DNA microarray analysis of hypothermia-exposed murine lungs for identification of forensic biomarkers.

We used DNA microarray technology to analyze the pulmonary transcriptome of mice killed by hypothermia. This analysis identified significant differential regulation of 4094 genes; specifically, 1699 genes were upregulated, and 2395 were downregulated in response to hypothermia. The gene encoding cathelicidin antimicrobial peptide was the most upregulated gene, and that encoding BAI1-associated protein 2-like 1 was the most downregulated. Gene-set analysis identified significant hypothermia-induced variations in 101 pathways, and we discovered that pathways related to immunity are involved in the pulmonary pathogenesis of hypothermia. The present findings demonstrate some of the acute pulmonary responses to hypothermia and indicate several pulmonary genes as candidate forensic biomarkers of hypothermia. Furthermore, the present findings suggest that host defense is induced in hypothermic lungs. The present microarray data may facilitate the development of protein analyses for human forensics by immunohistochemistry, western blotting and enzyme-linked immunosorbent assay and may be beneficial in clinical research of hypothermia.

An Autopsy Case of Sudden Death in Neurofibromatosis Type 1 With Pheochromocytoma and Myocarditis.

An autopsy case of sudden death in a 33-year-old man with neurofibromatosis type 1 (von Recklinghausen disease), pheochromocytoma, and myocarditis is reported. The decedent was found in his bedroom in cardiopulmonary arrest. Polypoid, elastic dermal papules on the neck, chest, abdomen, and back, and flat dark-brown macules on the chest and abdomen were observed. Flat, ovoid, dark-brown freckles were present in both axillae. Examination of the right adrenal gland revealed a tumor measuring 5 cm × 5 cm × 3 cm. Microscopic examination of the myocardium revealed moderate neutrophilic and lymphocytic infiltrates. In the right adrenal gland, tumor cells contained abundant granular eosinophilic cytoplasm and exhibited cell-nesting with an alveolar pattern (Zellballen). Polygonal cells were seen together with rich vascular networks. Immunohistochemical analyses showed cells diffusely positive for chromogranin A and dopamine ß-hydroxylase. Furthermore, blood from the right heart and the right common iliac vein contained high concentrations of serum epinephrine, norepinephrine, and dopamine. Death was attributed to adrenal crisis: circulatory failure caused by excessive catecholamines produced by the pheochromocytoma. In addition, myocarditis, which had been induced chronically by catecholamines, would have also contributed adversely to the clinical course. Pheochromocytoma and myocarditis should be considered when sudden death occurs in the setting of neurofibromatosis type 1.

Pediatric autopsy case of asphyxia due to salmon egg (ikura) aspiration.

Here we report an autopsy case of asphyxia due to aspiration of a salmon egg (ikura) into the airway. The patient was a 19-month-old girl. During breakfast, she put salmon eggs into her mouth, and began to walk. She slipped, fell down, and collapsed. She was pronounced dead following 2 h of resuscitation. The body was autopsied 28 h after death. The gastric contents consisted of rice, orange sections, and white salmon eggs. The lungs were deeply congested and over-inflated. In the right lung, areas of atelectasis in the upper and middle lobes were seen. A yellow salmon egg (8 mm in diameter) was found in the trachea. Although fish eggs are consumed throughout the world, reports of this sort are limited. The aspiration of fish eggs is under-acknowledged and underreported. The importance of preventive measures needs to be emphasized to parents and caregivers.

DNA microarray analysis of the mouse adrenal gland for the detection of hypothermia biomarkers: potential usefulness for forensic investigation.

We analyzed the adrenal gland transcriptome of mice killed by hypothermia using DNA microarray technology. A total of 4051 significantly expressed genes were identified; 2015 genes were upregulated and 2036 were downregulated.The FBJ osteosarcoma oncogene was the most upregulated,whereas stearoyl coenzyme A desaturase 3 was the most downregulated. Validation by quantitative polymerase chain reaction revealed that results obtained by both methods were consistent. In the gene set analysis, significant variations were found in nine pathways, and we suggest that transforming growth factor ß and tumor necrosis factor α would be involved in the pathogenesis of hypothermia. Gene functional category analysis demonstrated the most overexpressed categories in upregulated and downregulated genes were cellular process in biological process, binding in molecular function, and cell and cell part in cellular component. The present study demonstrated acute adrenal responses in hypothermia, and we suggest that understanding adrenal mRNA expression would be useful for hypothermia diagnosis. Furthermore, the present microarray data may also facilitate development of immunohistochemical analysis of human cases. In forensic practice, the combination of macroscopic and microscopic observations with molecular biological analyses would be conducive to more accurate diagnosis of hypothermia. Although this study is aimed at forensic practice, the present data regarding more than 20,000 genes of the adrenal gland would be beneficial to inform future clinical hypothermia research. From the viewpoint of adrenal gene activity, they could contribute to elucidating the pathophysiology of hypothermia.

