小澤 周二

Background: Deaths due to heat stroke have surged in recent years in Japan. While the long-term sequelae of heat stroke in its survivors are well understood, our previous studies suggest that heat exposure may cause fatal organ damage before the systemic inflammatory response is triggered. In this study we aimed to provide a comprehensive analysis of cytokines and other signaling factors in sera and myocardial tissues in a rat model of heat stress. Methods: Male Wistar rats were anesthetized and subjected to heat stress (37.0°C, 100% humidity; n = 8) to induce heat stroke (Heat group, n = 4) or normal temperatures (Control group, n = 4) for 90 min, followed by collection of blood sera and heart tissue samples. A multiplex assay system was used to analyze serum markers. Heart tissue was subjected to multiplex RNA sequencing and gene cascade analysis, and combined with iTRAQ (isobaric tags for relative and absolute quantitation) protein pathway analysis. Results: There were significantly higher levels of five interleukins (IL-1α, IL-2, IL-6, IL-17A, IL-18), fractalkine, interferon-γ-inducible protein-10, leptin, tumor necrosis factor α, and vascular endothelial growth factor, and significantly lower levels of LIX and RANTES, in the sera of the Heat group compared with the Control group. From the 2,741 genes that showed a significant difference in myocardial mRNA expression between the groups, we identified 48 transcription factors and 34 binding regions. Pathway analysis identified the involvement of key node proteins including protein kinase C isoforms, calmodulin-dependent protein kinase II, and protein interacting with C-kinase (PICK1). Conclusion: This model of heat stress in rats triggered changes in the mRNA and protein levels of many genes involved in cardiac function, primarily affecting the activity of ion channels and mitogen-activated protein kinase.

Glioblastoma multiforme (GBM) is the most common primary intracranial neoplasm. Although maternal mortality in Japan is among the lowest in the world, the main cause of maternal mortality is pregnancy-related intracranial hemorrhage. This case reports a 30-year-old female pregnant patient who suddenly died at 30 weeks of gestation. She had complained of headache from early stages during pregnancy and reported a headache and stiff neck starting two weeks before her death. Postmortem computed tomography revealed an intracerebral hematoma. The autopsy of the brain revealed severe swelling. The left frontal lobe contained a cavity filled with clear fluid, and a hematoma weighing 24 g. There was also widespread cerebral edema. The histological findings after evaluation of the cavity wall material were indicative of malignancy, and positive immunostaining for alpha thalassemia/mental retardation syndrome X-linked (ATRX), p53 and isocitrate dehydrogenase (IDH-1), was consistent with a diagnosis of GBM. There are studies that indicate pregnancy can promote the clinical and radiological advancement of glioma. The acceleration of tumor growth during pregnancy has been found to depend on several factors (e.g., hormonal factors, growth factors, and hemodynamic changes). Increased cardiac output and blood volume have been shown to promote the size of vascular tumors during pregnancy. Although headaches and neck stiffness are often considered pregnancy-related, this case highlights the importance of its prompt diagnosis, which can also save the fetus’ life. It also emphasizes the need for forensic pathologists to further evaluate intracerebral hematomas identified during investigations of maternal deaths during pregnancy.

法医解剖時に得た資料をもとに、睡眠中の乳幼児急死と関連する要因について調査した。多施設から報告された259例(男児145例、女児114例)を対象とした。発生頻度は1000出生当たり年0.3件程度であり、平均出生時体重は男児が2888±553g、女児が2750±370g、低出生体重児は44例(18%)を占めた。低出生体重児と早産は正常体重児と満期産に対して2倍程度の増強因子となった。母親の年齢について、30代を対照とした時に10代母親のオッズ比は11.1と10倍を超え、20歳代は2.1であった。出生順位については、第1子に対して第2および第3子は1.7、第4子以降は5.1と大きく上昇した。非喫煙者に対して喫煙者は4.5となった。また、異常発見時の添い寝は62%で、体位は仰向け52%、うつ伏せ40%、横臥位7%であった。10代の母親、下位の出生順位において特にリスクが高いと結論でき、発見時の睡眠体位等のデータも得られた。

STRUCTURED ABSTRACTPurpose/aim of the study: Dry eye syndrome is known to develop from several systemic inflammatory diseases. Although dry eye may develop due to extraintestinal complications of ulcerative colitis (UC), the pathogenesis is not well-known. This study aimed to investigate whether there was decrease in the tear secretion volume in a mice model with UC; the difference between the control and dextran sodium sulfate (DSS)-treated group was also determined.Materials and methods: This study included a mice model with UC induced by the oral administration of 5.0% DSS for 7 days. Following the DSS treatment, the tear volume was measured using the Schirmer's test. The colon and ocular tissues, including the lacrimal gland, were evaluated using histological and protein analyses. Additionally, tumor necrosis factor (TNF)-α and interleukin (IL)-6 in the plasma were determined. Differences between groups (DSS-treated versus control mice) were determined using Student's t-test.Results: The tear volume in DSS-treated mice was decreased compared to that in the control mice. Plasma levels of TNF-α and IL-6 in DSS-treated mice was higher than that of control. Morphological change was observed with the invasion of the inflammatory cell in the lacrimal gland of DSS-treated mice. Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells were increased in the lacrimal glands of DSS-treated mice compared with control group. The distribution of aquaporin-5 expressed in the lacrimal gland of DSS-treated mice was decreased compared to that in the control group.Conclusions: These findings suggest that a decrease in the tear volume in UC was associated with a functional decline in the inflamed lacrimal gland. This result therefore provides useful information that could contribute to the development of treatment approaches for dry eye associated with UC.

