A neutrophil elastase inhibitor (ONO-5046) reduces neurologic damage after spinal cord injury in rats.

In view of a cytoprotective effect of elastase inhibitor on chemokine-mediated tissue injury, we examined the neuroprotective effect of ONO-5046, a specific inhibitor of neutrophil elastase, in rats with spinal cord injury. Standardized spinal cord compression markedly increased cytokine-induced neutrophil chemo-attractant (CINC)-1 mRNA and protein. Their increases correlated with neurologic severity of injured rats. Immunohistochemically, CINC-1 protein was detected sequentially in vascular endothelial cells at 4 h, in perivascular neutrophils at 8 h, and in neutrophils infiltrating into cord substance at 12 h. Pretreatment with ONO-5046 (50 mg/kg) markedly ameliorated motor disturbance in injured rats, and reduced CINC-1 protein and mRNA expression. ONO-5046 also significantly reduced the increase of neutrophil accumulation or infiltration estimated by myeloperoxidase activity, and the extent of vascular permeability by Evans blue extravasation in the injured cord segment in comparison to control animals receiving vehicle. These results suggest that CINC-1 contributed to inflammation in rat spinal cord injury and ONO-5046 attenuated neurologic damage partly by blocking CINC-1 production of the chemoattractant, preventing neutrophil activation and vascular endothelial cell injury.

Possible involvement of interleukin-1 in cyclooxygenase-2 induction after spinal cord injury in rats.

A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A2 (detected as thromboxane B2) and prostaglandin I2 (detected as 6-ketoprostaglandin F1alpha). Thromboxane B2 was predominant during the first 1 h, whereas the 6-ketoprostaglandin F1alpha level exceeded that of thromboxane B2 at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1alpha and -1beta detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1alpha into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F1alpha resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1alpha injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1alpha.

Lung cancer: intermittent irradiation synchronized with respiratory motion--results of a pilot study.

PURPOSE: To test the feasibility of a system for intermittent irradiation synchronized with respiratory motion in a clinical setting. MATERIALS AND METHODS: A newly developed gate pulse controller that starts and stops irradiation at a chosen phase of the respiratory cycle by controlling a linear accelerator was used in six patients with lung cancer. A laser displacement sensor was used for the detection of respiratory motion. Three patients underwent radiation therapy during the cycles between 50% expiration and 50% inspiration (step 1), and three patients underwent radiation therapy during the cycles between 70% expiration and 30% inspiration (step 2). RESULTS: The system functioned well; irradiation was verified with portal verification radiography in all six patients. The range of the tumor position during synchronized irradiation was detectable with fast portal localization radiography. The treatment times for steps 1 and 2 were 1.38-1.71 and 2.03-2.18 times longer, respectively, than those for conventional irradiation. CONCLUSION: Synchronized irradiation with the authors' system allowed convenient and reliable reduction of the target volume. Further study is needed to standardize the system for clinical use.

[Is medical linac suitable for high-precision stereotactic irradiation?: investigations in geometrical accuracies of gantry and couch].

Linac-based radiosurgery has many advantages over the gamma knife, including low initial cost and no need of source replacement. On the other hand, most of the medical linacs currently in use were not originally designed to be applied for radiosurgery, and, therefore, careful quality assurance programs are required. In the gantry-head of a linac, a small CCD video camera is mounted in a position optically identical to that of the x-ray source. The video signal from the camera was digitalized to be evaluated for geometrical errors. A metal ball fixed to the stereotactic base frame via XYZ-sliding rods was used as a simulated target. Displacements of the target from the isocenter were measured during rotation of the gantry. Displacements in the gantry-rotation plane were satisfactorily small, while those perpendicular to it were maximal at gantry position angles of 0 degree and 180 degrees. This error night be caused by gravitational vending of the heavy gantry head. Although other major errors of the linac were within one millimeter, the center of coach rotation around the isocenter did not coincide with the center of gantry rotation, probably owing to gravitational vending. Special care should be taken when very small collimators are employed.

