Three Autopsy Cases of Non-Meningococcal Waterhouse-Friderichsen Syndrome with Hypoplastic Spleen or Post-Splenectomy Status.

We report three cases of Waterhouse-Friderichsen syndrome (WFS) that were confirmed during forensic autopsies. Case 1 involved a man in his 50s post-splenectomy. Bacteriological examination revealed Streptococcus pneumoniae (S. pneumonia). The patient was considered to have died of asphyxiation after aspirating vomit. Case 2 involved a man in his 40s. Bacteriological examination again revealed S. pneumoniae. Histopathological examination showed hypoplasia of the spleen. This patient was considered to have died of multiple-organ failure due to sepsis, disseminated intravascular coagulation, and WFS. Case 3 involved a post-splenectomy woman in her 60s with a history of systemic lupus erythematosus. Bacteriological examination revealed Streptococcus oralis. This patient was considered to have died of multiple-organ failure due to sepsis, disseminated intravascular coagulation, and WFS. These three cases were included among forensic autopsies conducted in the last 5 years. WFS has been considered a rare disease, but may be more frequent than previously assumed. If a mildly ill patient displays a sudden change in status and dies within a short period of time, we consider it necessary to perform not only bacteriological examinations, but also histopathological examination of the spleen during autopsy.

A novel method for sex determination by detecting the number of X chromosomes.

A novel method for sex determination, based on the detection of the number of X chromosomes, was established. Current methods, based on the detection of the Y chromosome, can directly identify an unknown sample as male, but female gender is determined indirectly, by not detecting the Y chromosome. Thus, a direct determination of female gender is important because the quality (e.g., fragmentation and amelogenin-Y null allele) of the Y chromosome DNA may lead to a false result. Thus, we developed a novel sex determination method by analyzing the number of X chromosomes using a copy number variation (CNV) detection technique (the comparative Ct method). In this study, we designed a primer set using the amelogenin-X gene without the CNV region as the target to determine the X chromosome copy number, to exclude the influence of the CNV region from the comparative Ct value. The number of X chromosomes was determined statistically using the CopyCaller software with real-time PCR. All DNA samples from participants (20 males, 20 females) were evaluated correctly using this method with 1-ng template DNA. A minimum of 0.2-ng template DNA was found to be necessary for accurate sex determination with this method. When using ultraviolet-irradiated template DNA, as mock forensic samples, the sex of the samples could not be determined by short tandem repeat (STR) analysis but was correctly determined using our method. Thus, we successfully developed a method of sex determination based on the number of X chromosomes. Our novel method will be useful in forensic practice for sex determination.

DJ-1 ameliorates ischemic cell death in vitro possibly via mitochondrial pathway.

DJ-1 is an important redox-reactive neuroprotective protein implicated in regulation of oxidative stress after ischemia. However the molecular mechanism, especially the mitochondrial function, by which DJ-1 protects neuronal cells in stroke remains to be elucidated. The aim of this study was to reveal whether DJ-1 translocates into the mitochondria in exerting neuroprotection against an in vitro model of stroke. Human neural progenitor cells (hNPCs) were initially exposed to oxygen-glucose deprivation and reperfusion injury, and thereafter, DJ-1 translocation was measured by immunocytochemistry and its secretion by hNPCs was detected by enzyme-linked immunosorbant assay (ELISA). Exposure of hNPCs to experimental stroke injury resulted in DJ-1 translocation into the mitochondria. Moreover, significant levels of DJ-1 protein were secreted by the injured hNPCs. Our findings revealed that DJ-1 principally participates in the early phase of stroke involving the mitochondrial pathway. DJ-1 was detected immediately after stroke and efficiently translocated into the mitochondria offering a new venue for developing treatment strategies against ischemic stroke.

Highly sensitive sex determination method using the exon 1 region of the amelogenin gene.

