A chromosome 16p13.11 microduplication causes hyperactivity through dysregulation of miR-484/protocadherin-19 signaling.

Chromosome 16p13.11 microduplication is a risk factor associated with various neurodevelopmental disorders such as attention-deficit/hyperactivity disorder, intellectual disabilities, developmental delay and autistic spectrum disorder. The underlying molecular mechanism of this genetic variation remained unknown, but its core genetic locus-conserved across mice and humans-contains seven genes. Here, we generated bacterial artificial chromosome-transgenic mice carrying a human 16p13.11 locus, and these mice showed the behavioral hyperactivity phenotype. We identified miR-484 as the responsible gene using a combination of expression and functional analyses. Mature miR-484 was expressed during active cortical neurogenesis, and overexpression of miR-484 decreased proliferation and increased neural progenitor differentiation in vivo. Luciferase screening identified the 3'-untranslated region of protocadherin-19 (Pcdh19) as a target of miR-484. The effect of miR-484 on neurogenesis was rescued by ectopic PCDH19 expression. These results demonstrate that miR-484 promotes neurogenesis by inhibiting PCDH19. Dysregulation of neurogenesis by imbalanced miR-484/PCDH19 expression contributes to the pathogenesis of 16p13.11 microduplication syndrome.

Morphological characteristics of olecranon fractures in adults : a Computed Tomography-based study.

The aim of this study was to identify the fragment's shape by evaluating olecranon fractures. We examined the CT images of 48 olecranon fractures (28 women and 20 men). Mean age was 59.9 years. On the olecranon's posterior surface, we measured the distance between the apex of the olecranon fragment and the radial edge of the flat spot on the short axis and the width of the flat spot on the same short axis. The tip radial ratio (i.e., the tip radial edge to the flat spot width) was derived from these parameters. The mean tip radial edge was 1.96 mm, and the flat spot width was 12.64 mm ; therefore, the tip radial ratio was 0.15 mm. Radial inclination on the articular surface was 30.55°. Our findings confirmed our hypothesis that the fracture lines run from the proximal ulnar side to the distal radial side on the olecranon's posterior and articular surfaces.

Production of three-dimensional tissue-engineered cartilage through mutual fusion of chondrocyte pellets.

In this study, the mutual fusion of chondrocyte pellets was promoted in order to produce large-sized tissue-engineered cartilage with a three-dimensional (3D) shape. Five pellets of human auricular chondrocytes were first prepared, which were then incubated in an agarose mold. After 3 weeks of culture in matrix production-promoting medium under 5.78g/cm(2) compression, the tissue-engineered cartilage showed a sufficient mechanical strength. To confirm the usefulness of these methods, a transplantation experiment was performed using beagles. Tissue-engineered cartilage prepared with 50 pellets of beagle chondrocytes was transplanted subcutaneously into the cell-donor dog for 2 months. The tissue-engineered cartilage of the beagles maintained a rod-like shape, even after harvest. Histology showed fair cartilage regeneration. Furthermore, 20 pellets were made and placed on a beta-tricalcium phosphate prism, and this was then incubated within the agarose mold for 3 weeks. The construct was transplanted into a bone/cartilage defect in the cell-donor beagle. After 2 months, bone and cartilage regeneration was identified on micro-computed tomography and magnetic resonance imaging. This approach involving the fusion of small pellets into a large structure enabled the production of 3D tissue-engineered cartilage that was close to physiological cartilage tissue in property, without conventional polyper scaffolds.

Lipopolysaccharide of Aggregatibacter actinomycetemcomitans up-regulates inflammatory cytokines, prostaglandin E2 synthesis and osteoclast formation in interleukin-1 receptor antagonist-deficient mice.

