Kazutoshi Ago

Multiplex amplified product-length polymorphism analysis for rapid detection of human mitochondrial DNA variations

A number of mutations in coding and noncoding regions of mitochondrial DNA (mtDNA) have previously been studied. In the present study, we simultaneously typed six mutation sites in the coding region by use of amplified product‐length polymorphism (APLP) analysis. The mtDNA variations of 2471 individuals from 20 populations of Japanese, Korean, Chinese, and German were examined and classified into 18 haplotypes. Two of these haplotypes, B1 (estimated ancestral haplotype) and C1, were distributed among all populations tested. However, the haplotypes A1, A2, B2, B3, and C2 were mostly restricted to the Mongoloid populations, whereas haplotypes B5 and C5 appeared almost exclusively in the German population. Phylogenetic analysis by the neighbor‐joining method revealed that the Japanese populations were more closely related to each other than to the other East Asian populations surveyed. The multiplex APLP method is suitable for large‐scale screening studies of mtDNA variability because it is both rapid and economical.

Temporal expression of wound healing-related genes in skin burn injury

Determination of the age of burns, as well as of wounds induced mechanically, is essential in forensic practice, particularly in cases of suspected child abuse. Here, we investigated temporal changes in the expression of 13 genes during wound healing after a burn. The expression of cytokines (IL-1β, IL-6, IL-10, TNF-α, and IFN-γ), chemokines (KC, MCP-1), proliferative factors (TGF-β, VEGF), proteases (MMP-2, 9, 13) and type I collagen in murine skin was examined by real-time PCR at 3, 6, 9, and 12 h and 1, 2, 3, 5, 7, and 14 days after a burn. Based on macroscopic and histological appearance, the healing process of a burn consists of 3 phases: inflammatory (from 3 h to 1 day after the burn), proliferative (from 1 to 7 days), and maturation (from 7 to 14 days). Expression of IL-1β, IL-6, TNF-α, IFN-γ and KC increased significantly in a biphasic pattern from 3 or 6 h to 12 h or 1 day and from 3 or 5 days to 7 days. Expression of MCP-1 increased significantly from 6 h to 5 days. Expression of both IL-10 and TGF-β increased significantly from 12 h to 7 days. Expression of VEGF, MMP-2, MMP-13 and type I collagen increased significantly from 3 days to 7 or 14 days. Expression of MMP-9 increased significantly from 6 h to 14 days. Our results suggest that evaluating the expression of a combination of these genes would enable the exact estimation of the age of a burn.

A fatal case of n-butane poisoning after inhaling anti-perspiration aerosol deodorant

We report a case of sudden death due to n-butane poisoning after the inhalation of anti-perspiration aerosol deodorant. The deceased was a 15-year-old boy who was found unresponsive on the road, and was pronounced dead after 1.25h. A spray can of anti-perspiration deodorant and vinyl bags were found in a thicket near the scene. An autopsy revealed pulmonary edema, cerebral edema and congestion of the organs. Using qualitative gas chromatography/mass spectrometry, the existence of n-butane was ascertained. The concentration of n-butane (in microl/ml or microl/g) was estimated to be 15.3 in the blood, 13.3 in the brain, 26.6 in the liver, 7.5 in the lung, and 13.6 in the kidney. These n-butane levels in the blood and in the tissues were higher than those of previous reports of death associated with n-butane inhalation. We concluded that the cause of death was n-butane poisoning and presumed that n-butane in the can of anti-perspiration aerosol deodorant induced fatal cardiac arrhythmia.

Two fatalities by hydrogen sulfide poisoning: Variation of pathological and toxicological findings

We report the simultaneous deaths of two individuals by inhalation of hydrogen sulfide (H(2)S), produced either by the putrefaction of a large quantity of sweet corn or by heavy oil that flowed out of the fuel tank of a large stranded cargo vessel. Ten workers went into a water ballast tank of the vessel to remove remaining heavy oil and suddenly felt unwell. Two of the ten workers (patient A, a male in his early thirties, and patient B, a male in his early sixties) died. Autopsies of the two patients revealed a partial green discoloration of the skin and pulmonary edema. Toxicological analysis revealed high levels of thiosulfate in the blood (0.089 mmol/L in patient A and 0.142 mmol/L in patient B). From these findings, we concluded that the cause of death in both patients was H(2)S poisoning. In addition, the autopsy of patient A revealed petechiae of the palpebral conjunctiva and the mucous membrane of the mouth and erosion of the respiratory tract. The autopsy of patient B failed to reveal these observations. We presumed that patient B may have been exposed to higher H(2)S levels, and that the circulation and respiration of patient B may have arrested faster than patient A. Thiosulfate levels in the blood may reflect the levels of H(2)S exposure. This case suggests that the pathological and toxicological findings of H(2)S poisoning vary from case to case.

