting. The interaction between HAoligos and their MMP-producing receptors was tested by blocking with anti-CD44 and anti-Toll-like receptor 4 (TLR-4). Phosphorylation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) was analyzed by immunoblotting.Endogenous HA decreased after treatment with HAoligos, while MMP-1 and MMP-3 expression increased in a dose-dependent manner. Pretreatment with anti-CD44 or anti-TLR-4 antibody significantly reduced the effect of HAoligos on MMP-1 and MMP-3 mRNA expression. NF-κB and p38 MAPK phosphorylation was enhanced by HAoligos pretreated with anti-TLR-4, and HAoligo-induced MMP production was blocked with an inhibitor of NF-κB and p38 MAPK pathways.Disruptive changes in CD44-HA interactions by HAoligos enhanced MMP-1 and MMP-3 production via activation of NF-κB and p38 MAPK signaling pathways in RSFs.

The Toll-like receptor (TLR) signaling pathway is activated in synovial fibroblast cells in patients with rheumatoid arthritis (RA). The receptor activator of nuclear factor-κB (RANK) and its ligand, RANKL, are key molecules involved in the differentiation of osteoclasts and joint destruction in RA. Hyaluronan (HA) is a major extracellular component and an important immune regulator. In this study, we show that lipopolysaccharide (LPS) stimulation significantly increases RANKL expression via a TLR-4 signaling pathway. We also demonstrate that HA suppresses LPS-induced RANKL expression, which is dependent on CD44, but not intercellular adhesion molecule-1 (ICAM-1). Our study provides evidence for HA-mediated suppression of TLR-4-dependent RANKL expression. This could present an alternative target for the treatment of destructed joint bones and cartilages in RA.

No clinically applicable drug is currently available to enhance neurite elongation after nerve injury. To identify a clinically applicable drug, we screened pre-approved drugs for neurite elongation in the motor neuron-like NSC34 cells. We found that zonisamide, an anti-epileptic and anti-Parkinson's disease drug, promoted neurite elongation in cultured primary motor neurons and NSC34 cells in a concentration-dependent manner. The neurite-scratch assay revealed that zonisamide enhanced neurite regeneration. Zonisamide was also protective against oxidative stress-induced cell death of primary motor neurons. Zonisamide induced mRNA expression of nerve growth factors (BDNF, NGF, and neurotrophin-4/5), and their receptors (tropomyosin receptor kinase A and B). In a mouse model of sciatic nerve autograft, intragastric administration of zonisamide for 1 week increased the size of axons distal to the transected site 3.9-fold. Zonisamide also improved the sciatic function index, a marker for motor function of hindlimbs after sciatic nerve autograft, from 6 weeks after surgery. At 8 weeks after surgery, zonisamide was protective against denervation-induced muscle degeneration in tibialis anterior, and increased gene expression of Chrne, Colq, and Rapsn, which are specifically expressed at the neuromuscular junction. We propose that zonisamide is a potential therapeutic agent for peripheral nerve injuries as well as for neuropathies due to other etiologies.

We hypothesized that patterns of CTNNB1 (β-catenin) mutations would affect the outcome of conservative therapy in patients with desmoid tumors. This study aimed to determine the significance of CTNNB1 (β-catenin) mutations in predicting the treatment outcome in patients with desmoid tumors treated with meloxicam, a cyclooxygenase-2 (COX-2) selective inhibitor. Between 2003 and 2012, consecutive thirty-three patients with extra-peritoneal sporadic desmoid tumors were prospectively treated with meloxicam as the initial systemic medical therapy. The efficacy of meloxicam was evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST). DNA was isolated from frozen tissue or formalin-fixed materials. CTNNB1 mutation analysis was performed by direct sequencing. Positivity of nuclear β-catenin staining by immunohistochemistry was compared with the status of CTNNB1 mutations. The correlation between the efficacy of meloxicam treatment and status of CTNNB1 mutations was analyzed. Of the 33 patients with meloxicam treatment, one showed complete remission (CR), 7 partial remission (PR), 12 stable disease (SD), and 13 progressive disease (PD). The following 3 point mutations were identified in 21 of the 33 cases (64%): T41A (16 cases), S45F (4 cases) and S45P (one case). The nuclear expression of β-catenin correlated significantly with CTNNB1 mutation status (p = 0.035); all four cases with S45F mutation exhibited strong nuclear expression of β-catenin. S45F mutation was significantly associated with a poor response (all cases; PD) (p = 0.017), whereas the other mutations had no impact on efficacy. The CTNNB1 mutation status was of significant prognostic value for meloxicam treatment in patients with sporadic desmoid tumors.

