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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

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.

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.

In order to elucidate the role of erythrocyte catalase in the accumulation of mercury in erythrocytes, labeled erythrocytes and plasma were prepared by exposing normal and acatalasemic mice to radioactive mercury vapor (203Hg0: 6.8mg/m3) for 30 min. Labeled erythrocytes (or plasma) were mixed with unlabeled plasma (or erythrocytes) of normal or acatalasemic mice and incubated at 0 degrees C for 1 h. After incubation, the radioactivity of mercury in the erythrocytes and the plasma was measured by a gammascintillation counter. When labeled erythrocytes were incubated with unlabeled plasma, the ratio of mercury transferred from acatalasemic erythrocytes to normal plasma (11.6%) or to acatalasemic plasma (13.3%) were significantly higher than that from normal erythrocytes to normal plasma (1.8%) or to acatalasemic plasma (2.2%). When labeled normal (or acatalasemic) plasma was incubated with unlabeled normal or acatalasemic erythrocytes, the uptake of mercury by acatalasemic erythrocytes from normal plasma was 2.0%, and 1.2% from acatalasemic plasma, which tended to be lower than that by normal erythrocytes from normal plasma (3.4%) or from acatalasemic plasma (2.2%). The results indicated impaired accumulation of mercury in acatalasemic erythrocytes, suggesting the importance of catalase in taking up mercury in erythrocytes and protecting other organs from toxic effects of metallic mercury.

A sharp rise in the number of patients with infectious gastroenteritis was observed in the 25th week of year 1996 in the Takahashi-Ashin district by researchers with the Infectious Disease Surveillance Program for tuberculosis and other infectious diseases in the Okayama Prefecture. This sharp rise occurred coincidentally with an outbreak of enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) infection in Niimi City of the Takahashi-Ashin district. However, this phenomenon of coincidental outbreaks was not observed during the outbreak of EHEC O157 infection in Oku Cho. By reviewing outpatients' charts in a sentinel hospital in Niimi City for the Infectious Disease Surveillance Program, it was noted that patients with acute gastrointestinal infection visiting the hospital during the increased incidence of infectious gastroenteritis may have been included as misclassified cases of EHEC O157 infection. On the other hand, the exponential probability plotting of symptomatic patients with EHEC O157 infection in Niimi City revealed a breaking point which suggested a dual exposure to contaminated food or an overlap with other acute gastrointestinal infections. The latter possibility was discounted, because stool culture-positive patients with EHEC O157 infection also exhibited a similar breaking point, and furthermore, the coincidental increase in infectious gastroenteritis in the same area was attributable to the EHEC O157 infection. The present study demonstrates the association between the sharp rise in gastroenteritis and the outbreak of EHEC O157 in the Takahashi-Ashin district. A careful analysis of the cases of infectious gastroenteritis by the Infectious Disease Surveillance Program would have predicted the outbreak of EHEC O157.

We developed a method for human identification of forensic biological materials by PCR-based detection of a human-specific sequence in exon 3 of the myoglobin gene. This human-specific DNA sequence was deduced from differences in the amino acid sequences of myoglobins between humans and other animal species. The new method enabled amplification of the target DNA fragment from 30 samples of human DNA, and the amplified sequences were identical with that already reported. Using this method, we were able to distinguish human samples from those of 21 kinds of animals: the crab-eating monkey, horse, cow, sheep, goat, pig, wild boar, dog, raccoon dog, cat, rabbit, guinea pig, hamster, rat, mouse, whale, chicken, pigeon, turtle, frog, and tuna. However, we were unable to distinguish between human and gorilla samples. This method enabled us to detect the target sequence from 25 pg of human DNA, and the target DNA fragment from blood stored at 37 degrees C for 6 months, and from bloodstains heated at 150 degrees C for 4 h or stored at room temperature for 26 years. Herein we also report a practical application of the method for human identification of a bone fragment.</P>

We have improved on conventional methods for HLA-DRB1 genotyping and devised a new method that is simple, cost-effective, and adequately applicable to routine forensic practice. This method consists of group-specific polymerase chain reaction (PCR) of the exon 2 region of the HLA-DRB1 gene and simultaneous detection of single nucleotide polymorphisms (SNPs) at multiple sites using multiplex primer extension reactions. With this method, we successfully detected HLA-DRB1 genotypes from the following materials: the peripheral blood of 142 donors, 6 aged saliva stains of known DRB1 genotype stored for 5-10 years at room temperature, 10 aged bloodstains of unknown DRB1 genotype stored for 29 years at room temperature, and minimal bloodstains and saliva stains from 3 donors of known DRB1 genotypes. Furthermore, we were able to type DRB1 alleles of the minor component in mixed samples at a proportion of 1/1,000 or 1/10,000. In a criminal case, DRB1 alleles detected from mixed bloodstains on a sword found at the scene enabled us to explain the case. This method is expected to be useful for forensic medicine.

Serum des-gamma-carboxy prothrombin (DCP) is commonly used to detect hepatocellular carcinoma (HCC). This review focuses on the clinical features of DCP-positive HCC and the molecular function of DCP in HCC. DCP-positive HCC demonstrates more aggressive clinicopathological features than DCP-negative HCC. Analysis of the biological effects of DCP revealed that DCP acts as a growth factor in both an autocrine and paracrine manner. DCP stimulates HCC cell proliferation through the Met-Janus kinase 1-signal transducer and activator of transcription 3 signaling pathway, whereas for vascular endothelial cells, it stimulates cell proliferation and migration through the kinase insert domain receptor-phospholipase C-gamma-mitogen-activated protein kinase signaling pathway.