Sex determination is a crucial factor in the identification of unidentified human remains. Sex determination by DNA analysis is particularly useful because it can be applied to samples for which morphological characteristics are unavailable. Because samples handled in forensic DNA typing are easily degraded by environmental factors and microorganisms, there is a need for a method that can accurately determine sex even in highly decayed samples. Previous studies mainly used sex differences in an intron of the amelogenin gene. However, this region is highly polymorphic, and there are cases where accurate sexing cannot be performed because of genetic mutations in the target region. Thus, for reliable sex determination, it is desirable to select loci with as few non-sexual polymorphisms as possible. In this study, we focused on the exon 1 region of the amelogenin gene, which has very little polymorphism other than sex differences. We developed a primer set for sex determination and compared it with the GlobalFiler™ PCR Amplification Kit (GF), which is widely used for forensic DNA typing. The results showed that the amount of DNA required for accurate sex determination was 25 pg for both methods, achieving equivalent sensitivity. Next, we compared the two methods using ancient human skeletons and found that the present method with its shorter amplicon was considerably superior to GF. The present method is simple, rapid, inexpensive, and suitable for analyzing highly degraded samples. Therefore, this method is expected to contribute to forensic sciences and physical anthropology.

Detection of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, and PV8 in blood in a forensic case using liquid chromatography-electrospray ionization linear ion trap mass spectrometry.

We present a case of fatal poisoning by 4-F-methcathinone (4-FMC; also called flephedrone), 4-methoxy-α-pyrrolidinopentiophenone (4-MeO-α-PVP), 4-fluoro-α-pyrrolidinopentiophenone (4-F-α-PVP), and α-pyrrolidinohepatanophenone (PV8). In this study, we compared the mass spectra of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, PV8, and α-pyrrolidinohexanophenone between LC-ESI-LIT-MS and GC-EI-MS analyses. Subsequently, we applied LC-ESI-LIT-MS for detection and quantification analyses of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, and PV8 in human authentic whole blood samples. More specific mass spectra for the target compounds were obtained with the LC-ESI-LIT-MS qualitative analyses than with the GC-EI-MS analyses, indicating that LC-ESI-LIT-MS was more suitable for the qualitative analysis of cathinones. The LC-ESI-LIT-MS validation data showed moderately good linearity and reproducibility for the compounds in the quantitative analyses at the range of 1-500 ng/mL. The detection limits of four cathinones ranged from 0.1 to 1 ng/mL. The concentrations of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, and PV8 in heart whole blood samples were 365, 449, 145, and 218 ng/mL, respectively. Those of the 4 cathinones in femoral vein whole blood samples were 397, 383, 127, and 167 ng/mL, respectively. We can then assume that the cause of death was acute poisoning by a combination of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, and PV8. In this article, we present a detailed LC-ESI-LIT-MS procedure for detection and quantification analyses of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, and PV8 in authentic human whole blood samples.

Hyperthermia combined with ethanol administration induces c-fos expression in the central amygdaloid nucleus of the mouse brain. A possible mechanism of heatstroke under the influence of ethanol intake.

Heatstroke is defined as a core body temperature that rises above 40.6 degrees C and is accompanied by mental status abnormalities such as delirium, convulsions, or coma resulting from exposure to environmental heat. There is fairly wide agreement that ethanol intake is a predisposing factor in heatstroke. This study was performed to identify the brain changes induced by heatstroke, using a mouse hyperthermia model with and without preceding ethanol administration. Exposure to heat of 42 degrees C until the core temperature reached to 43 degrees C followed by exposure to 37 degrees C for 15 min decreased the levels of partial pressures of O(2) in blood. Preceding ethanol administration and heat exposure induced hypotension, severe metabolic acidosis and respiratory failure, and, accordingly, produced heatstroke. Immunohistochemistry of the brains showed that preceding ethanol administration increased the number of c-fos-immunoreactive neurons, as a marker of neuronal activation, in the central amygdaloid nucleus, which is involved in thermoregulation. These results indicate that combined effects of ethanol and heat exposure induce heatstroke that is associated with activation of the central amygdaloid nucleus, implicating the pathophysiology and mechanisms of heatstroke under the influence of ethanol intake.

Brain germinoma in a young adult man with suspicion of fatal neglect.

