Immunohistochemical analysis of PDGFR-α for wound age determination.

Immunohistochemical analysis of platelet-derived growth factor receptor-α (PDGFR-α) was performed on human skin wounds obtained from forensic autopsy cases. Thirty human skin wounds were collected at different post-infliction intervals as follows: Group I, 4 h to 3 days (n = 16); Group II, 4 to 7 days (n = 7); Group III, 9 to 10 days (n = 3); and Group IV, 14 to 20 days (n = 4). Immunopositive reactions for PDGFR-α were not observed in the uninjured human skin specimens. In a semi-quantitative morphometrical analysis, the number of PDGFR-α-positive cells was observed increased in Group II, with the average number of PDGFR-α-positive cells being the highest in Group II. Additionally, in Group II, all specimens showed PDGFR-α-positive cells, with an average number of > 200 cells in five fields of view, suggesting a wound age of 4 to 7 days. Taken together, the immunohistochemical detection of PDGFR-α in human skin wounds can be a useful tool for wound age determination.

Forensic significance of intracardiac expressions of Nrf2 in acute myocardial ischemia.

When exposed to oxidative and electrophilic stress, a protective antioxidant response is initiated by nuclear factor erythroid 2-related factor 2 (Nrf2). However, the extent of its importance in the forensic diagnosis of acute ischemic heart diseases (AIHD), such as myocardial infarction (MI), remains uncertain. On the other hand, immunohistochemical analyses of fibronectin (FN) and the terminal complement complex (C5b-9) prove valuable in identifying myocardial ischemia that precedes necrosis during the postmortem diagnosis of sudden cardiac death (SCD). In this study, we investigated the immunohistochemical levels of Nrf2, FN, and C5b-9 in human cardiac samples to explore their forensic relevance for the identification of acute cardiac ischemia. Heart samples were obtained from 25 AIHD cases and 39 non-AIHD cases as controls. Nrf2 was localized in the nuclei of cardiomyocytes, while FN and C5b-9 were detected in the myocardial cytoplasm. The number of intranuclear Nrf2 positive signals in cardiomyocytes increased in AIHD cases compared to control cases. Additionally, the grading of positive portions of cardiac FN and C5b-9 in the myocardium was also significantly enhanced in AIHD, compared to controls. Collectively, these results indicate that the immunohistochemical investigation of Nrf2 combined with FN, and/or C5b-9 holds the potential for identifying early-stage myocardial ischemic lesions in cases of SCD.

A case of hemoperitoneum after percutaneous radiofrequency ablation in a patient with hepatocellular carcinoma.

We report a case of hemoperitoneum after percutaneous radiofrequency ablation in a patient with hepatocellular carcinoma. A 60-year-old female was hospitalized for the treatment of thrombasthenia and cirrhosis caused by chronic Hepatitis C, and computed tomography revealed hepatocellular carcinoma, which was treated by percutaneous radiofrequency ablation. After the ablation, hemoperitoneum was suspected because of the low hemoglobin level with abdominal pain. Approximately 6 h after the ablation treatment, the patient suddenly fell into a shock state and died. In this case, medical treatment-related death including malpractice was suspected, and forensic autopsy was performed. The abdominal cavity contained 910 mL of dark red fluid blood and 210 g of soft hemocoagula. Moreover, several puncture marks were observed on the liver surface and diaphragm, and there was no clear damage to the main arteries and veins. Considering the macroscopic and microscopic findings, the cause of death was assumed as hemorrhagic shock due to the hemoperitoneum caused by the damage to the liver by radiofrequency ablation. It is important to consider all the indications and adverse effects of radiofrequency ablation.

An autopsy case of fatal Japanese spotted fever in Wakayama.