BACKGROUND: Sudden unexpected death in infancy (SUDI) comprises both natural and unnatural causes of death. However, few epidemiological surveys have investigated SUDI in Japan. OBJECTIVE: This retrospective study was conducted to investigate the latest trends of circumstances and risk factors of SUDI cases in which collapse occurred during sleep. METHODS: Forensic pathology sections from eight universities participated in the selection of subjects from 2013 to 2018. Data obtained from the checklist form were analyzed based on information at postmortem. RESULTS: There were 259 SUDI cases consisting of 145 male infants and 114 female infants with a mean birth weight of 2888 ± 553 and 2750 ± 370 g, respectively. Deaths most frequently occurred among infants at 1 month of age (18%). According to population data as the control, the odds ratio (95% confidence interval) of mother's age ≤19 years was 11.1 (6.9-17.7) compared with ages 30-39. The odds ratio for the fourth- and later born infants was 5.2 (3.4-7.9) compared with the frequency of first-born infants. The most frequent time of day for discovery was between 7 and 8 o'clock, and the time difference from the last seen alive was a mean of 4.1 h. Co-sleeping was recorded for 61%, and the prone position was found for 40% of cases at discovery. Mother's smoking habit exhibited an odds ratio of 4.5 (2.9-5.8). CONCLUSION: This study confirmed the trends that have been observed for sudden infant death syndrome; particularly, very high odds ratios were evident for teenage mothers and later birth order in comparison with those in other developed countries. Neglect was suspected in some cases of the prolonged time to discovery of unreactive infants. To our knowledge, this is the first report of an extensive survey of SUDI during sleep in Japan.

This study presents an autopsy case of unexpected sudden death caused by mitochondrial disorders with a subdural hematoma suspected to have manifested from Pearson’s marrow pancreas syndrome in a four-year-old boy. The cause of death was not determined by his history and autopsy, but was later determined via a postmortem examination. The boy was unable to walk unaided four days before death. On the day of death, he developed a fever of 37.4 °C and coughed to the point of vomiting when given water by his mother, after which he became limp with fatigue. He was brought to the hospital by ambulance, but he died without recovering. The boy had many symptoms but no diagnosis. During the autopsy, a subdural hematoma was identified, and severe fatty changes were observed in the liver. Biochemical analysis and molecular testing revealed no particular metabolic disorders. The activity level of mitochondrial respiratory chain complex I was significantly decreased in the liver, whereas that of complex II was slightly decreased. Although the activity levels of complexes I to IV were all decreased in the cardiac muscle, a particularly marked decrease was observed for complex I. It was therefore concluded that a subdural hematoma without cerebral herniation in this case would not normally be a direct cause of death, but it suspected fatal in this case because of the deteriorated systemic condition resulting from the patient’s mitochondrial disorder suspected by Pearson’s marrow pancreas syndrome.

An autopsy case of sudden death due to pulmonary arterial hypertension (PAH) in a 5-year-old boy whose cause of death was not determined during autopsy, but was later determined by postmortem examination, is presented. The boy developed convulsions that subsequently stopped, but remained unconscious. He was transported to hospital by ambulance, but died soon after. The boy had been found to have right ventricular overload on ECG 2 weeks earlier. A plan had been made to consult a doctor for a specialist visit 2 months later. During autopsy, significant abnormalities or injuries were not observed on the body's external surface. Internal examination showed congested organs, and the blood remaining in the body was dark red with fluidity. The heart was significantly enlarged (146 g), with nearly equivalent thickness of the left and right ventricles, showing right ventricular hypertrophy. Obvious macroscopic abnormalities were not observed at the origin and main trunk of the pulmonary artery. The lungs were slightly swollen (right lung 100 g, left lung 95 g), severely congested, and edematous. A postmortem CT scan displayed some patchy shadows in both lungs; however, no significant abnormalities were detected. Histopathological examination suggested a diagnosis of PAH. Three genes (BMPR2, ALK1, and ENG) were tested, revealing a heterozygous insertion of five nucleotides, TTTCC, between nucleotides 2677 and 2678 within exon 12 of the BMPR2 gene. Therefore, the subject was considered to have had heritable PAH due to a BMPR2 gene mutation.

