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We describe a modified method for typing a polymorphic microsatellite D12S391 locus by PCR using a newly designed primer pair. This primer pair produces shorter D12S391 amplified fragments (104-156 bp) than the primer pair originally described by Lareu et al. (209-261 bp). The detection system for the D12S391 locus using the new primer pair and capillary electrophoresis (CE) analysis was evaluated using various forensic samples. The typing results from 70 DNA samples using the new primer pair and the original primer pair were completely identical. One hundred twenty-five amplified fragments from D12S391 alleles were sized correctly within +/- 0.25 bp of the D12S391 allelic ladder. A rare allele, 19.3, previously found only in Caucasians, was found for the first time in a Japanese subject, and it was clearly distinguished from allele 20 by the CE analysis. This detection system was sensitive and could detect D12S391 types from 16 pg of genomic DNA, and from a minor component at a ratio of 1:10 in mixed samples. This system was more useful for the analysis of degraded DNA than was the method using the original primer pair, and could detect D12S391 types from bloodstains that had been stored for 26 years. In addition, the specificity of the method was demonstrated using nonhuman DNA.

We previously reported that anthracyclines, which could generate reactive oxygen species (ROS), could induce the urokinase-type plasminogen activator (uPA) gene expression in human RC-K8 malignant lymphoma cells and in H69 small cell lung cancer (SCLC) cells. In screening other uPA-inducible anti-cancer agents, we found that camptothecin (CPT) and its derivative, SN38, could induce uPA in RC-K8 and H69 cells. CPT and SN38, which are also used for the treatment of lymphoma and SCLC, significantly increased the uPA accumulation in the conditioned media of both cells in a dose-dependent manner. The maximum induction of uPA mRNA levels was observed 24 h after stimulation. Pretreatment with pyrrolidine dithiocarbamate (PDTC), an anti-oxidant, inhibited the CPT-induced uPA mRNA expression. Thus, CPT induces uPA through gene expression, and, therefore, CPT may influence the tumor-cell biology by up-regulating the uPA/plasmin system.

A pilot study was conducted to assess the efficacy and feasibility of daily low-dose cisplatin with concurrent thoracic irradiation for clinically unresectable non-small-cell lung cancer (NSCLC). Patients with inoperable NSCLC who had poor risk factors such as advanced age, poor performance status, poor lung function, or concomitant active malignancy were entered into the study. Low-dose cisplatin (6 mg/m2) was administered daily before concurrent thoracic irradiation (2 Gy/day; total dose of 60 Gy) was given. Twenty-five patients were registered. The majority of the patients had either stage IIIA (24.0%) or stage IIIB (60.0%) disease. Fifteen patients (60.0%) completed the planned treatment. Both chemotherapy and radiotherapy were stopped in 3 patients (12.0%) due to poor response, and 7 patients (28.0%) partly received radiotherapy alone as a result of their toxicity response. The proportion of total administered dose to planned dose was 90.9% for chemotherapy and 99.3% for radiotherapy, which were comparable to those in previous studies for LA-NSCLC patients without poor risk factors. Grade 3 leukopenia and neutropenia developed in 14 patients (56.0%) and 10 patients (40.0%), respectively, but grade 4 toxicity was not encountered. Grade 3 pneumonitis and esophagitis were observed in 4 patients (16.0%) and 2 patients (8.0%), respectively. The overall response rate was 60.0%. The median survival time was 22 months, and the 2-year survival rate was 50.3%. Daily low-dose cisplatin and concurrent thoracic irradiation were well tolerated even by poor-risk patients with NSCLC, and showed a therapeutic efficacy similar to that for good-risk patients.

The morbidity of diabetes mellitus is increasing gradually in Japanese populations. It is important to clarify the risk factors of diabetes in Japanese populations in order to take adequate measures against the increasing morbidity of diabetes. In order to evaluate the link between past and concurrent obesity and diabetes in middle-aged Japanese men, we conducted a worksite-based historical cohort study in Okayama, Japan in 1999. Annual health examination data of middle-aged male workers in a worksite were collected. The relative risks of past and concurrent obesity for developing diabetes were calculated. Subjects with a past history of obesity at between 40 and 50 years of age had a significantly higher risk of developing diabetes by age 55 than did subjects in the normal weight group. These results suggest that, in order to prevent diabetes in middle-aged Japanese men, health guidance for normal weight maintenance should be provided not only for middle-aged men, but also for men under age 40.

