Antibacterial and Immunosuppressive Effects of OPS-2071, a Candidate Therapy for Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. Although many types of drug are used, clinical outcomes are still unsatisfactory. Previous studies have suggested that intestinal bacteria are involved in the pathogenesis of IBD. Accordingly, in an IBD model we evaluated the therapeutic effects of OPS-2071, a low-absorption quinolone antibacterial agent indicated for intestinal infection, and investigated its mechanism of action. METHODS: The therapeutic effects of OPS-2071 and comparison therapies were evaluated using naive CD4 + T cell-transfer IBD model mice. In vitro inhibition of LPS-induced TNF-α production and inhibitory effects on T cell responses stimulated using anti-CD3/CD28 antibody-loaded beads were evaluated using mouse splenocytes and human peripheral blood mononuclear cells. In addition, in vitro activities against bacteria implicated in IBD pathogenesis were tested. RESULTS: OPS-2071 dose-dependently decreased both colonic weight/length ratio and the colitis histological score as compared with the vehicle group. The therapeutic effect of OPS-2071 was equivalent to that of anti-IL-12/23 (p40) antibody. In vitro, OPS-2071 suppressed TNF-α production induced by LPS stimulation and T cell responses in a dose-dependent manner. At high concentrations, these effects were comparable to those of existing immunosuppressive agents, such as prednisolone, in both mouse and human cells. OPS-2071 also showed antibacterial activity against IBD-related bacteria. CONCLUSIONS: Our results suggest that OPS-2071 had both immunosuppressive and antibacterial effects. This dual effect makes OPS-2071 a unique and promising candidate for IBD.

Surgical treatment of a lumbar aneurysmal bone cyst using percutaneous endoscopic lumbar discectomy.

PURPOSE: Aneurysmal bone cysts of the lumbar spine are usually treated by curettage followed by bone or bioactive ceramics grafting. Here, we present the first case of an aneurysmal bone cyst of the lumbar spine treated by percutaneous endoscopic lumbar discectomy (PELD). METHODS: We describe the clinical characteristics of the patient including the radiological and pathological findings of the tumor and the surgical technique used. RESULTS: A 15-year-old boy presented with low back pain, and he was diagnosed with an aneurysmal bone cyst of the L3 vertebra based on radiological findings, including plain radiograph, computed tomography, and magnetic resonance imaging. The technique and equipment of PELD were used to perform curettage of the tumor cavity and fill it with hydroxyapatite granules. The skin incision was only 8 mm. The patient was discharged 1 day postoperatively and could walk without assistance. The postoperative course was uneventful and the symptoms improved following surgery. CONCLUSION: Endoscopic surgery via PELD can be a treatment option for ABCs of the lumbar spine.

Octacalcium phosphate suppresses chondrogenic differentiation of ATDC5 cells.

Implantation of octacalcium phosphate (OCP), a hydroxyapatite precursor, has been reported to induce chondrogenesis in vivo. In this study, we examined the effects of OCP on the chondrogenic differentiation of mouse chondroblastic ATDC5 cells in vitro. Contrary to our expectation, chondrogenic differentiation of ATDC5 cells evaluated by the mRNA expression of Col2a1, Acan and Col10a1 was suppressed by OCP. Among Sox9, Sox5 and Sox6, essential transcription factors for chondrogenesis, the expression of Sox6 mRNA was markedly lowered by OCP. Whereas ATDC5 cells dissolved OCP to liberate calcium and inorganic phosphorus, increased calcium or phosphate in the medium had little effect on the differentiation of these cells. Direct contact of ATDC5 cells with OCP was required to suppress the expression of Col2a1 and Sox6 mRNAs, whereas the introduction of Sox6 short interfering RNA lowered the expression of Col2a1 mRNA. On the other hand, the forced expression of Sox6 protein partially but significantly, restored the expression of Col2a1 mRNA suppressed by OCP. These results indicate that OCP suppresses the chondrogenic differentiation of ATDC5 cells, at least in part, at the Sox6 transcription level.

Painful lumbar spondylolysis among pediatric sports players: a pilot MRI study.

