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Keloids are disfiguring, hypertrophic scars with yet poorly understood pathomechanisms, which could lead to severe functional impairments. Here we analyzed the characteristics of keloidal cells by single cell sequencing and discovered the presence of an abundant population of Schwann cells that persisted in the hypertrophic scar tissue after wound healing. In contrast to normal skin, keloidal Schwann cells show a unique, pro-fibrotic phenotype. Our data support the hypothesis that keloidal Schwann cells contribute to the formation of the extracellular matrix and are able to affect M2 polarization of macrophages. Indeed, we show that macrophages in keloids predominantly display a M2 polarization and produce factors that inhibit Schwann cell differentiation. This study suggests the contribution of a Schwann cell - macrophage cross-talk to the continuous expansion of keloids, and that targeting Schwann cells might represent an interesting novel treatment option for keloids.

Trauma represents one of the leading causes of death worldwide. Traumatic injuries elicit a dynamic inflammatory response with systemic release of inflammatory cytokines. Disbalance of this response can lead to systemic inflammatory response syndrome or compensatory anti-inflammatory response syndrome. As neutrophils play a major role in innate immune defense and are crucial in the injury-induced immunological response, we aimed to investigate systemic neutrophil-derived immunomodulators in trauma patients. Therefore, serum levels of neutrophil elastase (NE), myeloperoxidase (MPO), and citrullinated histone H3 (CitH3) were quantified in patients with injury severity scores above 15. Additionally, leukocyte, platelet, fibrinogen, and CRP levels were assessed. Lastly, we analyzed the association of neutrophil-derived factors with clinical severity scoring systems. Although the release of MPO, NE, and CitH3 was not predictive of mortality, we found a remarkable increase in MPO and NE in trauma patients as compared with healthy controls. We also found significantly increased levels of MPO and NE on days 1 and 5 after initial trauma in critically injured patients. Taken together, our data suggest a role for neutrophil activation and NETosis in trauma. Targeting exacerbated neutrophil activation might represent a new therapeutic option for critically injured patients.

Antithymocyte globulins (ATG) are T cell depleting antibodies used in solid organ transplantation for induction therapy in sensitized patients with high risk of graft rejection. Previously described effects besides depletion of T cells suggest additional modes of action and identified further cellular targets. Here, we examined the transcriptional changes arising in immune cells from human blood after ex vivo stimulation with ATG on a single cell level to uncover additional mechanisms by which ATG regulates T cell activity and effector functions. Analysis of the paracrine factors present in plasma of ATG-treated whole blood revealed high levels of chemokines and cytokines including Interferon-γ (IFN-γ). Furthermore, we identify an increase of surface expression of programmed cell death 1 ligand 1 (PDL-1) on monocytes mediated by the released paracrine factors. In addition, we show that this induction is dependent on activation of JAK/STAT signaling via binding of IFN-γ to Interferon-γ receptor 1 (IFN-γR1). Lastly, we demonstrate that the modulation of the immune-regulatory axis of Programmed cell death protein 1 (PD1) on activated CD8+ T cells with PDL-1 found on monocytes mediated by ATG potently inhibits effector functions including proliferation and granzyme B release of activated T cells. Together our findings represent a novel mode of action by which ATG exerts its immunosuppressive effects.One Sentence SummaryATG increases PDL-1 on CD14+-monocytes and inhibits T cell effector functions.

Recently, a specific Schwann cell type with pro-fibrotic and tissue regenerative properties has been identified that contributes to keloid formation. In the present study, we have reanalysed published single cell RNA sequencing (scRNAseq) studies of keloids, healthy skin and normal scars to reliably determine the specific gene expression profile of the keloid-specific Schwann cell type in more detail.We were able to confirm the presence of the repair-like, pro-fibrotic Schwann cell type in the datasets of all three studies and identified a specific gene set for these Schwann cells. In contrast to keloids, in normal scars the number of Schwann cells was neither increased nor was their gene expression profile distinctly different from Schwann cells of normal skin. In addition, our bioinformatics analysis provided evidence for a role of transcription factors of the kruppel-like factor family and members of the immediate early response genes, in the de-differentiation process of keloidal Schwann cells.Together, our analysis strengthens the role of the pro-fibrotic Schwann cell type in the formation of keloids. Knowledge on the exact gene expression profile of these Schwann cells will facilitate their identification in other organs and diseases.