Japanese spotted fever (JSF) is potentially fatal infection transmitted by tick bites which vectors Rickettsia (R.) japonica. Since JSF was first described in 1984, the incidence has gradually been increased. We experienced a case of JSF of fatal outcome. A female in 70's was found dead on her bed, whose house was so called 'hoarding house' filled with many waists and unused items. The following day, the autopsy was performed. As representative symptom of external findings, skin rashes were seen on the trunk and extremities, and there were tick-bite eschars on the left upper arm. Internal findings showed no specific findings in each organ. Histopathological examination demonstrated massive inflammatory cell infiltrates mainly consisted of neutrophils in the dermis beneath the external eschar. Furthermore, destruction of glomeruli in kidney with microhemorrhage from mesangial regions was observed. The numerous inflammatory infiltrates were also observed in pulmonary interstitium, which were accompanied with histopathologic features of vasculitis. Biochemical examination showed severe systemic inflammation as monitored by elevated CRP of 16 mg/dL and renal dysfunction by BUN of 171.2 mg/dL and creatinine of 6.07 mg/dL. Subsequently polymerase chain reaction revealed specifically amplified signals for R. japonica from the samples of tick-bites eschar and blood. Thus, we diagnosed her cause of death as JSF which had been occurred multiorgan failure such as acute renal failure and possibly acute respiratory failure. (224 terms).

Unveiling the Dark Nexus: A systematic review on the interplay of mental health, substance abuse, and socio-cultural factors in femicide.

A shared definition of femicide would help to distinguish it from the murder of a woman and understand its root causes favoring prevention. We conducted a Systematic Literature Review (SLR) to assess how (and if) femicide cases were related to mental disorders. Articles papers that explicitly define or discuss femicides or articles that, albeit not expressly mention femicides, thoroughly compare generic homicides and homicides with female victims. We analyse 3546 articles were retrieved from the databases, and 75 studies fulfilled the eligibility criteria and were included in the SLR. Many forms of femicide emerge worldwide as people's values, beliefs, attitudes, and behaviours evolve (intimate partner femicide, femicide-suicide, religious femicide, honour, revolt femicide) and state of vulnerability. A tiny percentage of femicides occur at the hands of subjects with diagnosed mental disorders, and controversies exist regarding the possible link between femicide and the use of drugs and/or alcohol and other factors. The complex problem of violence against women must be addressed with a transdisciplinary approach and targeted interventions for both the victims and the perpetrators. The present SLR shows that it is not possible to link femicides to mental disorders and that socio and cultural factors appear to be more relevant. Further quantitative research is warranted to disentangle the root causes of this heinous phenomenon plaguing our times. Our studies show that using the proposed definition of feminicide would help to delimit and adequately recognise violence in courtrooms, promote the culture of equality, and identify adequate policy strategies for prevention.

Relationship between intrathrombotic appearance of HSP27 and HSP70 and thrombus ages in a murine model of deep vein thrombosis.

Heat shock proteins (HSPs) are molecular chaperones whose primary function is cytoprotection, supporting cell survival under (sub) lethal conditions. They have been implicated in various diseases such as inflammatory diseases and cancer due to their cytoprotective and immunomodulatory effects, and their biological mechanisms have been studied. Central family members include, HSP27, which is induced by various stimuli such as heat shock, hypoxia, hyperoxia, ultraviolet exposure, and nutritional deficiency, and HSP70, which is homeostatically expressed in many organs such as the gastrointestinal tract and has anti-cell death and anti-inflammatory effects. In this study, HSP27 and HSP70 were investigated during thrombus formation and dissolution in a deep vein thrombosis model by immunohistochemistry to determine their involvement in this process and whether their expression could be used as a forensic marker. In the process of thrombus formation and lysis, HSP27 and HSP70 were found to be expressed by immunohistochemical analysis. The role of inhibitors of HSP27 and HSP70 in the pathogenesis of thrombosis in mice was also investigated. When HSP27 or HSP70 inhibitors were administered, thrombi were significantly smaller than in the control group on day 5 after inferior vena cava ligation, indicating pro-thrombotic effects HSP27 and HSP70. If HSP27- or HSP70-positive cells were clearly visible and easily identifiable in the thrombus sections, the thrombus was presumed to be more than 10 days old. Thus, the detection of intrathrombotic HSP27 and HSP70 could forensically provide useful information for the estimation of thrombus ages. Collectively, our study implied that both HSP27 and HSP70 might be molecular targets for thrombus therapy and that the detection of HSP-related molecules such as HSP27 and HSP70 could be useful for the determination of thrombus ages.