INTRODUCTION: For children and adolescents who are very active athletes, fresh lumbar spondylolysis is the main pathologic cause of lower back pain (LBP). However, regarding the terminal-stage spondylolysis (pars defect), there have been few studies to clarify the pathomechanism of LBP. The purpose of this study is to clarify the cause of LBP associated with pars defects in athletes. This is the first report showing a possible pathomechanism of LBP in active athletes with painful pars defect. METHOD: Six pediatric athletes (5 boys and 1 girl) below 18 years old with painful bilateral lumbar spondylolysis were evaluated. In all cases, spondylolysis was identified as terminal stage (pseudoarthrosis) on CT scan. To evaluate the inflammation around the pars defects, short time inversion recovery (STIR) MRI was performed along with the sagittal section. Fluid collection, which is an indicator of inflammatory events, was evaluated in 12 pars defects as well as in 12 cranial and caudal adjoining facet joints. RESULTS: Inflammation (i.e., fluid collection) was observed in all 12 pars defects in six subjects at the pseudoarthrotic pars defects. In terms of facet joints, 7 of 12 (58%) pars defects showed fluid collection at the cranial and/or caudal adjoining joints on STIR MRI. CONCLUSION: The present study showed that inflammation was always present at the pars defects and in some cases at the adjoining facet joints. Thus, it is not difficult to understand how, during sports activity, inflammation may first occur at the pseudoarthrotic site and then spread to the adjoining facet joints. This mechanism could cause LBP associated with terminal-stage (pseudoarthrotics) spondylolysis in athletes.

Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart.

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell-, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0-Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells-and under the right conditions-differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.

Development of dual-curing type experimental composite resin cement for orthodontic bonding -effect of additional amount of accelerators on the mechanical properties-.

In this study, a dual-curing type composite resin cement that included a photo-initiator and two accelerators was designed. In particular, special emphasis was made on addressing questions on the effects from different amounts of additional accelerators on the flexural strength of the designed experimental composite resin cement, as well as on the tensile bond strength of the bracket bonded onto the enamel surface by the experimental composite resin cement. When 0.25 mass% of the p-tolydiethanolamine and sodium p-toluenesulfinate were added, the maximum flexural strength was obtained for the chemical-cured and dual-cured experimental composite resin cement. The dual-cured experimental composite resin cement's flexural strength value was in the mid-range of the values exhibited from the commercial resin cements. However, the dual-cured experimental composite resin cement exhibited noticeably high tensile bond strength when compared with the results obtained with the commercial resin cements.

In Vitro Study of the Effects of Denosumab on Giant Cell Tumor of Bone: Comparison with Zoledronic Acid.

Giant cell tumor of bone (GCTB) is a locally aggressive primary bone tumor that contains numerous osteoclasts formed from marrow-derived precursors through receptor activator of nuclear factor κ-B ligand (RANKL), an osteoclast differentiation factor expressed in neoplastic cells of GCTB. Denosumab, a fully human monoclonal antibody targeting RANKL, has recently been used for the treatment of GCTB, and superior treatment effects have been reported. The aim of this work was to elucidate the mechanism of action of denosumab, and the differences between denosumab and zoledronic acid at the level of GCTB cells. We isolated GCTB cells from 3 patients and separated them into osteoclasts, osteoclast precursors and proliferating spindle-shaped stromal cells (the true neoplastic component), and examined the action of denosumab on differentiation, survival and bone resorption activity of osteoclasts. Denosumab and zoledronic acid inhibited osteoclast differentiation from mononuclear cells containing osteoclast precursors. Zoledronic acid inhibited osteoclast survival, whereas an inhibitory effect of denosumab on osteoclast survival was not observed. The inhibitory effect on bone resorption by both agents was confirmed in culture on dentin slices. Furthermore, zoledronic acid showed dose-dependent inhibition of cell growth of neoplastic cells whereas denosumab had no inhibitory effect on these cells. Denosumab has an inhibitory effect on osteoclast differentiation, but no inhibitory effects on survival of osteoclasts or growth of neoplastic cells in GCTBs.

Accumulation of HLA-DR4 in Colonic Epithelial Cells Causes Severe Colitis in Homozygous HLA-DR4 Transgenic Mice.

