Hideo Tsurushima

Calcium phosphate composite layers for surface-mediated gene transfer

In this review, the surface-mediated gene transfer system using calcium phosphate composite layers is described. Calcium phosphate ceramics are osteoconductive bioceramics used typically in orthopedic and dental applications. Additionally, calcium phosphate particles precipitated by a liquid-phase process have long been used as a safe and biocompatible transfection reagent in molecular biology. Recently, calcium phosphate composite layers immobilizing DNA were fabricated on the surfaces of base materials through a biomimetic process using supersaturated solutions. These composite layers possess useful characteristics of both osteoconductive bioceramics and transfection reagents; they thus provide a biocompatible surface to support cell adhesion and growth, and can stimulate the cell effectively via surface-mediated gene transfer. By modifying the fabrication conditions, physicochemical and biological properties of the composite layers can be varied. With such an approach, these composite layers can be designed to have improved affinity for cells and to exhibit increased gene transfer efficiency over that of conventional lipid transfection reagents. The composite layers with the increased gene transfer efficiency induced specific cell differentiation and tissue regeneration in vivo. These composite layers, given their good biocompatibility and the potential to control cell behavior on their surfaces, have great potential in tissue engineering applications.

Reduction of end-stage malignant glioma by injection with autologous cytotoxic T lymphocytes.

Autologous cytotoxic T lymphocytes (CTL) against primary‐cultured malignant gliomas were generated from peripheral blood mononuclear cells in vitro in 4 patients. Activities of the CTL were highly specific to the corresponding autologous glioma and were inhibited, in one patient, with antibodies against CD3, CD8 and MHC‐class I molecules. When the CTL were injected 3 times into the primary‐tumor‐resected cavity via an Ommaya tube, reduction of the recurrent tumors with magnetic resonance imaging (MRI)‐measured volumes exceeding 45 cm3 was observed in 3 patients. In a patient with glioblastoma multiforme (GBM), the tumor volume (estimated, 130 cm3) was rapidly reduced to 1/3, although re‐recurrence of the tumor followed 40 days later. A slight but distinct rapid reduction of the tumor volume was observed in another GBM patient and in an anaplastic astrocytoma patient; essentially no change was observed in a further GBM patient. These results suggest that adoptive immunotherapy with autologous CTL will be clinically effective against end‐stage malignant gliomas.

Suprasellar ectopic pituitary adenoma : case report and review of the literature

The occurrence of a totally suprasellar ectopic pituitary adenoma in a 71-year-old man is described. The tumor was attached to the pituitary stalk, extending upward toward the third ventricle. No intrasellar lesion was observed. Histological examination revealed a pituitary adenoma with large numbers of eosinophilic cells with moderate nuclear polymorphism and rare mitosis. Immunohistochemical staining revealed that the tumor cells were strongly positive for anti-adrenocorticotropic hormone antibody. A review of five previously reported intracranial ectopic pituitary adenomas revealed that two were silent corticotropic tumors and two occurred with Cushings syndrome.

Newly Synthesized Radical-Containing Nanoparticles Enhance Neuroprotection After Cerebral Ischemia-Reperfusion Injury

BACKGROUND:Antioxidant nitroxyl radicals such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) have been investigated for their ability to scavenge free radicals produced by ischemia-reperfusion injury. However, the short in vivo half-life and toxicity of TEMPO have limited their clinical application. OBJECTIVE:We developed a core-shell-type nanoparticle, termed a radical-containing nanoparticle (RNP), to deliver nitroxyl radicals with prolonged in vivo half-life and pH-sensitivity. We evaluated the ability of RNP to deliver TEMPO radicals to the ischemic brain and scavenge free radicals in cerebral ischemia-reperfusion injury using rats. METHODS:When RNPs were administrated to middle cerebral artery occlusion rats, the delivery and clearance of RNPs were detected using electron paramagnetic resonance (EPR) assay. The production of superoxide anion in neuronal cells was observed with dihydroethidium staining. The treatment effects were evaluated by measuring the cerebral infarction volumes, lipid peroxidation and protein oxidation, and neurological symptom scoring. RESULTS:The TEMPO radicals contained in RNPs were detected for 6 hours after intravenous administration as a triplet EPR signal in the ischemic brain, and RNPs significantly reduced the production of superoxide anion in neuronal cells compared with saline and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyls (TEMPOL). The infarction volumes of rats treated by RNPs were significantly lower than those of rats treated by saline, micelles, and TEMPOL. In addition, RNP treatment suppressed lipid peroxidation and protein oxidation, and limited the adverse effects of TEMPO radicals such as hypotension. CONCLUSION:RNPs could be a promising neuroprotective agent with their enhanced ability to scavenge free radicals and reduced toxicity.

Formation of a FGF-2 and calcium phosphate composite layer on a hydroxyapatite ceramic for promoting bone formation

Fibroblast growth factor-2 (FGF-2) was immobilized on a hydroxyapatite (HAP) ceramic in supersaturated calcium phosphate solution prepared using solutions corresponding to clinically approved infusion fluids. To avoid the risk of FGF-2 denaturation, FGF-2 immobilization was carried out at 25 degrees C. FGF-2 was successfully immobilized on HAP ceramic surfaces by deposition with calcium phosphate to form a FGF-2 and calcium phosphate composite layer. A maximum of 2.72 +/- 0.01 microg cm(-2) of FGF-2 was immobilized in the composite layer formed on the HAP ceramic under the optimum condition. A FGF-2-immobilized HAP ceramic is likely to have the ability to release a sufficient amount of FGF-2 to promote bone formation. FGF-2 released from a FGF-2-immobilized HAP ceramic maintained its biological activity, since the proliferation of fibroblastic NIH3T3 was promoted. Therefore, the FGF-2-immobilized HAP ceramic is expected to be a useful material for promoting new bone formation.

