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Several specific assays have been developed for the measurement of etoposide in biological fluids. As large samples are required for high sensitivity, these systems are not appropriate for a pediatric practice. In the present study, however, an improved method for the determination of serum levels of the anticancer drug etoposide was developed, using high-performance liquid chromatography with fixed-wavelength ultraviolet detection. Etoposide was extracted from serum using dichloromethane. The efficiency of extraction from serum was 85.7 +/- 7.7% for etoposide and 81.1 +/- 8.4% for diphenylhydantoin, the internal standard. The serum concentrations of etoposide were measured in 0.2-ml serum samples. The lower limit of detection was 50 ng/ml. Each measurement was completed within 5 min. The linear quantitation range for etoposide was 0.05-50 microg/ml. This assay presents an alternative method for routine measurement of serum levels of etoposide in the pediatric oncology setting.

Increased activation of alveolar macrophage, neutrophil and mast cell has been proven in cigarette smoking (CS)-related lung disorders (CSLD). An increased production of cysteinyl-leukotrienes (LTs), which are mediators secreted from the mentioned cells, in response to CS has been shown in humans. The protective effect of LT1 receptor-1 antagonist (LTR-1AT) on CSLD is, however, not known. In this study we aimed to determine whether there is any protective effect of a LTR-1AT, montelukast (MK), on CSLD in Wistar rats. Nine controls and twenty-three smoke-exposed rats were enrolled into this study. Controls were exposed to non-filtered air, and the smoke-exposed rats were exposed to CS for 6 h/day, 6 days/week for three weeks. The CS-exposed rats were also treated with 0.1 mg/kg/day of MK or saline. Morphometric criteria for lung injury were determined as the mean linear intercept of alveolar septa (Lm), the volume density of alveolar septa (Vvspt) and the density of the alveolar surface area per unit volume of lung parenchyma (Sva.pa). Lung mast cells (LMC), which are a major source of LTs, were also counted. Results showed that Lm of the control group was significantly lower and Vvspt, Sva.pa of the controls were significantly higher compared to those of the CS-exposed groups. Animals treated with MK had significant protection against CSLD. Lm was significantly higher and Vvspt, Sva.pa were lower in the saline group than in the MK-treated group. The number of LMC in the CS-exposed groups was also significantly higher than that in the control group. Based on these results, one can suggest that some part of the pathogenesis of CSLD may be related to an enhanced LTs synthesis and LTR-1AT. Therefore, montelukast may protect against active or passive smoking-induced lung injury and related disorders.

In this study the clinical and laboratory findings of 48 children with purulent meningitis were examined, prospectively, to determine the prognostic factors in childhood meningitis in a developing country. Patients were examined for the following variables: history of antibiotic use; period between onset of symptoms and hospital admission; age at presentation; sex; fever; convulsion; level of consciousness; malnutrition; anemia; leukocyte and thrombocyte counts; erythrocyte sedimentation rate; serum C-reactive protein (CRP) level; and cerebrospinal fluid (CSF) including white blood cell count; glucose, protein, and CRP concentrations; antibiotic treatment; neurological sequelae; and fatality rate during the hospital stay. Most of these parameters were re-evaluated in all patients 36-48 h after admission. Patients were divided into 3 groups: surviving without sequelae, surviving with sequelae, and not surviving (deceased). A total of 48 children, 19 girls (39.5%) and 29 boys (60.5%), aged 2 months to 13 years, were included in the study. Of the 48 patients, 29 (60.5 %) survived without sequelae, 13 (27%) survived with sequelae and 6 (12.5%) died. In a comparison among groups, we found that absence of anemia, low (< 1,000) CSF white blood cell (WBC) count, and high CRP level at admission were the indicative of poor prognosis. Thirty-six to 48 h after admission, the presence of fever, depressed level of consciousness, high (> 1,000) CSF WBC count, and low CRP level were also poor prognostic factors. In addition, we observed that mortality rate was lower in the penicillin G + chloramphenicol group than in the ampicillin-sulbactam + cefotaxime group (P < 0.05). The mean period between onset of symptoms and hospital admission was longer in the surviving with sequelae and in the not surviving groups than in the surviving without sequelae group (P < 0.05).