The annual number of suicides in Japan increased sharply in 1998, and since that time it has consistently exceeded 30,000 per year. In this study, we analyze a database of personal and background characteristics of 824 cases (605 men, 219 women) who completed suicide in Okayama Prefecture in 2002 and 2003. The data were obtained with cooperation from the police. Using the methodologies in a previous European study as a model, we classified the suicide methods into 8 categories. To examine the generational and regional differences in the choice of methods, we stratified the sample into 4 age groups (<-24, 2544, 4564, and >-65) and 2 regional groups (Okayama/Kurashiki vs. other areas). Our results on gender differences in 7 of the suicide methods were mostly similar to the European data. However, our data showed a remarkably higher proportionate male-to-female mortality ratio for poisoning by other substances (ICD-10, X65-X69 codes) (1.83, 1.15-2.92). In terms of generational differences in the choice of suicide methods, the Mantel-Haenszel test of homogeneity was significant for most of the categories in our study, suggesting an impact of age on how people commit suicide. There were no remarkable regional differences in our sample. An epidemic curve for suicides via carbon monoxide poisoning using charcoal briquets revealed a trend of time clustering not observed in the other 6 means. The database constructed and used in this study contains richer information than conventional death statistics and is expected to provide helpful knowledge and insights for future epidemiological studies.

We encountered a rare case of suffocation by an advertising balloon filled with pure helium gas. Suffocation caused by inhalation of atmosphere lacking in oxygen is not exceptional, but reports of death by suffocation due to a pure inert gas such as helium are very rare. In this case, the balloon mooring on the ground was enclosed, warning signs were displayed, and it was clear that entering the balloon filled with an atmosphere lacking in oxygen was extremely dangerous and should not be done; the accident did, however, occur. Accidents of this kind may occur in the future unless appropriate education and countermeasures are taken.

Anti-idiotype antibodies (Ab2) play an important role in the homeostasis of immune responses and are related to the development and the disease activity of certain autoimmune diseases. The asialoglycoprotein receptor (ASGPR) is considered one of the target antigens in the pathogenesis of autoimmune chronic active hepatitis (AIH). We previously developed a mouse monoclonal antibody (clone 8D7) which recognizes rat and human ASGPR. In this study, to help investigate the anti-ASGPR antibody-anti-idiotype antibody network in patients with AIH, we developed a syngeneic mouse monoclonal Ab2 to the 8D7 anti-ASGPR antibody (Ab1). One clone, designated as 3C8, tested positive for specific reactivity to 8D7-Ab1 and did not bind to other irrelevant immunoglobulins. By competitive inhibition assays, the binding of 8D7-Ab1 to liver membrane extracts, i.e., the crude antigen preparation, was inhibited by 3C8-Ab2 in a dose-dependent manner, and the binding of 8D7-Ab1 to 3C8-Ab2 was inhibited by the liver membrane extracts. In the immunohistochemical analysis, 3C8-Ab2 blocked the specific staining of sinusoidal margins of rat hepatocytes by 8D7-Ab1. These results suggest that 3C8 anti-idiotype antibody recognizes the specific idiotypic determinants within the antigen-binding site of 8D7-Ab1.

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.

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.

We have previously developed an enzyme-linked immunosorbent assay (ELISA) to measure stool decay-accelerating factor (DAF) and found that stool DAF concentrations were significantly elevated in patients with colorectal cancer, suggesting that the measurement of stool DAF may be valuable for the detection of colorectal cancer. In order to refine the assay for the measurement of stool DAF, we investigated 1) effects of centrifugation of stool samples, 2) effects of detergents, and 3) adequate combination of various anti-DAF monoclonal antibodies for the ELISA system using only monoclonal antibodies. We found that high-speed centrifugation could be omitted and that only the removal of large undigested food residues by centrifugation of short duration in a low-speed benchtop microcentrifuge sufficed to adequately prepare the stool samples. Addition of 2 detergents, octyl beta-glucoside and sodium deoxycholate, known to solubilize glycosyl-phosphatidylinositol-anchored proteins such as DAF, did not influence stool DAF values. By using 2 mouse anti-DAF monoclonal antibodies (clone 4F11 and 1C6), we were able to achieve a stable ELISA for the measurement of stool DAF using a uniform source of antibodies. The results should allow us to consistently apply the DAF assay for routine use in the detection of colorectal cancer.

The preS2 region of the hepatitis B virus (HBV) has been reported to have human polymerized albumin receptor (PAR) activity, which correlates with viral replication. Here, we studied the genomic sequence of the preS region from rare patients lacking PAR activity, despite active viral replication. PAR and DNA polymerase activity was identified in 178 HBe antigen-positive HBV carriers, and a significant correlation between 2 markers was shown, except in 2 hepatitis patients lacking PAR activity. Nucleotide sequences of the preS region of HBV from both patients were examined by direct sequencing of PCR products. In one patient, a 45-base deletion was found to overlap half of the putative polymerized human albumin binding site in the preS2 region. In the other patient, a point mutation at the first nucleotide of the start codon of the preS2 region of HBV was found. There was no such genomic change in the 3 control HBV sequences. These results indicate that the preS2 region is necessary for binding of polymerized human albumin, and this is the first report of naturally existing mutant virus with no or low PAR activity.