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We report herein the results of anterior or posterior neural decompression with spinal stabilization in 16 patients with spinal metastases. Intractable back pain was relieved in 14 patients (87.5%) and 4 had complete pain relief. Neurologic recovery was observed in 8 out of 13 patients (61.5%) who had some neurologic deficits before surgery. The activities of daily living improved in 7 of 9 (77.7%), and 5 out of 8 patients (62.5%) who had been unable to walk before surgery became ambulatory after surgery. The average operation time was 3h 15 min with an average blood loss of 2150 ml. No patient died within 1 month after surgery and the median survival was 19.1 months. The results indicated that, if properly indicated, anterior or posterior neural decompression and spinal stabilization is a safe and effective treatment for patients with spinal metastases to improve the quality of life for the patients' remaining years.

APEX nuclease is a mammalian DNA repair enzyme having apurinic/apyrimidinic (AP) endonuclease, 3'-5'-exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities. It is also a redox factor (Ref-1), stimulating DNA binding activity of AP-1 binding proteins such as Fos and Jun. In the present paper, a cDNA for the enzyme was isolated from a rat brain cDNA library using mouse Apex cDNA as a probe and sequenced. The rat Apex cDNA was 1221 nucleotides (nt) long, with a 951-nt coding region. The amino acid sequence of rat APEX nuclease has 98.4% identity with mouse APEX nuclease. Using the rat Apex cDNA as a probe for Northern blot analysis, the size of rat Apex mRNA was shown to be approximately 1.5 kb. Its expression was compared in 9 rat organs on postnatal days 7 and 28. Although Apex mRNA was expressed ubiquitously, the levels varied significantly, suggesting organ- or tissue-specific expression of the Apex gene. The highest level was observed in the testis, relatively high levels in the thymus, spleen, kidney and brain, and the lowest level in the liver. The level of expression at postnatal day 28, with the exception of the testis, was almost the same as or lower in respective organs than that at postnatal day 7. Postnatal developmental changes of Apex mRNA expression in the testis and thymus were further studied. The expression in testis was markedly increased on postnatal days 21 and 28. The expression in thymus increased once at postnatal day 14, and then decreased. The developmental changes of Apex mRNA expression in testis and thymus suggest that APEX nuclease is involved in processes such as recombinational events.

This study was conducted to examine the effect of gamma-interferon (IFN-gamma) on experimental metastasis formation by murine colon 26 adenocarcinoma in BALB/c mice. We found that the number of experimental lung metastases was increased after colon 26 cells were pretreated for 1 h with as little as 1 OIU/ml of IFN-gamma. 5-[125I] iodo-2'-deoxyuridine-radiolabeled colon 26 cells pretreated with IFN-gamma remained at higher level in the lung at 24h after intravenous injection than when the cells were not pretreated. In vivo elimination of asialo GM1-positive cells increased the number of lung metastases and, in such mice, there was no longer a difference in metastatic ability between control and IFN-gamma-treated cells. Colon 26 cells were completely resistant to lysis by isolated splenocytes. Splenocytes incubated in vitro with interleukin 2 exhibited moderate cytotoxicity against colon 26 cells, but there were no significant differences between control and IFN-gamma-treated cells. Colon 26 cells pretreated with IFN-gamma demonstrated resistance to tumor necrosis factor alpha-mediated growth inhibition. The enhancement of metastases by IFN-gamma was dependent on de novo protein synthesis since the enhancement was abolished by cycloheximide. Taken together, the data suggest that the metastatic ability of colon 26 cells pretreated with IFN-gamma is significantly higher due to the resistance to asialo GM1-positive cells accompanied with de novo protein synthesis.