In considering the characteristics of the action of anxiolytic drugs and their mechanism in the brain, it may be necessary not only to study the behavioral pharmacology but also to perform brain site research. In the present study, the action of anxiolytic drugs was examined with respect to various behaviors that were induced by stimulating the brain areas related to emotions such as reward (pleasure) or aversion in rats. First, the low rate of response in lateral hypothalamic self-stimulation behavior was induced by schedules of low current brain stimulation, variable interval (VI) and differential reinforcement of low rate (DRL). Anxiolytic drugs such as benzodiazepines facilitated these low-rate responses. The drug susceptibility was highest in the low current stimulation, lower in the VI stimulation, and lowest in the DRL stimulation schedules. Furthermore, it was found by the auto-titration method in intracranial self-stimulation behavior that anxiolytic drugs decreased the threshold of stimulation reward. Second, it was recognized using the decremental lever pressing (DLP) paradigm that anxiolytic drugs increased the threshold of aversive stimulation of mesencephalic dorsal central gray (DCG), and this increasing effect of the drug was antagonized by GABA receptor blockers such as biccuculline. Finally, it was examined whether or not the conflict situation is established by combining brain stimulation reward and aversion, such as foot-shock or DCG stimulation. As a result, the conflict behavior was established by combining not only the brain stimulation reward and foot-shock aversion, but also the brain stimulation reward and DCG stimulation aversion. Further anxiolytic drugs exhibited anti-conflict action in both situations. The susceptibility of anxiolytic drugs was higher with respect to the conflict behavior induced by intracranial reward and aversion than to that induced by the conventional method based on milk reward and foot-shock aversion. These results suggest that behavioral methods using brain stimulation can examine the mechanisms of direct drug action at the brain stimulation site. Indeed, in the present brain stimulation behavioral study, anxiolytic drugs such as benzodiazepines increased the stimulation threshold in lateral hypothalamic self-stimulation and inhibited the DCG aversive stimulation, thus resulting in an anticonflict action of the drugs.

Radiation damage to normal brain tissue induced by interstitial irradiation with iridium-192 seeds was sequentially evaluated by computed tomography (CT), magnetic resonance imaging (MRI), and histological examination. This study was carried out in 14 mature Japanese monkeys. The experimental area received more than 200-260 Gy of irradiation developed coagulative necrosis. Infiltration of macrophages to the periphery of the necrotic area was seen. In addition, neovascularization, hyalinization of vascular walls, and gliosis were found in the periphery of the area invaded by the macrophages. All sites at which the vascular walls were found to have acute stage fibrinoid necrosis eventually developed coagulative necrosis. The focus of necrosis was detected by MRI starting 1 week after the end of radiation treatment, and the size of the necrotic area did not change for 6 months. The peripheral areas showed clear ring enhancement with contrast material. Edema surrounding the lesions was the most significant 1 week after radiation and was reduced to a minimum level 1 month later. However, the edema then expanded once again and was sustained for as long as 6 months. CT did not provide as clear of a presentation as MRI, but it did reveal similar findings for the most part, and depicted calcification in the necrotic area. This experimental model is considered useful for conducting basic research on brachytherapy, as well as for achieving a better understanding of delayed radiation necrosis.

The purpose of this study was to search for chromosomal susceptibility loci for comitant strabismus. Genomic DNA was isolated from 10mL blood taken from each member of 30 nuclear families in which 2 or more siblings are affected by either esotropia or exotropia. A genome-wide search was performed with amplification by polymerase chain reaction of 400 markers in microsatellite regions with approximately 10 cM resolution. For each locus, non-parametric affected sib-pair analysis and non-parametric linkage analysis for multiple pedigrees (Genehunter software, http://linkage.rockefeller.edu/soft/) were used to calculate multipoint lod scores and non-parametric linkage (NPL) scores, respectively. In sib-pair analysis, lod scores showed basically flat lines with several peaks of 0.25 on all chromosomes. In non-parametric linkage analysis for multiple pedigrees, NPL scores showed one peak as high as 1.34 on chromosomes 1, 2, 4, 7, 10, 15, and 16, while 2 such peaks were found on chromosomes 3, 9, 11, 12, 18, and 20. Non-parametric linkage analysis for multiple pedigrees of 30 families with comitant strabismus suggested a number of chromosomal susceptibility loci. Our ongoing study involving a larger number of families will refine the accuracy of statistical analysis to pinpoint susceptibility loci for comitant strabismus.</P>

