Cytokine storm and histopathological findings in 60 cases of COVID-19-related death: from viral load research to immunohistochemical quantification of major players IL-1\u3b2, IL-6, IL-15 and TNF-\u3b1

This study involves the histological analysis of samples taken during autopsies in cases of COVID-19 related death to evaluate the inflammatory cytokine response and the tissue localization of the virus in various organs. In all the selected cases, SARS-CoV-2 RT-PCR on swabs collected from the upper (nasopharynx and oropharynx) and/or the lower respiratory (trachea and primary bronchi) tracts were positive. Tissue localization of SARS-CoV-2 was detected using antibodies against the nucleoprotein and the spike protein. Overall, we tested the hypothesis that the overexpression of proinflammatory cytokines plays an important role in the development of COVID-19-associated pneumonia by estimating the expression of multiple cytokines (IL-1\u3b2, IL-6, IL-10, IL-15, TNF-\u3b1, and MCP-1), inflammatory cells (CD4, CD8, CD20, and CD45), and fibrinogen. Immunohistochemical staining showed that endothelial cells expressed IL-1\u3b2 in lung samples obtained from the COVID-19 group (p\u2009<\u20090.001). Similarly, alveolar capillary endothelial cells showed strong and diffuse immunoreactivity for IL-6 and IL-15 in the COVID-19 group (p\u2009<\u20090.001). TNF-\u3b1 showed a higher immunoreactivity in the COVID-19 group than in the control group (p\u2009<\u20090.001). CD8\u2009+\u2009T cells where more numerous in the lung samples obtained from the COVID-19 group (p\u2009<\u20090.001). Current evidence suggests that a cytokine storm is the major cause of acute respiratory distress syndrome (ARDS) and multiple organ failure and is consistently linked with fatal outcomes

Autoimmune polyglandular syndrome type 4: experience from a single reference center

Purpose: To characterize patients with APS type 4 among those affected by APS diagnosed and monitored at our local Reference Center for Autoimmune Polyglandular Syndromes. Methods: Monocentric observational retrospective study enrolling patients affected by APS diagnosed and monitored in a Reference Center. Clinical records were retrieved and analyzed. Results: 111 subjects (51 males) were affected by APS type 4, mean age at the onset was 23.1 ± 15.1 years. In 15 patients the diagnosis of APS was performed during the first clinical evaluation, in the other 96 after a latency of 11 years (range 1-46). The most frequent diseases were type I diabetes mellitus and celiac disease, equally distributed among sexes. Conclusions: The prevalence of APS type 4 is 9:100,000 people. Type I diabetes mellitus was the leading indicator of APS type 4 in 78% subjects and in 9% permitted the diagnosis occurring as second manifestation of the syndrome. Our data, showing that 50% of patients developed APS type 4 within the first ten years, don't suggest any particular follow-up time and, more importantly, don't specify any particular disease. It is important to emphasize that 5% of women developed premature ovarian failure

Cryptogenic Fibrosing Pleuritis

We report the case of a 46-year-old male patient who was referred for chest pain and bilateral pleural effusion. Despite treatment with antibiotics and steroids, the pleural effusion worsened over a few months until pulmonary function was halved. The CT scan showed bilateral pleural thickening with right basal opacity. Histology revealed extensive fibrotic tissue with focal collections of lymphocytes and giant cells without traces of asbestos bodies. Since no evidence of an infectious, embolic or occupational aetiology was found, this bilateral pleural effusion progressing to diffuse pleural thickening was diagnosed as cryptogenic fibrosing pleuritis, a rare pleural disease

Forensic DNA phenotyping: prediction of human externally visible traits in missing person identification