We report a forensic autopsy case of brain germinoma in a 26-year-old man who was severely wasted and initially suspected of fatal neglect. He had a history of nocturnal wandering and was confined by his parents. Neuropathological examination showed germinoma in the hypothalamus and neurohypophysis. The cause of death was certified as hypothalamo-hypophyseal insufficiency due to germinoma. Because hypothalamic lesions may dysregulate feeding behavior and sleeping rhythms, germinoma was considered the causative lesion of the anorexic wasting and nocturnal wandering. Confinement was a preventive measure of the patient's wandering. The findings in this case indicated that hypothalamic tumors should be recognized as a cause of wasting in autopsies suspected of fatal neglect.

Postmortem distribution of mepirapim and acetyl fentanyl in biological fluid and solid tissue specimens measured by the standard addition method.

PURPOSE: Mepirapim is a new synthetic cannabinoid. We previously reported that the concentrations of unchanged mepirapim in whole blood and urine were much higher than those of other synthetic cannabinoids. To determine the postmortem distribution of mepirapim and acetyl fentanyl in the deceased individual, we established a standard addition method for detailed analysis by liquid chromatography-mass spectrometry (LC-MS) for quantification of these drugs. METHODS: The LC-MS method was fully validated for linearity, extraction recovery, matrix effect and repeatability. RESULTS: Good linearities, extraction recoveries, matrix effects and repeatabilities were shown for both target compounds in all specimens. The concentrations of mepirapim and acetyl fentanyl in three body fluid specimens and 12 solid tissue specimens were measured. For mepirapim, the highest concentrations were found in the liver and kidney, and the concentrations in the blood and urine specimens were one order of magnitude lower than the high concentrations in the solid tissues except the psoas major muscle. For acetyl fentanyl, the highest concentrations were found in the myocardium, spleen and kidney, and the concentrations in the body fluid specimens were also one order of magnitude lower than the highest concentrations in the solid tissues. There were concentration differences of mepirapim and acetyl fentanyl among the regions of the brain. CONCLUSIONS: The concentration of unchanged mepirapim in urine was much higher than those of other synthetic cannabinoids; the higher dosage, urinary excretion, metabolisms and/or pharmacokinetics of mepirapim may be quite different from those of other synthetic cannabinoids.

Identification and quantification of mepirapim and acetyl fentanyl in authentic human whole blood and urine samples by GC-MS/MS and LC-MS/MS.

PURPOSE: We encountered a curious case in which two male subjects self-administered mepirapim plus acetyl fentanyl by different routes, i.e., intravenously and by inhalation. We have thus established a detailed procedure for quantification of mepirapim and acetyl fentanyl in whole blood and urine specimens using gas chromatography (GC)-tandem mass spectrometry (MS/MS). METHODS: The GC-MS/MS method was validated for linearity, extraction recovery, accuracy, and precision. Liquid chromatography-MS/MS was also used for identification of the target compounds. RESULTS: Good linearity and reproducibility were achieved in the range of 20-1000 ng/g for both target compounds in both matrices. The concentrations of mepirapim in heart whole blood, femoral vein whole blood, and urine of the deceased individual with administration by intravenous injection were 593, 567, and 527 ng/g, respectively; those of acetyl fentanyl were 155, 125, and 126 ng/g, respectively. The mepirapim and acetyl fentanyl concentrations in the urine specimen of the surviving individual who had administered them by inhalation were 4900 and 570 ng/g, respectively. CONCLUSIONS: To our knowledge, with the exception of a brief mention of a mepirapim concentration in a serum sample in emergency medicine, there are no reported data on the identification and quantification of mepirapim in biological samples. Mepirapim is a new synthetic cannabinoid. The concentration profiles of unchanged mepirapim in whole blood and urine were quite different and unique. A detailed clarification of such uniqueness is under way in our laboratory.

Genetic and Histological Alterations Reveal Key Role of Prostaglandin Synthase and Cyclooxygenase 1 and 2 in Traumatic Brain Injury-Induced Neuroinflammation in the Cerebral Cortex of Rats Exposed to Moderate Fluid Percussion Injury.