<P>In this study, 42 cases of spinal schwannomas are reviewed. We analyzed the therapeutic results of patients with spinal schwannomas in order to investigate the factors which affect the clinical outcomes. Early diagnosis and treatment could help procure a good result for the patient. The delay in diagnosis and the subsequent duration of symptoms was significantly longer in cases of lumbar lesions compared to cervical and thoracic lesions. Tumor recurrence was rare, but in some cases where complete resection was not possible, close follow-up of the patients postoperatively with MRI was indicated.</P>

A shortage of donor organs in clinical transplantation prompted us to study whether resuscitated dead hearts could be utilized for successful orthotopic heart transplantation. After 60 min of hypoxic cardiac arrest, one group of canine hearts was resuscitated (Res group, n = 6). The other group was harvested directly (Non-Res group, n = 6). In the Res group, cardiopulmonary bypass was utilized for resuscitation at 37 degrees C and the animals were then core-cooled to 15 degrees C. The hearts then were preserved in University of Wisconsin solution and orthotopically transplanted. Stable prostacyclin analogue (OP2507) and verapamil, a calcium antagonist, were added to the cardioplegia, and substrate-enriched warm blood cardioplegia and a hydroxy radical scavenger (EPC) were administered at the time of reperfusion of the transplanted heart. All animals in each group were successfully weaned from cardiopulmonary bypass with dopamine (5 micrograms/kg/min). Cardiac function without dopamine was better preserved in the Res group than the Non-Res group (Emax: 130.6 +/- 41.5% vs. 47.1 +/- 24.7%; mean +/- SD, as percent of postbrain death values, P < 0.01 by unpaired t-test). Cadaver hearts 60 min after anoxic arrest can be successfully re-animated and orthotopically engrafted. In addition, the core-cooling technique is useful. We believe this study serves as the key step in the clinical application of dead hearts to successful cardiac transplantation.

A method of genotyping IgA2 alleles in the human immunoglobulin alpha 2 heavy chain constant region (C alpha 2 gene) was developed by using the polymerase chain reaction (PCR). By this method, the genotype was determined by discriminating base substitution in the 3'-flanking region of alleles, A2m*1 and A2m*2, which manifest A2m serum types, by nested PCR using allele-specific primers. Three types, IgA2*1/IgA2*1, IgA2*2/IgA2*1, and IgA2*2/IgA2*2, were detected from DNA extracted from lymphocytes. Genotyping was possible from 100 pg of DNA by this method. The estimated allele frequency in 318 Japanese subjects was 0.561 for IgA2*1 and 0.439 for IgA2*2. Analysis of 29 cases of paternity tests suggested that the data follow Mendel's law of inheritance. This genotype could also be detected in whole blood, blood stains, saliva stains, and various organs and tissues. These results suggest the usefulness of the present method for paternity testing and individual identification in forensic medicine.

<P>Aggregation activity of platelets in umbilical blood is lower than that in adult blood, but the reason for this is not well understood. It has recently been clarified that calcium plays a role as a second messenger of platelet aggregation, and that glycoproteins of platelet surface membrane such as glycoprotein I b and IIb/IIIa are receptors for agonists inducing aggregation. We examined the concentrations of intracellular calcium and the membrane glycoproteins of platelets in umbilical and adult blood. The increase of intracellular calcium in umbilical platelets was lower than that in adult platelets when the aggregation was induced by ADP, collagen, thrombin and epinephrine. Only calcium ionophore A23187 induced aggregation of both umbilical and adult platelets. On the other hand, there were no qualitative differences between glycoproteins I b and IIb/IIIa of these two groups. Therefore, the low aggregation activity of umbilical platelets seems to be due to low responsiveness of the intracellular calcium system, not to the disorder of functional surface membrane glycoprotein.</P>

<P>Idiopathic interstitial pneumonia (IIP) is a progressive and often fatal pulmonary disorder, and evaluating the prognosis of patients with IIP has never been sufficient. Accordingly, factors including clinical features, laboratory data, cellular components in bronchoalveolar lavage (BAL) fluid and response to corticosteroid therapy were analyzed in 35 patients with IIP whose median age of respiratory onset was 60 years (range; 37-77 years). Nineteen patients (54.3%) were in the active stage of IIP and 16 of them were treated with corticosteroids. Significant prognostic factors were the neutrophil percentage in BAL fluid, interstitial shadows on chest radiograph, pulmonary function, blood oxygen level, grade of dyspnea, and disease activity at the initial examination. Patients in the active stage showed higher proportions of neutrophils and eosinophils in BAL fluid than those in the non-active stage. Despite corticosteroid therapy, the survival of patients in the active stage was significantly shorter than those in the non-active stage. Fifty percent of the patients treated with corticosteroids were regarded as responders at 1 month after the initiation of therapy; however, there was no significant difference between responders and non-responders in terms of survival time. In conclusion, disease activity and neutrophils in BAL fluid may be important predictors of the prognosis of IIP.</P>