IgE-mediated hypersensitivity is becoming increasingly prevalent and activation of mast cells and basophils represent key events in the pathophysiology of allergy. We have previously reported that the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCsec) exerts beneficial anti-inflammatory effects. Yet, its ability to alleviate allergic symptoms has not been investigated so far.Several experimental in vitro and in vivo models have been used in this basic research study. A murine ear swelling model was used to study the effects of PBMCsec on 48/80-induced mast cell degranulation in vivo. The transcriptional profile of murine mast cells was analysed by single cell RNA sequencing (scRNAseq). Mast cell activation was studied in vitro using primary skin mast cells. Basophils from individuals allergic to birch pollens were used to investigate basophile activation by allergens. Transcriptomic and lipidomic analyses were used to identify mRNA expression and lipid species present in PBMCsec, respectively.Topical application of PBMCsec on mouse ears (C57BL/6) significantly reduced tissue swelling following intradermal injection of compound 48/80, an inducer of mast cell degranulation. Single cell RNA sequencing of PBMCsec-treated murine dermal mast cells (Balb/c) revealed a downregulation of genes involved in immune cell degranulation and Fc-receptor signalling. In addition, treatment of primary human dermal mast cells with PBMCsec strongly inhibited compound 48/80- and α-IgE-induced mediator release in vitro. Furthermore, PBMCsec remarkably attenuated allergen driven activation of basophils from allergic individuals. Transcriptomic analysis of these basophils showed that PBMCsec downregulated a distinct gene battery involved in immune cell degranulation and Fc-receptor signalling, corroborating results obtained from dermal mast cells. Finally, we identified the lipid fraction of PBMCsec as the major active ingredient involved in effector cell inhibition.Collectively, our data demonstrate that PBMCsec is able to reduce activation of mast cells and basophils, encouraging further studies on the potential use of PBMCsec for treating allergy.Austrian Research Promotion Agency (852748 and 862068, 2015-2019), Vienna Business Agency (2343727, 2018-2020), Aposcience AG, Austrian Federal Ministry of Education, Science and Research (SPA06/055), Danube Allergy Research Cluster, Austrian Science Fund (I4437 and P32953).

ObjectivesBurn injuries elicit a unique and dynamic stress response which can lead to burn injury progression. Though neutrophils represent crucial players in the burn-induced immunological events, the dynamic secretion pattern and systemic levels of neutrophil-derived factors have not been investigated in detail so far.MethodsSerum levels of neutrophil elastase (NE), myeloperoxidase (MPO), citrullinated histone H3 (CitH3), and complement factor C3a were quantified in burn victims over 4 weeks post injury. Furthermore, the potential association with mortality, degree of burn injury, and inhalation trauma was evaluated. In addition, leukocyte, platelet, neutrophil, and lymphocyte counts were assessed. Lastly, we analyzed the association of neutrophil-derived factors with clinical severity scoring systems.ResultsSerum levels of NE, MPO, CitH3, and C3a were remarkably elevated in burn victims compared to healthy controls. Leukocyte and neutrophil counts were significantly increased on admission day and day 1, while relative lymphocytes were decreased in the first 7 days post burn trauma. Though neutrophil-derived factors did not predict mortality, patients suffering from 3rd degree burn injuries displayed increased CitH3 and NE levels. Accordingly, CitH3 and NE were elevated in cases with higher abbreviated burn severity indices (ABSI).ConclusionsTaken together, our data suggest a role for neutrophil activation and NETosis in burn injuries and burn injury progression. Targeting exacerbated neutrophil activation might represent a new therapeutic option for severe cases of burn injury.