Chronic Hepatitis C can progress to end-stage liver cirrhosis or hepatocellular carcinoma. Interferon (IFN) therapy is effective in clearing the hepatitis C virus and in improving liver histology, however, few patients maintain a sustained response (SR) after IFN withdrawal. Immediate retreatment with IFN is therefore considered to be both effective and necessary, especially for patients who do not respond to the initial course of IFN therapy. All 145 patients included in the present study underwent liver biopsy, followed by a first treatment course with various IFNs (alpha2a, alpha2b, alpha, OIF or beta). If hepatitis C virus (HCV) RNA was positive after the first treatment, the patient was assigned to one of 3 groups, depending on whether his or her alanine transaminase (ALT)level was normalized (incomplete response, IR), partially responsive(PR), or non-responsive (NR). After an observational interval of 6 to 76 months, a second IFN treatment was initiated with a higher dose or the same dose of the same IFN for the IR group, and with a different IFN for the PR and NR groups. At 6 months after retreatment with IFN, the overall efficacy of the retreatment was 29.7.% In the case of the IR group, who received the same IFN, the overall efficacy was 45.2%. In patients identified as non-SR after the first treatment, who received a different type of IFN for retreatment, the overall efficacy was 18.6%. Anti-IFN antibody was not detected in most of the breakthrough cases. For some IR patients, retreatment with the same IFN was effective. Anti-IFN antibody was mostly negative, indicating that the same IFN can be used in both the first treatment and retreatment to obtain an SR. Switching to a different IFN was effective for some PR and NR patients, suggesting that changing IFN for such cases is a good therapeutic choice.

In this study, we investigated the relationship between intimal thickening of the internal carotid artery (ICA) and immunological reaction, and between occlusion of the ICA and development of basal collateral vessels in moyamoya disease. Rod-shaped lactic acid-glycolic acid copolymer (LGA-50) and N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide: MDP), and immuno-embolic material, were injected into cats unilaterally via the common carotid artery. Histological changes of duplication of the internal elastic lamina could be seen mainly in the terminal portion of the ICA in the animals injected with rod-shaped LGA-50 containing MDP. No angiographic changes were seen in any of the animals. These findings suggest that the immunological reaction induced by MDP caused histological changes in the intima of the ICA similar to those observed in moyamoya disease. This experimental study, however, could not clarify the development of the basal collateral vessels.

Moyamoya disease is a progressive vascular disorder of unknown etiology. Theories of inflammatory and immunologic mechanisms have been proposed as the pathogeneses. We have designed a new method of administering N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) for experimental induction of moyamoya disease using an intravascular interventional technique combined with rod-shaped embolic materials made from lactic acid-glycolic acid copolymer. The embolic materials containing MDP were repeatedly injected into the right internal carotid artery of monkeys in the embolic group. Intravenous injections of MDP solution alone were performed in the intravenous group. Histological examination of the arteries demonstrated reduplication and lamination of the internal elastic laminae, which corresponded with findings of moyamoya disease in both groups. These histological changes occurred not only in the intracranial arteries on the embolization side, but also in the contralateral intracranial and even extracranial arteries. The changes were more prominent in the intravenous group than in the embolic group. We conclude that the systemic humoral factors induced by MDP in this study may be important in the pathogeneses of moyamoya disease. Our observations suggest that moyamoya disease is a systemic vascular disease and has an etiologic factor affecting both intracranial and extracranial arteries