BACKGROUND: Homozygous HLA-DR4/I-E transgenic mice (tgm) spontaneously developed colitis similar to human ulcerative colitis. We explored whether endoplasmic reticulum stress in colonic epithelial cells due to overexpression of HLA-DR4/I-E was involved in the pathogenesis of colitis. METHODS: Major histocompatibility complex class II transactivator-knockout (CIITAKO) background tgm were established to test the involvement of HLA-DR4/I-E expression in the pathogenesis of colitis. Histological and cellular analyses were performed and the effect of oral administration of the molecular chaperone tauroursodeoxycholic acid (TUDCA) and antibiotics were investigated. IgA content of feces and serum and presence of IgA-coated fecal bacteria were also investigated. RESULTS: Aberrantly accumulated HLA-DR4/I-E molecules in colonic epithelial cells were observed only in the colitic homozygous tgm, which was accompanied by upregulation of the endoplasmic reticulum stress marker Binding immunoglobulin protein (BiP) and reduced mucus. Homozygous tgm with CIITAKO, and thus absent of HLA-DR4/I-E expression, did not develop colitis. Oral administration of TUDCA to homozygotes reduced HLA-DR4/I-E and BiP expression in colonic epithelial cells and restored the barrier function of the intestinal tract. The IgA content of feces and serum, and numbers of IgA-coated fecal bacteria were higher in the colitic tgm, and antibiotic administration suppressed the expression of HLA-DR4/I-E and colitis. CONCLUSIONS: The pathogenesis of the colitis observed in the homozygous tgm was likely due to endoplasmic reticulum stress, resulting in goblet cell damage and compromised mucus production in the colonic epithelial cells in which HLA-DR4/I-E molecules were heavily accumulated. Commensal bacteria seemed to be involved in the accumulation of HLA-DR4/I-E, leading to development of the colitis.

Membranous osteogenesis system modeled with KUSA-A1 mature osteoblasts.

Several stromal cells were established from murine bone marrow cultures. One of the KUSA subclones, KUSA-A1 cells, displays osteogenic characteristics in vitro and in vivo. The calcium deposition, osteocalcin release, and parathyroid hormone (PTH) responsiveness of KUSA-A1 cells indicate that they are mature osteoblasts or osteocytes. Bone had formed in subcutaneous tissue 1 week after subcutaneous injection of cells into immunodeficient mice. The osteogenesis by KUSA-A1 was not mediated by chondrogenesis and thus was considered to be membranous ossification. These unique characteristics of KUSA-A1 cells provide an opportunity to analyze the process of membranous ossification in detail.

Solitary synovial chondromatosis arising in the gluteus maximus bursa: computed tomography and magnetic resonance imaging findings.

Chondral tumors in soft tissue are referred to as soft-tissue chondromas or extraskeletal chondromas, or as synovial chondromatosis if they arise in synovial tissue. We report the case of a 29-year-old man with synovial chondromatosis, also called synovial osteochondromatosis, which appeared in a solitary and extra-articular form. On magnetic resonance imaging (MRI) and computed tomography, the central portion of the tumor showed similar characteristics to bone marrow, despite the absence of any connection to adjacent bone. T2-weighted imaging displayed marked peripheral hyperintensity consistent with a cartilaginous area. These findings suggested the presence of enchondral ossification and were similar to those of skeletal osteochondroma, with the exception of the absence of attachment to bone. MRI is useful for distinguishing solitary synovial chondromatosis from other lesions, such as myositis ossificans, extraskeletal chondrosarcoma, and parosteal osteosarcoma.

Multilevel direct repair surgery for three-level lumbar spondylolysis.

A 45-year-old man presented to our clinic requesting evaluation for surgical treatment of chronic low back pain of more than 20 years duration. He was diagnosed with 3-level lumbar spondylolysis at L3-5. Direct repair using the pedicle screw and hook-rod system was conducted for all three levels. After the surgery, his low back pain completely disappeared. Six months later, he felt discomfort and heard a metallic sound as he twisted his trunk. Computed tomography and radiography indicated that the hook head for L3 and the screw head for L4 were interfering with each other, causing the sound. We confirmed bony union at L3 and removed the L3 system. Surgeons should be aware of such complications if direct repair using a pedicle screw and hook-rod system is conducted for multilevel spondylolysis.