Discordant microsatellite instability findings in two samples from a patient with biliary cancer that responded to pembrolizumab.

Microsatellite instability (MSI) is a key marker to predict response to immune checkpoint inhibitors; however, only 1-2% of biliary cancers have this genomic feature. In a patient with hilar biliary cancer, MSI was examined in two cancer specimens (forceps biopsy from the biliary stricture and endoscopic ultrasound-guided fine-needle aspiration biopsy [EUS-FNAB] from the adjacent lymph node). We observed discordant results, as high frequency of MSI was found only in the forceps biopsy. Although the FNAB sample was 10 times larger than that of the forceps biopsy, the tumor concentration was much lower, which is a possible reason for the discordance. Besides, immunohistochemistry of four mismatch-repair (MMR) proteins showed proficient MMR expressions. The tumor became refractory to gemcitabine, cisplatin, and S-1 but responded well to pembrolizumab. Caution is needed for sample selection and for interpretation of the test's results, to avoid missing rare chance for effective molecular target agents.

Essential Involvement of Neutrophil Elastase in Acute Acetaminophen Hepatotoxicity Using BALB/c Mice.

Intense neutrophil infiltration into the liver is a characteristic of acetaminophen-induced acute liver injury. Neutrophil elastase is released by neutrophils during inflammation. To elucidate the involvement of neutrophil elastase in acetaminophen-induced liver injury, we investigated the efficacy of a potent and specific neutrophil elastase inhibitor, sivelestat, in mice with acetaminophen-induced acute liver injury. Intraperitoneal administration of 750 mg/kg of acetaminophen caused severe liver damage, such as elevated serum transaminase levels, centrilobular hepatic necrosis, and neutrophil infiltration, with approximately 50% mortality in BALB/c mice within 48 h of administration. However, in mice treated with sivelestat 30 min after the acetaminophen challenge, all mice survived, with reduced serum transaminase elevation and diminished hepatic necrosis. In addition, mice treated with sivelestat had reduced NOS-II expression and hepatic neutrophil infiltration after the acetaminophen challenge. Furthermore, treatment with sivelestat at 3 h after the acetaminophen challenge significantly improved survival. These findings indicate a new clinical application for sivelestat in the treatment of acetaminophen-induced liver failure through mechanisms involving the regulation of neutrophil migration and NO production.

Forensic application of aquaporins.

Aquaporins (AQPs) are a family of water channel proteins that primarily elicit the basic functions of water transport and osmotic homeostasis. To date, at least 17 mammalian AQPs have been identified, AQP-0 to -12 have been found in higher orders including human, and AQP-13 to -16 have been described in older lineages. Moreover, these proteins have recently been shown to regulate many biological processes through unique activities, such as cell proliferation, migration, apoptosis, and mitochondrial metabolism. Several studies have focused on the involvement of AQPs in cell biology aspect, showing that they are involved in a variety of physiological processes and pathophysiological conditions. Furthermore, in the field of forensic medicine, studies on whether AQPs can be a useful marker for diagnosing various causes of death have been conducted using autopsy samples and animal experiments, which have produced interesting results. Herein, we review certain observations regarding AQPs and discuss their potential to contribute to the future practice of forensic research.

Application and limitation of a biological clock-based method for estimating time of death in forensic practices.