Wound age estimation by simultaneous detection of 9 cytokines in human dermal wounds with a multiplex bead-based immunoassay: an estimative method using outsourced examinations.

Wound age estimation for human dermal wounds was performed based on quantification of interleukin 1beta (IL 1beta), IL 5, IL 7, IL 12 p70, IL 13, IL 17, granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP 1), and macrophage inflammatory protein 1beta (MIP 1beta). IL 5, IL 12 p 70, IL 13, and IL 17 increased from the early phase, MCP 1 exclusively in the middle phase, and IL 1beta, G-CSF, and MIP 1beta from the middle phase to the late phase. IL 7 decreased from the early phase. Among the cytokines analyzed in the present study, MCP 1 was the most plentiful cytokine. In addition, an outsourced examination, which could be available to any forensic institute, was performed in two cases for confirmative purposes. Many factors have been proposed as markers for dermal wound age estimation, but the set of cytokines selected for the outsourced examination in the present study wound be useful in daily forensic practice.

Practical applications of molecular biological species identification of forensically important flies.

Species identification of the early colonizers on a corpse, such as Calliphoridae and Sarcophagidae, provides useful information for estimating the postmortem interval (PMI). However, the morphology of adult flies and nymphs is very similar among species of the same genus, and for many forensic scientists accurate identification is very difficult within their routine work. We present a simple and time-saving method for identification of forensically important flies by comparing sequenced 304 bp of mitochondrial DNA cytochrome oxidase gene subunit I (mtDNA COI). Eggs and larvae were recovered from the corpse during forensic autopsies and then reared in a growth chamber under controlled conditions. The emerging adults were killed by ethyl acetate and male specimens were observed under a stereomicroscope to identify the species based on the shape of the genitalia. Subsequently, DNA was extracted from identified specimens and sequenced mtDNA COI, resulting in the identification of 8 species of 4 genera of forensically important diptera. Moreover, the molecular biological species identification was useful in forensic practice, and there were cases in which analysis of DNA extracted from puparia supposed to be early colonizers of the corpse provided useful information for estimating the PMI. We also report sequencing of mtDNA COI of the green bottle fly, Lucilia ampullacea, newly collected from a corpse in Iwate prefecture.

An autopsy case related to a terrorist attack using a ball-bearing bomb.

We encountered an autopsy case related to a terrorist attack using a ball-bearing bomb. The decedent was a 51-year-old male without significant medical histories. During dinner in a restaurant, the perpetrator suddenly exploded a ball-bearing bomb, the blast from which blew the victim off his chair. The victim was found to be unresponsive, and pronounced dead. X-ray photographs taken before autopsy revealed six spherical shadows. Three penetrating wounds in the head, one in the neck and chest, and two in the left upper arm were observed in vivo. Six projectiles recovered from the body were identified as ball-bearings, one of which traveled through the midbrain, diencephalon, and left temporal lobe. Although blast injuries and penetrating wounds are often combined in bomb attack victims, penetrating brain injury would be the cause of death in this case. Lethal injuries to major organs can thus occur even though the destructive force of a ball-bearing bomb is weak. X-ray films were informative for detecting the ball-bearings in this case, suggesting that autopsy imaging is essential in cases of terrorism victims.

An autopsy case of aortitis resulting in sudden death due to a rupture of aneurysm of the aortic sinus.