After the initial insult in traumatic brain injury (TBI), secondary neurodegeneration occurs that is intimately associated with neuroinflammation. Prostaglandin (PG) synthases and cyclooxygenase (COX) 1 and 2 may contribute to inflammation in the brain. Temporal and spatial expression features of PG and COX1 and 2 following trauma may guide the development of antineuroinflammation strategies. Here, we examined PG synthase signaling and COX1 and 2 gene expression levels and COX-1- and 2-positive cell types and their temporal localization in TBI-induced brain in an effort to reveal their participation in the disease's evolving neuroinflammation. Using brain samples from the cerebral cortex of rats subjected to TBI model of lateral moderate fluid percussion injury (FPI), we sought to characterize the temporal (subacute TBI) and spatial (lateral cortical lesion) brain alterations accompanying the disease progression. Temporal gene expression changes of PG synthase signaling were compared between sham-operated and TBI-treated rats using microarray pathway analysis. Moreover, we examined COX1 and 2 expression patterns and their intracellular distribution in sham-operated and TBI-treated rats by immunohistochemistry. After FPI, COX1 and 2 gene expression levels, and PGE2 synthase increased while PGD2 synthase decreased, suggesting that PGE2 and PGD2 afforded contraindicative effects of inflammation and anti-inflammation, respectively. Immunohistochemical analyses showed that both COX1 and COX2 increased in a time-dependent manner in the brain, specifically in degenerating neurons of the cortex. Interestingly, the expression of COX cell type was cell-specific, in that COX1 was particularly increased in degenerating neurons while COX2 was expressed in macrophages. In view of the dynamic temporal and spatial expression of PG, COX1 and 2 gene expression and localization in the injured brain regulating PG synthase and COX1 and 2 activity will require a careful disease-specific tailoring of treatments to abrogate the neuroinflammation-plagued secondary cell death due to TBI.

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes.

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted "bidirectional analysis," which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples.

Changes in localization of synaptophysin following fluid percussion injury in the rat brain.

Traumatic brain injuries damage neurons and cause progressing dysfunctions of the brain. Synaptophysin (SYP), a major integral transmembrane protein of synaptic vesicles, provides a molecular marker for the synapse and serves as a functional marker of the brain. This study examined magnitude-dependent changes of SYP in the rat brain 2 days following low, moderate or high fluid percussion injuries and investigated time-dependent changes of SYP in the rat brain with moderate fluid percussion injury 2, 15 and 30 days after trauma using immunohistochemistry and Western blotting. SYP immunoreactivity increased in the lateral cortex and in the subcortical white matter, with increasing magnitude of injury and time after trauma. Increased SYP immunoreactivity was accompanied with degeneration of neuronal cell bodies, their processes and terminals as well as glial cell proliferations. Amounts of SYP measured by Western blotting remained unchanged in brains with moderate fluid percussion within 30 days after trauma. These findings indicate that trauma accumulates SYP at injured sites of neurons without changing SYP contents and that increased SYP immunoreactivity in the cerebral cortex following traumatic injury reflects an inhibition of synaptic vesicle transportation and dysfunction of synapses, thus providing a histological substrate for brain dysfunctions.

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.

Dural hemorrhage of the tentorium on postmortem cranial computed tomographic scans in children.

We analyzed four children with increased density of the tentorium on postmortem computed tomographic (CT) scans that suggested traumatic subarachnoid hemorrhage; but in which followup autopsy demonstrated an absence of subarachnoid hemorrhage. All cases had neither head injuries nor distinct intracranial abnormalities. Histological examinations showed extensive hemorrhage within the tentorium. These findings indicate that increased density of the tentorium on postmortem CT scan reflects 'dural hemorrhage' that may occur in children without intracranial abnormalities. Pediatricians and forensic pathologists should be aware of this phenomenon when using postmortem preautopsy CT scanning for diagnosis of child abuse.

A Unique Primer with an Inosine Chain at the 5'-Terminus Improves the Reliability of SNP Analysis Using the PCR-Amplified Product Length Polymorphism Method.

