Establishment of an IL-12-responsive T cell clone: its characterization and utilization in the quantitation of IL-12 activity.

We previously demonstrated that proliferation of terminally differentiated Th1 clones depends primarily on an interleukin-12 (IL-12)-paracrine mechanism mediated by their interactions with antigen-presenting cells (APC) rather than on an IL-2-autocrine mechanism. Such a Th1 clone (4-86, C57BL/6 origin) was cultured with recombinant IL-12 (rIL-12) in the absence of either antigen or APC. Some cells survived for several passages of culture with only rIL-12, and by limiting dilution, several clones highly reactive to rIL-12 alone were obtained. One of these clones, designated 2D6, was found to proliferate strongly in response to less than 1 pg/mL of rIL-12. This clone exhibited the following surface phenotypes: CD3+, T cell receptor (TCR) alpha beta+, Vbeta11+, NK-1.1-; CD4-CD8-; LFA-1+, ICAM-1+; and CD28+, CD80+, CD86+, CTLA-4-. In accordance with high responsiveness to IL-12, 2D6 cells were also found to express IL-12 receptor (IL-12R) as detected by incubation with rIL-12 and then staining with anti-IL-12 monoclonal antibody (mAb). Stimulation of 2D6 with rIL-12 resulted in the expression of interferon-gamma (IFN-gamma) and IL-10 mRNAs and production of these cytokines. The 2D6 clone responded to IL-2 (vigorously), IL-7 (moderately), and IL-4 (mildly) in addition to IL-12. However, the Ab capture assay using anti-IL-12 mAb enabled us to quantify IL-12-specific activity contained in a given sample. Thus, this study describes the unique features of the IL-12-responsive T cell clone and demonstrates the utilization of this clone in the quantitation of a specific IL-12 activity.

Regulation of T cell-dependent and -independent IL-12 production by the three Th2-type cytokines IL-10, IL-6, and IL-4.

The production of IL-12 by macrophages/dendritic cells (Mphi/DC) is mediated either by a T cell-dependent pathway that is induced primarily by the interaction of CD40 ligand (CD40L) on activated T cells with CD40 on IL-12-producing cells or by a T cell-independent pathway that is induced by bacteria or bacterial products and enhanced by interferon-gamma (IFN-gamma). In this study we investigated the ability of the Th2-type cytokines interleukin (IL)-10, IL-6, and IL-4 to modulate IL-12 production in Mphi/DC induced through the two pathways. IL-12 production was induced in Mphi/DC from normal mice by stimulation with the combination of IFN-gamma plus lipopolysaccharide (LPS) or Staphylococcus aureus Cowan I (a model for the T cell-independent pathway) or by co-culture with Chinese hamster ovary (CHO) cells transfected with the CD40L (a model for the T cell-dependent pathway). The effects of three Th2-type cytokines on IL-12 production by Mphi/DC through the two pathways were examined. IL-10 inhibited IL-12 production induced through both pathways, although the inhibitory effect was more potent on the (IFN-gamma + LPS)-induced pathway. IL-6 inhibited only (LPS + IFN-gamma)-induced IL-12 production. The effect of IL-4 was particularly noteworthy: this cytokine inhibited (LPS + IFN-gamma)-induced IL-12 production, whereas it potentiated the production of IL-12 induced by CD40L. Regulation of IL-12 protein production by IL-10 and IL-4 was found to correspond to the levels of mRNA accumulation for the p40 and p35 IL-12 genes, whereas the presence of IL-6 during stimulation decreased IL-12 protein production without affecting steady-state mRNA levels. These results indicate that IL-12 production in Mphi/DC induced through a T cell-dependent or -independent pathway is positively or negatively regulated by particular cytokines at various control levels.

Dynorphin mRNA expression in dorsal horn neurons after traumatic spinal cord injury: temporal and spatial analysis using in situ hybridization.

Dynorphin, an endogenous opioid, may contribute to secondary nervous tissue damage following spinal cord injury. The temporal and spatial distribution of preprodynorphin (PPD) mRNA expression in the injured rat spinal cord was examined by in situ hybridization. Rats were subjected to traumatic spinal cord injury at the T13 spinal segment using the weight-drop method. Motor function of these rats was evaluated by their ability to maintain their position on an inclined plane. Two double-labeling experiments revealed that increased PPD mRNA and dynorphin peptide expression were found exclusively in dorsal horn neurons. Neurons exhibiting an increase in the level of PPD mRNA were concentrated in the superficial laminae and the neck of dorsal horn within several spinal segments from the epicenter of the injury at 24 and 48 h after injury. A number of neurons showing increased PPD mRNA were found in gray matter adjacent to the injury areas. Segments caudal to the injury site exhibited a long-lasting elevation of PPD mRNA in neurons, compared to the rostral segments. The number of neurons expressing PPD mRNA in each rat was significantly positively correlated with its motor dysfunction. These findings suggest that increased expression of dynorphin mRNA and peptide in dorsal horn neurons occurs after traumatic spinal cord injury. This also supports the hypothesis that the dynorphin has a pathological role in secondary tissue damage and neurological dysfunction after spinal cord injury.