An autopsy case of spontaneous rupture of a clinically undiagnosed inflammatory aneurysm of the aortic sinus is presented. A 34-year-old man consulted a physician complaining of anterior chest pain and heartburn persisting for 2 days. Diagnoses of common cold and gastritis were made, but 3h later his condition deteriorated, leading to death. Autopsy revealed hemopericardium due to rupture of a saccular aneurysm of the aortic sinus. Histological examination of the aortic sinus disclosed aortitis characterized by fragmentation and loss of elastic tissues with fibrous thickening of the intima. Lymphocytes along with polymorphonuclear leucocytes and plasma cells infiltrated the adventitia and media at severely affected areas. These changes were limited to the aortic sinus, and other portions of the aorta and its main branches were essentially intact. An eccentric intimal thickening with medial degeneration was found within the anterior descending branch of the left coronary artery, and occasional collection of lymphocytes was noted in the epicardium. This case would be a rare and illustrative case for differentiation of aortitis, although we could not pinpoint the etiology of the aortitis because of non-specific histological findings and negative serological results for exogenous pathogens.

A study on mRNA expressions of interleukin 10 during fracture healing for wound age determination.

We investigated mRNA expressions of interleukin 10 (IL-10) during fracture healing for wound age determination. Macroscopically, calluses were formed at 144 h post-injury, and thickening of the fracture edge was observed at 240 h post-injury. Slight infiltration of neutrophils and macrophages was detected at 3 h post-injury, and moderate infiltration of T and B lymphocytes was detected at 72 h post-injury. In addition, osteoblasts proliferated at 24 h post-injury. IL-10 mRNA increased immediately after the intervention and peaked at 24 h, and the expressions were detected in osteocytes before and after injury, and in osteoblasts at 3 h post-injury. Although the intensity was stable in osteocytes, that in osteoblasts was strongest at 24 h post-injury. Among inflammatory cells, the expressions were exclusively detected in macrophages. These results indicated that examinations of macroscopic and microscopic changes in fracture edges, and mRNA expressions of IL-10, would be informative for fracture healing and dating.

Simultaneous detection of eight cytokines in human dermal wounds with a multiplex bead-based immunoassay for wound age estimation.

We performed quantification of IL 2, IL 4, IL 6, IL 8, IL 10, GM-CSF, IFN gamma, and TNF alpha in human dermal wounds for wound age estimation. The proliferation of dermal cells and infiltration of inflammatory cells were also analyzed. Neutrophils and macrophages were detected from 2 h post-injury, and strong infiltrations were seen at 33-49 h. T and B lymphocytes also infiltrated simultaneously from 71 h. Strong proliferation of fibroblasts were shown from 246 h, and thickening of the epidermis from 71 h. IL 10, GM-CSF, IFNgamma, and TNF alpha increased from the early phase of dermal wound healing, IL 6 exclusively in the middle phase, IL 2, IL 4, and IL 8 from the middle phase to the late phase. Among the cytokines analyzed in the present study, IL 6, IL 8, IFNgamma, and TNF alpha were strongly expressed. Results of the present study suggest that multiplex cytokine analysis at the wound site can be useful for wound age estimation. In addition, multiplex data obtained from the same sample with a single method would demonstrate more accurate interactions of cytokines during dermal wound healing. Although the present study was oriented to practical forensic pathology, the data obtained would be informative for various fields of medicine.

Simultaneous detections of 27 cytokines during cerebral wound healing by multiplexed bead-based immunoassay for wound age estimation.

Quantification of 27 cytokines following cerebral wounding was performed for wound age estimation. The cytokines evaluated included interleukin (IL)-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 p40, IL-12 p70, IL-15, IL-17, IL-18, basic fibroblast growth factor (bFGF), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), Interferon-gamma (IFN-gamma), keratinocyte derived cytokine (KC), leukemia inhibitory factor (LIF), macrophage-colony stimulating factor (M-CSF), monokine inducible by interferon gamma (MIG), macrophage inflammatory protein (MIP)-1 alpha, MIP 2, platelet-derived growth factor BB (PDGF BB), regulated upon activation, normal T-cell expressed, and secreted (Rantes), tumor necrosis factor-alpha (TNF-alpha), and vascular endothelial growth factor (VEGF). The proliferation of glial cells as well as the infiltration of inflammatory cells were also evaluated. Although astroglia proliferated from 72 hours post-injury, inflammatory cell dynamics were generally steady. Among cytokines analyzed in the present study, IL-1beta, IL-5, IL-6, IL-12 p40, G-CSF, IFN-gamma, KC, LIF, MIP2, and PDGF BB increased during the early phase of cerebral wound healing, and M-CSF increased during the middle phase, while IL-15, IL-18, and MIG increased during the late phase. In contrast, IL-1alpha, IL-10, IL-12 p70, and TNF-alpha were suppressed throughout the cerebral wound healing process. Based on our findings, quantitative cytokine analyses at the cerebral wound site may be a useful tool for wound age estimation. Further, this study suggests that multiplex data gained from the same sample using a single methodology demonstrates highly accurate cytokine interactions during the process of cerebral wound healing.

