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.

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.

The role of imaging modalities in the diagnosis of primary dedifferentiated parosteal osteosarcoma.

Dedifferentiated parosteal osteosarcoma (dd-POS) is defined as high-grade sarcomatous components coexisting with low-grade POS components. With regard to the histological diagnosis of dd-POS, the sampling of a small area of dedifferentiation through the densely mineralized POS can be a problem. In this situation, imaging is important to identify the area with the highest possibility of dedifferentiation. We report a patient in whom dedifferentiation was shown by computed tomography (CT) and magnetic resonance imaging (MRI). CT revealed a radiolucency in a highly mineralized area. T2-Weighted MRI showed a relatively high signal intensity, corresponding to the radiolucency, surrounded by a very low signal intensity area. Furthermore, gadolinium-enhanced T1-weighted MRI showed marked enhancement. Based on these imaging techniques, the condition was diagnosed as most likely to be a dd-POS, although a representative sample was not accessible by incisional biopsy. Neoadjuvant chemotherapy was administered, followed by wide resection and adjuvant chemotherapy. Four years after the surgery, partial lobulectomy was required because of a pulmonary metastasis. Three years after the second surgery, the patient remained well without evidence of metastases. Based on the initial diagnosis and, consequently, the optimal treatment of combined chemotherapy and wide resection, our patient showed a good clinical outcome.

Epstein-Barr virus-encoded poly(A)(-) RNA supports Burkitt's lymphoma growth through interleukin-10 induction.

Akata and Mutu cell lines are derived from Burkitt's lymphoma (BL) and retain the in vivo phenotype of Epstein-Barr virus (EBV) expression that is characterized by expression of EBV-determined nuclear antigen 1 (EBNA1), EBV-encoded RNAs (EBERs) and transcripts from the BAM:HI A region (BARF0). We found that EBV-positive Akata and Mutu cell clones expressed higher levels of interleukin (IL)-10 than their EBV-negative subclones at the transcriptional level. Transfection of an individual EBV latent gene into EBV-negative Akata cells revealed that EBERs were responsible for IL-10 induction. Recombinant IL-10 enabled EBV-negative Akata cells to grow in low (0.1%) serum conditions. On the other hand, growth of EBV-positive Akata cells was blocked by treatment either with an anti-IL-10 antibody or antisense oligonucleotide against IL-10. EBV-positive BL biopsies consistently expressed IL-10, but EBV-negative BL biopsies did not. These results suggest that IL-10 induced by EBERs acts as an autocrine growth factor for BL. EBERs, EBER1 and EBER2, are non-polyadenylated RNAs and are 166 and 172 nucleotides long, respectively. The present findings indicate that RNA molecules could regulate cell growth.

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.

Oncogenic role of Epstein-Barr virus-encoded RNAs in Burkitt's lymphoma cell line Akata.

Our previous reports indicated that Epstein-Barr virus (EBV) contributes to the malignant phenotype and resistance to apoptosis in Burkitt's lymphoma (BL) cell line Akata (N. Shimizu, A. Tanabe-Tochikura, Y. Kuroiwa, and K. Takada, J. Virol. 68:6069-6073, 1994; J. Komano, M. Sugiura, and K. Takada, J. Virol. 72:9150-9156, 1998). Here we report that the EBV-encoded small RNAs (EBERs) are responsible for these phenotypes. Transfection of the EBER genes into EBV-negative Akata clones restored the capacity for growth in soft agar, tumorigenicity in SCID mice, resistance to apoptotic inducers, and upregulated expression of bcl-2 oncoprotein that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. This is the first report which provides evidence that virus-encoded RNAs (EBERs) have oncogenic functions in BL cells.

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.

Effects of tension at the site of coaptation on recovery of sciatic nerve function after neurorrhaphy: evaluation by walking-track measurement, electrophysiology, histomorphometry, and electron probe X-ray microanalysis.

