Suzuyo Ohashi

Sequence‐specific gene silencing in murine muscle induced by electroporation‐mediated transfer of short interfering RNA

Post‐genomic biomedical research requires efficient techniques for functional analyses of poorly characterized genes in living organisms. Sequence‐specific gene silencing in mammalian organs may provide valuable information on the physiological and pathological roles of predicted genes in mammalian systems. Here, we attempted targeted gene knockdown in vivo in murine skeletal muscle through the electroporation‐mediated transfer of short interfering RNA (siRNA).

Successful genetic transduction in vivo into synovium by means of electroporation

This present study aims at establishing a novel in vivo gene delivery system for intra-articular tissues. Plasmid DNA (pDNA) carrying the firefly luciferase or enhanced green fluorescent protein (EGFP) genes as markers was injected into a joint space and electric stimuli were given percutaneously with a pair of electrodes. Injection with naked pDNA alone did not induce any detectable level of luciferase activity, whereas electroporation at 25-500 V/0.7 cm resulted in a significant expression of the marker gene in the synovium. The expression level depended on the voltage, the optimum transfection being achieved at 150 V/0.7 cm. When the Epstein-Barr virus (EBV)-based plasmid vectors harboring the EBV nuclear antigen 1 (EBNA1) gene and oriP sequence were substituted for conventional pDNA, the transfection efficiency was increased approximately 5-10 times. Histological examination of the EGFP gene-transfected joints revealed that the marker gene was expressed in the synovial membrane while other intra-articular tissues such as articular cartilage were negative for the transgene product. Transgene-specific mRNA was demonstrated in synovium but not in other organs as estimated by RT-PCR analysis. The present results strongly suggest that in vivo electroporation is a quite simple, safe, and effective gene delivery method that could be applicable to gene therapy against articular diseases.

Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein-Barr virus (EBV)-based plasmid vectors.

Naked plasmid DNA (pDNA) injection could become an alternative procedure to viral and nonviral gene delivery systems. We have previously shown that Epstein–Barr virus (EBV)-based plasmid vectors containing the EBV nuclear antigen 1 (EBNA1) gene and the oriP sequence enable quite high and long-lasting expression in various in vitro and in vivo transfection systems. The EBV-based plasmids were intravenously injected into mice via their tail vein under high pressure. A large amount of the marker gene product was expressed in the liver; as much as 320 μg of luciferase was demonstrated per gram of liver at 8 to 24 h after a single injection with 10 μg of DNA. More than 70% of liver cells stained with X-gal when β-gal gene was transferred. The expression level was significantly higher than that obtained by conventional pDNA lacking the EBNA1 gene and oriP. On day 35 after the transfection, the expression from the EBV-based plasmid was approximately 100-fold stronger than the conventional pDNA gene expression. Both the EBNA1 gene and oriP are a prerequisite for the augmentation of the transfection efficiency. These results suggest that the intravascular transfection with naked EBV-based plasmid may provide a quite efficient, simple and convenient means to transduce therapeutic genes in vivo into the liver. Gene Therapy (2001) 8, 1508–1513.

Alteration of Hindfoot Alignment After Total Knee Arthroplasty Using a Novel Hindfoot Alignment View

This study examined the coronal alignment of the hindfoot in varus osteoarthritis of the knee before and after total knee arthroplasty (TKA) in 100 legs using a novel imaging method. We categorized the preoperative hindfoot alignment into varus (30 legs) and valgus (70 legs) groups; imaging of the hindfoot was conducted preoperatively and postoperatively, and the varus-valgus angle (VVA) was measured as the hindfoot alignment. The femorotibial angle improved significantly after TKA. We found that the VVA improved significantly after TKA in the hindfoot valgus group (P<0.001), but not in the varus group (P=0.554), and we speculate that the hindfoot alignment in the valgus group improved as a result of a residual compensatory capacity in the hindfoot.

Interleukin 8 is produced by hydrostatic pressure in human osteoblast cell line, MG-63

Abstract To investigate the effects of hydrostatic pressure (HP) to cytokine productions, the osteoblast cell line, MG-63, was cultured in dishes until it reached a confluent level and then exposed to 1, 10 or 50 MPa of HP for 2 h in an apparatus which can load pressure under sterile conditions. At 12 and 24 h after giving the exposure, interleukin (IL)-1 α , IL-1 β , IL-6, IL-8, TNF- α , IFN- α and IFN- γ levels in culture supernatants were measured with ELISA. The same procedure was repeated for the human fibroblast cell line, TIG-3, as the control. After the HP exposure, levels of all the cytokines in MG-63, except IL-8, were lower than 20 pg/ml and immeasurable. However, at 12 and 24 h after exposure to 50 MPa, IL-8 levels increased significantly compared to the levels in the cells without HP and this increase was confirmed by Northern blot hybridization. In the control TIG-3 cells, levels of all the cytokines after HP exposure were lower than 20 pg/ml and immeasurable. Because high HP induced IL-8 production in the osteoblast-like cell line, mechanical stress could induce inflammatory reaction in bone tissues and then result in bone destruction.