Estimating time of death is one of the most important problems in forensics. Here, we evaluated the applicability, limitations and reliability of the developed biological clock-based method. We analyzed the expression of the clock genes, BMAL1 and NR1D1, in 318 dead hearts with defined time of death by real-time RT-PCR. For estimating the time of death, we chose two parameters, the NR1D1/BMAL1 ratio and BMAL1/NR1D1 ratio for morning and evening deaths, respectively. The NR1D1/BMAL1 ratio was significantly higher in morning deaths and the BMAL1/NR1D1 ratio was significantly higher in evening deaths. Sex, age, postmortem interval, and most causes of death had no significant effect on the two parameters, except for infants and the elderly, and severe brain injury. Although our method may not work in all cases, our method is useful for forensic practice in that it complements classical methods that are strongly influenced by the environment in which the corpse is placed. However, this method should be applied with caution in infants, the elderly, and patients with severe brain injury.

Forensic application of epidermal expression of HSP27 and HSP70 for the determination of wound vitality in human compressed neck skin.

Estimating the age and vitality of human skin wounds is essential in forensic practice, and the use of immunohistochemical parameters in this regard remains a challenge. Heat shock proteins (HSPs) are evolutionarily conserved universal proteins that protect biological systems from various types of stress. However, its importance in forensic pathology for determining wound activation in neck compression skin remains unclear. The expression of HSP27 and HSP70 in neck skin samples was immunohistochemically examined to understand its forensic applicability in determining wound vitality. Skin samples were obtained from 45 cases of neck compression (hanging, 32 cases; strangulation, 10 cases; manual strangulation, 2 cases; other, 1 case) during forensic autopsies; intact skin from the same individual was used as a control. HSP27 expression was detected in 17.4% of keratinocytes in the intact skin samples. In the compressed region, the frequency of HSP27 expression in keratinocytes was 75.8%, which was significantly higher than that in intact skin. Similarly, HSP70 expression was 24.8% in intact skin samples and 81.9% in compressed skin samples, significantly higher in compressed skin than in intact skin samples. This increase in case compression cases may be due to the cell defence role of HSPs. From a forensic pathology perspective, the immunohistochemical examination of HSP27 and HSP70 expression in neck skin could be considered a valuable marker for diagnosing traces of antemortem compression.

B cell receptor repertoire analysis from autopsy samples of COVID-19 patients.

Neutralizing antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being developed world over. We investigated the possibility of producing artificial antibodies from the formalin fixation and paraffin-embedding (FFPE) lung lobes of a patient who died by coronavirus disease 2019 (COVID-19). The B-cell receptors repertoire in the lung tissue where SARS-CoV-2 was detected were considered to have highly sensitive virus-neutralizing activity, and artificial antibodies were produced by combining the most frequently detected heavy and light chains. Some neutralizing effects against the SARS-CoV-2 were observed, and mixing two different artificial antibodies had a higher tendency to suppress the virus. The neutralizing effects were similar to the immunoglobulin G obtained from healthy donors who had received a COVID-19 mRNA vaccine. Therefore, the use of FFPE lung tissue, which preserves the condition of direct virus sensitization, to generate artificial antibodies may be useful against future unknown infectious diseases.

Immune Mechanisms of Pulmonary Fibrosis with Bleomycin.

Fibrosis and structural remodeling of the lung tissue can significantly impair lung function, often with fatal consequences. The etiology of pulmonary fibrosis (PF) is diverse and includes different triggers such as allergens, chemicals, radiation, and environmental particles. However, the cause of idiopathic PF (IPF), one of the most common forms of PF, remains unknown. Experimental models have been developed to study the mechanisms of PF, and the murine bleomycin (BLM) model has received the most attention. Epithelial injury, inflammation, epithelial-mesenchymal transition (EMT), myofibroblast activation, and repeated tissue injury are important initiators of fibrosis. In this review, we examined the common mechanisms of lung wound-healing responses after BLM-induced lung injury as well as the pathogenesis of the most common PF. A three-stage model of wound repair involving injury, inflammation, and repair is outlined. Dysregulation of one or more of these three phases has been reported in many cases of PF. We reviewed the literature investigating PF pathogenesis, and the role of cytokines, chemokines, growth factors, and matrix feeding in an animal model of BLM-induced PF.

Femicide and forensic pathology: Proposal for a shared medico-legal methodology.