The effects of tension at the site of coaptation on recovery of sciatic nerve function after neurorrhaphy were studied by evaluating walking-track measurements, nerve conduction velocity measurements, histomorphometry, and electron probe X-ray microanalysis. Forty adult male Lewis rats underwent right sciatic nerve (SN) transection followed by one of four different nerve repair procedures (N = 10 rats per group). In Group 1, the gap was repaired by end-to-end epineural coaptation. In Group 2, a 5-mm segment of SN was resected, and the defect was repaired under high tension by epineural neurorrhaphy. In Group 3, a 5-mm segment of SN was resected, and the defect was repaired with a 5-mm interposition nerve graft. In Group 4, a 5-mm segment of SN was resected. Then, to lessen the tension that follows neurorrhaphy, an anchoring suture was added. Finally, end-to-end coaptation was performed. Walking-track analysis showed better functional recovery in Group 1 than in Group 2, and better recovery in Group 3 than in Group 2. Group 4 showed a tendency toward better recovery comparing with Group 2. Electron probe X-ray microanalysis revealed higher Na, Cl, and K peaks in axoplasm accompanied by increase in the endoneural fluid pressure (EFP) in Group 2 than those of Group 1. This higher level of Na, Cl and K may be due to impairment of axonal sodium and potassium transport mechanism in Group 2. Increase in EFP may affect nerve regeneration.

Clinical usefulness of serum tartrate-resistant fluoride-sensitive acid phosphatase activity in evaluating bone turnover.

This study was carried out to evaluate the clinical validity and usefulness of serum tartrate-resistant fluoride-sensitive acid phosphatase (TrFsACP) activity using 2,6-dichloro-4-acetylphenyl phosphate as substrate at pH 6.2 in metabolic bone diseases. The mean Z-scores of TrFsACP activity in patients on hemodialysis were higher than in healthy subjects (male: 2.04+/-1.98, n = 49, P < .05; female: 1.49+/-2.43, n = 39, P < .05) and increased with duration of hemodialysis (r = .516, P < .01). Bone alkaline phosphatase also was found to be significantly higher in hemodialysis patients (male: 0.93+/-1.49, P < .05; female: 1.66+/-2.42, P < .05) compared with normal subjects: but had lower correlation with duration of hemodialysis than TrFsACP (r = .277, P < .05). Ulcerative colitis (1.37+/-2.21, n = 15) in males showed a significantly higher Z-score of TrFsACP compared with control subjects (P < .05). The relationship of TrFsACP activity and ultrasound findings (stiffness; speed of sound [SOS]; broadband ultra sound attenuation [BUA]) in healthy women aged 30-75 years (n = 95) were inversely and significantly correlated with stiffness (r = -.465, P < .01 ), SOS (r = -.484, P < .01), and BUA (r = -.366, P < .01), but were age dependent. TrFsACP activity significantly correlated with stiffness (r = -.521, P < .05) and SOS (r = -.527, P < .05) only in the age group of 46-55 years. BUA (r = -.313, P > .05) did not correlate significantly in any subject in the present study. We conclude that serum TrFsACP activity is useful in the diagnosis and monitoring of bone turnover.

Monoclonal expansion of synoviocytes in rheumatoid arthritis.

OBJECTIVE: To examine whether synoviocytes from patients with rheumatoid arthritis (RA) have a stronger growth ability than those from patients with osteoarthritis (OA), and to determine whether these synoviocytes clonally expand in situ. METHODS: Synovial tissues from 13 RA patients and 4 OA patients were cultured, and their ability to form colonies in soft agarose was examined. RA and OA synoviocytes were also examined in varying concentrations of fetal calf serum (FCS)-containing medium to test the effects of FCS on colony formation. DNA was extracted from clones with colony-forming ability in nonpannus lesions and from synoviocytes in pannus lesions. Restriction fragment length polymorphism (RFLP) analysis was used to examine phosphoglycerate kinase 1 (PGK-1) gene patterns. Production of cytokines by these cells was also assessed. RESULTS: All 13 RA synoviocytes exhibited colony formation, whereas none of the 4 OA synoviocytes did. This tendency was also seen with all of the concentrations of FCS examined, although growth varied in a dose-dependent manner. In contrast to OA synovial clones, cloned RA synoviocytes obtained from colonies exhibited a partial RFLP PGK-1 gene pattern, suggesting that the clones originated from monoclonal cells. Of note, 3 of 7 noncloned synoviocytes from pannus lesions exhibited a monoclonal pattern. Pannus cells produced high levels of transforming growth factor beta and platelet-derived growth factor. CONCLUSION: These findings suggest that synoviocytes with a strong growth ability are present in the rheumatoid synovium, and that these cells expand monoclonally, particularly in pannus lesions.