Changes in the axonal membrane potential and Ca2+ concentration associated with peripheral nerve grafting after spinal cord injury.

We performed peripheral nerve allografting in rats with spinal cord injury, and measured motor function and axonal membrane potential as well as Ca(2+) concentration of the nerve grafting spinal cord area by using a behavior observation system and a confocal laser-scanning microscope, respectively. In our experiments, we produced a model of peripheral nerve grafting after spinal cord injury by peripheral nerve allografting (sciatic nerve) in rats with spinal cord injury (thoracic cord hemisection). The group with spinal cord injury that underwent peripheral nerve grafting showed improvement in motor function, a significant increase in the axonal action potential, and a slight increase in the Ca(2+) concentration compared with the group that did not undergo nerve grafting.

Bone and soft tissue tumours of the foot: review of 83 cases.

PURPOSE: To review cases of bone and soft tissue tumours of the feet managed at the Hyogo College of Medicine, Nishinomiya and Takarazuka Municipal Hospital, Takarazuka, Japan. METHODS: Retrospective analysis of 83 patient records treated for bone and soft tissue tumours of the feet between 1974 and 2000. RESULTS: There were 33 benign bone tumours, one primary malignant bone tumour, and 2 metastatic bone cancer. Marginal resection was performed in cases of osteochondroma and curettage in cases of other benign bone tumour. Despite below-knee amputation in the case of chondrosarcoma, the patient died because of pulmonary metastasis. Two patients with metastatic cancer also died, and 2 cases of osteochondroma and one of benign chondroblastoma recurred. There were 47 cases of soft tissue tumour. Treatment for benign soft tissue tumours was marginal resection; no cases recurred. In contrast, all patients with soft tissue sarcoma died after surgery. The majority of bone tumours were located in the toe and hindfoot areas, in the first and second decades of life, whereas soft tissue tumours occurred mainly in the midfoot area and in patients aged between 20 and 50 years. The sex distribution was almost even for bone tumours (male: female ratio, 19:17), whereas about half as many males as females had soft tissue tumours (14:33). CONCLUSION: Bone and soft tissue tumours of the feet are uncommon. Most bone tumours are chondrogenic, but differential diagnosis of malignant from benign disease is difficult and prognosis is poor. Management of benign tumours by marginal resection has good prognosis, whereas prognosis of soft tissue sarcomas is very poor.

[Population pharmacokinetic analysis of indocyanine green retention rate at 15 minutes].

Using the population pharmacokinetic (Bayesian) method, we investigated the indocyanine green (ICG) retention rate at 15 minutes (R 15) in patients with liver disease. For the Bayesian analysis, the mean and variance of parameters, the disappearance rate (K) and the distribution volume, were calculated by the one compartment analysis with data of inpatients. The mean value of the disappearance rate (K) was shown to be smaller and the variances of parameters of the patients were larger than normal values reported, so that it was suggested that the studied group included patients with various kinds of liver diseases. Accordingly, the Bayesian analysis was performed using above statistical results. The distribution volumes, calculated from three points measurements by Bayesian method, were estimated smaller and the initial concentrations higher than those obtained by the one compartment analysis. However, the retention rate (R 15) were well correlated the results of one compartment analysis. The Bayesian analysis using one point measurement at 15 minutes, resulted that the values of retention rate (R 15) were shown about 17% lower, but well correlated with the coefficient gamma = +0.9972 to those obtained by usual method. The disappearance rate (K), which calculated simultaneously from the one point analysis, showed the lowest value on liver cirrhosis. So that, the application of Bayesian analysis to the one point measurement of ICG test was useful to clinical evaluation of liver disease.

Ceramic unicompartmental knee arthroplasty for spontaneous osteonecrosis of the knee joint.

Unicompartmental replacement with a ceramic knee prosthesis was performed in 10 patients with spontaneous osteonecrosis. The follow-up period averaged 42 months, with a range of 24 to 68 months. All of the knees received a Kyocera unicompartmental knee arthroplasty and all patients were evaluated using The Knee Society Clinical Rating System. The mean knee score increased from 50.6 to 90.8 at the last follow-up, showing no deterioration with time. All of the patients showed marked improvement of pain and excellent or good results were obtained in all cases. None of the patients had progressive radiolucency wider than 2 mm at the final follow-up. Ceramic unicompartmental knee arthroplasty is an acceptable treatment for spontaneous osteonecrosis of the knee joint; a satisfactory postoperative range of motion can be achieved, there are few complications, and a high initial success rate.