Circulating hepatitis C virus (HCV) particles can be fractionated by means of differential flotation centrifugation. It is reported that in the bottom fraction HCV is in the form immune complexes, whereas in the top, it is free of antibodies. We evaluated the significance of circulating complex and free HCV in chronic hepatitis C, and assessed the relationship in terms of the response to interferon (IFN) therapy. We examined sera before, just after, and 1 year after administering IFN to 18 patients with chronic hepatitis C, 10 of whom responded (group CR), and 8 did not (group NR). The amounts of virus were similar between both groups before therapy. After differential flotation centrifugation with 1.063 g/ml of NaCl, the top and bottom fractions were assayed for HCV RNA. Before therapy, HCV RNA was detected in the top fraction in 1 of 10 in group CR, and in 6 of 8 in group NR (P < 0.05, chi-square test). HCV RNA was positive in the bottom fraction of all samples. In a follow-up study of group NR, HCV RNA was detected in the top fraction in 3 of 8 just after IFN therapy, and in 7 of 8 after 1 year. This study suggests that the presence of HCV in the top fraction can predict a poor response to IFN therapy.

<P>Microdetermination of alpha-fetoprotein (AFP) glycoforms by lectin affinity electrophoresis followed by chemiluminescence reaction using horseradish peroxidase (POD) or alkaline phosphatase (ALP) in antibody-affinity blotting was developed. The intensity of chemiluminescence obtained by ALP was greater than that by POD; however, the coefficient of variation with POD was less than that with ALP. The optimized sensitivity of the chemiluminescence method with POD was two times that of the most sensitive colorimetric method currently available in terms of the chemiluminescence intensity per unit AFP concentration. The lower detection limit by the chemiluminescence method with POD (0.5 ng/ml) was much lower than that by the colorimetric method (3 ng/ml). Both methods gave identical percentages of lentil lectin- and erythroagglutinating phytohemagglutinin-reactive minor bands using a serum with 52 ng/ml AFP. This result indicates that microdetermination of AFP glycoforms by chemiluminescence after lectin-affinity electrophoresis was more sensitive than currently available methods and that it is potentially useful for clinical application</P>

We purified an apurinic/apyrimidinic (AP) endonuclease from mouse ascites sarcoma (SR-C3H/He) cells. The enzyme showed nicking activity on acid-depurinated DNA but not on untreated, intact DNA. It also showed priming activity for DNA polymerase on both acid-depurinated and bleomycin-damaged DNA. The priming activity on bleomycin-damaged DNA was two times higher than that on an acid-depurinated DNA. The enzymatic properties indicate that the enzyme is a class II AP endonuclease having DNA 3' repair diesterase activity. The purified enzyme has a molecular weight of 39,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal pH for AP endonuclease activity was 8.0 in 50 mM Tris-HCl buffer. The AP endonuclease activity depended on divalent cation such as Mg2+ and Co2+ ions, and was inhibited by 2 mM EDTA with no addition of the divalent cation. An appropriate concentration of sodium or potassium salt stimulated the activity. Partial digestion of the AP endonuclease with Staphylococcus aureus V8 protease produced 4 major peptide fragments which may be used for protein sequencing.