Two patterns of β-amyloid precursor protein (APP) immunoreactivity in cases of blunt head injury

Immunostaining for beta-amyloid precursor protein (APP) is widely recognized as an effective tool for detecting diffuse traumatic axonal injury (TAI). APP selectively labels injured axons, such as axonal bulbs and varicose axons. However, it has been reported that axonal bulbs are detected in cases of cerebral hypoxia without head injury. Therefore, we examined whether there are differences in the morphological pattern of axonal bulbs between trauma and hypoxia. Sections of the corpus callosum from 25 cases of head injury and 23 control cases were immunostained for APP. APP staining detected axonal bulbs in 14 cases of head injury, who survived more than several hours, although it failed to label axons in control cases. In addition, two patterns of immunoreactivity were identified in several cases of head injury. The first pattern showed that labeled axons were oriented along with white matter bundles; the second demonstrated that the axons were scattered irregularly. The first pattern alone was found in 5 of 14 cases, while cases of the second pattern alone were not observed. Both patterns were detected in 5 cases and in the remaining 4 cases, clear patterns were not found. From these findings, we speculated that the first pattern may represent TAI. Further examinations are required for determining whether these two patterns are identical with patterns of trauma and hypoxic brain damage as indicated by [Oehmichen M, Meissner C, Schmidt V, Pedal I, König HG, Saternus KS. Axonal injury--a diagnostic tool in forensic neuropathology? A review. Forensic Sci Int 1998;95:67-83] and [Graham DI, Smith C, Reichard R, Leclercq PD, Gentleman SM. Trials and tribulations of using beta-amyloid precursor protein immunohistochemistry to evaluate traumatic brain injury in adults. Forensic Sci Int 2004;146:89-96].

Positional asphyxia or diabetic ketoacidosis? A case report

We describe an autopsy case in which a patient with diabetic ketoacidosis (DKA) was found in a head-down position. A female in her late 70s was found dead in her home in a supine position on the kitchen floor. The upper part of her body was hanging down over the edge of the kitchen floor to the backyard through the open window. External examination revealed congestion of the head and upper region of the face and neck. There were numerous petechiae on the superior palpebral conjunctivae and upper part of the oral mucosa. On internal examination, extensive hemorrhages in the subcutaneous fat tissues and muscles were observed at the upper part of the neck, although there were no external injuries on the neck. Histopathological examination revealed that hemorrhages were accompanied with infiltration of polymorphonuclear leukocytes both within and around the hemorrhages on the neck skin. Nodular glomerulosclerosis and many fat droplets in the cytoplasm of proximal tubule cells were found in the kidney. Postmortem blood analysis showed acetone (204.2 μg/ml), HbA1c (10.8%), acetoacetate (<2.0 μmol/l), 3-hydroxybutyrate (11,844 μmol/l), blood urea nitrogen (128.9 mg/dl), and creatinine (3.11 mg/dl). The glucose and acetone levels in the urine were 876.7 mg/dl and 201.4 μg/ml, respectively, suggesting that she suffered severe DKA. However, since hemorrhage of the neck could have developed only when she was still alive, asphyxia should have arisen antemortem. Based on these findings, we concluded that the direct cause of her death is positional asphyxia, which was resulted from DKA. It is difficult to diagnose the cause of death when the victim is in an unusual posture. To confirm a suspicion of positional asphyxia, photographs of the undisturbed scene are useful in addition to a precise autopsy and accurate examinations.

Two different immunostaining patterns of beta-amyloid precursor protein (APP) may distinguish traumatic from nontraumatic axonal injury.

