Royal jelly does not prevent bone loss but improves bone strength in ovariectomized rats.

OBJECTIVE: Royal jelly (RJ) has been used for medical and nutritional purposes, and previous studies have indicated that it may have estrogenic activity. The present study investigated the effects of RJ on bone metabolism in ovariectomized (OVX) rats. METHODS: Rats (12 weeks old) were randomly divided into four groups, namely Baseline, Sham, OVX, and OVX + RJ groups. Rats in the Baseline group were killed immediately, whereas rats in the OVX and OVX + RJ groups underwent bilateral ovariectomy and those in the Sham group underwent sham operation. RJ was administered to rats in the OVX + RJ group daily for 12 weeks. At the end of the 12-week period, bone mass, bone histomorphometry, and bone mechanics were analyzed. RESULTS: Femur bone mineral density (BMD) was significantly lower in the OVX group than in the Sham group, and this decrease in BMD was not ameliorated by RJ administration. However, femur stiffness, as evaluated by a three-point bending test, was significantly higher in the OVX + RJ group than in the OVX group. CONCLUSION: The findings of the present study suggest that RJ does not prevent bone loss, but does improve bone strength in OVX rats.

Developmental and adult-specific processes contribute to de novo neuromuscular regeneration in the lizard tail.

Peripheral nerves exhibit robust regenerative capabilities in response to selective injury among amniotes, but the regeneration of entire muscle groups following volumetric muscle loss is limited in birds and mammals. In contrast, lizards possess the remarkable ability to regenerate extensive de novo muscle after tail loss. However, the mechanisms underlying reformation of the entire neuromuscular system in the regenerating lizard tail are not completely understood. We have tested whether the regeneration of the peripheral nerve and neuromuscular junctions (NMJs) recapitulate processes observed during normal neuromuscular development in the green anole, Anolis carolinensis. Our data confirm robust axonal outgrowth during early stages of tail regeneration and subsequent NMJ formation within weeks of autotomy. Interestingly, NMJs are overproduced as evidenced by a persistent increase in NMJ density 120 and 250 days post autotomy (DPA). Substantial Myelin Basic Protein (MBP) expression could also be detected along regenerating nerves indicating that the ability of Schwann cells to myelinate newly formed axons remained intact. Overall, our data suggest that the mechanism of de novo nerve and NMJ reformation parallel, in part, those observed during neuromuscular development. However, the prolonged increase in NMJ number and aberrant muscle differentiation hint at processes specific to the adult response. An examination of the coordinated exchange between peripheral nerves, Schwann cells, and newly synthesized muscle of the regenerating neuromuscular system may assist in the identification of candidate molecules that promote neuromuscular recovery in organisms incapable of a robust regenerative response.

Improvement of neurological disorders in postmenopausal model rats by administration of royal jelly.

OBJECTIVE: Royal jelly (RJ) from honeybees (Apis mellifera) has estrogenic activity. Estrogen deficiency after menopause leads to a high risk of memory impairment and depression as well as metabolic syndrome and osteoporosis. We here investigated the effect of RJ on memory impairment and depression-like behaviors in ovariectomized (OVX) rats. METHODS: OVX rats were administered with RJ for 82 days. Hippocampus-dependent spatial memory and depression-like behaviors were assessed by the Morris water maze test and the forced swimming test, respectively. The weights of body, brain and uterus and the contents of protein and myelin galactolipids including galactosylceramide and sulfatide were measured. RESULTS: Memory impairment and depression-like behaviors in OVX rats were recovered to the levels of sham-operated rats by RJ administration. Increased body weight and decreased uterine weight in OVX rats were recovered to the levels of sham-operated rats by 17ß-estradiol (E2) administration but not by RJ administration. In contrast, brain weight was slightly increased by RJ administration but not by E2 administration. The contents of protein and myelin galactolipids were higher in the brains of RJ-administered OVX rats than in the brains of E2-administered OVX rats. CONCLUSION: The results suggest that RJ has a beneficial effect on neurological symptoms of a menopausal disorder.

Prospective study of deep vein thrombosis in patients with spinal cord injury not receiving anticoagulant therapy.