Two-directional arthrographic findings made during conservative treatment of developmental dislocation of the hip were compared with the femoral-head configurations and radiological results obtained from long-term follow-up examinations in this retrospective study. Sixty hips were followed until at least age 14. Arthrography was carried out according to Terazawa's method. The shape of the superior, anterior, and posterior limbus was evaluated based on a modified Fujii's classification. The femoral-head configuration was classified into 4 groups, and the radiological results were evaluated using Severin's classification at the final observation. There was a statistically significant relationship between the shape of the anterior limbus, the number of portions of deformed limbus (superior, anterior, posterior), and the femoral-head configuration. Also, a statistically significant relationship between the shape of the limbus and Severin's classification was observed. These results suggest that the deformed limbus seems to play an important role in triggering femoral-head deformities, possibly via mechanical compression, and negatively affects development of the hip joint.

Pathologic features of Mycoplasma pneumoniae infection (M. pneumonia) are generally non-specific, and the literature regarding the pathologic features of M. pneumonia with intraalveolar exudates is limited. Clinical and histopathological studies were performed in 3 patients with M. pneumonia which did not respond to erythromycin and minocycline, but all rapidly recovered after corticosteroid therapy. In pathologic findings, we observed intraalveolar exudates and focal organization in M. pneumonia, and its intraalveolar lesions were compared between M. pneumonia and bronchiolitis obliterans organizing pneumonia containing fibrin (BOOP). Immunohistochemical studies were performed using the streptavidin biotin peroxidase complex method with anti-alpha-smooth muscle actin antibody and anti-pancytokeratin AE1/AE3 antibody. In pathologic findings, more fibrin deposits in intaalveolar lesions were observed in M. pneumonia than in BOOP. In intaalveolar lesions of M. pneumonia, a larger amount of nuclear debris, more neutrophils, and more erythrocytes were noted. Myofibroblasts were observed in the organization of BOOP, while in the intaalveolar lesions of M. pneumonia, myofibroblasts were not observed. These results suggest that M. pneumonia with intraalveolar exudates responds well to corticosteroid and its intraalveolar lesions apparently differed from those in BOOP.

Genetic and molecular biological methodologies are being applied to the study of patients with epilepsy at an ever-increasing pace. Accurate classification of epilepsy within large families has allowed identification of genes through linkage analysis and then isolation of gene products. Mutations causing ion channel abnormalities coupled with clinical patterns of focal epilepsy syndromes are beginning to change our thinking about the etiology of recurrent seizures in all patients. Molecular methodology is beginning to have impact on understanding of the mechanisms of actions of drugs used to treat epilepsy and will have an impact on how future treatments are designed.

Leu-7 positive lymphocytes, including natural killer cells, play an important role in the immune system's surveillance function to prevent the development of cancer. The incidence of lung cancer is significantly high in patients with end-stage pulmonary fibrosis. We hypothesized that the number of Leu-7 positive cells may be decreased in areas of severe pulmonary fibrosis. To demonstrate this, Leu-7 positive cells were immunohistochemically stained in 41 lung specimens obtained from patients with idiopathic pulmonary fibrosis and pulmonary fibrosis associated with collagen vascular disorders. The number of Leu-7 positive cells was evaluated according to the pathological findings. In pathologically normal lung, Leu-7 positive cells were mostly found within the capillaries of the septa and rarely in the alveolar space or the stroma. The number of Leu-7 positive cells was 0.69 +/- 0.15 in areas of advanced fibrosis (n = 41), 2.39 +/- 0.60 in areas that had newly developeing fibrosis (n = 41), 1.14 +/- 0.57 in bronchiolitis obliterans organizing pneumonia (n = 9), and 1.35 +/- 0.87 in diffuse alveolar damage (DAD) (n = 11). The number of Leu-7 positive cells in areas of newly developing fibrosis (2.39 +/- 0.60) was significantly higher than that in areas of established fibrosis (0.69 +/- 0.15, P < 0.05). Our present study demonstrates a significant decrease in the number of Leu-7 positive cells in areas of advanced fibrosis. This evidence may partly explain the high incidence of lung cancer associated with pulmonary fibrosis.