BACKGROUND AND OBJECTIVE: The interleukin (IL)-1 receptor antagonist (Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is unclear whether the IL-1Ra plays a protective role in periodontal disease. The purpose of this study was to compare IL-1Ra knockout (KO) and wild-type (WT) mice in regard to proinflammatory cytokine production, osteoclast formation and bone resorption in response to periodontal bacterial lipopolysaccharide (LPS). MATERIAL AND METHODS: Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10 µg/mL of Aggregatibacter actinomycetemcomitans LPS for 24 h. The levels of IL-1alpha (IL-1α), IL-1beta (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-6 were measured in periotoneal Mφs supernatant fluid (PM-SF) using an ELISA. Bone marrow cells were obtained from the mice and stimulated with PM-SF for 9 d, then stained with TRAP. The frequency of TRAP-positive multinucleated giant cell formation was calculated based on a fusion index. PM-SF-stimulated calvarial bone resorption was analyzed using micro-computed tomography, and calvarial histological analysis was performed using hematoxylin and eosin and TRAP staining. The expression of cyclooxygenase-2 (Cox2), prostanoid receptor EP4 (Ep4) and Rank mRNAs in bone marrow cells were measured using real-time quantitative PCR, while prostaglandin E2 (PGE2 ) production was determined by ELISA. RESULTS: The levels of IL-1α, IL-1ß, TNF-α and IL-6 in IL-1Ra KO mice PM-SF stimulated with A. actinomycetemcomitans LPS were significantly increased by approximately 4- (p < 0.05), 5- (p < 0.05), 1.3- (p < 0.05) and 6- (p < 0.05) fold, respectively, compared with the levels in WT mice. Moreover, osteoclast formation, expression of Rank, Ep4 and Cox2 mRNAs and production of PGE2 were significantly increased by approximately 2- (p < 0.05), 1.6- (p < 0.05), 2.5- (p < 0.05), 1.6- (p < 0.05) and 1.9- (p < 0.05) fold, respectively, in IL-1Ra KO mice stimulated with A. actinomycetemcomitans LPS compared with WT mice. CONCLUSION: IL-1Ra regulates IL-1 activity and appears to reduce the levels of other inflammatory cytokines, including TNF-α and IL-6, while it also reduces expression of the EP4 receptor related to prostanoid sensitivity and osteoclast formation. These results suggest that IL-1Ra is an important molecule for inhibition of inflammatory periodontal bone resorption.

Selection of highly osteogenic and chondrogenic cells from bone marrow stromal cells in biocompatible polymer-coated plates.

To enrich the subpopulation that preserves self-renewal and multipotentiality from conventionally prepared bone marrow stromal cells (MSCs), we attempted to use 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer-coated plates that selected the MSCs with strong adhesion ability and evaluated the proliferation ability or osteogenic/chondrogenic potential of the MPC polymer-selected MSCs. The number of MSCs that were attached to the MPC polymer-coated plates decreased with an increase in the density of MPC unit (0-10%), whereas no significant difference in the proliferation ability was seen among these cells. The surface epitopes of CD29, CD44, CD105, and CD166, and not CD34 or CD45, were detectable in the cells of all MPC polymer-coated plates, implying that they belong to the MSC category. In the osteogenic and chondrogenic induction, the MSCs selected by the 2-5% MPC unit composition showed higher expression levels of osteoblastic and chondrocytic markers (COL1A1/ALP, or COL2A1/COL10A1/Sox9) at passage 2, compared with those of 0-1% or even 10% MPC unit composition, while the enhanced effects continued by passage 5. The selection based on the adequate cell adhesiveness by the MPC polymer-coated plates could improve the osteogenic and chondrogenic potential of MSCs, which would provide cell sources that can be used to treat the more severe and various bone/cartilage diseases.

Involvement of fibroblast growth factor 18 in dedifferentiation of cultured human chondrocytes.