<P>We purified an apurinic/apyrimidinic (AP) endonuclease from mouse ascites sarcoma (SR-C3H/He) cells. The enzyme showed nicking activity on acid-depurinated DNA but not on untreated, intact DNA. It also showed priming activity for DNA polymerase on both acid-depurinated and bleomycin-damaged DNA. The priming activity on bleomycin-damaged DNA was two times higher than that on an acid-depurinated DNA. The enzymatic properties indicate that the enzyme is a class II AP endonuclease having DNA 3' repair diesterase activity. The purified enzyme has a molecular weight of 39,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal pH for AP endonuclease activity was 8.0 in 50 mM Tris-HCl buffer. The AP endonuclease activity depended on divalent cation such as Mg2+ and Co2+ ions, and was inhibited by 2 mM EDTA with no addition of the divalent cation. An appropriate concentration of sodium or potassium salt stimulated the activity. Partial digestion of the AP endonuclease with Staphylococcus aureus V8 protease produced 4 major peptide fragments which may be used for protein sequencing.</P>

<P>We investigated the effect of estrogens, 17 beta-estradiol, estradiol-3-benzoate and estrone, on 2-amidinopropane hydrochloride (AAPH)-provoked, free radical-dependent hemolysis in vitro. Incubation experiment was performed by mixing AAPH (400 mM) and washed human erythrocyte suspension with or without various sex hormones and radical scavengers. After 170 min of incubation, 50% hemolysis was detected in the control group (incubation without sex hormones or radical scavengers), whereas after the addition of estrogens (5 mM), hemolysis was nearly completely inhibited until 180 min of incubation. It was found that the inhibitory activities of estrogens on oxidative hemolysis were stronger than that of alpha-tocopherol and had nearly identical to that of N-acetyl-L-cysteine. Testosterone had no inhibitory effects. The elevation of thiobarbituric acid-reactive substances, a marker for lipid peroxidation, was also inhibited by estrogens. These results add further evidence that estrogens are strong radical scavengers in humans.</P>

To study the pathology of muscle atrophy in rheumatoid arthritis (RA), we examined the vastus medialis in rheumatoid patients histologically. The relationship of the findings to their ambulatory ability and long-term steroid therapy was investigated. The muscles of the RA patients were also compared with those of patients with osteoarthritis (OA). Specimens of the vastus medialis were collected from 29 knees of 23 patients with RA and 16 knees of 13 patients with OA during total knee arthroplasty. Muscle fibers were classified according to their type, and the ratio between the area of single type I and type II fibers as well as the ratio between the total area of these fibers was calculated. The total area of type II fibers in the RA group was significantly greater than in the OA group (P < 0.05). In the RA group, the mean proportion of the type II fibers relative to the total muscle fiber area tended to increase with the decline of ambulatory ability, while there was no such increase in the OA group. The proportion of type II fibers was increased significantly in RA patients on long-term steroid therapy when compared to those without therapy. In the ratio of the area of a single fiber, there was no clear relationship to ambulatory ability and long-term steroid therapy. It is considered that muscle atrophy in RA is not solely disuse atrophy, but also has a close relationship to steroid therapy and the pathology of the disease itself.

The sensitivity of five kinds of cytotoxicity assays using ethanol on human hepatoblastoma cells (HUH-6 line), which were cultured as monolayers or spheroids, was compared. Ethanol was chosen as a test because it acts on cell membranes directly without being metabolized and exerts its cytotoxicity. The assay methods used were as follows: 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), colony formation, cell growth and DNA assays. The sensitivity of the assays was: LDH < DNA < cell growth < MTT < colony formation. LDH assay had the advantage that the same culture could be used for multiple assays, but when a small number of cells were assayed, no significant increase in the release of LDH was detected in the assay cultures compared with the control cultures. Although the DNA and cell growth assays were more sensitive than the LDH assay, the extent of cell damage may be underestimated because the damaged cells and DNA present in the cultures are included in the assay samples. On the other hand, both MTT and colony formation assays showed a high sensitivity. The MTT assay was done within 24 h after ethanol was added to the cultures and was applicable to both monolayer and spheroid cultures, while the colony formation assay required 1-2 weeks and it was applicable only to monolayer cultures. Taken together, the MTT assay was the most suitable method to evaluate the cytotoxic effects of ethanol on HUH-6 cells cultured as either monolayers or spheroids.