Enhanced bone formation using hydroxyapatite ceramic coated with fibroblast growth factor-2

Our objective was to develop a bone substitute coated with fibroblast growth factor-2 (FGF-2) that subsequently releases FGF-2. We investigated the use of our system of bone substitutes to induce bone formation. Hydroxyapatite ceramic buttons (HAP-CBs) were coated with FGF-2 by precipitation in supersaturated calcium phosphate solution. HAP-CBs were coated with high or low doses of FGF-2, denoted as FGF-H and FGF-L. The release of FGF-2 from FGF-H and FGF-L was evaluated using its release profile and bioactivity. The efficacy of the subsequent bone formation was quantified using rats with round-shaped bone defects (5mm in diameter) of the right parietal bone. Group 1 was treated only with HAP-CBs, group 2 with HAP-CBs and drops of FGF-2 solution, group 3 with FGF-L and group 4 with FGF-H. To detect the release of FGF-2 in vivo, the expression of bone morphogenic protein-2 (BMP-2) was measured in the defective bone tissue. FGF-2 was released in vitro from FGF-H and FGF-L, and maintained its bioactivity. Rats treated with FGF-L showed better bone formation than rats from the other groups. BMP-2 expression was detected in the defective bone tissues of group 3 at 14 days, which might indicate in vivo FGF-2 release during this period. A specific FGF-2 concentration may be needed for bone formation, and our system can release FGF-2 at adequate concentrations to induce bone formation.

Highly efficient gene transfer system using a laminin–DNA–apatite composite layer

We have recently developed a safe and efficient gene transfer system using a laminin–DNA–apatite composite layer. The objectives of the present study were to fully characterize and optimize the laminin–DNA–apatite composite layer in relation to the efficiency of gene transfer and to demonstrate the feasibility of the composite layer in the induction of cell differentiation.

Magnetic resonance imaging of spinal cord injury without radiologic abnormality

A 3-year-old girl with spinal cord injury without radiologic abnormality (SCIWORA) is reported. Magnetic resonance imaging (MRI) showed an abnormal intensity of the spinal cord at the level of C7 to Th1 in the acute stage and severe atrophic change in the chronic stage. This is the first report of SCIWORA with positive diagnostic imaging. The mechanism of SCIWORA is discussed based on the positive MRI findings.

BMP-2 gene-fibronectin-apatite composite layer enhances bone formation.

BackgroundSafe and efficient gene transfer systems are needed for tissue engineering. We have developed an apatite composite layer including the bone morphogenetic protein-2 (BMP-2) gene and fibronectin (FB), and we evaluated its ability to induce bone formation.MethodsAn apatite composite layer was evaluated to determine the efficiency of gene transfer to cells cultured on it. Cells were cultured on a composite layer including the BMP-2 gene and FB, and BMP-2 gene expression, BMP-2 protein concentrations, alkaline phosphatase (ALP) activity, and osteocalcin (OC) concentrations were measured. A bone defect on the cranium of rats was treated with hydroxyapatite (HAP)-coated ceramic buttons with the apatite composite layer including the BMP-2 gene and FB (HAP-BMP-FB). The tissue concentration of BMP-2, bone formation, and the expression levels of the BMP-2, ALP, and OC genes were all quantified.ResultsThe apatite composite layer provided more efficient gene transfer for the cultured cells than an apatite composite layer without FB. The BMP-2 concentration was approximately 100~600 pg/mL in the cell-culture medium. Culturing the cells on the apatite composite layer for 27 days increased ALP activity and OC concentrations. In animal experiments, the tissue concentrations of BMP-2 were over 100 pg/mg in the HAP-BMP-FB group and approximately 50 pg/mg in the control groups. Eight weeks later, bone formation was more enhanced in the HAP-BMP-FB group than in the control groups. In the tissues surrounding the HAP button, the gene expression levels of ALP and OC increased.ConclusionThe BMP-2 gene-FB-apatite composite layer might be useful for bone engineering.

Application of liposomes incorporating doxorubicin with sialyl Lewis X to prevent stenosis after rat carotid artery injury

Restenosis remains a serious complication that can occur after angioplasty. This study investigated the efficiency of an active targeting chemotherapy using liposomes, including doxorubicin, whose surface was decorated with sialyl Lewis X (SLX) (Dox-Lipo-SLX) to prevent stenosis after angioplasty. Its delivery was controlled via the affinity between SLX and E-selectin proteins, which are expressed on vessel walls with injury. In vitro experiments confirmed the accumulation of doxorubicin as a consequence of Dox-Lipo-SLX adhering to E-selectin-positive cells. Significant doxorubicin accumulation was observed on injured vessel walls in rats treated with Dox-Lipo-SLX. In contrast, there was little accumulation using free doxorubicin or a liposome containing doxorubicin (Dox-Lipo), but without SLX. Rats were assigned to one of four groups: Dox-Lipo-SLX, Dox-Lipo, free doxorubicin, or no treatment. Dox-Lipo-SLX, Dox-Lipo, and free doxorubicin, including a dose of 0.08mg/kg doxorubicin, were intravenously administered three times in each group after angioplasty. The residual lumen area of rats in the group treated with Dox-Lipo-SLX was significantly larger than those in all other groups. These results demonstrate that an active targeting drug delivery system utilizing Dox-Lipo-SLX effectively prevents stenosis after angioplasty.