STUDY DESIGN: Prospective cross-sectional study. OBJECTIVES: To investigate the timing of deep vein thrombosis (DVT) onset secondary to spinal cord injury without anticoagulant therapies. SETTING: Spinal Cord Injury Center in Hokkaido, Japan. METHODS: Between November 2012 and June 2013, patients with spinal cord injury who were admitted to our hospital within 1 day after the injury and treated surgically within 24 h underwent a neurological examination, leg vein ultrasonography and D-dimer test 1, 3, 7, 14 and 28 days after surgery. All patients received treatment with intermittent pneumatic compression and elastic stockings, but without any anticoagulant. RESULTS: DVT developed in 12 patients (11 men and 1 women), with a mean age of 62.2 years (range, 41-80 years; mean age of total sample, 63.2 years (range, 25-78 years)), all distal to the popliteal vein. DVT occurred more often with a more severe paralysis (66.3%, AIS A and B). The median (± standard error) length of time from the operation to DVT detection was 7.5±2.2 days. The mean D-dimer level upon DVT detection was 14.6±11.8 µg ml(-1), with no significant differences between those who developed DVT and those who did not at any of the time points. CONCLUSION: These results suggest that DVT can develop at the very-acute stage of spinal cord injury and the incidence increases with a more severe paralysis. DVT detection was more reliable with ultrasonography, which should be used with DVT-preventive measures, beginning immediately after the injury, for the management of patients with spinal cord injury.

Transcriptomic analysis of tail regeneration in the lizard Anolis carolinensis reveals activation of conserved vertebrate developmental and repair mechanisms.

Lizards, which are amniote vertebrates like humans, are able to lose and regenerate a functional tail. Understanding the molecular basis of this process would advance regenerative approaches in amniotes, including humans. We have carried out the first transcriptomic analysis of tail regeneration in a lizard, the green anole Anolis carolinensis, which revealed 326 differentially expressed genes activating multiple developmental and repair mechanisms. Specifically, genes involved in wound response, hormonal regulation, musculoskeletal development, and the Wnt and MAPK/FGF pathways were differentially expressed along the regenerating tail axis. Furthermore, we identified 2 microRNA precursor families, 22 unclassified non-coding RNAs, and 3 novel protein-coding genes significantly enriched in the regenerating tail. However, high levels of progenitor/stem cell markers were not observed in any region of the regenerating tail. Furthermore, we observed multiple tissue-type specific clusters of proliferating cells along the regenerating tail, not localized to the tail tip. These findings predict a different mechanism of regeneration in the lizard than the blastema model described in the salamander and the zebrafish, which are anamniote vertebrates. Thus, lizard tail regrowth involves the activation of conserved developmental and wound response pathways, which are potential targets for regenerative medical therapies.

Gefitinib and luteolin cause growth arrest of human prostate cancer PC-3 cells via inhibition of cyclin G-associated kinase and induction of miR-630.

Cyclin G-associated kinase (GAK), a key player in clathrin-mediated membrane trafficking, is overexpressed in various cancer cells. Here, we report that GAK expression is positively correlated with the Gleason score in surgical specimens from prostate cancer patients. Embryonic fibroblasts from knockout mice expressing a kinase-dead (KD) form of GAK showed constitutive hyper-phosphorylation of the epidermal growth factor receptor (EGFR). In addition to the well-known EGFR inhibitors gefitinib and erlotinib, the dietary flavonoid luteolin was a potent inhibitor of the Ser/Thr kinase activity of GAK in vitro. Co-administration of luteolin and gefitinib to PC-3 cells had a greater effect on cell viability than administration of either compound alone; this decrease in viability was associated with drastic down-regulation of GAK protein expression. A comprehensive microRNA array analysis revealed increased expression of miR-630 and miR-5703 following treatment of PC-3 cells with luteolin and/or gefitinib, and exogenous overexpression of miR-630 caused growth arrest of these cells. GAK appears to be essential for cell death because co-administration of gefitinib and luteolin to EGFR-deficient U2OS osteosarcoma cells also had a greater effect on cell viability than administration of either compound alone. Taken together, these findings suggest that GAK may be a new therapeutic target for prostate cancer and osteosarcoma.

Effect of the ethanol extract of Pleurotus eryngii on bone metabolism in ovariectomized rats.