Alcohol intake induces cardiovascular effects. We performed a comprehensive analysis of the gene expression profiles of myocardial tissues from a mouse model of alcohol feeding by microarray to clarify the effect of alcohol on myocardium from the perspective that the acute phase of alcohol use causes changes to myocardial injury related gene expression profiles, while the withdrawal phase is associated with changes in gene expression profiles related to myocardial protection. A model of single alcohol feeding was generated using pathogen-free, 7-week-old, male C57BL/6N mice administered a peroral injection of 7.0 g/kg ethanol (single alcohol-fed group group) or saline (control group). A repeated alcohol feeding group was generated following Lieber’s method. To investigate the effects of alcohol in the alcohol-fed group, the left ventricles of hearts were extirpated 1 h (alcohol group) or 24 h (withdrawal group) after the last alcohol feeding. Myocardial tissues were then subjected to RNA extraction and gene expression analysis by real-time reverse transcription polymerase chain reaction. Sera were also analyzed for various signal transducers, including 22 cytokines. Using RNA extracted from extirpated myocardia, mRNA expressions were comprehensively analyzed by one-color microarray (n=4) using SurePrint G3 Mouse Gene Expression 8x60K (Agilent) containing 38,640 gene probes as the microchip, and gene ontology and gene cascade analyses were performed. Differing gene profiles were seen in the repeated alcohol intake group. In the acute phase of alcohol intake, there were changes in gene profiles related to activation of the STAT pathway and blood cytokine-mediated inflammatory responses to hypercardia. In the withdrawal from alcohol phase, changes in gene profiles related to STAT activity did not persist, although gene profile changes related to apoptosis and the development of sudden death were observed. Further detailed investigations of the effects of alcohol on cardiomyocytes are planned.

Heatstroke damages various organs, such as the brain and heart, either directly by heat exposure or by systemic inflammatory responses because of hypercytokinemia. Electrolyte drink intake before heat exposure might suppress heat exposure induced cardiac stress. This study aimed to determine how electrolyte drink intake before heat exposure modulates heat exposure-induced damage to the diencephalon and brain stem in a rat model. After giving rats an electrolyte drink or water for 1 week, they were exposed to heat in a CO2 incubator for 90 min at 37°C. Following this, they were decapitated and their diencephalon and brain stem immediately harvested. Total RNA was isolated from these tissues and cDNA was synthesized by reverse transcription. In addition, relative mRNA expression of the Finkel–Biskis–Jinkinsosteosarcoma oncogene (c-fos), heat shock 70 kD protein 1A (Hspa1a), heat shock 70 kD protein 1B (Hspa1b), and heat shock protein B1 (Hspb1) were determined using real-time polymerase chain reaction. Results showed that heat exposure significantly pregulates each gene in the diencephalon and brain stem, indicating that heat stress affects these brain areas. Electrolyte drink intake before heat exposure did not modify gene expressions in the brain stem but suppressed the upregulation of c-fos, Hspa1a, and Hspb1 in the diencephalon, the body’s thermoregulatory center. These findings suggested that electrolyte drink intake before heat exposure alleviates heat stress in the diencephalon and might consequently suppress the upregulation of Fos (which regulates biological functions as a transcription factor) and genes related to heat shock proteins, which play a role in cellular protection. Therefore, electrolyte drink intake before heat exposure reduces the intensity of heat stress to the body and improves heat tolerance.

A case of sudden cardiac arrhythmia causing death following volatile substance misuse is presented. A 17-year-old man was found in his dormitory room having suffered a cardiac pulmonary arrest. He was brought to an emergency medical center where he was confirmed dead. The police investigation suggested butane gas aspiration since cans for portable cooking stoves were found in his room, but whether they were involved in his death was unclear. Thus, a judicial autopsy was performed 8 hours after his death. The autopsy found no severe injuries that could have led to death. Inhalation of butane gas was strongly suspected, and a screening test of intratracheal gas and femoral venous blood samples collected at autopsy detected butane. Histopathologically, all organs examined showed congestion. Endocardial hyperplasia on the left ventricular wall was confirmed on cardiac examination, and the lungs showed the transformation of the lumina of the tracheal branches due to constriction, indicating repeated stimuli. Thus, the autopsy and histological findings did not indicate any pre-existing pathological findings as a possible cause of death. Gas chromatography-mass spectrometry showed trace levels of isobutene and n-butane, while gas chromatography analysis showed trace levels of 2-butanone, which is only detected in decedents with a history of chronic butane misuse, in samples of femoral blood, cerebrum, brain stem, heart, lungs, liver, stomach contents, iliopsoas muscle, and fat tissues (from the abdomen). Thus, the autopsy findings led to the conclusion that the decedent habitually inhaled butane gas and died due to butane gas poisoning.