We estimated the number of stray dogs in Kathmandu, Nepal, where human rabies cases still occur, and in Shimotsui, Okayama Prefecture, Japan. In Kathmandu, the stray dog density was 2,930 stray dogs/km2, and the ratio of stray dogs to humans was 1:4.7. In Shimotsui, the density was 225 stray dogs/km2, and the ratio was 1:5.2. Since the stray dog population in Nepal is very large, one of the measures used to prevent dog bites and dog-acquired infections such as rabies is an effort to capture stray dogs. Another such measure is an effort to decrease the availability of food for stray dogs. We also organized health education programs in both Nepal and Okayama Prefecture, Japan, which involved a course on the prevention of dog bites and subsequent infections. After each course, a questionnaire survey was conducted. The results suggest that the course participants understood these important preventive methods. In addition to the measures mentioned above and the routine vaccination of dogs, this health education course is recommended as a long-term preventive program

Controversy exists over whether the predominant cell death of hepatocytes is due to apoptosis or necrosis after ischemia/reperfusion injury. In this study we investigated the predominant cell death of hepatocytes after cold ischemia/reperfusion injury using the Annexin V-based assay, and evaluated the anti-apoptotic effect of ascorbic acid 2-glucoside (AA-2G) added to the University of Wisconsin solution (UW solution) in rat liver transplantation. The retrieved liver was preserved in 4 UW solution for 24 h, and then transplanted orthotopically to the syngeneic Wistar recipient. The animals were divided into 2 groups, a control group (n=10), in which liver grafts were preserved in UW solution (4), and an AA-2G group (n=10), in which liver grafts were preserved in UW solution (4) with AA-2G (100 ug/ml). The serum AST level 4 h after reperfusion in the control group was significantly suppressed in the AA-2G group, and the bile production of the liver graft in the AA-2G group was well recovered. The mean survival time in the AA-2G group was significantly improved compared with that in the control group. Annexin-V and Propidium iodide staining 4 h after reperfusion showed a significantly higher percentage of viable hepatocytes in the AA-2G group compared with the control group (93.4 +/- 2.0 vs. 80.3 +- 2.1%, P<0.05). In the control group, the main cell death of hepatocytes was apoptosis (early apoptosis: 10.0 +- 4.7%, late apoptosis: 6.4 +/- 1.7%). The addition of AA-2G to the UW solution significantly inhibited both early and late apoptotic cell death 4 h after reperfusion (early apoptosis: 0.98 +/- 0.88%, late apoptosis: 2.2 +/- 1.1%). The expression of caspase 9 in the immunostaining of the liver graft was suppressed in the AA-2G group compared with in the control group. Our study using the Annexin V-based assay provided evidence that the predominant cell death of hepatocytes was apoptosis after 24 h cold ischemia/reperfusion injury in rat liver transplantation. The addition of AA-2G to the UW solution attenuated 24 h cold ischemia/reperfusion injury by inhibiting the apoptosis of hepatocytes.

To improve the efficacy of interferon (IFN) treatment for chronic hepatitis C, we have proposed the twice-daily administration of IFN-beta as a promising induction therapy. In this study, we demonstrated differences between the clearance of circulating HCV-RNA and the induction of anti-viral actions during the first 2 weeks of treatment. Nine patients with a high viral load and genotype 1b were randomly assigned to 3 groups: group A received 3MU of IFN-beta twice a day at intervals of 5 and 19 h; group B received 3MU of IFN-beta twice a day at intervals of 10 and 14 h; group C received 6MU of IFN-alpha once a day with ribavirin. The expression of OAS2, PKR, and MxA in peripheral blood mononuclear cells (PBMCs) were quantified by real-time polymerase chain reaction method. The viral clearance showed a bi-phasic pattern, and those in the second phase of groups A and B were significantly steeper than that of group C. The peak level of OAS2 during the first phase was correlated with the first phase decay. The MxA expression tended to be higher in group A and B than in group C. The expression of these 3 proteins tended to decrease at day 6 in group C, but increase in groups A and B. These might make differences in the viral decay during the second phase