OBJECTIVE: Chondrocytes inevitably decrease production of cartilaginous matrices during long-term cultures with repeated passaging; this is termed dedifferentiation. To learn more concerning prevention of dedifferentiation, we have focused here on the fibroblast growth factor (FGF) family that influences chondrocyte proliferation or differentiation. MATERIALS AND METHODS: We have compared gene expression between differentiated cells in passage 3 (P3) and dedifferentiated ones in P8 of human cultured chondrocytes. We also performed ligand administration of the responsive factor or its gene silencing, using small interfering RNA (siRNA). RESULTS: FGFs 1, 5, 10, 13 and 18 were higher at P8 compared to P3, while FGFs 9 and 14 were lower. Especially, FGF18 showed a 10-fold increase by P8. Ligand administration of FGF18 in the P3 cells, or its gene silencing using siRNA in the P8 cells, revealed dose-dependent increase and decrease respectively in type II collagen/type I collagen ratio. Exogenous FGF18 also upregulated expression of transforming growth factor beta (TGF-beta), the anabolic factor of chondrocytes, in P3 chondrocytes, but P8 cells maintained a low level of TGF-beta expression, suggesting a decrease in responsiveness of TGF-beta to FGF18 stimulation in the dedifferentiated chondrocytes. CONCLUSION: FGF18 seems to play a role in maintenance of chondrocyte properties, although its expression was rather high in dedifferentiated chondrocytes. Upregulation of FGF18 in dedifferentiated chondrocytes implied that it may be a marker of dedifferentiation.

Gene transfer of bFGF to recipient bed improves survival of ischemic skin flap.

BACKGROUND: The recipient bed is a promising target of angiogenic therapy to treat ischemic skin flaps. We delivered basic fibroblast growth factor (bFGF) gene to the recipient bed by a plasmid-based method with electroporation, and assessed the effects on flap viability in a rat dorsal skin flap model. METHODS: A 25 x 90 mm(2) axial skin flap was elevated on the back of male Sprague-Dawley rats. Two days before flap elevation, an expression plasmid vector containing the bFGF gene with the signal sequence was injected into the dorsal muscles beneath the skin flap, and then electroporation was delivered (FGF-E(+) group). As control, rats were injected with a plasmid vector containing LacZ gene (LacZ-E(+) group), instead of bFGF gene. Other groups of animals received plasmid vector containing bFGF (FGF-E(-) group) or LacZ (LacZ-E(-) group) gene without electroporation. Seven days later, the area of necrosis and neovascularisation of the skin flap were evaluated. RESULTS: The bFGF gene was successfully transferred to the dorsal muscles, and bFGF was expressed in muscle tissue. The area of flap necrosis (%) in the FGF-E(+) group (21.7+/-5.3%) was significantly smaller than that in the LacZ-E(+) (28.3+/-4.1%), FGF-E(-) (29.7+/-3.3%), and LacZ-E(-) (28.1+/-2.5%) groups. Postmortem angiograms and histological analyses showed that vascularisation in the distal part of the skin flap was significantly increased in the FGF-E(+) group compared with the other groups. CONCLUSION: These findings suggested that gene delivery of bFGF to the recipient bed muscles enhanced vascularity and viability of an ischemic skin flap, and that plasmid-based gene delivery with electroporation was a suitable delivery method.

Novel 2-amino-1,4-dihydropyridine calcium antagonists. II. Synthesis and antihypertensive effects of 2-amino-1,4-dihydropyridine derivatives having N,N-dialkylaminoalkoxycarbonyl groups at 3- and/or 5-position.

Novel 2-amino-1,4-dihydropyridine derivatives I, which contain N,N,-dialkylaminoalkoxycarbonyl groups at the 3- and/or 5-position, were synthesized and their antihypertensive effects were evaluated in spontaneously hypertensive rats. Remarkably prolonged duration of antihypertensive action was observed when a tertiary amino group was introduced into either the 3- and/or 5-ester side-chain of the 1,4-dihydropyridine ring. In particular, the compounds containing cyclic amino moieties at the 3-position showed greater potency than those with acyclic amino moieties. Chemical modification studies indicated that the two ester side-chains of 1,4-dihydropyridine at the 3- and 5-position might function in a different manner in relation to the antihypertensive activities. 3-(1-Benzhydrylazetidinited potent and long-lasting antihypertensive effects with gradual onset of action, and is a promising candidate as an antihypertensive drug.

Evidence for lipopolysaccharide as the predominant proinflammatory mediator in supernatants of antibiotic-treated bacteria.

