PD-L1 Overexpression in the Lungs of Subjects Who Died from COVID-19: Are We on the Way to Understanding the Immune System Exhaustion Induced by SARS-CoV-2?

Knowledge of the pathogenic mechanisms of severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) is certainly a priority for the scientific community. Two main elements are involved in the biology of the most severe forms of coronavirus disease 2019 (COVID-19): the direct cytopathic effect of the virus against the host tissues, and a dysfunction of the immune system, characterized by the exhaustion of T lymphocytes. The exhaustion of T cells in COVID-19 is poorly understand, but some data could suggest a possible role of PD-1/PD-L1 axis. The aim of this study was to evaluate the possible role of PD-L1 expression in the pulmonary tissue in subjects affected by COVID-19. The presence of SARS-CoV-2 in the pulmonary tissue, and its exact location, was indagated by in situ hybridization; the expression of PD-L1 and CD8 in the same tissue was indagated by immunohistochemistry. Overall, PD-L1 resulted diffusely expressed in 70% of the cases, and an intense expression was observed in 43.5% of cases. Diffuse and intense presence of SARS-CoV-2 by in situ hybridization significantly correlated with an intense PD-L1 expression, and with expression of PD-L1 by pneumocytes. PD-L1 is overexpressed in the pulmonary tissue of subjects died from COVID-19, and mainly in subjects with a high viral load. These data suggest a possible role of PD-L1 in the immune system exhaustion at the basis of the severe forms of the disease.

Postmortem interval assessment by MALDI-TOF mass spectrometry analysis in murine cadavers.

AIMS: This study assessed the use of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry as an alternative method to identify species associated with the thanatomicrobiota and epinecrotic communities. METHODS AND RESULTS: The study was conducted on 10 murine cadavers, and microbiological swabs were collected from five external anatomical sites (eyes, ears, nose, mouth and rectum) and four internal organs (brain, spleen, liver, heart), during 16 and 30 days, for the thanatomicrobiota and epinecrotic communities, respectively. Our results revealed that the postmortem microbiota associated with the external cavities showed changes over time and reduced taxonomic diversity. The internal organs, initially sterile, showed signs of microbial invasion at 3 and 10 days postmortem for the liver-spleen and heart-brain, respectively. The postmortem microbiota was mainly dominated by Firmicutes and Proteobacteria. CONCLUSIONS: MALDI-TOF is a promising method for estimating postmortem interval (PMI), associated with rapid sample handling, good reproducibility and high productivity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study investigated microbial changes during the decomposition process and proposed a simple strategy for PMI estimation. Results introducing the application of the MALDI-TOF method in the field of forensic.

Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte.

RATIONALE: About 50% of hospitalized coronavirus disease 2019 (COVID-19) patients with diabetes mellitus (DM) developed myocardial damage. The mechanisms of direct SARS-CoV-2 cardiomyocyte infection include viral invasion via ACE2-Spike glycoprotein-binding. In DM patients, the impact of glycation of ACE2 on cardiomyocyte invasion by SARS-CoV-2 can be of high importance. OBJECTIVE: To evaluate the presence of SARS-CoV-2 in cardiomyocytes from heart autopsy of DM cases compared to Non-DM; to investigate the role of DM in SARS-COV-2 entry in cardiomyocytes. METHODS AND RESULTS: We evaluated consecutive autopsy cases, deceased for COVID-19, from Italy between Apr 30, 2020 and Jan 18, 2021. We evaluated SARS-CoV-2 in cardiomyocytes, expression of ACE2 (total and glycosylated form), and transmembrane protease serine protease-2 (TMPRSS2) protein. In order to study the role of diabetes on cardiomyocyte alterations, independently of COVID-19, we investigated ACE2, glycosylated ACE2, and TMPRSS2 proteins in cardiomyocytes from DM and Non-DM explanted-hearts. Finally, to investigate the effects of DM on ACE2 protein modification, an in vitro glycation study of recombinant human ACE2 (hACE2) was performed to evaluate the effects on binding to SARS-CoV-2 Spike protein. The authors included cardiac tissue from 97 autopsies. DM was diagnosed in 37 patients (38%). Fourth-seven out of 97 autopsies (48%) had SARS-CoV-2 RNA in cardiomyocytes. Thirty out of 37 DM autopsy cases (81%) and 17 out of 60 Non-DM autopsy cases (28%) had SARS-CoV-2 RNA in cardiomyocytes. Total ACE2, glycosylated ACE2, and TMPRSS2 protein expressions were higher in cardiomyocytes from autopsied and explanted hearts of DM than Non-DM. In vitro exposure of monomeric hACE2 to 120 mM glucose for 12 days led to non-enzymatic glycation of four lysine residues in the neck domain affecting the protein oligomerization. CONCLUSIONS: The upregulation of ACE2 expression (total and glycosylated forms) in DM cardiomyocytes, along with non-enzymatic glycation, could increase the susceptibility to COVID-19 infection in DM patients by favouring the cellular entry of SARS-CoV2.

