Cytokines as new biomarkers of skin wound vitality.

BACKGROUND: The diagnosis of skin wound vitality is currently based on standard histology, but histological findings lack sensitivity in case of a short survival time. New reliable biomarkers of vitality are therefore strongly needed. We assessed the ability of 10 candidate cytokines (IFN-γ, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-α) to discriminate between vital and early post-mortem wounds. METHODS: Twenty-four cadavers with a recent open skin wound (< 3 h) were included (20 men, 4 women, mean age = 51.0 ± 24.3 years). An early post-mortem wound was performed in an uninjured skin area, and both wounds were sampled at the autopsy (post-mortem interval (PMI) = 66.3 ± 28.3 h). Needle-puncture sites related to resuscitation cares were included as very early post-mortem wounds (n = 6). In addition to standard histology, cytokines levels were simultaneously measured in each sample using a multiplex sandwich immunoassay, then normalized on healthy skin levels. A quantitative evaluation of IL-8-positive cells in ante- and post-mortem wound samples was also performed. RESULTS: In the training set of samples (n = 72), cytokine levels were significantly higher in vital wounds (mean age = 47 ± 53 min) than in post-mortem wounds (mean PMI = 6.9 ± 9.0 h) (p < 0.2), except for two cytokines (IFN-γ and IL-2). IL-8 was the best discriminatory cytokine (Se = 54%, Sp = 100%, AUC = 0.79), while a multivariate model combining IL-4 and IL12p70 was a bit more discriminant (Se = 55%, Sp = 100%, AUC = 0.84). In the validation set (n = 72), the discriminatory power of the cytokines and the predictive model was slightly lower, with IL-8 remaining the best cytokine (Se = 46%, Sp = 96%, AUC = 0.75). The predictive model remained highly specific (Sp = 100%). Both the cytokines and the predictive model allowed the iatrogenic injuries to be correctly classified as post-mortem wounds. Standard histology and immunohistochemistry showed 21% sensitivity and a specificity of 79% and 100%, respectively. Only two iatrogenic wounds could be properly categorized histologically. CONCLUSION: This study suggests that cytokines could be useful biomarkers of skin wound vitality and that the immunoassay method could be more sensitive than immunohistochemistry to identify wounds with a short survival time. Further research is underway to confirm these preliminary data.

Control of nerve cord formation by Engrailed and Gooseberry-Neuro: A multi-step, coordinated process.

One way to better understand the molecular mechanisms involved in the construction of a nervous system is to identify the downstream effectors of major regulatory proteins. We previously showed that Engrailed (EN) and Gooseberry-Neuro (GsbN) transcription factors act in partnership to drive the formation of posterior commissures in the central nervous system of Drosophila. In this report, we identified genes regulated by both EN and GsbN through chromatin immunoprecipitation ("ChIP on chip") and transcriptome experiments, combined to a genetic screen relied to the gene dose titration method. The genomic-scale approaches allowed us to define 175 potential targets of EN-GsbN regulation. We chose a subset of these genes to examine ventral nerve cord (VNC) defects and found that half of the mutated targets show clear VNC phenotypes when doubly heterozygous with en or gsbn mutations, or when homozygous. This strategy revealed new groups of genes never described for their implication in the construction of the nerve cord. Their identification suggests that, to construct the nerve cord, EN-GsbN may act at three levels, in: (i) sequential control of the attractive-repulsive signaling that ensures contralateral projection of the commissural axons, (ii) temporal control of the translation of some mRNAs, (iii) regulation of the capability of glial cells to act as commissural guideposts for developing axons. These results illustrate how an early, coordinated transcriptional control may orchestrate the various mechanisms involved in the formation of stereotyped neuronal networks. They also validate the overall strategy to identify genes that play crucial role in axonal pathfinding.

Organotypic culture of human brain explants as a preclinical model for AI-driven antiviral studies.