Chronic alcohol consumption remains a global health problem and has been implicated in many chronic and acute diseases. Alcohol consumption is regarded as a major risk factor for sudden cardiac death, with chronic alcohol consumption contributing to collagen accumulation, myocardial fibrosis, and arrhythmias. Although various signal transducers are believed to be involved in the instigation and progression of alcohol-induced cardiac dysfunction, the mechanism remains poorly understood. In the present study, mRNA and protein levels of sarco/endoplasmic reticulum Ca2+ ATPase type 2a (SERCA2a), connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinase-1 (TIMP-1), and matrix metalloproteinase-3 (MMP-3) were measured in a repeated-ethanol-exposed mouse model to investigate the effect of chronic alcohol consumption on cardiac tissue. We hypothesized that chronic alcohol consumption brings about an imbalance in the expression of MMPs and TIMPs, and also alters the regulation of proteins like SERCA2a and CTGF, resulting in myocardial remodeling and cardiac dysfunction. Cardiac tissue isolated from ethanol-exposed mice and non-exposed mice, after 6 weeks on diet, were assayed for SERCA2a, CTGF, TIMP-1 and MMP-3 expression by western blotting (for protein expression) and quantitative reverse transcription-polymerase chain reaction (for mRNA expression). Our data revealed that ethanol exposure in C57BL/6N mice yielded a significant accumulation of the transcripts of Ctgf and Mmp3, without significantly affecting the levels of the corresponding proteins. TIMP-1 mRNA and protein expression were not changed by ethanol exposure. Notably, expression of the SERCA2a protein was reduced significantly in the ethanol group compared to the control group, despite the lack of significant changes in accumulation of the corresponding mRNA. Our findings on mRNA expression of the Ctgf, MMP3, and TIMP1 encoding genes are consistent with previous reports on protein expression of these signal transducers, given that the data for the western blots did not reveal significant changes in accumulation of these proteins. Therefore, we conclude that the MMP/TIMP imbalance induced by extended alcohol exposure may contribute significantly to myocardial remodeling and cardiac dysfunction, which is in turn responsible for sudden cardiac death in ethanol abusers.

Acute gastric volvulus resulting in abdominal compartment syndrome was determined to be the cause of death in a 4-year-old girl who presented with abdominal distension. At about 1AM on the day of her death, she was brought to our emergency medical center. Physical examination and plain abdominal X-ray revealed pronounced gastric dilatation. A decompression procedure was performed, followed by observation. She went into cardiopulmonary arrest around 1PM on the same day and died. Postmortem investigation, including an autopsy and computed tomography (CT), was performed to determine the cause of death. The findings included that the stomach was severely distended. Evidence was seen of mucosal hemorrhage in the gastric mucosa on the greater curvature side, which was thinned in places but without perforation. No necrosis of the gastric mucosa was observed; reversible changes were evident on histopathological examination. The postmortem CT images suggested that the pyloric region was positioned cranioventrally to the cardiac region. None of the findings indicated sudden blockage, and the cause of death was determined to be acute gastric volvulus resulting in abdominal compartment syndrome. The abnormal placement of the organs was difficult to determine based on physical examination alone; postmortem CT and careful examination were helpful in conducting the autopsy in this case.

We developed a novel tool for concluding drowning as a cause of death. We designed nine primer pairs to detect representative freshwater or marine bacterioplankton (aquatic bacteria) and then used real-time PCR with TaqMan probes to rapidly and specifically detect them. We previously cultured the genus Aeromonas, which is a representative freshwater bacterial species, in blood samples from 94% of victims who drowned in freshwater and the genera Vibrio and/or Photobacterium that are representative marine bacteria in 88% of victims who drowned in seawater. Based on these results, we simultaneously detected eight species of bacterioplankton (Aeromonas hydrophila, A. salmonicida; Vibrio fischeri, V. harveyi, V. parahaemolyticus; Photobacterium damselae, P. leiognathi, P. phosphoreum) using three sets of triplex real-time PCR assays and TaqMan probes labelled with fluorophores (FAM, NED, Cy5). We assayed 266 specimens (109 blood, 157 tissues) from 43 victims, including 32 who had drowned in rivers, ditches, wells, sea or around estuaries. All lung samples of these 32 victims were TaqMan PCR-positive including the lung periphery into which water does not readily enter postmortem. On the other hand, findings in blood and/or closed organs (kidney or liver) were PCR-positive in 84% of the drowned victims (except for those who drowned in baths) although the conventional test detected diatoms in closed organs in only 44% of the victims. Thus, the results of the PCR assay reinforced those of diatom tests when only a few diatoms were detectable in organs due to the low density of diatoms in the water where they were found. Multiplex TaqMan PCR assays for bacterioplankton were rapid, less laborious and high-throughput as well as sensitive and specific. Therefore, these assays would be useful for routine forensic screening tests to estimate the amount and type of aspirated water.