We studied the pressure exerted by hands during push-ups in 21 paraplegic and 2 tetraplegic patients employing 4 different hand positions. In the fingers-spread position, the initial force exerted was a vertical force (Fz), followed by a medio-lateral force (Fy) and then an antero-posterior force (Fx). In the other 3 positions, the order of force type exertion was Fz, Fx, and then Fy. All subjects with neurological injury levels above T4 and subjects between T5 and T10 without spinal instrumentation could not push themselves up in the fingers-spread position. The fact that Fy is initiated before Fx in the fingers-spread position indicates that lateral balancing of the trunk is critical in this position, thus explaining why subjects without spinal instrumentation with neurological injury at a level higher than T10 could not control their spinal columns while performing push-ups. In contrast, antero-posterior balancing takes priority in the other hand positions. We believe that spinal instrumentation helps balance the trunk in the lateral direction, converting the thoracic spine into a rigid body in subjects with neurological injury at levels above T10.

A 55-year-old Japanese woman presented at our hospital complaining of hematochezia 4 months after surgery for a rectal carcinoma. A proctoscopy revealed 2 protuberant lesions in the rectum, 5 mm anally from the anastomotic suture line. The diagnosis of adenocarcinoma was confirmed by biopsy. It was considered that these lesions were caused by intraluminal implantation from the primary rectal carcinoma. The patient underwent an endoscopic resection for these recurrent lesions and has remained stable, with neither recurrence nor metastasis, in the 7 years since the resection. For rectal carcinoma, we propose early follow-up by proctoscopy, namely within 4 months after surgery.

Two kinds of surgical models of small intestinal transplantation (SITx) in rats, namely heterotopic (HIT) and orthotopic transplantion (OIT), have been reviewed. In OIT, the small intestine of the recipient is removed and the transplanted intestine replaces it in continuity. On the other hand, in the HIT model, the small intestinal grafts are rendered dysfunctional without alimentary tract continuity. Histological evidence showed that acute rejection appeared earlier in HIT as compared to OIT. Hyperplasia and hypertrophy of the muscularis externa produced in the chronic rejection process were more pronounced in HIT allografts. The HIT grafts showed severe mucosal atrophy due to the lack of intraluminal trophic factors, because oral feedings can stimulate tropic hormones for mucosal growth, and provide nutrients for enterocytes. Intestinal permeability was consistently higher after HIT than after OIT. The HIT grafts demonstrated less contractility and less response to chemical stimulation than did OIT grafts. The OIT models are advantageous in studies of intraluminal nutrients, and intestinal secretions in these models might modulate the intestinal immune status and possibly delay rejection. The superior intestinal barrier function and the delayed onset of rejection in OIT rats suggest that nutrients and other factors in the succus entericus are important for the maintenance of intestinal graft function.

Based on recent LightCycler techniques developed for the quantitation of serum HCV RNA, we have developed a quantitative method for the intracellular hepatitis C virus (HCV) RNA using LightCycler PCR. A simple real-time PCR assay, based on the SYBR Green I dye and LightCycler fluorimeter and with no probe requirement, is described. In the presence of 0.5 microg of cellular RNA, it was demonstrated that as few as 25 copies of HCV RNA could be specifically detected with a set of primers that amplify a 144-base pair sequence unique to the 5'-noncoding region of HCV RNA. We demonstrated that this method was useful for the evaluation of antiviral reagents using HCV-infected human cultured cells.

In autoimmune chronic active hepatitis (AIH) and primary biliary cirrhosis (PBC), various autoantibodies including anti-asialoglycoprotein receptor (ASGPR) antibodies have been found in patients' sera. We have previously developed a mouse monoclonal antibody against rat and human ASGPR. In this study, we developed a capture enzyme-linked immunosorbent assay (ELISA) for detection of anti-ASGPR antibodies using this monoclonal antibody and investigated the occurrence of anti-ASGPR antibodies in the sera of patients with various liver diseases. Serum samples were obtained from 123 patients with various liver diseases, including 21 patients with AIH and 40 patients with PBC. In this capture ELISA, the target antigen in the crude rat liver membrane extracts was captured on the ELISA wells by the ASGPR-specific mouse monoclonal antibody. Thus, the cumbersome process of antigen purification was rendered unnecessary. Using this capture ELISA, we detected the anti-ASGPR antibody in 67% of the patients with AIH, in 100% of the patients with PBC, and in 57% of the patients with acute hepatitis type A. However, the anti-ASGPR antibody was rarely detected in patients with other liver diseases such as primary sclerosing cholangitis and obstructive jaundice. Our findings suggest that this capture ELISA would be useful for the detection of anti-ASGPR antibodies in autoimmune liver diseases.