After attending this poster presentation, attendees will better understand the emerging role assumed by externally visible characteristics (EVCs) prediction from forensic samples, or from body parts, in police investigations towards finding unknown individuals, particularly when conventional DNA profiling fails to provide informative leads. The poster presentation will impact the forensic science community by prove the usefulness of forensic DNA phenotyping (FDP), highlighted by missing person identification casework in which our laboratory successfully tested the method proposed. Predicting externally visible characteristics (EVCs) using informative DNA molecular markers has started to become a rapidly developing area in forensic DNA phenotyping (FDP). The most relevant forensic cases for DNA-based EVCs prediction would be those in which the evidence DNA is useful for describing a person’s physical appearance from skeletal remains. Here we evaluate the HIrisPlex DNA test multiplex for the simultaneous prediction of eye, hair, and skin colour in 20 italian skeletal remains deal with 20 different missing person identification caseworks in which identification was performed using the conventional STR-based method. In order to test the reliability and the accuracy of the DNA-based EVCs prediction, victim pictures or portraits were available as reference document. DNA-based system used consists of a SNaPshot multiplex assay targeting a total of 41 SNPs, involved in skin, eye and heir colour prediction1. A total of 20 different bone samples (14 femurs and 6 tibias) were evaluated and processed. Bone samples were cleaned chemically, using diluited bleach, and irradiated with UV light for 30 minute prior to grinding into a fine powder. Genomic DNA was obtained from 0.5 g of bone powder using the QIAamp DNA Investigator Kit (Qiagen). The DNA extracts were quantified using the Investigator Quantiplex HYres Kit (Qiagen). The reactions were carried out in an RotorGene 5-plex System (Qiagen) according to the manufacturer’s instructions. PCR amplification of all 17 SNPs was performed in a single multiplex PCR assay, in a final volume of 10 μl, as described by Chaitanya et al1. The amplified PCR products were purified with ExoI (Thermo Scientific) and incubated at 37°C for 45min and 80°C for 15min. SBE was carried out for all PCR products simultaneously in a single multiplex reaction using 2 μl of the purified PCR products. Finally, the purified SBE products were analysed on the ABI 310 HID Genetic Analyser (Thermo Scientific). Gene Mapper ID-X v1.0 software program (Thermo Scientific) was used for the allele calling and analysis of the results. Prediction of eye, hair, and skin colour was perfomed using a web interface at https://hirisplex.erasmusmc.nl/ allowing the retrieval of individual prediction probabilities for 3 eye colour, 4 hair colour, and now 5 skin colour categories from HIrisPlex genotype input data of 41 SNPs. Each victim picture’s iris, hair, and skin colour were subjectively and objectively determined for all 20 skeletal remains analysed. For the subjective determination, examiners classified victim’s pictures in the same color category (iris, hair, and skin colour) for 84% of the document examined. Results showed an overall prediction accuracies of 91.6%, 90,4% and 91,2% rispectively for iris, hair, and skin colour, at the 0,7 threshold. Our sample set had only 2 inconclusive results as compared to the IrisPlex database. This is likely due to that these samples showed an intermediate eye and hair colour. The results demonstrate the robustness and reliability of the HIrisPlex genotyping system and may encourage its use for the prediction of eye, hair, and skin traits from DNA in missing person cases and outside the in anthropological applications to bringing back eye, hair and skin colour of deceased persons from analysing their skeletal remains or body parts. Reference(s): Chaitanya L., Breslin K., Zuñiga S., Wirken L., Pośpiech E., Kukla-Bartoszek M., Sijen T., et al. The HIrisPlex-S system for eye, hair and skin colour prediction from DNA: Introduction and forensic developmental validation. For. Sci. Int.: Genet. 2018, 35, 123-135

Autoimmune Diseases in Patients with Cushing’s Syndrome after Resolution of Hypercortisolism: Case Reports and Literature Review

Introduction. Cushing’s syndrome (CS) is a clinical condition characterized by excessive cortisol production, associated with metabolic complications, such as diabetes mellitus, dyslipidemia, metabolic syndrome, hypertension, and cardiovascular diseases. Nowadays, the occurrence of autoimmune diseases in CS have not been completely evaluated in the previous studies. Objective. The aim of this study was to evaluate the occurrence of autoimmune diseases in CS patients after successfully treated. Materials and Methods. From January 2001 to December 2017, in our Secondary Hypertension Unit, we evaluated 147 CS patients (91 with ACTH-independent disease, 54 with ACTH-dependent disease, and 2 patients with ectopic ACTH production. Results. 109 CS patients (74.1%) were surgically treated (67 ACTH-independent CS patients (61.5%) undergone adrenalectomy and 42 ACTH-dependent CS (38.5%) undergone transsphenoidal surgery) and evaluated after 6, 12, and 24 months after clinical and biochemical remission of disease. In 9 (8.3%) of overall treated CS patients (8.3%), during follow-up, we observed the onset of some manifestations of autoimmune diseases. In particular, one patient had a systemic lupus erythematosus, one patient had rheumatoid arthritis, 4 patients reported autoimmune thyroiditis (Basedow-Graves’ disease and Hashimoto’s thyroiditis), one patient had clinical features of psoriasis, one patient showed myasthenia gravis, and one patient had giant cell arteritis. Conclusions. Our results demonstrate that patients successfully treated for CS could develop autoimmune diseases. Therefore, after treatment, CS patients need to be strictly monitored in order to evaluate the possible onset of autoimmune diseases