Viral neuroinfections represent a major health burden for which the development of antivirals is needed. Antiviral compounds that target the consequences of a brain infection (symptomatic treatment) rather than the cause (direct-acting antivirals) constitute a promising mitigation strategy that requires to be investigated in relevant models. However, physiological surrogates mimicking an adult human cortex are lacking, limiting our understanding of the mechanisms associated with viro-induced neurological disorders. Here, we optimized the Organotypic culture of Post-mortem Adult human cortical Brain explants (OPAB) as a preclinical platform for Artificial Intelligence (AI)-driven antiviral studies. OPAB shows robust viability over weeks, well-preserved 3D cytoarchitecture, viral permissiveness, and spontaneous local field potential (LFP). Using LFP as a surrogate for neurohealth, we developed a machine learning framework to predict with high confidence the infection status of OPAB. As a proof-of-concept, we showed that antiviral-treated OPAB could partially restore LFP-based electrical activity of infected OPAB in a donor-dependent manner. Together, we propose OPAB as a physiologically relevant and versatile model to study neuroinfections and beyond, providing a platform for preclinical drug discovery.

Brain exposure to SARS-CoV-2 virions perturbs synaptic homeostasis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with short- and long-term neurological complications. The variety of symptoms makes it difficult to unravel molecular mechanisms underlying neurological sequalae after coronavirus disease 2019 (COVID-19). Here we show that SARS-CoV-2 triggers the up-regulation of synaptic components and perturbs local electrical field potential. Using cerebral organoids, organotypic culture of human brain explants from individuals without COVID-19 and post-mortem brain samples from individuals with COVID-19, we find that neural cells are permissive to SARS-CoV-2 to a low extent. SARS-CoV-2 induces aberrant presynaptic morphology and increases expression of the synaptic components Bassoon, latrophilin-3 (LPHN3) and fibronectin leucine-rich transmembrane protein-3 (FLRT3). Furthermore, we find that LPHN3-agonist treatment with Stachel partially restored organoid electrical activity and reverted SARS-CoV-2-induced aberrant presynaptic morphology. Finally, we observe accumulation of relatively static virions at LPHN3-FLRT3 synapses, suggesting that local hindrance can contribute to synaptic perturbations. Together, our study provides molecular insights into SARS-CoV-2-brain interactions, which may contribute to COVID-19-related neurological disorders.

Myeloperoxydase and CD15 With Glycophorin C Double Staining in the Evaluation of Skin Wound Vitality in Forensic Practice.

Background: The determination of skin wound vitality based on tissue sections is a challenge for the forensic pathologist. Histology is still the gold standard, despite its low sensitivity. Immunohistochemistry could allow to obtain a higher sensitivity. Upon the candidate markers, CD15 and myeloperoxidase (MPO) may allow to early detect polymorphonuclear neutrophils (PMN). The aim of this study was to evaluate the sensitivity and the specificity of CD15 and MPO, with glycophorin C co-staining, compared to standard histology, in a series of medicolegal autopsies, and in a human model of recent wounds. Methods: Twenty-four deceased individuals with at least one recent open skin wound were included. For each corpse, a post-mortem wound was performed in an uninjured skin area. At autopsy, a skin sample from the margins of each wound and skin controls were collected (n = 72). Additionally, the cutaneous surgical margins of abdominoplasty specimens were sampled as a model of early intravital stab wound injury (scalpel blade), associated with post-devascularization wounds (n = 39). MPO/glycophorin C and CD15/glycophorin C immunohistochemical double staining was performed. The number of MPO and CD15 positive cells per 10 high power fields (HPF) was evaluated, excluding glycophorin C-positive areas. Results: With a threshold of at least 4 PMN/10 high power fields, the sensitivity and specificity of the PMN count for the diagnostic of vitality were 16 and 100%, respectively. With MPO/glycophorin C as well as CD15/glycophorin C IHC, the number of positive cells was significantly higher in vital than in non-vital wounds (p < 0.001). With a threshold of at least 4 positive cells/10 HPF, the sensitivity and specificity of CD15 immunohistochemistry were 53 and 100%, respectively; with the same threshold, MPO sensitivity and specificity were 28 and 95%. Conclusion: We showed that combined MPO or CD15/glycophorin C double staining is an interesting and original method to detect early vital reaction. CD15 allowed to obtain a higher, albeit still limited, sensitivity, with a high specificity. Confirmation studies in independent and larger cohorts are still needed to confirm its accuracy in forensic pathology.