This study was conducted to determine the gestational age-related reference range of the preload index [peak velocity during atrial contraction (A)/peak velocity during ventricular systole (S)] for the inferior vena cava (IVC), the right hepatic vein, the middle hepatic vein and the left hepatic vein. The slope and the intercept of the regression line for each preload index were compared among the 4 veins using analysis of covariance. Doppler measurements were obtained for the 4 veins of 316 normal fetuses at 22-40 weeks of gestation. A and S values were measured from the recorded flow velocity waveform of each vein and the A/S ratio was calculated as the preload index. The regression lines for the preload index of the 4 veins decreased gradually throughout gestation. Analysis of covariance revealed no significant differences in the slopes of the regression lines for the 4 veins. However, the intercepts of the regression lines for all hepatic veins were significantly higher than that of the regression line for the IVC (P<0.0001), with the difference ranging from 0.024 to 0.033. There were no significant differences among the intercepts of the regression lines for different hepatic veins. We concluded that the relationship between the preload index and the duration of gestation was statistically similar for all hepatic veins, and strongly resembled that for the IVC.

The aim of the present study was to evaluate the effects of three different anti-androgenic drug-therapy regimens, Diane 35 (cyproterone acetate (CPA) [2 mg] and ethinyl estradiol [35 microg]) plus CPA, Diane 35 plus spironolactone, and spironolactone alone, in patients with hirsutism. In this prospective, randomized clinical study, 79 subjects with idiopathic hirsutismus were studied. The patients were divided into 3 groups. Group I patients (n=32) were treated with Diane 35 plus CPA, group II patients (n=25) with Diane 35 plus spironolactone [100 mg], and group III patients (n=22) with spironolactone [100 mg] alone. Serum FSH, LH, testosterone (T), and DHEAS levels were analyzed before and after treatment at 6 and 12 months. Hirsutism scores were graded according to the Ferriman-Gallwey scoring system, and side effects were monitored. All treatment regimens were found to be efficient and well-tolerated, and none of the patients stopped therapy due to any adverse event. However, in hormone screening, only patients on the Diane 35 plus CPA regimen revealed a decrease in serum T levels after therapy. As such, treatment of each hirsute patient should be planned individually, but with regard to both cost-efficiency and potential side effects, we recommend spironolactone alone in the treatment of hirsutismus.

Allele and genotype frequencies for 15 short tandem repeat (STR) polymorphisms--D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX and FGA--in a Japanese population were estimated. No deviations of the observed allele frequency from Hardy-Weinberg equilibrium expectations were found for any of the systems studied. Between 2 new pentanucleotide STR loci, Penta E and Penta D, for which there is only limited data regarding the allelic distribution in Japanese, the Penta E locus was found to be highly polymorphic and exhibited a tri- or tetra-modal distribution pattern having allelic peaks with 5, 11, 15 and 20 repeats. The distribution was significantly different from that of the other ethnic groups. Statistical parameters of forensic importance, the power of discrimination (PD), observed and expected heterozygosity values (H), polymorphism information content (PIC), power of discrimination (PD), matching probability (pM), power of exclusion (PE), and typical paternity index (PI), were calculated for the loci. These parameters indicated the usefulness of the loci in forensic personal identification and paternity testing among Japanese. The systems Penta E, FGA, D18S51 and D8S1179 were the most informative. This method was successfully applied to forensic personal identification and paternity testing among Japanese, thereby confirming its efficacy for forensic practice.

Healthy subjects 40 years old were used as controls in a study of stellate cells (S-100 protein-containing cells, or S-100 cells) in subjects with chronic alcoholism and fatty liver or fatty cirrhosis. S-100 cells were sparsely found in the adenohypophysis of control subjects, and these cells sometimes formed small clusters. However, in chronic alcoholics with fatty liver or fatty cirrhosis, the number of stellate cells in the anterior pituitary tended to be 17 times higher than it was in the control group. No increase in the number of S-100 positive cells that constitute the large and small follicles in the intermediate pituitary. The physiological function of the S-100 protein has not yet been identified. The fact that an increase in prolactin-secreting and growth hormone-secreting cells, as well as a decrease in gonadotrophs were observed in the hypophysis of alcoholics suggests that the function of stellate cells may be closely related to these phenomena. Our results also imply that the stellate cells found in the anterior and intermediate pituitary differ in function although they both produce S-100 proteins.