Haplotype analysis of 17 Y-STR loci in a Japanese population.

We analyzed 17 Y-STR loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS438, DYS439, DYS456, DYS458 and DYS464a/b/c/d) in 252 Japanese males using three multiplex PCR typing systems. Two variants were found at DYS385a/b. A total of 244 different haplotypes were observed, of which 239 were found in single individuals. The haplotype diversity for the 17 loci was 0.996.

A case of carbamazepine overdose with focal myocarditis.

An autopsy case of carbamazepine overdose with focal myocarditis is reported. The decedent was a 33-year-old female with a history of schizophrenia and bipolar disorder, who reportedly took 5-day dose of prescribed medications at around midnight. Although she stayed home following the direction of the physician, she was pronounced dead 8h after the intake. At autopsy she was obese, and her face was slightly swollen. The 420 g heart was free of coronary atherosclerosis, and the myocardium had no obvious scars. Both the left and right lungs were markedly congested and edematous. Strong congestion was also noted in the brain and visceral organs. Microscopic examination disclosed focal infiltration of inflammatory cells, most of which were lymphocytes, into the myocardium. In the toxicological analyses, carbamazepine concentration in the blood was 9.9 microg/ml, and other medications were below the toxic levels. It was considered that under the compromised cardiac function due to myocarditis presumably induced by some prescribed medications, and obesity, the carbamazepine overdose deteriorated her condition by triggering critical arrhythmia or congestive heart failure.

Species identification of the forensically important flies in Iwate prefecture, Japan based on mitochondrial cytochrome oxidase gene subunit I (COI) sequences.

Molecular biological species identification of forensically important flies distributed in Iwate prefecture, Japan, was carried out, and the utility of this method was evaluated. The dipteran nymphs that were early colonizers on the corpse were reared to adult and morphologically identified. Meanwhile they were sequenced over a 304 base pair region of the mitochondrial cytochrome oxidase gene subunit I (COI). Six species belonging three genera of Calliphoridae (Calliphora lata, C. vicina, Lucilia cuprina, L. illustris, L. sericata, Chrysomya pinguis) and two species of Sarcophagidae (Parasarcophaga crassipalpis, P. similis) were collected and identified. Each fly had somewhat different ecological features such as seasonal dominance and habitat. The COI sequences of each species were unique and distinguishable from each other, although they showed high homology. Species identification from immature diptera by the DNA sequences was simple and time-saving because there was no need to wait for adult emergence or knowledge of morphological keys, and it provided information about not only postmortem interval but also seasonal and environmental conditions surrounding the corpse.

The applicability of holography in forensic identification: a fusion of the traditional optical technique and digital technique.

In this study, the applicability of holography in the 3-dimensional recording of forensic objects such as skulls and mandibulae, and the accuracy of the reconstructed 3-D images, were examined. The virtual holographic image, which records the 3-dimensional data of the original object, is visually observed on the other side of the holographic plate, and reproduces the 3-dimensional shape of the object well. Another type of holographic image, the real image, is focused on a frosted glass screen, and cross-sectional images of the object can be observed. When measuring the distances between anatomical reference points using an image-processing software, the average deviations in the holographic images as compared to the actual objects were less than 0.1 mm. Therefore, holography could be useful as a 3-dimensional recording method of forensic objects. Two superimposition systems using holographic images were examined. In the 2D-3D system, the transparent virtual holographic image of an object is directly superimposed onto the digitized photograph of the same object on the LCD monitor. On the other hand, in the video system, the holographic image captured by the CCD camera is superimposed onto the digitized photographic image using a personal computer. We found that the discrepancy between the outlines of the superimposed holographic and photographic dental images using the video system was smaller than that using the 2D-3D system. Holography seemed to perform comparably to the computer graphic system; however, a fusion with the digital technique would expand the utility of holography in superimposition.

Studies on mRNA expression of tissue-type plasminogen activator in bruises for wound age estimation.