This study involved the examination of 1,006 chest x-ray films of workers from the industries devoted to shipyard welding, stone grinding, and refractory crushing in southern Okayama prefecture. Of the reviewed films, analysis was focused on subjects with a profusion rate of 0/1 as well as pneumoconiotic subjects (exhibiting profusion rates of 1/0 or greater) in order to discover cases in the beginning stages. One-hundred-and-seventy-four films illustrated a profusion rate of 0/1 or greater, and the proportion of this profusion rate was revealed to be highest in shipyard welders. Even some workers under 40 years of age were found to have already developed pneumoconiosis. Of these 1,006 subjects, 30 volunteers permitted us to measure their personal dust exposure concentrations. The measured concentration of the shipyard welders' dust exposure (respirable dust; 3.3 86.3 mg/m3, total dust; 7.5-117.0 mg/m3) was higher than those of the other 2 industries. Statistical differences among the industries were observed in the respirable dust concentrations. A statistically significant positive correlation was demonstrated between the working duration in dusty environments and the rate of profusion. The present findings suggest the need for taking adequate measures in Okayama in order to prevent workers from developing, or to help retard the progression of, pneumoconiosis.

Tremendous excitement has been generated by the use of botulinum toxin for the treatment of various types of urethral and bladder dysfunction over the past several years. Botulinum toxin is the most lethal naturally occurring toxin known to mankind. Why, then, would an urologist want to use this agent to poison the bladder or urethral sphincter? In this review article we will examine the mechanisms underlying the effects of botulinum toxin treatment. We will discuss the current use of this agent within the urologic community and will provide perspectives on future targets of botulinum toxin.

Leukotrienes, one of the mediators of inflammation in asthma, have a strong bronchoconstrictive effect. L-carnitine has been reported to influence respiratory functions. It has also been reported that L-carnitine inhibits leukotriene synthesis. To evaluate the effects of L-carnitine on oxygen saturation, urine leukotriene E4 levels and lung histopathology in a murine model of asthma, high IgE responder BALB/c mice (n = 24) were systemically sensitized to ovalbumin and chronically challenged with low particle mass concentrations of aerosolized ovalbumin, and then they were divided into 3 groups (study groups A, B, and C) each including eight mice. After methacholine-induced bronchoconstriction, the mice in groups A and B were given intraperitoneal L-carnitine (250 and 125 mg/kg, respectively), while the mice in group C were given placebo. Oxygen saturation of the mice was measured by pulse oxymeter before and after methacholine and after L-carnitine/ placebo application. In addition, urine leukotriene E4 levels were measured before asthma development, and 24-h after L-carnitine injection in asthmatic mice. Inflammation in the lung tissues of the sacrificed animals was scored histopathologically to determine the effect of L-carnitine on tissue level. A control group of non-sensitized mice (n = 8) treated with placebo only was used for comparison of urine leukotriene E4 levels and of histopathological parameters. Oxygen saturation of the mice in the study groups tended to decrease after methacholine and to improve after L-carnitine injection, although these changes were not significant at all time points. Urine leukotriene E4 levels of all 3 study groups increased significantly after asthma development. The rate of increment was smallest in the group given the highest L-carnitine dose (group A). Inflammation at the tissue level was also mildest in group A, and severest in the group that was not given carnitine (group C). All of the study groups and the control group differed significantly with respect to inflammation scores. In conclusion, L-carnitine improved oxygen saturation, and decreased urine leukotriene E4 levels and inflammation in lung tissues in the present murine model of asthma.