OBJECTIVE: Postmenopausal bone loss and the possible progression to osteoporosis are a major health concern. Mushrooms have been recognized as functional foods. Pleurotus eryngii extract has been reported to have estrogenic activity, suggesting that its consumption may mitigate postmenopausal bone loss. The aim of the present study was to determine the effect of supplementation with an ethanol extract of P. eryngii on bone metabolism in a postmenopausal osteoporosis rat model. METHODS: Female 12-week-old Wistar rats were subjected to either sham operation or bilateral ovariectomy. The ovariectomized rats were then subdivided into two groups: one fed the extract and the other not. Twelve weeks after surgery, indices of bone mass, bone histomorphometry, and bone mechanics were measured. RESULTS: The right femur bone mineral content and density of the ovariectomized group were significantly lower than in the Sham group, and extract supplementation did not have any significant effect on these differences. Furthermore, ovariectomy significantly increased measures of mineralizing surface and bone formation rates; again, extract supplementation again had no significant effect. CONCLUSION: Our findings suggest that the ethanol extract of P. eryngii does not alter bone metabolism in ovariectomized rats, suggesting that consumption of P. eryngii may not be beneficial in slowing bone loss after menopause.

CAWS administration increases the expression of interferon γ and complement factors that lead to severe vasculitis in DBA/2 mice.

BACKGROUND: Candida albicans water-soluble fraction (CAWS), a mannoprotein-ß-glucan complex obtained from the culture supernatant of C. albicans NBRC1385, causes CAWS-mediated vasculitis (CAWS-vasculitis) in B6 and DBA/2 mice with mild and lethal symptoms, respectively. Why CAWS is lethal only in DBA/2 mice remains unknown. RESULTS: We performed DNA microarray analyses using mRNA obtained from peripheral blood mononuclear cells (PBMCs) of B6 and DBA/2 mice and compared their respective transcriptomes. We found that the mRNA levels of interferon-γ (Ifng) and several genes that regulate the complement system, such as C3, C4, Cfb, Cfh, and Fcna, were increased dramatically only in DBA/2 mice at 4 and 8 weeks after CAWS administration. The dramatic increase was confirmed by quantitative real-time polymerase chain reactions (qRT-PCR). Moreover, mRNA levels of immune-related genes, such as Irf1, Irf7, Irf9, Cebpb, Ccl4, Itgam, Icam1, and IL-12rb1, whose expression levels are known to be increased by Ifng, were also increased, but only in DBA/2 mice. By contrast, the mRNA level of Dectin-2, the critical receptor for the α-mannans of CAWS, was increased slightly and similarly in both B6 and DBA/2 mice after CAWS administration. CONCLUSIONS: Taken together, our results suggest that CAWS administration induces Dectin-2 mediated CAWS-vasculitis in both B6 and DBA/2 mice and the expression of Ifng, but only in DBA/2 mice, which led to increased expression of C3, C4, Cfb, Cfh, and Fcna and an associated increase in lethality in these mice. This model may contribute to our understanding of the pathogenesis of severe human vasculitis.

Ficolin-1 is up-regulated in leukocytes and glomeruli from microscopic polyangiitis patients.

Microscopic polyangiitis (MPA) is a systemic autoimmune disease that often has a fatal outcome. Although delineating the molecular pathogenesis is essential for its remedy, an understanding of its molecular mechanism has remained elusive. To search for new markers of active lesions that might help better understand the molecular basis of MPA and aid in its diagnosis, we here performed DNA microarray analysis with peripheral blood mononuclear cells (PBMCs). Compared to normal control, several genes were up- or down-regulated in MPA patients, including up-regulation of the mRNA level of ficolin-1 (FCN1 or M-ficolin), an innate pattern recognition complement molecule. The amount of ficolin-1, as detected by immunohistochemistry, was higher in the glomeruli of another group of MPA patients than in the glomeruli of control patients who harbored almost normal glomeruli. Many of the ficolin-1 dots were also positive for CD68, suggesting that the ficolin-1-positive cells were monocytes, such as macrophages or dendritic cells. This is not due to the difference in the number of neutrophil or monocytes in the blood samples of MPA and control patients. Taken together, we conclude that increased ficolin-1 expression could serve as a new marker for the characterization of MPA, especially when it is associated with local active lesions.

Cell adhesion molecule 1 is a new osteoblastic cell adhesion molecule and a diagnostic marker for osteosarcoma.