Lipopolysaccharide (LPS), purified from gram-negative bacteria, is well known to induce proinflammatory responses in monocytes and macrophages, and release of LPS from the microbial surface has been suggested to be an important initiating event in the sepsis syndrome. However, numerous studies have documented that a variety of constituents present in the outer cell membrane of gram-negative bacteria have the capacity to activate cells of the immune system. Given that the majority of immunotherapeutic approaches designed to intervene in gram-negative sepsis have to date targeted the LPS molecule, it would be of value to assess the relative proinflammatory properties of LPS and other gram-negative structures. Experiments were therefore undertaken to assess stimulation of human monocytes by components released from Escherichia coli following bacteriolysis by the cell wall-active antibiotic ceftazidime. As assessed by both induction of procoagulant activity and release of tumor necrosis factor, bacterial culture supernatants contain significant proinflammatory activity. When culture supernatants are fractionated via either velocity sedimentation in sucrose gradients or isopycnic density gradient ultracentrifugation in cesium chloride, the predominant monocyte-stimulating activity is identified in LPS-containing fractions. Further, such activity can be readily abrogated by the addition of polymyxin B. These results provide support for the hypothesis that LPS may be responsible for the majority of the proinflammatory activity released from E. coli following bacteriolysis in vitro.

A protective effect of glutathione-dextran macromolecular conjugates on acetaminophen-induced hepatotoxicity dependent on molecular size.

Glutathion (GSH) was covalently attached to dextrans with various molecular weights of 2, 5, 10, 40, and 70 kDa by the cyanogen bromide activation method. The conjugates obtained synthetically were white or pale yellowish powders containing 6-10% (w/w) of GSH. The average molecular weights of the conjugates were estimated to be larger and the molecular weight distribution was a little broader than that of each original dextran. The conjugates significantly stabilized GSH and liberated it gradually under physiological conditions (t1/2 = 0.99-1.6h). Mice depleted of GSH by treatment with buthionine sulfoximine, a potent inhibitor of gamma-glutamylcysteine synthetase, exhibited a significant increase in hepatic GSH level after intravenous injection of the conjugates. In mice given a hepatotoxic dose of acetaminophen, the survival rate increased progressively with coadministration of the conjugates, whereas a small improvement was found when free GSH was given. The conjugate of GSH attached to dextran with the molecular weight of 40 kDa exhibited the highest prophylactic effect on acetaminophen-induced hepatotoxicity in mice. The prolonged retention of the conjugates of larger molecular weight in the circulation would cause a higher hepatic accumulation. These results suggested that molecular size would be the most critical factor in the delivery of GSH, as a dextran conjugate, into the liver.

Differential antibiotic-induced release of endotoxin from gram-negative bacteria.

Treatment of log phase cultures of Escherichia coli with cell wall active antibiotics results in increased exposure of immunologically reactive lipid A epitopes of lipopolysaccharide (LPS) and release of soluble LPS into culture supernatants. Comparison of the efficacy of two cell wall active antibiotics, ceftazidime, a penicillin-binding protein 3 selective antibiotic, and imipenem, a penicillin-binding protein 2 selective antibiotic, for their relative efficacy in mediating LPS release indicated quantitative but not qualitative differences, with the former antibiotic manifesting a significantly broader range of concentrations at which LPS release could be demonstrated. Comparison of the relative efficacy of these two antibiotics in a mouse bacteraemia model in which animals were made hypersensitive to the lethal effects of endotoxin by treatment with D-galactosamine indicated that the latter antibiotic may provide a greater level of protection. These studies suggest that the release of endotoxin mediated by antibiotic treatment may contribute to the pathogenesis of disease in infectious due to gram-negative organisms.

Monoclonal anti-lipid A IgM antibodies HA-1A and E-5 recognize distinct epitopes on lipopolysaccharide and lipid A.