The association between depressed mood and condom use was examined among incarcerated male adolescents. One hundred and eighty male adolescents who were detained in Birmingham, Alabama in the United States were interviewed during a period of incarceration. Contrary to patterns generally found in adult samples, nearly 50% of this adolescent sample that did not use condoms regularly actually recognized the advantages of condom use. This behavior pattern was deemed "inconsistent," and those engaging in this "inconsistent" behavior pattern were found to have a higher score of depressed mood compared to participants with a "consistent" behavior pattern. As a result, a relationship between depressed mood and decisional balance for condom use within adolescents was evident. These findings suggest that assessment and treatment of depressed mood within this high-risk population could potentially contribute to a reduction in high-risk sexual behaviors.

In this study, we tested brain surface cooling as a new method of inducing selective brain hypothermia, and evaluated its effects on focal cerebral ischemia using a cat model of transient middle cerebral artery (MCA) occlusion. Cats underwent 1 h of MCA occlusion followed by 5 h of reperfusion. Brain surface cooling was induced for 4 h during and after MCA occlusion in the hypothermia group, but not in the normothermia group. Brain surface cooling was performed using saline perfusion into the subdural space. Rectal temperature, brain surface temperature, and deep brain temperature were monitored, and regional cerebral blood flow (rCBF) and somatosensory evoked potential (SEP) were serially measured. After 5 h of reperfusion, water content was also measured. Although the rectal temperature was maintained at about 37 degrees C, the brain surface temperature decreased rapidly to 33 degrees C and was maintained at that temperature. For 3 h following reperfusion, the rCBF was lower in the hypothermia group than in the normothermia group. At 4 and 5 h after reperfusion, the recovery of SEP amplitude was significantly more enhanced in the hypothermia group than in the normothermia group. In the gray matter, the water content was significantly more diminished in the hypothermia group than in the normothermia group. These results demonstrate that our method is useful for protecting the ischemic brain from a transient MCA occlusion. This method may be adapted for neurological surgery.

Levels of alpha-fetoprotein (AFP), its glycoforms AFP-L3 and AFP-P4, and proteins induced by vitamin K absence or antagonist-II (PIVKA-II) were determined in sera obtained from patients in Yangon General Hospital (20 with hepatocellular carcinoma (HCC), 29 with chronic liver diseases, including 3 with chronic hepatitis and 26 with cirrhosis of the liver, and 9 with other hepatobiliary diseases). Forty-five percent of the patients with HCC had serum AFP levels above 10,000 ng/ml, indicating that nearly half of the HCC patients were at an advanced stage of the disease. Thus, the AFP sensitivity was as high as 70% with 100% specificity for a cutoff level of 200 ng/ml. The sensitivity of AFP-L3 was 75% and a specificity 90% for a cutoff level of 15%. AFP-P4 showed a higher sensitivity of 80% and a similar specificity of 86% for a cutoff level of 12%. Combined evaluation of AFP-L3 and/or AFP-P4 increased the sensitivity to 90% with the same specificity of 86%, indicating that AFP-L3 and AFP-P4 are useful as adjuncts for diagnosis of HCC in the present population. PIVKA-II had a high sensitivity of 90%, although the specificity was lower than 45%, probably due to the low cutoff level, as some cholestatic patients were included in the control group.

Transforming growth factor-beta1 (TGF-beta1) exerts potent immunosuppressive effects. In this study, we investigated the potential role of TGF-beta1 produced by hepatocellular carcinoma (HCC) cell lines in immunosuppression mechanisms. Using the Mv1Lu cell-growth inhibition assay and an enzyme-linked immunosorbent assay (ELISA), we detected optimal levels of TGF-beta1 in the culture supernatants conditioned by the HCC cell lines PLC/PRF/5, Hep3B, and HepG2. To determine the biological activity of TGF-beta1 in the supernatants, we examined the effects of the culture supernatants on the production of interferon (IFN)-gamma induced during the culture of peripheral blood mononuclear cells (PBMCs) stimulated with interleukin (IL)-12. IFN-gamma production of IL-12-stimulated PBMCs in the 1:1 dilution of the acid-activated conditioned medium of PLC/PRF/5, Hep3B, and HepG2 reduced to 14.7 +/- 0.8, 17.3 +/- 9.0, and 35.9 +/- 14.6%, respectively, compared with the value in the culture with control medium (complete culture medium). These results suggest that HCC cells producing TGF-beta1 may reduce the generation or activation of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, and thus could enhance their ability to escape immune-mediated surveillance.