Current 454-pyrosequencing technology enables massive parallel sequencing. We used this technology to investigate the diversity of aquatic microbes in 14 specimens (blood and organs) of two drowning victims and in two water samples taken from the discovery sites. The 16S ribosomal RNA (rRNA) genes of microbes, which are often used to identify species (or genera), have nine highly variable regions (V1-V9), each of which is surrounded by conserved regions. Some parts within the conserved regions are common over domains of microbes, such as between bacteria and algae (16S rRNA genes on algal chloroplast genomes). We therefore simultaneously amplified the target regions (V7 and V8) of various microbes in the blood and organs of drowning victims using PCR with custom-designed primers that were based on the conserved regions. We then exhaustively analyzed the PCR products by pyrosequencing using the Genome Sequencer FLX Titanium system (Roche-454 Life Sciences). This approach identified a wide array of bacteria including cyanobacteria and algae including Bacillariophyceae (diatom), Cryptophyceae, Dictyochophyceae, Chrysophyceae and Trebouxiophyceae in the blood and organs of the victims and water at discovery sites. Our data further indicated that when conventional diatom testing of lungs yielded insufficient evidence of water aspiration, the detection of various exogenous microbes by 454-pyrosequencing is very useful to support a conclusion of death by drowning. To the best of our knowledge, this is the first attempt to use a new generation sequencer to investigate diverse aquatic microbes in the blood and closed organs of drowning victims.

We previously applied our method of detecting marine or freshwater bacterioplankton (bacteria) in the blood of immersed victims as a marker of drowning. However, we did not confirm the absence of post-mortem bacterial invasion during immersion. Here we examined the nature of bacterioplankton in blood samples from 21 immersed and 4 non-immersed cadavers. We found only freshwater bacterioplankton in the blood of two victims that were retrieved from the sea or an estuary inhabited by marine bacterioplankton even though one victim was highly putrefied. The results of diatom testing suggested that these two victims had drowned in fresh or brackish water with low salinity and then flowed out to the estuary or the sea. Two others were submerged in water, but representative bacterioplankton were undetectable in their blood although one victim was highly putrefied. Autopsy findings and the results of diatom tests did not indicate that the cause of death was drowning. As in previous studies, we identified freshwater bacterioplankton in the blood of seven other victims that had drowned in freshwater, marine bacterioplankton in the blood of four victims that had drowned in seawater and none in four victims found on land that had died by means other than drowning. Bacterioplankton in the blood of drowned victims appears to reflect the type of water aspirated and blood does not become easily contaminated with bacteria post-mortem even in decomposed bodies.

We collected 68 fresh, brackish, and seawater samples from various sites around the estuaries of 2 rivers at high and low tides. Seawater flowed approximately 2.4 (salinity, 2.2% at the site) and 1.2 km (1.8%) upstream of the estuaries, but the surface comprised essentially fresh water up to the mouth. Sites contained 69 to 22,200 diatoms/50 mL of water, and the numbers varied by depth and at sites separated by only approximately 1.2 km. Diatoms ranged from 2.8 to 429 μm (mean range, 16.1-59.2 μm) in size. Large pennate diatoms populated fresh water areas, and most sedimented before reaching the sea. Numbers of pennate diatoms of less than 20 μm were decreased in areas of seawater. Numbers of centric diatoms tended to increase nearer the sea, and seawater contained large centric diatoms. Brackish water containing large volumes of seawater was easily discriminated by assemblages of marine diatoms, unlike that containing a little seawater, because marine diatoms could be found in fresh water around estuaries. Tides and the nature of the river often altered diatomaceous assemblages at the same estuarial sites. Caution is recommended for forensic interpretation of aqueous media to deduce drowning sites.

Numbers and types of bacterioplankton proliferating in blood samples mixed with water of various salinity levels were examined to determine the characteristics of species associated with salinity. Water samples (total n=88) were collected from the midstream of two rivers (freshwater; n=10; salinity <0.05%), from around their estuaries (areas of freshwater, n=20, salinity <0.05%; areas of brackish water, n=20, salinity <0.05-3.1%; areas of marine water beyond the mouths of the rivers, n=28, salinity 2.4-3.3%), and from the coast (areas of marine water; n=10; salinity 3.3-3.5%). Freshwater bacteria were identified in 41 of 42 blood samples mixed with water at ≤1.3% salinity, and the genus Aeromonas, which is universally distributed in freshwater environments, was predominant. Marine bacteria were identified in all of 46 blood samples mixed with water at ≥1.8% salinity, and most comprised the genera Vibrio and Photobacterium that are universally distributed in seawater environments. Aeromonas was undetectable in all blood samples mixed with brackish or sea water at ≥1.8% salinity although they are detectable even in seawater environments. Thus, the present results showed that bacterioplankton capable of proliferating in human blood reflects the salinity of water.

A decomposed female body with an open abdomen and pleural cavity washed up on a beach after a powerful typhoon. Autopsy findings could not determine the cause of death because of leaching and putrefaction. Numbers and types of diatoms in organs overall, suggested the aspiration of fresh or brackish water with low salinity. However, this could not be confirmed because of contamination via the open cavities. We simultaneously investigated the presence of bacterioplankton in liver, kidney and lung homogenates using a modification of our reported bacteriological method. The freshwater bacterioplankton Plesiomonas shigelloides was identified in each of these organs, but marine bacterioplankton were undetectable despite the circumstances under which the body was discovered. The presence of freshwater bacterioplankton reinforced the results of the diatom test, and we concluded that this victim had died of drowning in fresh or brackish water with low salinity.