Effect of Repeated Administration of ɣ-Valerolactone (GVL) and GHB in the Mouse: Neuroadaptive Changes of the GHB and GABAergic System

Background: Gamma-hydroxybutyric acid (GHB) at low dosages has anxiolytic effects and promotes REM sleep and low-wave deep sleep. In the U.S., the legal form of GHB is prescribed to adults suffering from narcolepsy-associated cataplexy; the sodium salt of GHB is reserved for alcohol-addiction treatment. GHB is also a molecule of abuse and recreational use, it is a controlled substance in several countries, so gamma-valerolactone (GVL) has frequently been used as a legal substitute for it. GHB’s abuse profile is most likely attributable to its anxiolytic, hypnotic, and euphoric properties, as well as its widespread availability and inexpensive/low cost on the illicit market. Methods: Our study is focused on evaluating the potential effects on the mouse brain after repeated/prolonged administration of GHB and GVL at a pharmacologically active dose (100 mg/kg) through behavioral study and immunohistochemical analysis using the markers tetraspanin 17 (TSPAN17), aldehyde dehydrogenase 5 (ALDH5A1), Gamma-aminobutyric acid type A receptor (GABA-A), and Gamma-aminobutyric acid type B receptor (GABA-B). Results: Our findings revealed that prolonged administration of GHB and GVL at a pharmacologically active dose (100 mg/kg) can have effects on a component of the mouse brain, the intensity of which can be assessed using immunohistochemistry. The findings revealed that long-term GHB administration causes a significant plastic alteration of the GHB signaling system, with downregulation of the putative binding site (TSPAN17) and overexpression of ALDH5A1, especially in hippocampal neurons. Our findings further revealed that GABA-A and GABA-B receptors are downregulated in these brain locations, resulting in a greater decrease in GABA-B expression. Conclusions: The goal of this study, from the point of view of forensic pathology, is to provide a new methodological strategy for better understanding the properties of this controversial substance, which could help us better grasp the unknown mechanism underlying its abuse profile

Nonfunctioning Adrenal Pheochromocytoma Incidentally Discovered Associated with Renal Oncocytoma

Pheochromocytoma (PHEO), a rare catecholamine-producing tumor, is a disease entity with variable clinical manifestations. The classic triad of symptoms are palpitations, headaches, and diaphoresis, but the signs and symptoms are wide ranging and mainly reflect the hemodynamic and metabolic actions of the catecholamines produced and secreted by the tumor. We describe the rare case of a patient affected by nonfunctioning and asymptomatic PHEO associated with the incidental discovery of renal oncocytoma, and review the literature

Forensic DNA Phenotyping (FDP): A Prediction of Human Externally Visible Traits in Missing Person Identification

No abstract availabl

Wound Vitality in Decomposed Bodies: New Frontiers Through Immunohistochemistry

Background: The question about wound vitality and the estimation of wound age of production are two of the classic investigation fields of forensic sciences. To answer this, the techniques most frequently used in research studies are immunohistochemistry (IHC), molecular biology, and biochemistry. Despite the great data on the literature about the usefulness of IHC in forensic pathology, there is always a request for further studies, especially on tissues altered by putrefactive phenomena. In fact, the degradation of the tissues is intended as the main limiting factor to the use of this technique.Scope: The aim of this pilot study was to evaluate the immunohistochemical behavior of samples collected from decomposed bodies (in different putrefaction phases) and to relate these findings to wound vitality and postmortem interval.Materials and Methods: Samples of skin and soft tissues were collected during autopsies, which were executed on decomposed bodies, whose cause of death was concluded to be traumatic. An immunohistochemical study was performed using antibodies against CD15, CD45, IL-15, tryptase, and glycophorin-A MMPs (endopeptidases involved in degrading extracellular matrix proteins: MMP-9 and MMP-2). An immunohistochemistry (IHC) reaction was evaluated according to a qualitative method as the following legend: (0): not expressed, (+): isolated and disseminated expression, (++): expression in groups or widespread foci, and (+++): widespread expression.Results: Most of the tested markers (tryptase, glycophorin, IL15, CD 15, CD 45, and MMP9) showed to be highly expressed in the tissue of putrefied skin for 15 days.Discussion and Conclusion: Although certainly inconclusive, this experimental application demonstrated that a nonexclusive but combined use of multiple antibodies is appropriate to verify wound vitality in decomposed bodies. Among them, GPA exhibited major reliability