Matricide and psychiatric evaluation: An update.

Matricide is an infrequent crime that has often raised to the suspicion that the offender could suffer from a pathological mental status. Although it is hard to establish a relationship between mental disorders and specific forms of homicide, several studies suggest that matricide offender frequently suffers from schizophrenia or other psychotic disorders. Aim of the study was to review the literature in order to explore epidemiologic and psychiatric characteristics of matricide offenders with a focus on the cause of death and crime circumstances. According to the PRISMA statement, 16 out of 225 studies were included. Main findings were the following: 80 victims were reported in total, killed by 81 offenders. In one case two brothers were responsible for the matricide. 81.5% of the offenders were young males. The most represented psychiatric pattern of the offenders was schizophrenia and psychotic disorders (43.2%). 6.2% of victims had also psychiatric disorders. Sharp force injuries were the first cause of death (55%), followed by blunt trauma (15%) and asphyxia (15%). In 12% of cases overkilling was also reported. 13.6% of offenders were considered not guilty for reason of insanity while 25.9% of the offenders had diminished criminal responsibility. A case study of a young homicide offender suffering from personality disorder is reported. In this case the victim also suffered from psychiatric disorders and an overkilling occurred as she was stabbed multiple times post-mortem. In a second case of matricide, the victim was dismembered and the human remains were concealed in several plastic bags.

Helium Suicide, a Rapid and Painless Asphyxia: Toxicological Findings.

Suicide by helium inhalation has become increasingly common in the last few decades in Europe and the US because it produces a quick and painless death. Inhaled-gas suicides can easily be assessed through death scene investigation and autopsy. However, helium is a colorless and odorless inert gas that unfortunately cannot be detected using standard toxicological analysis. A successful gas analysis was performed following the suicide of a 17-year-old female. For the detection of helium, central/peripheral blood samples and gaseous samples from the esophagus, stomach, and upper and lower respiratory airways (from the trachea and the primary left and right bronchia) were collected with a gastight syringe, ensuring minimal dilution. Qualitative analyses were positive in all gaseous samples. Quantitative analyses were performed using a special gas-inlet system with a vacuum by which the sample can be transferred to a mass spectrometer, reducing the risk of contamination. Helium concentrations were 20.16% from the trachea, 12.33% from the right lung, and 1.5% from the stomach. Based on the high levels of helium, the cause and manner of death were assessed as asphyxia suicide by inhalation of helium. Therefore, toxicological analyses should always be applied in order to gain evidence of inhaled gas in gaseous samples.

Suicide by Pesticide (Phorate) Ingestion: Case Report and Review of Literature.

It has been estimated that approximately one in seven of all global suicides is due to pesticide self-poisoning, mostly in rural areas of developing countries. Organophosphorus (OP) compounds are a group of pesticides exerting their toxicological effects through non-reversible inhibition of the enzyme acetylcholinesterase (AChE). Among these compounds, phorate (thimet) is one of the most dangerous compounds, the use of which is restricted in many countries. A case of intentional suicide after phorate ingestion in a 24-year-old Bengali male is described. This is the second case of suicidal ingestion of phorate reported in the forensic literature, and the first presenting complete toxicological findings.