A kinematic and kinetic analysis of the hip and knee joints in patients with posterior tibialis tendon dysfunction; comparison with healthy age-matched controls

BACKGROUND The biomechanical abnormalities in patients with posterior tibial tendon dysfunction (PTTD) have been described, but few studies have investigated biomechanical chains of adjacent joints. Therefore, we examined the gait pattern of the lower extremity in subjects with PTTD, focusing on the hip and knee joints. METHODS We compared 19 PTTD patients (average age: 67.1) with 30 age-matched control subjects (average age: 65.1). Gait analysis was performed with a nine-camera motion-capture system and four force plates, using the Vicon Plug-In-Gait and Vicon Nexus software. Temporal-spatial parameters were compared between PTTD and control subjects, and motion and ground reaction force data were compared between the affected limb, the contralateral limb, and the right limb in control subjects. RESULTS Subjects with PTTD had increased stance phase ratio and decreased stride length, cadence, and gait speed. The limbs of subjects with PTTD showed increased knee internal rotation at lording response, which was biased to abduction in the knee joint during the gait cycle, and irregular hip flexion and knee extension moment in the terminal stance, even under control of gait speed. SIGNIFICANCE We believe that the subjects with PTTD have an increased risk of knee osteoarthritis in both the affected and contralateral limbs.

Magnetic resonance imaging findings of chronic plantar fasciitis before and after extracorporeal shock wave therapy

INTRODUCTION The objective of this study is to examine the relationships between treatment outcome and changes in magnetic resonance (MR) imaging findings after extracorporeal shock wave therapy (ESWT) for chronic plantar fasciitis. METHODS The subjects were 23 feet of 23 patients of refractory plantar fasciitis. The mean age was 53.7 years. The thickness of the plantar fascia (PF) and findings of a high-signal intensity area (HSIA) inside the PF, edema around the PF, and bone marrow edema (BME) of the calcaneus were investigated on MR images. The Japanese Society for Surgery of the Foot (JSSF) ankle-hindfoot scale and a visual analogue scale (VAS) were used. Correlations between an improvement in symptoms and one in the MRI findings were analyzed. RESULTS The mean thickness of the PF was 4.4±1.6mm before ESWT and 4.6±1.8mm six months after ESWT. After ESWT, there was a decrease in the numbers of feet showing HSIA inside the PF from 15 to 6, in edema around the PF from 16 to 2, and in BME of the calcaneus from 11 to 4. Clinical outcomes improved with ESWT from 70.3±5.5 to 88.6±9.1 points (JSSF), 74.1±25.3 to 28.5±24.4 points (VAS), respectively. Improvements in symptoms according to the JSSF and VAS scores and improvement in edema around the PF on MR images showed a significant correlation. CONCLUSIONS Edema around the PF improved significantly in association with an improvement in symptoms after ESWT.

Examination of the Correlation Between Foot Morphology Measurements Using Pedography and Radiographic Measurements

Abstract Pedography provides excellent visualization of the footprint. However, the correlation between the footprint images and radiographic measures has not been thoroughly evaluated. Therefore, the objectives of our study were to examine the correlation between the pedography‐based measures of foot morphology and radiographic measurements and to propose reference values for the diagnosis of flatfoot using footprint imaging. The plantar footprints of 100 right feet were photographed using a pedography standing platform. The sole and arch areas were measured to calculate the footprint index (FPI). The lateral talar–first metatarsal angle (LTM) and calcaneal pitch angle (CP) were measured on standing lateral radiographs, and the talonavicular coverage angle was measured on frontal radiographs. The Pearson moment correlation between the FPI and radiography‐based measures was calculated. The area under the receiver operating characteristic curve was calculated using an LTM of <−4° as the identifying criterion of flatfoot. The sensitivity and specificity of FPI were calculated for LTM values <−4°. The FPI correlated with the LTM (y = −17.964 ± 52.644x, R = 0.588) and CP (y = 9.2304 ± 27.739x, R = 0.659) but not with the talonavicular coverage angle (y = 26.01 ± 15.78x, R = 0.207). The area under the receiver operating characteristic curve was 0.753, with a cutoff FPI of 0.208, yielding a sensitivity of 0.462 and specificity of 0.934 for flatfoot identification. Pedography could provide an easy screening tool for flatfoot, with an FPI cutoff of 0.208, yielding a specificity of 93.4%. &NA; Level of Clinical Evidence: 3

Relationship Between Grading With Magnetic Resonance Imaging and Radiographic Parameters in Posterior Tibial Tendon Dysfunction

ABSTRACT The objective of the present study was to elucidate the relationship between the state of the posterior tibial tendon (PTT) on magnetic resonance images and foot deformity. The cases included 34 feet in 27 patients with PTT deformity and the controls included 18 feet in 12 patients who had undergone magnetic resonance imaging for other foot diseases. The PTT was closely examined on the magnetic resonance images and classified using the Conti classification. The control feet with no injury to the PTT were classified as grade 0. The talonavicular coverage angle, lateral talo‐first metatarsal angle, medial cuneiform to fifth metatarsal height, calcaneal pitch angle, and varus–valgus angle were measured as radiographic parameters for flatfoot deformation, and the relation between the Conti classification and each parameter was examined statistically. A significant difference was observed in the talonavicular coverage angle between grade 0 and the other grades; the lateral talo‐first metatarsal angle between grade 0 and the other grades and between grades 1 and 3; the medial cuneiform to fifth metatarsal height among grades 0, 2, and 3 and grades 1, 2, and 3; the calcaneal pitch angle between grades 1 and 3; and the varus–valgus angle among grades 0, 2, and 3 and between grades 1 and 3. Eversion of the forefoot was observed, along with an advanced collapse in the medial longitudinal arch, from an early stage of PTT injury. &NA; Level of Clinical Evidence: 4