In this study we used paraffin-embedded human pituitary obtained from 248 autopsy cases and identified mixed cell follicles by the immunohistochemical method. We examined the number and size of the mixed cell follicles, and the ratio of each component cell of these follicles, in the anterior pituitary at various age groups. The number of follicles increased with age, and the size of the follicles also tended to enlarge with age. Statistical analysis showed that a high correlation existed between age and the number or the size of the mixed cell-follicles formed by various adenohypophyseal cells. In addition, when the proportions of the different cell types that formed the follicles were examined, sex differences were observed with aging for the GH cells, the PRL cells, and the gonadotroph (GTH) cells, while no changes were observed with aging in both men and women for the ACTH cells and TSH cells. These results indicate that the number, size, and ratio of each component cell of follicles in the anterior pituitary are adequately applicable for the purpose of age estimation in routine forensic medicine.

The aim of this study was to reveal the clinical features of anterior urethral recurrence in patients with superficial bladder cancer, and to determine the appropriate treatment. Three hundred and three patients with superficial bladder cancer, who were newly diagnosed and initially treated conservatively in our hospital between 1965 and 1990, were followed for at least 5 years and their clinical outcomes were analyzed. Clinical factors, including anterior urethral recurrence, were evaluated statistically regarding tumor progression. Eight patients (2.6%) had anterior urethral recurrence following superficial bladder cancer. Twenty-four patients (7.9%) had tumor progression and 149 (49.2%) had tumor recurrence. In a multivariate analysis using a logistic model, anterior urethral recurrence was the most important factor, followed by histological grade. Four of 5 patients who were treated for anterior urethral recurrent tumors by transurethral resection showed progression and died of the cancer within one year. Two of the remaining three patients who underwent radical cysto-urethrectomy at the time of anterior urethral recurrence survived. Anterior urethral recurrence following superficial bladder cancer is a predictor for rapid subsequent malignant progression. Once there is anterior urethral recurrence, radical intensive therapy, including radical cysto-urethrectomy, should be carried out immediately.

In patients with hepatocellular carcinoma (HCC), natural killer (NK) cell activity decreases significantly, and the reduced activity may be associated with the progression of HCC. In this study we evaluated the effects of pulsing with interleukin (IL)-2 and/or IL-12 on the activation of freshly isolated peripheral blood lymphocytes (PBL) derived from patients with HCC. PBL obtained from 9 HCC patients, 4 liver cirrhosis patients, and 9 normal subjects were cultured in the presence of IL-2 and/or IL-12. After 24 h of incubation, the levels of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha presented in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA). The IFN-gamma and TNF-alpha production of PBL pulsed by a combination of IL-2 and IL-12 was significantly higher than those of PBL stimulated by either IL-2 or IL-12 alone. The mRNA encoding perforin, granzyme B, as well as IFN-gamma and TNF-alpha, were markedly enhanced in PBL stimulated with a combination of IL-12 and IL-2. The pulsing procedure of IL-12 in combination with IL-2 resulted in the increase of IFN-gamma and TNF-alpha, and the expression of perforin and granzyme B mRNA in PBL obtained from HCC patients, as well as in those obtained from normal subjects. These results indicate that adoptive immunotherapy based on PBL pulsed with a combination of IL-2 and IL-12 may be a promising adjunctive strategy for HCC treatment.

Germline mutations of the LKB1 gene are associated with Peutz-Jeghers syndrome (PJS), which is characterized by mucocutaneous pigmentation and gastrointestinal hamartoma with an increased risk of cancer development. In this study, we have employed polymerase chain reaction and DNA sequencing analysis to characterize the LKB1 gene in a 25-year-old Japanese PJS patient. Direct sequence analyses revealed a novel single base deletion at nucleotide 844 in exon 6 (844delC) in one LKB1 allele, resulting in a frame shift and in the introduction of a premature termination codon in this mutated allele.