Specific binding of two monoclonal IgM antibodies previously investigated as therapeutic agents for treating gram-negative septic shock, HA-1A and E5, was assessed with respect to lipid A and lipopolysaccharide (LPS). Both antibodies bound to lipid A; however, binding of HA-1A was significantly greater than that of E5 to LPS derived from rough strains of bacteria. Reciprocal competitive inhibition experiments supported the concept that HA-1A and E5 bind to distinct epitopes on lipid A. Further, competitive inhibition studies using a monoclonal anti-idiotype antibody with specificity for the variable region of HA-1A suggested that HA-1A and E5 do not share a common idiotype. Finally, studies using double-stranded DNA as antigen indicated that E5 but not HA-1A will bind to DNA. Collectively, these data indicate that HA-1A and E5 are different lipid A-specific antibodies that bind to distinct epitopes on lipid A.

Characterization of specific binding of a human immunoglobulin M monoclonal antibody to lipopolysaccharide and its lipid A domain.

The human immunoglobulin M monoclonal antibody HA-1A was first described as an antibody which bound specifically to the lipid A region of lipopolysaccharide (LPS) (N. N. H. Teng, H. S. Kaplan, J. M. Herbert, C. Moore, H. Douglas, A. Wunderlich, and A. Braude, Proc. Natl. Acad. Sci. USA 82:1790-1794, 1985) and provided significant protection when administered to patients with gram-negative bacteremia and shock (E. J. Ziegler, C. J. Fisher, Jr., C. L. Sprung, R. C. Straube, J. C. Sadoff, G. E. Foulke, C. H. Wortel, M. P. Fink, R. P. Dellinger, N. N. H. Teng, I. E. Allen, H. J. Berger, G. L. Knatterud, A. F. LoBuglio, C. R. Smith, and the HA-1A Sepsis Study Group, New Engl. J. Med. 324:429-436, 1992). Since that original report, questions have arisen in the scientific literature concerning the specificity of this antibody in LPS and/or lipid A binding. Experiments have, therefore, been carried out with a variety of assay formats to determine the capacity of this HA-1A antibody to bind to lipid A and LPS. Direct binding experiments with a sensitive enzyme-linked immunosorbent assay (ELISA) system have established that HA-1A will bind to purified lipid A from both Escherichia coli and Salmonella spp. These results have been confirmed by using a fluid-phase antigen-antibody competitive inhibition assay with purified lipid A and an antibody-antibody competitive inhibition assay with a monoclonal antibody with known specificity for lipid A. The HA-1A monoclonal antibody has also been shown to bind to a panel of R-chemotype LPS by ELISA and, unlike many other previously reported anti-lipid A antibodies, binding of HA-1A to R-chemotype LPS and lipid A is comparable. Although binding of HA-1A to S-LPS (smooth, wild-type LPS) could not be detected by direct ELISA, competitive inhibition experiments with some preparations of S-LPS have been able to show specific HA-1A binding. Collectively, these data confirm the binding specificity of HA-1A for the lipid A component of LPS and provide evidence that this monoclonal antibody manifests a relatively uncommon profile in its capacity to bind lipid A and R-chemotype LPS as well as some preparations of S-LPS.

[Clinical evaluation of stent placement for tracheal and bronchial stenosis].

Expandable metallic stents were successfully introduced in 7 patients, including 4 with left main bronchial stenosis caused by bronchopulmonary tuberculosis, 2 with main bronchial stenosis caused by lung cancer and one with tracheal stenosis caused by adenoid cystic carcinoma. The length of stenosis was 1.5-5 cm. The stents were 1.5-2.5 cm long with barbs, and their full expanded diameter was 1.5 cm. Balloon dilatation was performed before stenting in all cases. The stents were inserted by using a 10-12 Fr catheter. In all patients except the one with tracheal stenosis, stents were introduced under local anesthesia without any difficulties. No migration of stents occurred. After stent placement, there were no respiratory difficulties, and radionuclide lung perfusion scan and chest radiographic findings such as lung atelectasis showed marked improvement in three cases. Combined therapy of stent placement and bronchial arterial infusion chemotherapy showed marked effectiveness in one case with lung cancer. Expandable metallic stents were very useful in eliminating tracheobronchial stenosis symptoms.

[Experimental study of water jet angioplasty].