Four-rod stabilization of severely destabilized lumbar spine caused by metastatic tumor.

We report a case of a 67-year-old female with severely destabilized lumbar spine caused by metastatic malignant tumor. The primary lesion was a thyroid follicular adenocarcinoma. Complete destruction of the L3, L4, and L5 vertebrae had resulted in severe instability, which left the patient with severe back pain and bed-ridden. Since the vertebrae were so severely damaged at 3 levels, 4 rods were used to stabilize the spine. Following stabilization, the pain was alleviated and the patient's quality of life improved. We introduce here the 4-rod technique to stabilize the spine over 3 vertebral levels following severe destruction by metastatic tumor.

Lumbar spondylolysis in juveniles from the same family: a report of three cases and a review of the literature.

Spondylolysis is reported as a stress fracture of the pars interarticularis with a strong hereditary basis. Three cases of lumbar spondylolysis in juveniles from the same family are reported, and the genetics of the condition are reviewed. The first boy, a 13-year-old soccer player, was diagnosed with terminal stage L5 bilateral spondylolysis with grade 1 slippage. The second boy, a 10-year-old baseball player, had terminal stage right side unilateral spondylolysis. The third boy, also a 10-year-old baseball player, was diagnosed with early stage bilateral L5 spondylolysis. The second and third boys are identical twins, and all three cases exhibited concomitant spina bifida occulta. Lumbar spondylolysis has a strong hereditary basis and is reported to be an autosomal dominant condition.

V-rod technique for direct repair surgery of pediatric lumbar spondylolysis combined with posterior apophyseal ring fracture.

We report a pediatric baseball player having both a fracture of the posterior ring apophysis and spondylolysis. He was presented to a primary care physician complaining of back pain and leg pain. Despite conservative treatment for 3 months, the pain did not subside. He was referred to our clinic, and surgical intervention was carried out. First, a bony fragment of the caudal L5 apophyseal ring was removed following fenestration at the L5-S interlaminal space, bilaterally: and decompression of the bilateral S1 nerve roots was confirmed. Next, pseudoarthrosis of the L5 pars was refreshed and pedicle screws were inserted bilaterally. A v-shaped rod was inserted beneath the L5 spinous process, which stabilized the pars defects. After the surgery, back pain and leg pain completely disappeared. In conclusion, the v-rod technique is appropriate for the spondylolysis direct repair surgery, especially, in case the loose lamina would have a partial laminotomy.

Establishment of HLA-DR4 transgenic mice for the identification of CD4+ T cell epitopes of tumor-associated antigens.

Reports have shown that activation of tumor-specific CD4(+) helper T (Th) cells is crucial for effective anti-tumor immunity and identification of Th-cell epitopes is critical for peptide vaccine-based cancer immunotherapy. Although computer algorithms are available to predict peptides with high binding affinity to a specific HLA class II molecule, the ability of those peptides to induce Th-cell responses must be evaluated. We have established HLA-DR4 (HLA-DRA*01:01/HLA-DRB1*04:05) transgenic mice (Tgm), since this HLA-DR allele is most frequent (13.6%) in Japanese population, to evaluate HLA-DR4-restricted Th-cell responses to tumor-associated antigen (TAA)-derived peptides predicted to bind to HLA-DR4. To avoid weak binding between mouse CD4 and HLA-DR4, Tgm were designed to express chimeric HLA-DR4/I-E(d), where I-E(d) α1 and ß1 domains were replaced with those from HLA-DR4. Th cells isolated from Tgm immunized with adjuvant and HLA-DR4-binding cytomegalovirus-derived peptide proliferated when stimulated with peptide-pulsed HLA-DR4-transduced mouse L cells, indicating chimeric HLA-DR4/I-E(d) has equivalent antigen presenting capacity to HLA-DR4. Immunization with CDCA155-78 peptide, a computer algorithm-predicted HLA-DR4-binding peptide derived from TAA CDCA1, successfully induced Th-cell responses in Tgm, while immunization of HLA-DR4-binding Wilms' tumor 1 antigen-derived peptide with identical amino acid sequence to mouse ortholog failed. This was overcome by using peptide-pulsed syngeneic bone marrow-derived dendritic cells (BM-DC) followed by immunization with peptide/CFA booster. BM-DC-based immunization of KIF20A494-517 peptide from another TAA KIF20A, with an almost identical HLA-binding core amino acid sequence to mouse ortholog, successfully induced Th-cell responses in Tgm. Notably, both CDCA155-78 and KIF20A494-517 peptides induced human Th-cell responses in PBMCs from HLA-DR4-positive donors. Finally, an HLA-DR4 binding DEPDC1191-213 peptide from a new TAA DEPDC1 overexpressed in bladder cancer induced strong Th-cell responses both in Tgm and in PBMCs from an HLA-DR4-positive donor. Thus, the HLA-DR4 Tgm combined with computer algorithm was useful for preliminary screening of candidate peptides for vaccination.