In a previous work, authors have proposed a medico-legal definition of femicide as the murder due to the failure to recognize the right of self-determination of women. The aim of this paper was to apply the proposed definition to a cohort of cases to characterise femicides and female homicides and assess whether femicides can be considered a distinct entity or not. A comparison between female and male homicides was performed to assess common and distinctive features. Femicides were identified and compared to the cohort of non-femicide female murder. Results were compared to those reported in published forensic studies. Significant associations between female and male homicides were found for sex and partner/ex-partner offender, sex and indoor homicide and sex and asphyxia as dynamic of death emerged. A higher prevalence of indoor homicides and asphyxiation and of partner relationships were documented in female homicides. Gunshot, blunt injuries and cut wounds are well represented in both types of homicides. Most affected sites are back and chest in male homicides, and head, breasts, pubis, and limbs in female homicides. When comparing femicides and female homicides, a positive association between strangulation as harmful mean and a negative one between femicides and indoor homicides were found. Male and female homicides can be considered as two distinct victimological phenomena. Focusing on femicide allows to establish injuries and circumstantial patterns, that could represent evidence of a specific murder. More studies with a standardized data collection are needed to corroborate the theory of this paper.

Macrophage polarity and wound age determination.

We investigated the dynamics of the gene expression of M1 and M2 macrophage markers during skin wound healing in mice. Expression of M1-macrophage markers, such as Il12a, Tnf, Il6, Il1b, and Nos2 was upregulated after wounding and peaked at 1 or 3 days after injury, and that of M2-macrophage markers such as Mrc1, Cd163, Ccl17, Arg, and Tgfb1, peaked at 6 days after injury. Consistent with these findings, using triple-color immunofluorescence analysis revealed that F4/80+CD80+ M1 macrophages were more abundant than F4/80+CD206+ M2 macrophages on day 3 in mouse wound specimens, and that M2 macrophages were prominently detected in day 6 wounds. For application in forensic practice, we examined macrophage polarization using human wound specimens. The average ratios of CD68+iNOS+ M1 macrophages to CD68+CD163+ M2 macrophages (M1/M2 ratios) were greater than 2.5 for the wounds aged 2-5 days. Out of 11 wounds aged 1-5 days, five samples had the M1/M2 ratios of > 3.0. These observations propose that the M1/M2 ratios of 3.0 would indicate a wound age of 1-5 days as the forensic opinion. This study showed that M1 and M2 macrophages in human skin wound might be a promising marker for wound age determination.

Forensic Application of Epidermal Ubiquitin Expression to Determination of Wound Vitality in Human Compressed Neck Skin.

Ubiquitin is a member of the heat shock protein family and is rapidly induced by various types of stimuli, including ischemic and mechanical stress. However, its significance in determining wound vitality of neck compression skin in forensic pathology remains unclear. We immunohistochemically examined the expression of ubiquitin in the neck skin samples to understand its forensic applicability in determining wound vitality. Skin samples were obtained from 53 cases of neck compression (hanging, 42 cases; strangulation, 11 cases) during forensic autopsies. Intact skin from the same individual was used as the control. Ubiquitin expression was detected in 73.9% of keratinocytes in intact skin samples, but only in 21.2% of keratinocytes in the compression regions, with statistical differences between the control and compression groups. This depletion in the case of neck compression may be caused by the impaired conversion of conjugated to free ubiquitin and failure of de novo ubiquitin synthesis. From a forensic pathological perspective, immunohistochemical examination of ubiquitin expression in the skin of the neck can be regarded as a valuable marker for diagnosing traces of antemortem compression.

Immune Cells Profiles in the Different Sites of COVID-19-Affected Lung Lobes in a Single Patient.