Immunostaining for beta-amyloid precursor protein (APP) is recognized as an effective tool for detecting traumatic axonal injury, but it also detects axonal injury due to ischemic or other metabolic causes. Previously, we reported two different patterns of APP staining: labeled axons oriented along with white matter bundles (pattern 1) and labeled axons scattered irregularly (pattern 2) (Hayashi et al. (Leg Med (Tokyo) 11:S171-173, 2009). In this study, we investigated whether these two patterns are consistent with patterns of trauma and hypoxic brain damage, respectively. Sections of the corpus callosum from 44 cases of blunt head injury and equivalent control tissue were immunostained for APP. APP was detected in injured axons such as axonal bulbs and varicose axons in 24 of the 44 cases of head injuries that also survived for three or more hours after injury. In 21 of the 24 APP-positive cases, pattern 1 alone was observed in 14 cases, pattern 2 alone was not observed in any cases, and both patterns 1 and 2 were detected in 7 cases. APP-labeled injured axons were detected in 3 of the 44 control cases, all of which were pattern 2. These results suggest that pattern 1 indicates traumatic axonal injury, while pattern 2 results from hypoxic insult. These patterns may be useful to differentiate between traumatic and nontraumatic axonal injuries.

Postmortem wounds caused by cookie-cutter sharks (Isistius species): an autopsy case of a drowning victim

A woman in her late 50s was found deceased and floating in an ocean bay in a temperate region (N31 3304800, E130 3305500) surrounded by wave dissipation blocks. The woman’s appearance was orderly, except for her sweater, which was turned up to her face. Her jacket, shoes, and bag were found on a nearby block. The police investigation ascertained that she suffered from depression and had previously contemplated suicide. No suicide note or farewell letters were found. She was last seen alive, by relatives, approximately 6 days earlier. Medico-legal autopsy revealed several antemortem abrasions and bruises on the head, one of which was accompanied by mild subarachnoid hemorrhages without cerebral contusions on the right parietal lobe. In addition, the victim showed the typical findings of drowning such as ballooning of the lungs (weight: left, 310 g; right, 380 g) with impressions created by the ribs and so-called ‘‘Paltauf spots’’ on the pleural surface, bilateral pleural effusions (left, 160 ml; right, 170 ml), and froth in the airway, despite moderately advanced putrefaction. In the diatom test, marine planktons were detected not only in the digested lungs, but also in the spleen and left kidney. Toxicological investigations were negative. From all of these findings, the cause of death was determined to be drowning. However, the most notable finding was that circular and elliptical wounds, ranging from soybean-size to 3.7 9 5.1 cm, were found on the exposed (undressed) parts of the body such as the anterior cervical region (Fig. 1) and bilateral antebrachial regions. The edges of these wounds were relatively sharp and were not associated with clear subcutaneous hemorrhages. Their spoon like concave bottom reached the fatty tissue or superficial layer of the muscle.

Forensic diagnosis of ante- and postmortem burn based on aquaporin-3 gene expression in the skin

In order to diagnose death associated with fire, it is essential to show that the person was exposed to heat while still alive. We investigated both AQP1 and AQP3 expression in the skin of an experimental burn model, as well as in forensic autopsy cases, and discuss its role in the differential diagnosis of ante- and postmortem burns. In animal experiments, there was no difference in AQP1 gene expression among four groups (n=4): antemortem burn, postmortem burn, mechanical wound, and control. However, AQP3 expression in the antemortem burn was increased significantly compared with that of the other groups even at 5min after burn. Water content of the skin was decreased significantly by the burn procedure. Consistent with animal experiments, AQP3 gene expression in the skin of antemortem burn cases was increased significantly compared with postmortem burns, mechanical wounds, and controls (n=12 in each group). These observations suggest that dermal AQP3 gene expression was increased to maintain water homeostasis in response to dehydration from burn. Finally, our results suggest that AQP3 gene expression may be useful for forensic molecular diagnosis of antemortem burn.

Thymus and Adrenal Glands in Elder Abuse

Abstract Endogenous glucocorticoid-induced thymic involution is generally considered to be an important finding for determining child abuse. The present study investigated the weight of the thymus and the adrenal glands in elder abuse cases to identify a potential marker for elder abuse. There was no significant difference in the thymus and the adrenal weight between elder abuse and control cases. However, the elder abuse cases in which the duration of abuse was less than 3 months showed a significant increase in the adrenal weight in comparison to control cases. In such cases, histopathological findings showed a loss of intracellular light granules from the zona fasciculata, which might indicate a loss of cholesterol due to the overproduction of glucocorticoid. These results might imply that the elderly, who were maltreated for less than 3 months, were in the early phase of a long-term stress state during which stress-induced overproduction of glucocorticoid was observed in adrenal glands as indicated by Selye. Our results suggest that an increase in adrenal weight may be a potential marker for elder abuse of relatively short periods, especially less than a few months.