A vitamin D receptor gene polymorphism in the translation initiation codon: effect on protein activity and relation to bone mineral density in Japanese women.

The effect of a T-C transition polymorphism at the translation initiation codon of the human vitamin D receptor (VDR) gene on the biological function of the encoded protein was investigated. Of 239 Japanese women volunteers subjected to genotype analysis for this polymorphism, 32 (13%) were genotype MM (the M allele is ATG at the putative translation start site), 75 (31%) were genotype mm (the m allele is ACG at the putative translation start site), and 132 (55%) were genotype Mm. The bone mineral density (BMD) in the lumbar spine (L2-L4) was determined for 110 healthy premenopausal women from the volunteers and was shown to be 12.0% greater (p < 0.05) for mm homozygotes than for MM homozygotes. Synthesis of the proteins by the M and m alleles from the cloned cDNAs in vitro and in transfected COS-7 cells revealed them to have a size of 50 and 49.5 kD, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This size difference is consistent with initiation of translation of the M allele-encoded protein from an ATG codon located at nucleotides +10 to +12 in the conventional open reading frame. The extent of vitamin D-dependent transcriptional activation of a reporter construct under the control of a vitamin D response element in transfected HeLa cells was approximately 1.7-fold greater for the m type VDR than for the M type protein. These results suggest that the polymorphism at the translation start site of the VDR gene may modulate BMD in premenopausal Japanese women.

Site-specific modification of rabbit muscle creatine kinase with sulfhydryl-specific fluorescence probe by use of hydrostatic pressure.

We investigated the effect of pressure on the reactivity of cysteine residues of rabbit muscle creatine kinase (CK). Performing the fluorescent modification under high pressure, a unique sulfhydryl group (Cys-253) of CK was labeled, in addition to Cys-282, which is known as a single reactive sulfhydryl under ambient conditions. CK is composed of two identical subunits, containing four cysteine residues in each subunit. Cys-282 plays an important role in enzymatic activity. In the pressure range from 0.1 MPa to 300 MPa, only one sulfhydryl group for each subunit of CK reacted with the reagents. However, at 400 MPa 2 sulfhydryl groups were modified. The 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage method revealed that both Cys-282 and Cys-253 were modified at 400 MPa. The chemical modification of Cys-282 induced a loss of enzymatic activity. By taking advantage of the modification under high pressure, selective modification of Cys-253 with 5-[N-(iodoacetamidoethyl)amino]-naphthalene-1-sulfonate (IAEDANS) was performed. A reversible blocking of Cys-282 at atmospheric pressure was followed by the reaction of Cys-253 with the fluorescent probe at 400 MPa. After the decompression, Cys-282 was unblocked, and obtained Cys-253-modified CK retained up to 64% of the catalytic activity of the intact CK. The fluorescent properties of IAEDANS covalently bound at Cys-253 were not significantly different from those of IAEDANS covalently bound at Cys-282.

Experimental and clinical studies of eicosanoids in cerebrospinal fluid after spinal cord injury.

OBJECTIVE: In an attempt to elucidate a possible role for eicosanoids in the pathogenesis of spinal cord injury (SCI), we measured the concentration of leukotriene (LT) C4, thromboxane B2, and 6-keto-prostaglandin F1 alpha in cerebrospinal fluid in both a canine experimental model and 11 patients with SCIs. METHODS: The eicosanoid concentration in cerebrospinal fluid was measured by radioimmunoassay. Neurological severity was assessed according to the grading system of Frankel et al.. Control samples were obtained from 20 patients without SCIs. RESULTS: In the canine model, a significant increase in all eicosanoid levels was found on Days 1 to 7, which subsequently returned to the control levels. In the clinical study, the highest mean (+/- standard error of the mean) concentrations of LTC4, thromboxane B2, and 6-keto-prostaglandin F1 alpha in the acute stage of SCI were 95.9 +/- 10.7, 175.2 +/- 38.2, and 167.5 +/- 39.9 pg/ml, respectively. These concentrations were five to nine times higher than control levels. There was a good correlation between cerebrospinal fluid LTC4 levels and the neurological severity. The time-dependent change in LTC4 concentrations in seven patients with SCIs was similar to that observed in the canine model. In addition, the highest mean concentrations of the eicosanoids measured in patients with complete paralysis was also similar to those of the canine model. The eicosanoid concentrations in five patients with SCI were measured more than 6 months after the onset of injury. Although all eicosanoid levels had elevated in the acute stage of injury, they were not elevated and showed the same levels as the controls at the chronic stage. CONCLUSION: The findings suggest that enhanced arachidonate metabolism occurs in humans and support the evidence from animal experiments that emphasizes the importance of eicosanoids in the secondary processes mediating ischemia and edema.