Minimally invasive and simultaneous removal of herniated intracanal and extracanal lumbar nucleus pulposus with a percutaneous spinal endoscope.

Herniated nucleus pulposus (HNP) in the lumbar spine is usually found in the neural canal (in the intracanal space) and occasionally in the extracanal space, where it is known as a lateral HNP. HNP is rarely found simultaneously in both spaces. However, we experienced such a case in a 48-year-old man who presented with right leg pain and lower back pain that had lasted for more than a year. MRI revealed HNP in both the right intracanal and extracanal spaces at L2-L3. A transforaminal approach was used to complete a percutaneous endoscopic discectomy. An 8-mm incision was made with the patient under local anesthesia, and the percutaneous endoscope was inserted at the affected disc space. First, the HNP fragments in the intracanal space were removed, and then the cannula and endoscope were extracted to the extracanal space where the extracanal fragments were removed. Two hours after the surgery, the patient stood and walked. Right leg pain and lower back pain had disappeared. Unlike other techniques such as Love's procedure and the microendoscopic discectomy technique, the use of a transforaminal approach with the percutaneous endoscopic technique enables the HNP fragments in the intracanal and extracanal spaces to be removed at the same time with a single approach.

R848, a toll-like receptor 7 agonist, inhibits osteoclast differentiation but not survival or bone-resorbing function of mature osteoclasts.

R848, also known as resiquimod, acts as a ligand for toll-like receptor 7 (TLR7) and activates immune cells. In this study, we examined the effects of R848 on differentiation, survival, and bone-resorbing function of osteoclasts. R848 inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) and human peripheral blood-derived monocytes induced by receptor activator of NF-κB ligand in a dose-dependent manner. In addition, it inhibited mouse osteoclast differentiation induced in cocultures of bone marrow cells and osteoblasts in the presence of dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, R848 did not affect the survival or bone-resorbing activity of mouse mature osteoclasts. R848 also upregulated the mRNA expression levels of interleukin (IL)-6, IL-12, interferon (IFN)-γ, and inducible nitric oxide synthase in mouse BMMs expressing TLR7. IFN-ß was consistently expressed in the BMMs and addition of neutralizing antibodies against IFN-ß to the cultures partially recovered osteoclast differentiation inhibited by R848. These results suggest that R848 targets osteoclast precursors and inhibits their differentiation into osteoclasts via TLR7.

Novel and efficient synthesis of 4-dimethylamino-2-glycosylaminoquinazolines by cyclodesulfurization of glycosyl thioureas with dimethylcyanamide.

4-dimethylamino-2-glycosylaminoquinazoline derivatives were synthesized by cyclodesulfurization of N-aryl-N'-glycosyl thioureas with dimethylcyanamide in the presence of silver triflate in good yields.

Novel quinazoline ring synthesis by cycloaddition of N-arylketenimines with N,N-disubstituted cyanamides.

The reaction of N-aryl-substituted ketenimines with N,N-disubstituted cyanamides or (MeS)2C=N-CN under high pressure afforded 4-(N,N-disubstituted amino) or 4-(MeS)2C=N-substituted quinazoline derivatives, respectively. These products were formed by [4+2] cycloaddition between the aza-diene moieties of the N-arylsubstituted ketenimines and cyano groups. A 4-(unsubstituted amino)quinazoline derivative was synthesized by hydrolysis of the latter product.