Whereas the COVID-19 disease pathophysiology is under investigation, it is important to identify the pathways of viral transmission and inflammation from the pre-illness to the disease-onset stages. We analyzed five lung lobes from a patient with COVID-19 who finally died after prolonged lung protective ventilation. Pathological examination revealed moderate inflammation in upper lung lobes and uneven yet severe inflammation and diffuse alveolar damage in lower lung lobes. SARS-CoV-2 was detected at higher levels not in severely, but rather moderately inflamed middle lung lobes, and immunohistochemistry and bulk RNA-sequencing results showed that immune cells were detected at higher levels in lower lung lobes. The mRNA expression of cytokine families varied. We found an increase in keratin 5- or aquaporin 3-expressing basal cells in the severely inflamed lower lung lobes, and the alveolar stromal tissues were filled with them. Thus, this analysis of lung samples from a patient helps to determine the COVID-19 pathophysiology at a specific time point, and the virus localization and inflammatory responses at each site of the lungs provide various important indications.

Bone Marrow-Derived Cells and Wound Age Estimation.

Appropriate technology as well as specific target cells and molecules are key factors for determination of wound vitality or wound age in forensic practice. Wound examination is one of the most important tasks for forensic pathologists and is indispensable to distinguish antemortem wounds from postmortem damage. For vital wounds, estimating the age of the wound is also essential in determining how the wound is associated with the cause of death. We investigated bone marrow-derived cells as promising markers and their potential usefulness in forensic applications. Although examination of a single marker cannot provide high reliability and objectivity in estimating wound age, evaluating the appearance combination of bone marrow-derived cells and the other markers may allow for a more objective and accurate estimation of wound age.

No-reflow phenomenon and in vivo cholesterol crystals combined with lipid core in acute myocardial infarction.

BACKGROUND: The release of lipid-laden plaque material subsequent to ST-segment elevation myocardial infarction (STEMI) may contribute to the no-reflow phenomenon. The aim of this study was to investigate the association between in vivo cholesterol crystals (CCs) detected by optical coherence tomography (OCT) and the no-reflow phenomenon after successful percutaneous coronary intervention (PCI) in patients with acute STEMI. METHODS: We investigated 182 patients with STEMI. Based on the thrombolysis in myocardial infarction (TIMI) flow grade after PCI, patients were divided into a no-reflow group (n = 31) and a reflow group (n = 151). On OCT, CCs were defined as thin, high-signal intensity regions within a plaque. A multivariable logistic regression analysis was performed to determine predictors for the no-reflow phenomenon. RESULTS: The prevalence of CCs was higher in the no-reflow group than the reflow group (no-reflow group, 77% vs. reflow group, 53%; p = 0.012). The multivariable logistic model showed that the CC number, lipid arc and ostial lesions were positive independent predictors of no-reflow. The combination of a lipid arc ≥ 139°and CC number ≥ 12 showed good predictive performance for the no-reflow phenomenon (sensitivity, 48%; specificity, 93%; and accuracy, 86%). CONCLUSION: In vivo CCs at the culprit plaque are associated with the no-reflow phenomenon after PCI in patients with STEMI. The combination of the number of CCs and lipid arc can predict the no-reflow phenomenon after PCI with a high accuracy of 86%.

A case of necrotizing fasciitis following intra-articular injections - Iatrogenic or spontaneous?

Here, we report a case of necrotizing fasciitis following intra-articular injection of hyaluronic acid. A 73-year-old female received intra-articular injections of hyaluronic acid due to arthralgia at the left shoulder and knee, and was found dead in her living room at one day. At the forensic autopsy, injection marks with bullae and erythema were found at the left shoulder and knee and liquefactive necrosis of muscle tissues was observed in the left but not right extremities. Histopathological examinations of the left upper arm and thigh revealed severe rhabdomyolysis with lots of bacterial clusters. Bacteriological examinations detected group A Streptococcus from intracardiac blood and affected muscle tissues. Postmortem biochemical analysis of blood showed escalated blood urea nitrogen (133.8 mg/dL), creatinine (4.57 mg/dL) and C-reactive protein (45.0 mg/dL). The cause of her death was diagnosed as streptococcal toxic shock syndrome (STSS). Moreover, it was suggested that the injection was inappropriately conducted and served as a portal of bacterial entry.