We investigated mRNA expression of tissue-type plasminogen activator (tPA) and inflammatory cell dynamics for wound age estimation of bruises in mice. Neutrophils were detected from 1 h post-injury. Up to 8 h, they accumulated in subcutaneous tissue and the lower part of the dermis, and thereafter they extended to all the layers. Macrophages became detectable 3 h post-injury, and moderate infiltration of lymphocytes was seen from 144 h. In addition, epidermal thickening was also seen from 72 h. tPA mRNA expression peaked at 1 h, and increased slightly at 72 h post-injury. tPA mRNA was detected in epidermal cells, fibroblasts, and endothelial cells before and after injury, from 3 h in neutrophils and from 72 h in macrophages, respectively. This study presents the time-dependent expression of tPA mRNA in bruises in relation to temporal histologic characteristics during wound healing, which was considered to be useful for wound age estimation. Furthermore, it is suggested that tPA plays an important role in the first step of tissue remodeling.

A case of acute gasoline intoxication at the scene of washing a petrol tank.

We encountered a case of acute gasoline intoxication at the scene of washing the inner wall of a petrol tank. The decedent was a 50-year-old male, who was the supervisor. Two young workers wearing mask respirators began to wash the inner wall of the gasoline tank under poor ventilation. About 1 h later, because one of the workers lost consciousness, the supervisor entered the tank, without a mask respirator, to rescue the worker. However, the supervisor immediately fainted, and died 26 h after the accident. In the autopsy, blisters were observed on the skin of the face, neck, anterior chest, upper extremities, and back. The heart contained hemolyzed blood. Histologically, hemorrhagic pulmonary edema, pneumonia, and proximal tubular necrosis were found. In the toxicological analyzes, toluene, xylene, and trimethylbenzene were detected in the blood, brain, and gastric contents. Toluene concentrations in the blood and brain were 0.3 mug/ml and 3.7 mug/g, respectively. Since pathological findings were consistent with the reported findings concerning gasoline intoxication, and constituents of gasoline were in the body, death was attributed to acute gasoline intoxication. It was considered that sufficient ventilation and proper use of a mask respirator were essential for this kind of work.

Studies on mRNA expression of basic fibroblast growth factor in wound healing for wound age determination.

We investigated the mRNA expression of basic fibroblast growth factor (bFGF) for wound age determination during dermal, cerebral, hepatic and renal wound healing in mice. The bFGF mRNA expression in the injured skin peaked at 1 h and was detected in epidermal cells, fibroblasts, endothelial cells and neutrophils. In the injured cerebrum the expression increased from 1 h and peaked at 48 h. In the intact cerebrum, bFGF was detected exclusively in the endothelial cells, whereas it was also detected in astrocytes during wound healing. Time-dependent expression of bFGF mRNA in skin and cerebrum was considered to be useful for wound age determination. On the other hand it was suggested that bFGF mRNA in astrocytes could be a vital sign of the acute phase. In hepatic and renal injuries, however, bFGF mRNA expression increased slightly in endothelial cells at 24 h, in neutrophils of the liver and in the glomeruli of the kidneys.

Immunohistochemical study of basic fibroblast growth factor and vascular endothelial growth factor expression for age determination of cutaneous wounds.

The authors investigated the expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) during skin wound healing using immunohistochemical techniques. After a full-thickness incision was made on the dorsal skin, mice were killed 0.5, 1, 3, 8, 24, 72, 144, or 240 hours after incision, and the wound was excised. To evaluate the influences of postmortem degeneration, cutaneous wound excision was performed 1 to 5 days after the mice were killed. The excised wounds were stained by the conventional streptoavidin-biotinylated peroxidase complex method, using specific antibodies, and the ratio of the number of positive cells to total cells was determined. Expression of bFGF was detected in the nuclei of epidermal cells and fibroblasts in the early 0.5- to 1-hour phases and the late 24- to 144-hour phases. Expression of VEGF was detected in the cytoplasm of epidermal cells in the 24- to 144-hour phases. Immunoreactivity of both cytokines was detected 1 day post mortem and was especially well preserved in the fibroblasts. Time-dependent expression of both factors suggested that they would be useful markers for the determination of wound age. However, bFGF should be superior to VEGF because of its earlier expression and because of its more persistent expression in dermal fibroblasts with increasing postmortem interval.