Increased production of eicosanoids, TXA2, PGI2 and LTC4 in experimental spinal cord injuries.

Arachidonate metabolites have many kinds of bioactivities. Thromboxane A2 (TXA2) stimulates platelet aggregation and vasoconstriction, whereas prostaglandin I2 (PGI2) antagonises its activities. Thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) are determined in biological materials. Production of TXB2, 6-keto-PGF1 alpha and leukotriene C4 (LTC4), which have potent vascular permeability, was measured by radioimmunoassay in experimental spinal cord injured animals. TXB2 level in the rat spinal cord reached a peak concentration of 133.6 +/- 3.8 pmol/g cord, and 6-keto-PGF1 alpha increased to 26.2 +/- 11.7 pmol/g cord 5 minutes after the injury. There was good correlation between TXB2 production and vascular damage as monitored by fluorescein uptake. When OKY-046 ((E)-3-[4-(1-imidazolylmethyl) phenyl]-2-propenoic acid), which selectively inhibits TXA2 synthetase activity, was administered 10 minutes before injury, the increase in TXB2 production was inhibited by more than 80%, but the degree of vascular damage was reduced by only 40%. In the guinea pig spinal cord, LTC4 levels reached a peak concentration of 2.2 +/- 0.4 pmol/g cord 10 minutes after compression, while that of TXB2 reached 146.8 +/- 6.2 pmol/g cord. The increased production of TXB2 was correlated with the degree of compression injury while that of LTC4 production did not. These findings suggest that vasoactive eicosanoids, TXA2, PGI2 and LTC4, play important roles in secondary damage following spinal cord injury, although their roles may be different among species of animals.

[Alteration of leukotriene C4 levels in experimental spinal cord injury].

Leukotriene C4 (LTC4) production in the guinea pig spinal cord following compression injury was determined by radioimmunoassay, in the same way thromboxane B2 (TXB2), a stable metabolite of thromboxane A2 (TXA2), was also measured. When the spinal cords were compressed under a 20 gram weight for 10 minutes, LTC4 levels reached peak values (2.2 +/- 0.4 pmol/g cord) 10 minutes after release, then gradually decreased until being undetectable 60 minutes later. TXB2 levels reached peak values (146.8 +/- 6.2 pmol/g cord) 5 minutes after the release from compression, the TXB2 level then gradually decreased, but remained at about 1/2 of the peak level even 60 minutes after the release. When the spinal cords were compressed with various weights, TXB2 production depended on the degree of compression, while LTC4 production was not affected by the compression injury. The LTC4 production confirmed in the injured spinal cords is suggestive of its relation to secondary disorders after spinal cord injury, spinal edema, in particular.

Clinical considerations and biochemical basis of prognosis of cervical spinal cord injury.

A total of 118 patients with cervical spinal cord injury were studied to determine the neurologic improvement achieved by either conservative or surgical treatment. Useful recovery was observed in 55% of the patients with incomplete cord injuries, but in none of those with complete cord injuries. There was no significant difference between the treatment groups regarding neurologic improvement. In experimental studies on rats, the increased levels of lipid peroxides and thromboxane after spinal cord injury were found to be proportional to the magnitude of injury. These evidences suggest that spinal cord injury is directly related with the magnitude of injury, and the prognosis is determined entirely at the time of injury.