Two newborn infants (one male and one female) were discovered dead and frozen in a home freezer. Although thawing is expected to lead to changes that may hamper postmortem investigations, the victims could not be examined in the frozen state and were thus immersed in saline at 37 degrees C to completely thaw them over about 90 min. Autolysis and putrefaction were not evident, postmortem changes were slight, and the internal organs were soft, allowing a thorough examination, including an autopsy and a histological investigation. Autopsy showed that both infants were full-term at the time of death. Hydrostatic tests of the lung and stomach indicated that the infants had been born alive, and based on histological analyses of pulmonary alveoli and bronchioles, they had started breathing. Malformations, pathological findings, and signs of suspected asphyxia were absent, and we assume that the infants were very likely to have been murdered. However, the cause of death is not yet fully understood. Although a police investigation revealed that the infants were smothered by occlusion of the mouth and nose and then frozen, there were no injuries on the skin around the nose and mouth. Nonetheless, we concluded that none of the postmortem findings were in conflict with this suspected mechanism of death. As these infants had been kept frozen since their death and thawed to body temperature as rapidly as possible in saline at 37 degrees C, their bodies were well preserved, which was helpful for the postmortem investigation.

We measured bacterioplankton in blood from cadavers retrieved from the sea (n=12), near estuaries (n=4), rivers (fresh water, n=8) and from bathtubs (n=4) as well as from non-drowned victims (n=10) discovered near aquatic environments. Blood from 11 victims drowned in seawater developed bioluminescent and/or blue colonies (oxidase test positive) on selective media containing 2-4% NaCl. Homology analyses of the 16S rRNA gene showed that all of them were marine bacteria (genera: Photobacterium, Vibrio, Shewanella, Psychrobacter). Blood from all victims drowned in rivers generated blue colonies on plates containing 3%, but not 4% NaCl. Homology analyses showed that the blue colonies were generated from bacteria that inhabit fresh water (Aeromonas). None of the blood samples from victims that drowned in bathtubs generated bioluminescent and blue colonies. However, all cadavers contained bacteria that produced unstained colonies (Staphylococcus, Bacillus, Enterobacter, Escherichia, etc.). Among non-drowned victims, blood from two gave rise to blue colonies on plates containing < or =3% NaCl (Pseudomonas). Of the cadavers found near estuaries, bioluminescent and blue colonies developed from two of them on media containing 2-4% NaCl (Photobacterium, Vibrio, Listonella), but not from two others on plates containing 4% NaCl (at < or =3%; blue colonies, Aeromonas; unstained colonies, Citrobacter, Vagococcus, Proteus, Enterobacter). These results suggested that the presence of numerous bacterioplankton in immersed cadavers could support a conclusion of death by drowning.

A 34-year-old man was discovered by his coworkers in a tank filled with 35% (w/w) hydrochloric acid. Despite undergoing intensive treatment, he died one and a half days later. An autopsy revealed generalized high tensity, overall grayish brown skin color, heavy gastric submucosal hemorrhage and heavy pulmonary edema. We concluded that death was caused by burn shock due to wide, generalized chemical burn. Microscopic investigation of the burn in the area with grayish brown skin considered coagulation necrosis of full-thickness of the skin (third-degree or deep burn), revealed that the burn was judged to cover the partial thickness of the skin (second-degree or dermal burn). These findings suggest that chemical burn by hydrochloric acid results in a change of skin color due to chemical reaction so that the appearance of the chemical burn is more severe than the degree assigned by histological examination.

We detected numerous bioluminescent bacteria in blood samples from two cadavers that had been immersed in estuarine environments. Autopsy, diatomaceous and toxicological findings indicated death by drowning, which agreed with environmental aspects and the findings of police investigations. Bioluminescent bacteria appeared in blood samples cultured on selective agar containing 2%, 3% and 4% NaCl after about 18h. Blood from the left side of the heart, the right side of the heart and the femoral vein generated 7.0 x 10(2), 2.0 x 10(4) and 8.0 x 10(2) cfu/ml of blood (case 1), and 1.8 x 10(4), 1.1 x 10(3) and 2.5 x 10(1) cfu/ml (case 2) of bioluminescent colonies, respectively, in agar containing 4% NaCl. Homologous analysis based on the 16S rRNA gene also identified the bioluminescent colonies as Vibrio fischeri and V. harveyi, which normally inhabit seawater. This simple assay might serve as an additional indicator to support a conclusion of death by drowning together with the diatom test.