B cell-mediated down-regulation of IFN-gamma and IL-12 production induced during anti-tumor immune responses in the tumor-bearing state.

Unfractionated spleen cells taken from tumor-bearing mice contained tumor-primed T cells which produced lymphokines such as IFN-gamma and IL-2 through collaboration with antigen-presenting cells (APC) binding tumor antigens when cultured in vitro. Here, we investigated the regulatory mechanisms underlying IFN-gamma production by T-APC interactions. Elimination of B cells from a splenic population of tumor-bearing mice resulted in enhanced IFN-gamma production. Adding B cells back into cultures down-regulated IFN-gamma production to almost the same levels as those induced by unfractionated spleen cells. IL-2 production was not enhanced by B cell depletion, but rather was significantly suppressed. IFN-gamma-selective up-regulation was due to an enhancement of IL-12 production because IL-12 was detected in B cell-depleted cultures and enhanced IFN-gamma production was prevented by addition of anti-IL-12 mAb or anti-CD40 ligand (CD40L) mAb capable of inhibiting CD40L-induced IL-12 production. These results indicate that B cells interfere with IFN-gamma production induced through interactions between anti-tumor T cells and APC, and this suppressive effect is based on the capacity of CD40+ B cells to down-regulate the CD40L-induced IL-12 production by APC.

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.

A critical role for IL-18 in the proliferation and activation of NK1.1+ CD3- cells.

Like IL-12, IFN-gamma-inducing factor/IL-18 has been shown to stimulate T cells for IFN-gamma production and growth promotion. Considering the NK-stimulatory capacity of IL-12, we investigated the effect of IL-18 on NK lineage cells. A CD4- CD8- surface Ig- Ia- fraction of freshly prepared C57BL/6 spleen cells proliferated strikingly in response to combinations of IL-12 + IL-18 or IL-2 + IL-18, but not to the individual cytokines or IL-2 + IL-12. Cells proliferating in response to IL-2 + IL-18 were NK1.1+ CD3-, whereas IL-12 + IL-18-responsive cells were NK1.1- CD3-. Restimulation of the former cells with IL-12 + IL-18 or the latter cells with IL-2 + IL-18 resulted in the generation of NK1.1- CD3- or NK1.1+ CD3- cells, respectively. Moreover, a NK1.1+ CD3- CD4- CD8- surface Ig- Ia- population isolated from spleen cells was found to form NK1.1+ CD3- or NK1.1- CD3- blasts by stimulation with IL-2 + IL-18 or IL-12 + IL-18, respectively, and the NK1.1 positivity on these blasts was again reversed after restimulation with an alternative combined stimulus. Both types of blasts produced enormously large amounts of IFN-gamma in response to IL-12 + IL-18 and exhibited strikingly high levels of NK activity. These results indicate that IL-18 plays an obligatory role in inducing proliferation and activation of NK1.1+ CD3- CD4- CD8- cells and that the expression of the NK1.1 marker is reversible, depending on the cytokine used for stimulation in combination with IL-18.

Differential capacities of CD4+, CD8+, and CD4-CD8- T cell subsets to express IL-18 receptor and produce IFN-gamma in response to IL-18.

IL-12 and IL-18 have the capacity to stimulate IFN-gamma production by T cells. Using a T cell clone, we reported that IL-18 responsiveness is generated only after exposure to IL-12. Here, we investigated the induction of IL-18 responsiveness in resting CD8+, CD4+, and CD4-CD8- T cells. Resting T cells respond to neither IL-12 nor IL-18. After stimulation with anti-CD3 plus anti-CD28 mAbs, CD8+, CD4+, and CD4-CD8- T cells expressed IL-12R, but not IL-18R, and produced IFN-gamma in response to IL-12. Cultures of T cells with anti-CD3/anti-CD28 in the presence of rIL-12 induced IL-18R expression and IL-18-stimulated IFN-gamma production, which reached higher levels than that induced by IL-12 stimulation. However, there was a substantial difference in the expression of IL-18R and IL-18-stimulated IFN-gamma production among T cell subsets. CD4+ cells expressed marginal levels of IL-18R and produced small amounts of IFN-gamma, whereas CD8+ cells expressed higher levels of IL-18R and produced more IFN-gamma than CD4+ cells. Moreover, CD4-CD8- cells expressed levels of IL-18R comparable to those for CD8+ cells but produced IFN-gamma one order higher than did CD8+ cells. These results indicate that the induction of IL-18R and IL-18 responsiveness by IL-12 represents a mechanism underlying enhanced IFN-gamma production by resting T cells, but the operation of this mechanism differs depending on the T cell subset stimulated.