AIMS: An immunohistochemical screen for mouse embryos showed that cell adhesion molecule 1 (CADM1), which is an immunoglobulin superfamily member, was expressed in developing bones. Here, we determined the cell types expressing CADM1 and examined its usefulness in the differential diagnosis of osteosarcoma. MAIN METHODS: Serial sections of murine developing mandibles were stained with anti-CADM1 antibody, by a coloring substrate reactive to alkaline phosphatase (ALP), a broad osteoblastic marker for preosteoblasts to osteoblasts, and by in situ hybridization for osteopontin (OPN), a marker for mature osteoblasts. CADM1 immunohistochemistry was also performed on human remodeling bones, osteosarcomas and other soft tissue tumors. KEY FINDINGS: CADM1 immunohistochemistry for the mandible revealed that morphologically identifiable osteoblasts expressed CADM1 on their plasma membranes, but neither osteocytes nor bone lining cells did. At the mandibular margin, not only OPN-positive cells but also OPN-negative, ALP-positive cells were CADM1-positive, whereas inside the mandible, OPN-positive cells were often CADM1-negative. Clear membranous staining was detected in the majority of osteosarcomas (46/57), whereas only 13% (6/46) of the other soft tissue tumors were CADM1-positive (P<0.001). SIGNIFICANCE: These results indicated that CADM1 was a novel osteoblastic adhesion molecule that is expressed transiently during osteoblastic maturation, and a useful diagnostic marker for osteosarcoma cells.

Adhesion molecule CADM1 contributes to gap junctional communication among pancreatic islet α-cells and prevents their excessive secretion of glucagon.

Cell adhesion molecule-1 (CADM1) is a recently identified adhesion molecule of pancreatic islet α-cells that mediates nerve-α-cell interactions via trans-homophilic binding and serves anatomical units for the autonomic control of glucagon secretion. CADM1 also mediates attachment between adjacent α-cells. Since gap junctional intercellular communication (GJIC) among islet cells is essential for islet hormone secretion, we examined whether CADM1 promotes GJIC among α-cells and subsequently participates in glucagon secretion regulation. Dye transfer assays using αTC6 mouse α-cells, which endogenously express CADM1, supported this possibility; efficient cell-to-cell spread of gap junction-permeable dye was detected in clusters of αTC6 cells transfected with nonspecific, but not with CADM1-targeting, siRNA. Immunocytochemical analysis of connexin 36, a major component of the gap junction among αTC6 cells, revealed that it was localized exclusively to the cell membrane in CADM1-non-targeted αTC6 cells, but diffusely to the cytoplasm in CADM1-targeted cells. Next, we incubated CADM1-targeted and non-targeted αTC6 cells in a medium containing 1 mM glucose and 200 mM arginine for 30 min to induce glucagon secretion, and found that the targeted cells secreted three times more glucagon than did the non-targeted. We conducted similar experiments using pancreatic islets that were freshly isolated from wild-type and CADM1-knockout mice, and expressed glucagon secretion as ratios relative to baseline values. The increase in ratio was larger in CADM1-knockout islets than in wild-type islets. These results suggest that CADM1 may serve as a volume limiter of glucagon secretion by sustaining α-cell attachment necessary for efficient GJIC.

Long-term results of conventional varus half-wedge proximal femoral osteotomy for the treatment of osteonecrosis of the femoral head.

We have previously described the mid- to long-term results of conventional simple varus intertrochanteric osteotomy for osteonecrosis of the femoral head, showing that 19 of the 26 hips had good or excellent results. We extended the follow-up to a mean of 18.1 years (10.5 to 26) including a total of 34 hips in 28 patients, with a mean age at surgery of 33 years (19 to 53). There were 18 men and ten women and 25 hips (74%) had a satisfactory result with a Harris hip score ≥ 80. In all, six hips needed total hip replacement (THR) or hemiarthroplasty. The collapse of the femoral head or narrowing of the joint space was found to have progressed in nine hips (26%). Leg shortening after osteotomy was a mean of 19 mm (8 to 36). With conversion to THR or hemiarthroplasty as the endpoint, the ten-year survival rate was 88.2% (95% confidence interval (CI) 82.7 to 93.7) and the 20-year survival rate was 79.7% (95% CI 72.1 to 87.3); four hips were converted at ten years and other two hips were converted at 20 years. Shortening of the leg after osteotomy remains a concern; however, the conventional varus half-wedge osteotomy provides favourable long-term results in hips with less than two-thirds of the medial part of the femoral head affected by necrotic bone and with normal bone superolaterally.

Neonatal lethality in knockout mice expressing the kinase-dead form of the gefitinib target GAK is caused by pulmonary dysfunction.