Some patients with rheumatoid arthritis (RA) as well as those with other collagen diseases are positive for antinuclear antibody (ANA). We investigated the frequency of positivity for ANA in 104 patients with RA and evaluated the clinical features and laboratory data in the ANA-positive and -negative groups. The presence of ANA in sera was studied by indirect immunofluorescence using HEp-2 cells as the antigen substrate. Sera with a positive fluorescence at a dilution of 1:20 were considered to be positive for ANA. Of the 104 patients, 39 (37.5%) were positive for ANA. The staining pattern in the positive cases varied, but most were speckled (64.1%) and homogeneous (48.7%). A small number showed a nucleolar (20.5%) or a centromere (10.3%) pattern. None showed a shaggy pattern. The ANA titer was lower in RA patients compared with those with other collagen-related diseases such as systemic lupus erythematosus or progressive systematic sclerosis. None of the patients positive for ANA with either a nucleolar or centromere staining pattern had progressive systemic sclerosis or the CREST syndrome. One patient each had Raynaud's phenomenon and pulmonary fibrosis. There was no correlation between ANA positivity and indicators of joint inflammation. The prevalence of ANA positivity in patients with advanced or prolonged disease was higher than those with early stages or short durations. There was no correlation with drug therapy.

We experienced a patient with traumatic neuroma of the gallbladder with no history of gallbladder surgery or cholelithiasis. A 74-year-old man was referred to our department after a gallbladder tumor was incidentally discovered during a preoperative screening examination for prostate hypertrophy. Ultrasonography, MRI, CT and endoscopic retrograde cholangiography revealed a protuberant lesion of the gallbladder. Laparoscopic cholecystectomy was attempted but adhesion between the liver and duodenum forced us to convert to open laparotomy. Cholecystectomy and adjacent liver tissue resection was performed. Diagnosis was made by frozen histology during operation. It revealed no malignancy. Postoperative pathological examination revealed traumatic neuroma associated with inflammation. To our knowledge, this is the first reported case of gallbladder neuroma without a history of gallstones or surgery in the English and Japanese literature since 1980. This traumatic neuroma should be considered in a differential diagnosis in treating gallbladder neoplasm, even in the absence of an operative history or cholelithiasis.

To characterize primary sclerosing cholangitis (PSC) in Japanese patients and its association with inflammatory bowel disease (IBD), 155 reported cases of PSC, including 6 cases of our own, were reviewed. The prevalence of IBD was less in Japanese PSC patients than in Western patients (23% versus 62-100%). Japanese PSC patients with IBD were younger (mean age, 33.1 versus 51.8 years) and were more often women (51% versus 36%) than those without IBD. Seventy-four percent of PSC patients with IBD had extensive colonic lesions, and 89% of those developed IBD simultaneously, with or prior to PSC. There were 3 cases of neutrophilic cholangitis among the PSC patients with IBD but none in those without IBD. Based on these observations, we speculate that there may be subtypes of PSC which differ pathophysiologically.

To diagnose hepatocellular carcinoma (HCC) functionally and immediately, we examined the usefulness of indocyanine green (ICG) injection during ultrasound-guided liver biopsy. Liver specimens were obtained after intravenous ICG injection by ultrasound-guided biopsy from 251 space-occupying lesions (SOL) in 136 patients. The tissues were immediately examined for ICG uptake using an infrared Vidicon camera and were also subjected to histopathological examinations. Of the 112 ICG-negative biopsy specimens, 105 were histologically diagnosed as HCC, 6 as dysplastic nodules (DN) and 1 as a regenerative nodule (RN). Of the 139 ICG-positive specimens, 18 were diagnosed as HCC, 1 as DN and 120 as RN. The sensitivity of the absence of ICG uptake (SEAIU), the specificity of the absence of ICG uptake (SPAIU), and the positive predictive value of the absence of ICG uptake (PPAIU) for the diagnosis of HCC were 85.3%, 94.5% and 93.8%, respectively. Of the 251 SOLs, 184 were less than 2 cm. SEAIU, SPAIU and PPAIU for the diagnosis of these small HCC were 85.3%, 94.5% and 91.4%, respectively. These results support the reliability of ICG injection during ultrasound-guided liver biopsy to diagnose even small HCC.