In past years, the canonical Wnt/β-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. FRZB, a soluble antagonist of Wnt signaling, has been studied in osteoarthritis (OA) animal models and OA patients as a modulator of Wnt signaling. We screened for FDA-approved drugs that induce FRZB expression and suppress Wnt/β-catenin signaling. We found that verapamil, a widely prescribed L-type calcium channel blocker, elevated FRZB expression and suppressed Wnt/β-catenin signaling in human OA chondrocytes. Expression and nuclear translocation of β-catenin was attenuated by verapamil in OA chondrocytes. Lack of the verapamil effects in LiCl-treated and FRZB-downregulated OA chondrocytes also suggested that verpamil suppressed Wnt signaling by inducing FRZB. Verapamil enhanced gene expressions of chondrogenic markers of ACAN encoding aggrecan, COL2A1 encoding collagen type II α1, and SOX9, and suppressed Wnt-responsive AXIN2 and MMP3 in human OA chondrocytes. Verapamil ameliorated Wnt3A-induced proteoglycan loss in chondrogenically differentiated ATDC5 cells. Verapamil inhibited hypertrophic differentiation of chondrocytes in the explant culture of mouse tibiae. Intraarticular injection of verapamil inhibited OA progression as well as nuclear localizations of β-catenin in a rat OA model. We propose that verapamil holds promise as a potent therapeutic agent for OA by upregulating FRZB and subsequently downregulating Wnt/β-catenin signaling.

Biopolymers consist of three major classes, i.e., polynucleotides (DNA, RNA), polypeptides (proteins) and polysaccharides (sugar chains). It is widely accepted that polynucleotides and polypeptides play fundamental roles in the pathogenesis of neurodegenerative diseases. But, sugar chains have been poorly studied in this process, and their biological/clinical significance remains largely unexplored. Amyotrophic lateral sclerosis (ALS) is a motoneuron-degenerative disease, the pathogenesis of which requires both cell autonomous and non-cell autonomous processes. Here, we investigated the role of keratan sulfate (KS), a sulfated long sugar chain of proteoglycan, in ALS pathogenesis. We employed ALS model SOD1(G93A) mice and GlcNAc6ST-1(-/-) mice, which are KS-deficient in the central nervous system. Unexpectedly, SOD1(G93A)GlcNAc6ST-1(-/-) mice exhibited a significantly shorter lifespan than SOD1(G93A) mice and an accelerated appearance of clinical symptoms (body weight loss and decreased rotarod performance). KS expression was induced exclusively in a subpopulation of microglia in SOD1(G93A) mice, and became detectable around motoneurons in the ventral horn during the early disease phase before body weight loss. During this phase, the expression of M2 microglia markers was transiently enhanced in SOD1(G93A) mice, while this enhancement was attenuated in SOD1(G93A)GlcNAc6ST-1(-/-) mice. Consistent with this, M2 microglia were markedly less during the early disease phase in SOD1(G93A)GlcNAc6ST-1(-/-) mice. Moreover, KS expression in microglia was also detected in some human ALS cases. This study suggests that KS plays an indispensable, suppressive role in the early phase pathogenesis of ALS and may represent a new target for therapeutic intervention.