Gefitinib (Iressa) is an inhibitor of the epidermal growth factor receptor (EGFR) that has shown promising activity in the treatment of patients with non-small cell lung cancer (NSCLC). However, adverse side effects of gefitinib treatment, such as respiratory dysfunction, have limited the therapeutic benefit of this targeting strategy. The present results show that this adverse effect can be attributed to the inhibition of the novel gefitinib target GAK (Cyclin G-associated kinase), which is as potently inhibited by the drug as the tyrosine kinase activity of EGFR. Knockout mice expressing the kinase-dead form of GAK (GAK-kd) died within 30 min after birth primarily due to respiratory dysfunction. Immunohistochemical analysis revealed that surfactant protein A (SP-A) was abundant within alveolar spaces in GAK-kd(+/+) mice but not in GAK-kd(-/-) pups. E-cadherin and phosphorylated EGFR signals were also abnormal, suggesting the presence of flat alveolar cells with thin junctions. These results suggest that inhibition of GAK by gefitinib may cause pulmonary alveolar dysfunction, and the present study may help prevent side effects associated with gefitinib therapy in NSCLC patients.

The Chiari pelvic osteotomy for patients with dysplastic hips and poor joint congruency: long-term follow-up.

We report the mid- to long-term (mean 20.3 years, 10 to 32.5) results of the Chiari pelvic osteotomy in patients with pre- to advanced stage osteoarthritis in dysplastic hips. We followed 163 Japanese patients (173 hips) with a mean age at surgery of 20 years (9 to 54). Overall, 124 hips (72%) had satisfactory results, with Harris hip scores ≥ 80. Satisfactory results were seen in 105 of 134 hips with pre- or early osteoarthritis (78%) and 19 of 39 hips with advanced osteoarthritis (49%). A total of 15 hips (9%) underwent a total hip replacement (THR) with a mean interval between osteotomy and THR of 16.4 years. With conversion to THR as the endpoint, the 30-year survival rate was 85.9% (95% confidence interval 82.3 to 89.5). It was 91.8% for patients with pre- or early osteoarthritis and 43.6% for those with advanced osteoarthritis (p < 0.001). We now perform the Chiari osteotomy for patients with dysplastic hips showing poor joint congruency and who prefer a joint-conserving procedure to THR.

Atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knee: a prospective multicenter clinical trial in Japan.

BACKGROUND: New tissue-engineering technology was developed to create a cartilage-like tissue in a three-dimensional culture using atelocollagen gel. The minimum 2-year followup outcome of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in knees was reported from the single institution. The present multicenter study was conducted to determine clinical and arthroscopic outcomes in patients who underwent atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knees. METHODS: At six medical institutes in Japan, we prospectively evaluated the clinical and arthroscopic outcomes of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in 27 patients (27 knees) with cartilage lesions on a femoral condyle or on a patellar facet over 24 months. RESULTS: The Lysholm score significantly increased from 60.0 +/- 13.7 points to 89.8 +/- 9.5 points (P = 0.001). Concerning the ICRS grade for arthroscopic appearance, 6 knees (24%) were assessed as grade I (normal) and 17 knees (68%) as grade II (nearly normal). There were few adverse features, except for detachment of the graft in two cases. CONCLUSIONS: We concluded that transplanting chondrocytes in a newly formed matrix of atelocollagen gel can promote restoration of the articular cartilage of the knee.

Minimally invasive surgical treatment for tuberculous spondylodiscitis.

The authors report the cases of 3 patients with tuberculous spondylodiscitis. All patients suffered from severe back or low back pain. Posterolateral endoscopic debridement and irrigation were performed followed by retention of a drainage tube at the affected sites. Additional puncture and drainage were conducted at the same time when extensive cold abscesses were identified around the paravertebral muscle. All patients experienced immediate pain relief postoperatively. This technique is effective for rapid pain relief and in obtaining neurological resolution for patients in the early stages of tuberculous spondylodiscitis and may also be a good method for preventing further vertebral collapse and kyphotic spinal deformity such as Gibbus vertebrae.

Results of tenolysis for flexor tendon adhesion after phalangeal fracture.

There are only three reports of the surgical outcomes of flexor tendon tenolysis after phalangeal fractures. The fracture type, the time to mobilisation following injury and the time between the injury and tenolysis did not affect the results of tenolysis. The outcome only correlated significantly to the total passive range of motion before tenolysis.