LIFE BEYOND LIFE - An Easy Way to Derive Lung Fibroblasts from Cadavers.

Several protocols have illustrated the possibility of deriving cells, such as fibroblasts, from different organs. These techniques generally concern organs sampled from living persons, but have already been described for cadavers, especially concerning the skin and tendons. We present, for the first time, an easy way to derive pulmonary fibroblasts from a lung tissue sampled from a cadaver and directly culture plated. The fibroblast output was checked daily. We obtained lung fibroblasts from 3 (60%) cadavers and 2 (100%) living persons. The fibroblast output took about 3 days for cells from living persons and took up to 39 days for those from cadavers. We did not clearly identify any parameters that could explain these differences. Nevertheless, these derived cells had the same features as the source cells, especially in terms of morphology and proliferation, and could potentially be used in different research domains such as forensic or regeneration medicine.

Technical note: The two step procedure (TSP) for the determination of age at death of adult human remains in forensic cases.

This paper presents the principles and results of TSP (the two step procedure), a comprehensive (combined) method of age estimation in mature human skeletal remains. The first step consists of the examination of the pubic symphysis using the Suchey-Brooks system for a "pre-choice". Then for SBS phases I, II, III, (young adults up to about 40) the age estimate is given using the chronological interval corresponding to each phase. For SBS phase is IV, V or VI (mature adults, about 40 to 60), then (second step) the dental method of Lamendin (using single rooted tooth) will be applied alone. Both methods are fast, easy to learn and to use (requiring no preparation except cleaning soft tissues from the pubic bone) and are not expensive, making TSP usable by all pathologists or anthropologists in any Forensic unit. It is also of great practical use in mass disaster and mass grave situation. After 15 years of use, a literature review and four evaluation studies we confirm that TSP is more accurate than any single method for aging adults and at least as good as more complicated combined methods. Despite its advantages TSP is, like all other aging methods, not efficient in adults over 65 years of age.

A concerted action of Engrailed and Gooseberry-Neuro in neuroblast 6-4 is triggering the formation of embryonic posterior commissure bundles.

One challenging question in neurogenesis concerns the identification of cues that trigger axonal growth and pathfinding to form stereotypic neuronal networks during the construction of a nervous system. Here, we show that in Drosophila, Engrailed (EN) and Gooseberry-Neuro (GsbN) act together as cofactors to build the posterior commissures (PCs), which shapes the ventral nerve cord. Indeed, we show that these two proteins are acting together in axon growth and midline crossing, and that this concerted action occurs at early development, in neuroblasts. More precisely, we identified that their expressions in NB 6-4 are necessary and sufficient to trigger the formation of the PCs, demonstrating that segmentation genes such as EN and GsbN play a crucial role in the determination of NB 6-4 in a way that will later influence growth and guidance of all the axons that form the PCs. We also demonstrate a more specific function of GsbN in differentiated neurons, leading to fasciculations between axons, which might be required to obtain PC mature axon bundles.

Polycomb group-dependent Cyclin A repression in Drosophila.

Polycomb group (PcG) and trithorax group (trxG) proteins are well known for their role in the maintenance of silent and active expression states of homeotic genes. However, PcG proteins may also be required for the control of cellular proliferation in vertebrates. In Drosophila, PcG factors act by associating with specific DNA regions termed PcG response elements (PREs). Here, we have investigated whether Drosophila cell cycle genes are directly regulated by PcG proteins through PREs. We have isolated a PRE that regulates Cyclin A (CycA) expression. This sequence is bound by the Polycomb (PC) and Polyhomeotic (PH) proteins of the PcG, and also by GAGA factor (GAF), a trxG protein that is usually found associated with PREs. This sequence causes PcG- and trxG-dependent variegation of the mini-white reporter gene in transgenic flies. The combination of FISH with PC immunostaining in embryonic cells shows that the endogenous CycA gene colocalizes with PC at foci of high PC concentration named PcG bodies. Finally, loss of function of the Pc gene and overexpression of Pc and ph trigger up-regulation and down-regulation, respectively, of CycA expression in embryos. These results demonstrate that CycA is directly regulated by PcG proteins, linking them to cell cycle control in vivo.