A newly invented angioplasty by using water jet energy was investigated for evaluating its safety and effectiveness in 3 dogs. The water jet was produced from a small tip nozzle (0.2 mm in diameter) of catheter by injecting 20 ml of diluted contrast medium. By using this method recanalization of femoral arterial occlusion produced by fresh thrombi was achieved in all 3 dogs. Post recanalization angiography showed no apparent small vessel occlusions. Injection apart more than 5 mm from the inner surface of human aorta provoked no apparent changes histopathologically. Water jet angioplasty may be useful in treating vascular occlusive disease.

Synergistic action of vitamin E and vitamin C in vivo using a new mutant of Wistar-strain rats, ODS, unable to synthesize vitamin C.

It is well known that vitamins E and C exhibit synergistic action in in vitro systems, but with regard to in vivo systems, much of the available data are confusing. To elucidate this problem we used a new mutant of Wistar-strain rats that cannot synthesize vitamin C, namely, ODS rats. Two experiments were planned: (1) during development of vitamin E deficiency, whether vitamin C could spare the consumption of vitamin E; and (2) under conditions of a regular level of vitamin E intake, whether different dose levels of vitamin C can affect vitamin E concentration in tissues. The results obtained show that with vitamin C intake, higher levels of vitamin E were deposited in tissues in both experiments. With the development of vitamin E deficiency, rats in the group with a higher dose of vitamin C deposited higher concentrations of alpha-tocopherol. With simultaneous administration of vitamin E and vitamin C to the same mutant rats, the rats in the group with a higher dose of vitamin C deposited higher levels of vitamin E in all tissues tested. Thus, we concluded that vitamin C can spare the consumption of vitamin E in vivo as well as in vitro.

[Experimental study of endoesophageal and intestinal expandable metallic stent placement].

Gianturco expandable metallic stents were implanted into the esophagus and small intestine of 10 rabbits in order to evaluate the influence of wire stents on the gastrointestinal tract. The stents were constructed of 0.010 inch stainless steel wire. The relaxed diameter of the stents was 12-14 mm and the length was 10 mm. Except for one stent placed in the small intestine, the stents did not migrate and were covered with thickening mucosal epithelium. The mucosal inflammatory changes were slight, but severe intestinal adhesions were noted. The findings in the two groups were not significantly different at three and six weeks. Five rabbits died within three weeks of intestinal disorders caused by severe intestinal adhesions and/or perforations. The experimental data showed that implantation of metallic wire stents into the gastrointestinal tract resulted in severe damage to the esophagus and small intestine of rabbits.

[Clinical experience of percutaneous transluminal angioplasty and expandable metallic stent placement for Budd-Chiari syndrome with massive thrombus in the inferior vena cava; report of a case].

A 72-year-old female who had suffered from lower extremity edema and liver cirrhosis due to Budd-Chiari syndrome with massive thrombus in IVC was treated by PTA and placement of self-expandable metallic stent. Thrombolysis therapy after PTA was very effective. Five months later, she had no clinical symptom due to IVC stenosis.

[Experimental study of the intraurethral placement of expandable metallic stents].

Various types of Gianturco expandable metallic stents were implanted into the urethra of four dogs in order to evaluate the suitable expandability of the stents in urethra and also to determine the effect of the stents on the urethral wall. The stent of 1.5 cm in length and constructed of 0.010 inch round stainless steel wire with twelve zigzag bends showed the suitable expandability on the canine urethra compared to the other stents. The urethra remained patent and the inflammatory changes on the urethral wall were moderately noted. No hematuria or calcifications around the stents were noted in any dogs. The experimental data showed a potential clinical application.

[In vitro study of radiofrequency thermal angioplasty].

We performed some in vitro experiments using a radiofrequency thermal angioplasty instrument. The distribution of heat emitted from the hot tip of the device was evaluated in an agar phantom by thermography and it produced a concentric circular pattern. The temperature of the hot tip was about 290 degrees C when measured by thermocouples in the air. When the RF probe approached perpendicularly to the cadaver arterial wall, a crater with charring and coagulating necrosis was formed. However, when the RF probe traveled parallel to the arterial wall, as in normal practice, it did not seem to perforate the artery. The device could ablate non-calcified atheromatous plaque.