To investigate the effectiveness of marine bacteria as a new marker of drowning in seawater, we determined the optimal conditions of media required to selectively detect marine bacteria and applied the technique to drowned cadavers. We incubated model blood samples (n=20 per group) mixed with seawater, river, tap or muddy water on agar plates (Todd Hewitt, TH; Marine 2216, M2216) and determined the NaCl concentration required to selectively detect marine bacteria. We also used TCBS agar plates without manipulation to isolate Vibrio spp. Among the culture media, TH agar was superior. Bioluminescent colonies were detected only in blood mixed with seawater. Blue colonies stained using the cytochrome oxidase test (COT), were detected in blood mixed with both sea and river water. However when the NaCl concentration was above 4%, COT stained colonies were detectable only in blood mixed with seawater. We subsequently used 2, 3 and 4% NaCl in TH and TCBS agar to examine blood from victims who had drowned in seawater (n=8) and in fresh water (n=7), as well as from victims who died near aquatic environments (non drowned; dry-land control, n=7). Bioluminescent colonies were detectable on 2-4% NaCl TH agar only from two victims that drowned in seawater. Bioluminescent colonies did not grow on TCBS agar. Blue colonies from all cadavers that had drowned in seawater (8/8) and in four of those that had drowned in fresh water (4/7) proliferated on TH agar containing 2% and/or 3% NaCl, but at 4% NaCl such colonies were detected only from cadavers that had drowned in seawater (8/8). Colonies from only one cadaver from seawater grew on TCBS agar. Furthermore, neither bioluminescent nor blue colonies were detected on TH agar containing 4% NaCl in samples from two cadavers found in an estuary (brackish water) who were thought to have been carried from areas of fresh water. Homologous analyses of the 16S rRNA gene revealed that the dominant colonies on TH agar containing 4% NaCl were marine bacteria (Photobacterium, Vibrio, Shewanella, Psychrobacter). Thus, proliferating bioluminescent and/or blue colonies detected in the blood of immersed cadavers using 4% NaCl TH agar, could help to establish drowning in seawater.

症例は65歳女性で、統合失調症にて長期入院中に胸痛が出現し、呼吸困難となり、酸素投与で一旦は回復したが、その後死亡した。臨床経過からは死因が判然としないため、承諾解剖を実施した。外表検査では前胸上部から顔面のうっ血、眼瞼眼球結膜の溢血点を認めた。解剖の結果、死因は機能性イレウスにより多量の腸管ガスが貯留し、横隔膜が挙上されて腹腔内圧が上昇するで呼吸運動が妨げられ窒息死したものと判断された。本症例では病理組織学的に大腸メラノーシスが認められ、その発生要因としてsennosideの長期服用が示唆された。

We studied the effects of chronic alcohol intake on the disposition of alcohol and its metabolites in the rat. We used male Wistar rats for all of the experiments in this study. Using a pair-feeding process, rats were fed a liquid diet containing alcohol or without alcohol for 6 weeks. Ethanol solutions (0.5, 1.0, 1.5, and 2.0 g/kg body weight [BW]) were administered as a bolus, intravenously. We then measured blood ethanol and acetate concentrations. Simultaneous multiline fitting was performed using mean blood alcohol concentration (BAC)-time curves fitted to the one-compartment open model with parallel first-order and Michaelis-Menten elimination kinetics. At low doses (0.5, 1.0, and 1.5 g/kgBW), no differences were observed between the alcohol group and the control group with respect to ethanol elimination rate, area under the curve of ethanol (AUC(EtOH)), and mean residence time of ethanol (MRT(EtOH)). At higher doses (2.0 g/kgBW), ethanol elimination rate in the alcohol group was significantly higher than in the control group (P<.5%). These findings were also substantiated by corresponding changes in AUC(EtOH) and MRT(EtOH). At low doses, no differences were observed between the alcohol group and the control group with respect to plateau concentration of acetate (AcT) (concentration of steady state=C(ss)AcT), area under the curve of AcT (AUC(AcT)), and mean residence time of AcT (MRT(AcT)). However, at higher doses, although there were no differences in C(ss)AcT, both AUC(AcT) and MRT(AcT) were significantly lower in the alcohol group when compared to the control group (P<.5%). Chronic alcohol consumption increases ethanol oxidation and AcT metabolism in rats, as observed at high blood alcohol concentrations (BACs). These effects were observed at BACs of 3.5-4.5 mg/ml, and were not observed at lower doses. Thus, with general alcohol consumption, interindividual differences and intra-individual changes in alcohol metabolism may not take into account increased or accelerated metabolism due to alcohol tolerance.

低出生体重児で精神障害をもつ3歳男児が、睡眠薬トリクロールシロップを誤飲し死亡した。解剖の結果、顔面及び四肢などに打撲傷が、気管内に吐物が認められた。慢性硬膜下血腫も認められ、その後半部に新しい出血が認められたことから、慢性硬膜下血腫から再出血をきたし、そのため脳浮腫・脳圧亢進が生じて嘔吐し、吐物の吸引により窒息死したものと判断した。本屍は前期破水・切迫早産のため約30週に1740gで出生し、生後は夜間の不眠が続き、母親から「情緒のコントロールがきかない」「言い出したら聞かない」「他の子供に暴力をふるう」「店の商品である食品や、夜中に冷蔵庫の物(生魚など)を手当り次第に食べる」などの訴えがあったが、遺伝子検査を含めた各種検査でも原因は判明しなかった。本件は親による虐待も疑われ、前記の訴えも「代理によるMunchausen症候群」による詐話の疑いがあったが、明らかにはならなかった。