A flavonoid inhibitor of 5-lipoxygenase inhibits leukotriene production following ischemia in gerbil brain.

Leukotrienes C4 and D4 are arachidonic acid metabolites that constrict blood vessels and enhance vascular permeability; their biosynthesis is initiated by the reaction of arachidonic acid with 5-lipoxygenase enzyme. After bilateral carotid artery occlusion for 15 minutes and reperfusion of the gerbil brain for 15 minutes, we determined the brain tissue concentrations of leukotrienes C4 and D4 by radioimmunoassay; they had increased from a baseline concentration of less than 1 to a mean +/- SEM concentration of 12.8 +/- 3.9 pmol/g brain. We also studied the effect of a flavonoid 5-lipoxygenase inhibitor on leukotriene production in the reperfused gerbil brain. A water-soluble flavonoid (5-hexyloxy-3',4'-dihydroxy-6,7-dimethoxyflavone 4'-disodium phosphate) was administered intravenously at a dose of 200 mg/kg body wt; 15 minutes later, both carotid arteries were occluded. The enhanced production of leukotrienes C4 and D4 in the reperfused brain was reduced by approximately 80% (from a mean +/- SEM of 12.8 +/- 3.9 to 2.2 +/- 1.3 pmol/g brain) in the presence of the 5-lipoxygenase inhibitor. The flavonoid did not affect the production of prostaglandin D2, the concentration of which also increased in the reperfused ischemic brain.

Arachidonate 5-lipoxygenase of porcine leukocytes studied by enzyme immunoassay using monoclonal antibodies.

The cytosol fraction of porcine leukocytes contained 5-lipoxygenase, the activity of which was masked by a predominant activity of 12-lipoxygenase. The 5-lipoxygenase was partially purified to a specific activity of about 10 nmol of arachidonic acid oxygenated/min/mg of protein and given to mice as an antigen to prepare monoclonal antibodies against the enzyme. Two species of antibodies recognized separate sites of the 5-lipoxygenase protein and did not cross-react with 12-lipoxygenase. They were utilized to develop a peroxidase-linked immunoassay of sandwich-type, which allowed a quantitative determination of the 5-lipoxygenase protein. The assay was applied to a screening of the 5-lipoxygenase content in various porcine tissues. By far the highest content of 5-lipoxygenase was found in leukocytes. About one-tenth the amount of the enzyme was found in lung, pancreas, ileum, and thymus, which could not be attributed to the contaminating leukocytes in these tissues.

Enzyme immunoassay of 6-ketoprostaglandin F1 alpha in a solid phase.

A solid-phase enzyme immunoassay of 6-ketoprostaglandin F1 alpha (a stable degradation product of prostaglandin I2) was developed in which the hapten molecule was labeled with beta-galactosidase. The antiserum was bound to a polystyrene tube, and the enzyme-labeled and unlabeled 6-ketoprostaglandin F1 alpha were allowed to react in a competitive manner with the immobilized antibody. The activity of the immunologically bound beta-galactosidase was assayed by fluorometry, and correlated with the amount of unlabeled 6-ketoprostaglandin F1 alpha. According to a calibration curve 6-ketoprostaglandin F1 alpha was detectable in a range of 30 fmol-10 pmol. When 6-ketoprostaglandin F1 alpha was extracted from human serum by using an octadecylsilyl silica column (Sep-Pak C18) and the crude extract was subjected to the enzyme immunoassay, the content of 6-ketoprostaglandin F1 alpha was 50-60 pmol/ml of human serum. An endogenous substance(s) which disturbed the immunoreaction and gave such an apparently high concentration of 6-ketoprostaglandin F1 alpha separated from the endogenous 6-ketoprostaglandin F1 alpha by HPLC. With the purified 6-ketoprostaglandin F1 alpha fraction there was a good correlation (r = 0.994) between enzyme immunoassay and radioimmunoassay. The validity of the enzyme immunoassay was confirmed by gas chromatography-selected ion monitoring.