Improved method for measuring tartrate-resistant acid phosphatase activity in serum.

We describe an improved method for the kinetic measurement of tartrate-resistant acid phosphatase (TrACP; EC 3.1.3.2) activity in serum. Of the TrACP derived from erythrocytes, platelets, and macrophages (osteoclasts and others), that from the first two sources is also resistant to fluoride, whereas skeletal TrACP is sensitive to fluoride. Thus, osteoclast-derived TrACP can be measured specifically by exploiting its sensitivity to fluoride. We measured the activity of tartrate-resistant and fluoride-sensitive acid phosphatase (TrFsACP) by using 2,6-dichloro-4-acetylphenyl phosphate as substrate at pH 6.2. The activity of TrFsACP in serum was increased by adding hexadimethrine bromide (Polybrene) to the reaction mixture. This method was not influenced by hemolysis with hemoglobin concentrations as great as 0.9 g/L. The mean +/- SD values of TrFsACP activity by this method were 20.4 +/- 2.8 and 16.4 +/- 2.3 U/L for young (ages 20-29 years) men (n = 34) and women (n = 50), respectively. The highest mean TrFsACP activity was found among children younger than 15 years, followed by that in elderly subjects (older than 60).

A mechanism underlying synergy between IL-12 and IFN-gamma-inducing factor in enhanced production of IFN-gamma.

IL-12 and IFN-gamma-inducing factor (IGIF) have the capacity to stimulate IFN-gamma production by T cells. Using an IL-12-responsive T cell clone, 2D6, we investigated how these two cytokines collaborate for IFN-gamma production. 2D6 obtained from cultures containing rIL-12 produced IFN-gamma in response to rIGIF. 2D6 from cultures deprived of IL-12 for 24 h produced only marginal levels of IFN-gamma production following stimulation with either rIL-12 or rIGIF alone. However, simultaneous stimulation of these 2D6 cells with both cytokines resulted in strikingly enhanced levels of IFN-gamma production. 2D6 could also be maintained in the presence of rIL-2 instead of rIL-12. 2D6 lines maintained with rIL-12 (2D6(IL-12)) or rIL-2 (2D6(IL-2)) exhibited differential IGIF responsiveness: both lines responded similarly to rIL-2 or rIL-12, whereas the 2D6(IL-12) or 2D6(IL-2) exhibited high or marginal IGIF responsiveness, respectively. The 2D6(IL-12) line expressed IGIF receptor (IGIFR), whereas the 2D6(IL-2) did not. Overnight exposure of the 2D6(IL-12) to rIL-2 reduced IGIFR expression and conversely, exposure of the 2D6(IL-2) to rIL-12 restored IGIFR expression. IGIFR expression by these 2D6 lines correlated with the capacity to produce IFN-gamma in response to rIGIF. Purified naive T cells stimulated with anti-CD3 plus anti-CD28 mAb and subsequently cultured with rIL-12 were also found to express IGIFR and induce enhanced IFN-gamma production following IGIF stimulation. These results indicate that the induction of IGIFR by IL-12 represents one of the mechanisms underlying the synergy between IL-12 and IGIF in IFN-gamma production.

Resolution enhancement printing with a variable spot-size laser diode.

We demonstrate enhanced resolution printing using a variable spot-size laser diode. The near-field spot size of the laser diode can be changed by controlling the refractive-index distribution in the laser stripe through the injected current. The ratio of the minimum-to-maximum spot size is 2.1:1. This technology provides high-resolution printing without increasing the scanning frequency. Smoother character outlines that consist of finer steps are produced with this laser diode. An effective resolution of 1200 dots /in. (dpi) can be obtained by a printer system with 600-dpi resolution.

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.