MALDI-TOF Mass Spectrometry Analysis and Human Post-Mortem Microbial Community: A Pilot Study.

Introduction: The human post-mortem microbiome (HPM) plays a major role in the decomposition process. Successional changes in post-mortem bacterial communities have been recently demonstrated using high throughput metagenomic sequencing techniques, showing great potential as a post-mortem interval (PMI) predictor. The aim of this study is to verify the application of the mass spectrometry technique, better known as MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry), as a cheap and quick method for microbe taxonomic identification and for studying the PM microbiome. Methods: The study was carried out on 18 human bodies, ranging from 4 months to 82 years old and with a PMI range from 24 h up to 15 days. The storage time interval in the coolers was included in the final PMI estimates. Using the PMI, the sample study was divided into three main groups: seven cases with a PMI < 72 h; six cases with a PMI of 72−168 h and five cases with a PMI > 168 h. For each body, microbiological swabs were sampled from five external anatomical sites (eyes, ears, nose, mouth, and rectum) and four internal organs (brain, spleen, liver, and heart). Results: The HPM became increasingly different from the starting communities over time in the internal organs as well as at skin sites; the HPM microbiome was mostly dominated by Firmicutes and Proteobacteria phyla; and a PM microbial turnover existed during decomposition, evolving with the PMI. Conclusions: MALDI-TOF is a promising method for PMI estimation, given its sample handling, good reproducibility, and high speed and throughput. Although several intrinsic and extrinsic factors can affect the structure of the HPM, MALDI-TOF can detect the overall microbial community turnover of most prevalent phyla during decomposition. Limitations are mainly related to its sensitivity due to the culture-dependent method and bias in the identification of new isolates.

Variable levels of spike and ORF1ab RNA in post-mortem lung samples of SARS-CoV-2-positive subjects: comparison between ISH and RT-PCR.

Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2; thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR; however, it is able to localize the virus in the cellular context since it preserves the morphology.

A Descriptive Study on Causes of Death in Hospitalized Patients in an Acute General Hospital of Southern Italy during the Lockdown due to Covid-19 Outbreak.

(1) Background: All deaths that occurred in a hospital of Southern Italy ("San Giuseppe Moscati" Hospital of Avellino) with medium jurisdiction (up to 425,000 citizens approximately) in the period from 9 March to 4 May 2020 were analyzed. The primary endpoint of the study was to analyze the causes of death in the period study. Secondary endpoints included: (1) the assessment of overall mortality in the emergency period compared with the same period of the past years (2018-2019) in the jurisdiction area; (2) the assessment of the amounts of deaths with positive and negative reverse transcription-polymerase chain reaction (RT-PCR) of nasopharyngeal and oropharyngeal swabs; (3) the frequency of clinical and radiological features consistent with Covid-19 infection in negative RT-PCR cases. (2) Methods: Patients' information and laboratory data were collected through the computerized medical record system (My Hospital, Italy) used for the clinical management of all referring patients. Epidemiological, clinical, and radiological data were reviewed along with the results of nasopharyngeal and oropharyngeal swabs. (3) Results: From 9 March to 4 May 2020, 140 deaths (87 males, 53 females) from all causes occurred in total at "San Giuseppe Moscati" Hospital, of which 32 deaths were Covid-19 related. (4) Conclusions: The excess of mortality could be higher than the one reported in the official epidemiological surveys. False negative cases can have a distorting effect on the assessment of the real mortality rate and the excess mortality. Furthermore, many who died from Covid-19 were likely never tested or they had false negative RT-PCR results. Other victims probably died from causes indirectly related to Covid-19.

Electrocution Stigmas in Organ Damage: The Pathological Marks.