A novel method for quantitation of brain neurosteroid levels using HPLC with UV detection is described. In this simple and reliable method, testosterone from the brain and whole blood, and the internal standard, 17alpha-methyl testosterone, were extracted in 20% acetonitrile-phosphate buffer (pH 2.8), followed by solid phase extraction (SPE). The calibration curve was linear in concentration ranges from 0.1 to 10 ng from 0.2 g of tissue. We successfully applied this method to the analysis of endogenous testosterone in the male offspring of rats exposed to alcohol in utero. The concentration of testosterone at 21 post delivery in fetal alcohol exposure (FAE) group was significantly greater than the concentrations in either pair-fed or the ad libitum controls. These results support the usefulness of this method as a means of quantitating neurosteroids, and illustrate its applicability to fetal alcohol exposure.

An established approach to forensic bloodstain examinations is to first confirm that the suspected bloodstain is human blood and then to perform tests to determine individual identity. Although the confirmation of human blood step may be skipped in order to secure sufficient sample size for individual identification when the amount of bloodstain available for analysis is very limited, the validity of such partial examinations is likely to be challenged in court trials. Therefore, in order to perform complete examination of minute bloodstains, we developed a one-tube method in which hemoglobin and DNA were simultaneously extracted for use in confirmation of human blood and individual identification, respectively. Blood was diluted in 5 steps to produce 1- to 500-fold dilutions, and 1-μl aliquots were applied to tips of cotton-wool swabs. Prepared swabs were held at room temperature for periods up to 28 days. All swabs emitted light in a darkened room after being sprayed with luminol. Using 1.5-ml microcentrifuge tubes, the sprayed swabs were soaked in 100 μl of blood extraction buffer, one of the reagents in a DNA extraction kit designated for forensic samples. Following incubation, a 3-l aliquot of the resulting blood extraction fluid (bloodstain dissolved in blood extraction buffer) was subjected to immunoblotting against human hemoglobin A in order to confirm the presence of human blood. The remaining blood extraction fluid was used for DNA extraction. After the quantification of DNA, TH01, F13A01 and amelogenin loci, and mtDNA were amplified by PCR. All samples tested positive for human blood by immunoblotting. The amount of DNA extracted using this method was comparable with that of other extraction protocols. TH01 and the amelogenin loci gave similar results in that both loci were amplified in up to 5- or 20-fold dilutions of blood that had been held for 7 days and in 1- or 5-fold dilutions held for 28 days. F13A01 was less successful in that the locus was amplified in 1- or 5-fold dilutions held for 7 days, while amplification was only possible for 40% of samples at 1-fold dilution (no dilution) held for 28 days. In contrast, amplification was possible for mtDNA at concentrations as low as 500-fold dilution held for 28 days. Thus, the one-tube method allowed both confirmation of human blood and individual identification from minute bloodstains on single swabs; however, only mtDNA can be amplified in aged and/or diluted bloodstains.

Species-specific differences in a non-polymorphic region of the mitochondrial cytochrome b gene appear to be large enough to allow human-specific amplification of forensic DNA samples. We therefore developed a PCR-based method using newly designed primers to amplify a 157-bp portion of the human mitochondrial cytochrome b gene. The forward and reverse primers were designed to hybridize to regions of the human mitochondrial cytochrome b gene with sequences differing from those of chimpanzee by 26% (7 bp/27 bp) and 26% (6 bp/23 bp), respectively. Using this primer pair, we successfully amplified DNA extracted from blood samples of 48 healthy adults. All these human samples produced a single band of the expected size on agarose gel electrophoresis, and the sequence of the single band was shown to be identical to that of the target region (157 bp) by sequence analysis. On the other hand, no visible bands were amplified from DNA extracted from blood samples of animals including non-human primates (chimpanzee, gorilla, Japanese monkey, crab-eating monkey) and other species (cow, pig, dog, goat, rat, chicken and tuna). Thus, DNA producing a single band following PCR amplification using this primer pair can be reasonably interpreted as being of human origin. In addition, aged biological specimens comprising bloodstains, hair shafts and bones were successfully identified as being of human origin, illustrating the applicability of the present method to forensic specimens.

直腸温(Tr)からの死後経過時間の推定には,Henssgeの式が世界的に用いられている.Henssgeの式は,2つの指数関数からなるため,解析的に解くことは不可能であるため,コンピュータプログラムおよびノモグラムによる解法を開発した.これまでノモグラムを使用してきたが,ノモグラム上で正確に線を引かなければならないという作業に煩わしさを感じていた.そこで,Tr=f(t)における直腸温Trと死後経過時間tとの関係を,Microsoft Excelでテーブルにし,死後経過時間tの概算値を簡便を試みた.各環境温を3度間隔とし,6ページの表に納めた.直腸温の測定値から死後経過時間を読みとるだけでなく,逆に,例えば死後12時間経過しているとすればこの程度の直腸温になるはずであるといった使い方もできた