Polymerase chain reaction-amplified product length polymorphism (PCR-APLP) is one of the most convenient and reliable methods for single nucleotide polymorphism (SNP) analysis. This method is based on PCR, but uses allele-specific primers containing SNP sites at the 3'-terminus of each primer. To use this method at least two allele-specific primers and one "counter-primer", which serves as a common forward or reverse primer of the allele-specific primers, are required. The allele-specific primers have SNP sites at the 3'-terminus, and another primer should have a few non-complementary flaps at the 5'-terminus to detect SNPs by determining the difference of amplicon length by PCR and subsequent electrophoresis. A major disadvantage of the addition of a non-complementary flap is the non-specific annealing of the primer with non-complementary flaps. However, a design principle for avoiding this undesired annealing has not been fully established, therefore, it is often difficult to design effective APLP primers. Here, we report allele-specific primers with an inosine chain at the 5'-terminus for PCR-APLP analysis. This unique design improves the competitiveness of allele-specific primers and the reliability of SNP analysis when using the PCR-APLP method.

Inhibition mechanisms of hematophagous invertebrate compounds acting on the host blood coagulation and platelet aggregation pathways.

To facilitate blood feeding, hematophagous invertebrates have evolved a sophisticated array of physiological compounds that counteract homeostatic systems and inflammatory reactions of the vertebrate host. For this reason, hematophagous invertebrates possess a variety of anticoagulation components that are inhibitors of coagulant factors or antagonists of the platelet receptor. The examination of kinetic data and the crystal structure analysis have exposed the inhibition mechanisms for many of these anticoagulant reagents. Here, we attempt to classify the antihemostatic molecules and to focus on the kinetic approaches that have been instrumental in defining these mechanisms.

Traumatic basal ganglia hematoma in a fatal traffic accident victim.

A 41-year-old male driver involved in a fatal traffic accident presented with a basal ganglia hematoma on postmortem computed tomographic imaging. Clinicians could not exclude the possibility of non-traumatic intracerebral hemorrhage as the underlying cause of the traffic accident. However, analysis of the head injuries showed that the hematoma was caused by an impact to the front-parietal region directed toward the tentorium. The absence of hypertensive arteriolar changes and hemosiderin deposits in the brain supported the traumatic origin of the hematoma.

Accidental fatal hypothermia in elderly people with Alzheimer's disease.

We report two forensic autopsy cases of fatal accidental hypothermia in an 89-year-old woman and a 76-year-old man who were found dead and unclothed. In both cases, Alzheimer's disease (AD) was diagnosed by neuropathological examination. Wandering due to AD was determined as the cause of these accidents. Although paradoxical undressing in hypothermic victims is known to occur as a result of cold exposure, in our patients, undressing was attributed to dementia due to AD before they became hypothermic. These cases indicate that neuropathological examination is crucial to determining the cause of such accidents and that undressing is not always the result of hypothermia in elderly victims.

Incipient Alzheimer's disease as the underlying cause of a motor vehicle crash.

A 75-year-old female driver died in a single vehicle crash. A forensic autopsy diagnosed the cause of death as blood loss due to laceration of the liver caused by the crash. Although she had neither clinical history of dementia nor gross abnormalities of the brain, neuropathological examinations showed a substantial number of senile plaques indicative of Alzheimer's disease (AD). An interview with a family member revealed that she had symptoms of dementia. Cognitive dysfunction due to AD was proposed as the underlying cause of the crash. It is recommended that forensic autopsy of older drivers should include neuropathological examinations to identify incipient AD. Recognition of AD as an underlying cause of crashes is also important to promote the health care of older people.

Milk aspiration in an infant during supine bottle feeding: a case report.

A two-month-old infant died during bottle feeding in the supine position in the caregiver's absence. Scene investigations and autopsy examinations, including alpha-lactalbumin immunohistochemistry of the lungs, revealed the cause of death to be asphyxia due to aspiration of milk pooled in the naso-oral cavity, as a result of unsupervised supine feeding. This case emphasizes the need for an investigation into feeding positions and immunohistochemical examinations for the diagnosis of asphyxia due to milk aspiration.