Keloids are disfiguring, hypertrophic scars with yet poorly understood pathomechanisms, which could lead to severe functional impairments. Here we analyzed the characteristics of keloidal cells by single cell sequencing and discovered the presence of an abundant population of Schwann cells that persisted in the hypertrophic scar tissue after wound healing. In contrast to normal skin, keloidal Schwann cells possess a repair-like phenotype and high cellular plasticity. Our data support the hypothesis that keloidal Schwann cells contribute to the formation of the extracellular matrix and are able to affect M2 polarization of macrophages. Indeed, we show that macrophages in keloids predominantly display a M2 polarization and produce factors that inhibit Schwann cell differentiation. Our data suggest a contribution of this cross-talk to the continuous expansion of keloids, and that targeting Schwann cells might represent an interesting novel treatment option for keloids.

Background Tendon pathologies are common and several data suggests that the peripheral nervous system is involved in this disorder. Immunohistochemistry (IHC) is one of the pillars to characterize nervous structures and their implication in the pathogenesis of chronic tendon pain. Most commonly, formalin-fixed, paraffin-embedded (FFPE) tendons are used for immunohistochemical characterization of the innervation. However, FFPE specimens exhibit major disadvantages: First, antigens (proteins) are masked and antigen retrieval is necessary to restore antigenicity. Second, FFPE specimens involve immunolabeling with enzyme-conjugated antibodies but this approach has limitations when multiple antigens are of interest simultaneously. Consequently, there is a demand in the orthopedic community for an alternative immunohistochemical approach to visualize tendon innervations. Results Here, we present a guide how to visualize tendon innervation. This guide couples paraformaldehyde fixation, cryo-embedding, immunofluorescence, and confocal laser scanning microscopy. We demonstrate the utility of our approach in the long head of the biceps tendon. For nerve fiber characterization, we used different neuronal markers including antibodies against neurofilament, protein gene product 9.5, calcitonin gene related peptide, and substance P. We show that it is possible to collect high quality, multicolor images of the innervation pattern of tendons. To map immunolabeled structures and the anatomical structures of the tendon fluorescence images and bright field images were merged. Conclusion For the orthopedic community our approach might be a convenient research tool to simultaneously utilize multiple neuronal markers on the same tissue section and to define with greater accuracy the heterogeneity of tendon innervation.

Micro‐RNAs (miRNAs) are highly evolutionarily conserved short‐length/noncoding RNA molecules that modulate a wide range of cellular functions in many cell types by regulating the expression of a variety of targeted genes. miRNAs have also recently emerged as key regulators of neuronal genes mediating the effects of psychostimulant drugs and memory‐related neuroplasticity processes. Smoking is a predominant addictive behaviour associated with millions of deaths worldwide, and nicotine is a potent natural psychoactive agonist of cholinergic receptors, highly abundant in cigarettes. The influence of miRNAs modulation on cholinergic signalling in the nervous system remains however poorly explored. Using miRNA knockout mice and biochemical, electrophysiological and pharmacological approaches, we examined the effects of miR‐132/212 gene disruption on the levels of hippocampal nicotinic acetylcholine receptors, total ERK and phosphorylated ERK (pERK) and MeCP2 protein levels, and studied the impact of nicotine stimulation on hippocampal synaptic transmission and synaptic depression and strengthening. miR‐132/212 deletion significantly altered α7‐nAChR and pERK protein levels, but not total ERK or MeCP2, and resulted in both exacerbated synaptic depression and virtually abolished memory‐related synaptic strengthening upon nicotine stimulation. These observations reveal a functional miRNAs/nicotinergic signalling interplay critical for nicotinic‐receptor expression and neuroplasticity in brain structures relevant for drug addiction and learning and memory functions., Micro‐RNAs are key regulators of neuronal genes mediating the effects of psychostimulant drugs and neuroplasticity. Using miRNA knockout mice and biochemical, electrophysiological and pharmacological approaches, this work describes that miR‐132/212 gene deletion alters molecular elements from the cholinergic signalling pathway (α7‐nAChR and pERK protein levels) and not only exacerbates synaptic depression but also virtually abolishes memory‐related synaptic strengthening upon nicotine stimulation.