Clinical experience of ceramic cementless total knee arthroplasty in RA and a histological study of the bone-ceramic interface in revision cases.

The Kyoto total knee cementless prosthesis (KC-1) was used in 23 patients with rheumatoid arthritis. Its femoral component is made of alumina ceramics which articulated with a HDP plate supported by a ceramic plate. Although the followup period is short, the postoperative result is not satisfactory, because remarkable sinking of the tibial component was noticed in those patients who have bone atrophy. We had 6 cases of revision so far. Since 1986, we have been using bone cement in all cases of RA in doing TKA.

Spinal fractures resulting from the 1995 Great Hanshin Earthquake of the Kobe-Osaka area of Japan.

One of the worst earthquakes hit the Hanshin area between Kobe and Osaka, in the early morning at 5:46 AM on January 17th 1995. The destructive force with MG 7.2 severely damaged buildings, houses, roads and railways, leaving 6500 dead, and 34,900 injured. Hyogo College of Medicine located in this area was also severely damaged, thus there was a major challenge to provide post-quake medical support. A post-quake investigation in this area was done by 50 affiliated hospitals. More than 15,000 victim-patients were treated at these hospitals during the first 3 days after the quake. Major injuries were spinal fractures, and other trunk fractures, including rib or pelvis fractures, but fractures of long bones were uncommon, because the quake hit this area in the early morning when most people were asleep. In this study, the mechanisms of these major injuries were analysed by direct interview soon after the quake, with 230 victim-patients who had 140 spinal fractures, and 100 with rib or pelvis fractures. Most of those who had a spinal fracture had either sat up or stood up on their 'Futon' mattresses without bed frames and were struck on their backs by falling furniture or ceilings. On the other hand, patients who had fractures of the ribs or the pelvis had been lying in the supine or lateral position and were hit on their chest or pelvis. This characteristic lifestyle pattern of the Japanese people to lie down on the floor directly beside furniture, resulted in these injuries. From these results, we will emphasize the following precautions:- If an earthquake occurs during sleeping hours at home, do not stand up or sit up. The best position is to crouch on the 'Futon' mattress.

[Paraplegia caused by vertebral compression fractures in senile osteoporosis]. / Paraplegie infolge von Wirbelkompressionsfrakturen bei seniler Osteoporose.

Paraplegia followed spontaneous deformed osteoporotic spine is very rare. Our own 3 cases treated conservatively in two and operatively in one with success and 11 cases reported in the literatures were analyzed. All patients were recovered from paraplegia. One patient was died one and a half year after the recovery of her paralytic episode. Thorough autopsy study of this patient showed no organic lesion in the regarded spinal cord due to such kind of spine deformity.

Replacement of the Epstein-Barr virus plasmid with the EBER plasmid in Burkitt's lymphoma cells.

Transfection of an Epstein-Barr virus (EBV)-encoded plasmid containing EBER caused a substantial decrease in the level of plasmid containing EBV in Akata and Mutu Burkitt's lymphoma (BL) lines, but failed to do so in other BL lines. The results suggest that EBER could replace the role of EBV, but other EBV products also play a role in the growth of BL.

CD21-mediated entry and stable infection by Epstein-Barr virus in canine and rat cells.

We developed an adenovirus vector for transduction of the human CD21 gene (Adv-CD21), the Epstein-Barr virus (EBV)-specific receptor on human B lymphocytes, to overcome the initial barrier of EBV infection in nonprimate mammalian cells. Inoculation of Adv-CD21 followed by exposure to recombinant EBV carrying a selectable marker resulted in the successful entry of EBV into three of seven nonprimate mammalian cell lines as evidenced by expression of EBV-determined nuclear antigen (EBNA). The EBV-susceptible cell lines included rat glioma-derived 9L, rat mammary carcinoma-derived c-SST-2, and canine kidney-derived MDCK. Subsequent selection culture with G418 yielded drug-resistant cell clones. In these cell clones, EBV existed as an episomal form, as evidenced through the Gardella gel technique. Among the known EBV latency-associated gene products, EBV-encoded small RNAs, EBNA1 and transcripts from the BamHI-A rightward reading frame (BARF0), and latent membrane protein 2A were expressed in all EBV-infected cell clones. The viral lytic events could be induced in these cell clones by simultaneous treatment with 12-O-tetradecanoylphorbol-13-acetate and n-butyric acid, but they were abortive, and infectious virus was not produced. These results indicate that once the initial barrier for attachment is overcome artificially, EBV can establish a stable infection in some nonprimate mammalian cells, and they raise the possibility that transgenic animals with the human CD21 gene could provide an animal model for EBV infection.

Three-dimensional microfabrication with two-photon-absorbed photopolymerization.