BACKGROUND: Diagnostic criteria for electrocution related death are still a challenge in forensic pathology and it seems that the electrical mark is the only reliable evidence. METHODS: A comparison of histological and morphological findings of skin and internal organs from an autopsy series of electrocution deaths with those mostly reported in literature as representative for electrocution. RESULTS: The morphological changes of heart, brain and other main internal organs are still unspecific. Organ's damage observed in electrocution deaths shows a wide variability, not reliable for a certain diagnosis of electrocution. The electrical mark is still the golden standard for diagnosis of electrocution. CONCLUSIONS: In electrocution related deaths, pathological findings of the main internal organs are not enough evidence to support with certainty a post-mortem diagnosis that a victim suffered an electrical damage. Although the organ histological changes are undoubtedly the starting point for a better understanding of the fatal even, the diagnosis of death from electrical damage is still a dark and unsolved chapter. The electrical mark still represents a fundamental indicator above all in the medical-legal field, but the identification of pathognomonic elements and signs not limited to the skin alone could be a valid help in the future, especially in unclear cases.

Conocimiento, actitudes y percepciones sobre VIH/SIDA e infecciones de transmisión sexual en estudiantes ingresados a odontología y medicina de una universidad venezolana / Knowledge, attitudes and perceptions about HIV/AIDS and sexually transmitted infections in new dental and medical students in a venezuelan university

En la lucha contra las infecciones de transmisión sexual (ITS) y la infección por el Virus de Inmunodeficiencia Humana (VIH)/Síndrome de Inmunodeficiencia Adquirida (SIDA) la formación y capacitación desde etapas tempranas de la carrera profesional requiere, entre otras cosas, conocer el nivel de conocimientos, actitudes y percepciones (CAP), que los estudiantes de las ciencias de la salud tienen al respecto, abordando no solo la carrera de medicina, sino otras, como es el caso de odontología. Por estas razones el objetivo de la presente investigación fue evaluar el nivel de CAP de una muestra de estudiantes de pregrado de primer año de ambas carreras de una universidad venezolana (Universidad Central de Venezuela), con respecto a las ITS e infección VIH/SIDA. Del total (n = 120), 63,3% correspondió al sexo femenino; la edad promedio fue de 18,64 años. Con respecto a la proporción de respuestas correctas o en acuerdo de toda la muestra estudiada, se encontró que del total de preguntas, el rango de respuestas correctas o en acuerdo en la población evaluada estuvo entre 60% y 100,0%, respondiendo correctamente o en acuerdo en promedio 82,6% de las respuestas (± 8,46), siendo significativamente mayor en estudiantes de medicina (84,92% ± 7,78%) que en estudiantes de odontología (80,29% ± 8,54%) (t = 3,101; p = 0,002). En términos generales se observó que los estudiantes evaluados tanto de odontología como de medicina de la principal universidad venezolana presentan un buen nivel de conocimiento básico como actitudes y percepciones adecuadas sobre el VIH/SIDA e ITS. Es importante tomar en consideración los resultados para futuros estudios y especialmente para intervenciones que permitan con ello tener una correcta actitud y percepción sobre el VIH/SIDA e ITS por parte de ellos.

In the fight against sexually transmitted infections (STI) and the Human Immunodeficiency Virus (HIV)/Acquired Immunodeficiency Syndrome (AIDS), training and capacitating since early stages of the professional career require, among other things, to address the level of knowledges,attitudes and perceptions (KAP), that health sciences students have on it; taking on consideration not just medicine, but also other areas such as dental studies. For theses reasons theobjective of this study was to assess the KAP level of a sample undergraduate studies, at 1º year of both programs from a Venezuelan university (Universidad Central de Venezuela), in regard to STI and HIV/AIDS. From the total (n=120),63.3% were females; mean age was 18.64 years old. Regard the proportion of correct or in-agreement answers, the range of them in the assessed population ranged between 60% and100.0%, being correctly or in-agreement responded, in mean, at 82.6% of the questions (±8.46), being significantly higherin medical students (84.92%±7.78%) than in dental ones(80.29%±8.54%) (t=3.101; p=0.002). In general terms, it was observed that those evaluated students, in both programs,from the main Venezuelan university presented a good level of basic knowledge as well appropriate attitudes and perceptions about STI and HIV/AIDS. It is important to consider these results in future studies and particularly for interventions in order to have a correct attitude and perception on STI and HIV/AIDS from them.