We propose a method for three-dimensional microfabrication with photopolymerization stimulated by two-photon absorption with a pulsed infrared laser. An experimental system for the microfabrication has been developed with a Ti:sapphire laser whose oscillating wavelength and pulse width are 790 nm and 200 fs, respectively. The usefulness of the proposed method has been verified by fabrication of several kinds of microstructure by use of a resin consisting of photoinitiators, urethane acrylate monomers, and urethane acrylate oligomers.

Evanescent-wave holography by use of surface-plasmon resonance.

We report a method for evanescent-wave holography using surface-plasmon resonance from the illumination light. The device we have made consists of three layers: a prism of high refractive index, a thin metallic film, and a grating. Evanescent waves generated by the surface plasmons are diffracted with a prerecorded grating to reconstruct a three-dimensional image. The possibility of white-light illumination and the application to a flat display system with waveguides in the proposed method are discussed.

Multibeam scanning optics with single laser source for full-color printers.

In the novel optical system described here, four-color toners can be developed in one rotation of the photoconductor, and the color control information is given when the intensities of the laser power levels are changed and the two polarization directions are switched. A polarizing beam splitter between the common scanning optics and the photoconductor enables the laser beam to pass through a common scanning system and to illuminate two positions on the photoconductive material. The laser beam polarization direction is controlled by an electro-optical device immediately behind the laser. In each illuminated position, two-color toners are developed by a three-level (trilevel) photographic process. This simplified optical system eliminates the registration errors that occur with four-color information items and can be useful in high-speed printing systems.

Immunocytochemical localization of a cell adhesion molecule, integrin alpha5beta1, in nerve growth cones.

It is well-known that nerve growth cones, the growing tips of nerves, play a central role in determining the direction taken by regenerating peripheral nerves though neurotropism and contact guidance. In order to identify the molecules expressed on growth cones that are responsible for contact guidance, we investigated the possible involvement of integrins as sensors for the extracellular matrix. In cultured rat PC-12i cells and chick dorsal root ganglion cells, we found that integrin alpha5beta1 was concentrated in the filopodia and central regions of the growth cones. These integrin alpha5beta1-rich regions corresponded well with the sites where the growth cones came into contact with and adhered to the extracellular matrix. Integrin alpha5beta1 has been reported to bind with fibronectin in the extracellular matrix. When examined by triple staining and confocal laser scanning microscopy, we found that the distribution of integrin alpha5beta1 in the growth cones was very similar to that of actin filaments. Integrin alpha5beta1 was also expressed by Schwann cells. On immuno-electron microscopy, integrin alpha5beta1 was also identified in regenerating axons in vivo. These results suggest that integrin alpha5beta1 expressed on growth cones may function as a sensor for the extracellular matrix and Schwann cells, and thus mediate functionally important interactions in the development and regeneration of peripheral nerves.

[The pathologico-anatomic study of the metastatic tumor of the spine (author's transl)].

The radiological and histological studies were performed on the three spinal column obtained at autopsy from cases with metastatic tumors to the spine with complete paralysis treated surgically. These three cases consisted of a maxillary carcinoma with metastasis to the cervical spine, a seminoma with metastasis to the thoracic spine and a metastasized renal cell carcinoma to the thoracic spine. All patients died without recovery from the paralysis, in spite of the immediate tumor resection and spinal fusion were done immediately after the appearance of complete paralysis. The histological examination of cross section of these spinal column showed that tumor cells infiltrated in the vertebral body and spinal canal, and spinal cord was remarkably compressed by the tumor. Therefore, various degrees of degeneration were seen in the spinal cord. In addition, many tumor-cell emboli were seen in veins of vertebral body and spinal canal in two cases, and also tumor cell infiltration to an intradural space and marked deformity of the spinal cord were seen in one case. These above findings would suggest that tumor cells, which metastasized to vertebral body through venous plexus from the primary lesion, infiltrated into the spinal canal, compressed spinal cord and caused paralysis.

Biomechanical behavior of the posterior elements of the lumbar spine under loading--a study of stress analysis by means of three dimensional photoelastic experiments.

To investigate the biomechanical behavior of the lumbar posterior elements under loading, stress analysis was carried out. The three dimensional photoelastic method was used for stress analysis in this study. The forces applied to the superior and the inferior articular facets were concentrated on the pars interarticularis and transmitted to the pedicle. However, almost no stress was generated in the lamina. Highly stressed areas were found in the pars interarticularis and around the lateral recess. The highest compressive stress was present in the pars interarticularis. The deformation pattern of the lumbar spinal canal was recognized and it became flat as the lateral recess was narrowed and deepened. These results very interestingly correspond to the fact that spondylolysis and lumbar canal stenosis are found in the above-mentioned areas clinically.