While the consumption of caffeine and cocoa has been associated with a variety of health benefits to humans, some authors have proposed that excessive caffeine intake may increase the frequency of epileptic seizures in humans and reduce the efficiency of antiepileptic drugs. Little is known, however, about the proconvulsant potential of the sustained, excessive intake of cocoa on hippocampal neural circuits. Using the mouse as an experimental model, we examined the effects of the chronic consumption of food enriched in cocoa-based dark chocolate on motor and mood-related behaviours as well as on the excitability properties of hippocampal neurons. Cocoa food enrichment did not affect body weights or mood-related behaviours but rather promoted general locomotion and improved motor coordination. However, ex vivo electrophysiological analysis revealed a significant enhancement in seizure-like population spike bursting at the neurogenic dentate gyrus, which was paralleled by a significant reduction in the levels of GABA-α1 receptors thus suggesting that an excessive dietary intake of cocoa-enriched food might alter some of the synaptic elements involved in epileptogenesis. These data invite further multidisciplinary research aiming to elucidate the potential deleterious effects of chocolate abuse on behaviour and brain hyperexcitability.

The innervation of the long head of the biceps tendon (LHBT) is not sufficiently documented. This is a drawback since pathologies of the LHBT are a major source of shoulder pain. Thus, the study aimed to characterize structurally and molecularly nervous elements of the LHBT. The proximal part of 11 LHBTs was harvested intraoperatively. There were 8 female and 3 male specimens. Age ranged from 66 to 86 years. For structural analyses, nervous elements were viewed in the transmission electron microscope. For molecular characterization, we used general neuronal markers including antibodies against neurofilament and protein gene product 9.5 (PGP9.5) as well as specific neuronal markers including antibodies against myelin basic protein (MBP), calcitonin gene-related product (CGRP), substance P (SP), tyrosine hydroxylase (TH), and growth-associated protein 43 (GAP43). Anti-neurofilament and anti-PGP9.5 visualized the overall innervation. Anti-MBP visualized myelination, anti-CGRP and anti-SP nociceptive fibers, anti-TH sympathetic nerve fibers, and anti-GAP43 nerve fibers during development and regeneration. Immunolabeled sections were analyzed in the confocal laser scanning microscope. We show that the LHBT contains unmyelinated as well as myelinated nerve fibers which group in nerve fascicles and follow blood vessels. Manny myelinated and unmyelinated axons exhibit molecular features of nociceptive nerve fibers. Another subpopulation of unmyelinated axons exhibits molecular characteristics of sympathetic nerve fibers. Unmyelinated sympathetic fibers and unmyelinated nociceptive fibers express proteins that are found during development and regeneration. Present findings support the hypothesis that ingrowth of nociceptive fibers are the source of chronic tendon pain.

BACKGROUND: Viral reduction and inactivation of cell-derived biologicals is paramount for patients’ safety and so viral reduction needs to be demonstrated to regulatory bodies in order to obtain marketing authorisation. Allogeneic human blood-derived biological medicinal products require special attention. APOSEC(TM), the secretome harvested from selected human blood cells, is a new biological with promising regenerative capabilities. We evaluated the effectiveness of inactivation of model viruses by methylene blue/light treatment, lyophilisation, and gamma irradiation during the manufacturing process of APOSEC(TM). MATERIALS AND METHODS: Samples of intermediates of APOSEC(TM) were acquired during the manufacturing process and spiked with bovine viral diarrhoea virus (BVDV), human immunodeficiency virus type 1 (HIV-1), pseudorabies virus (PRV), hepatitis A virus (HAV), and porcine parvovirus (PPV). Viral titres were assessed with suitable cell lines. RESULTS: Methylene blue-assisted viral reduction is mainly effective against enveloped viruses: the minimum log(10) reduction factors for BVDV, HIV-1, and PRV were ≥6.42, ≥6.88, and ≥6.18, respectively, with no observed residual infectivity. Viral titres of both HAV and PPV were not significantly reduced, indicating minor inactivation of non-enveloped viruses. Lyophilisation had minor effects on the viability of several enveloped model viruses. Gamma irradiation contributes to the viral safety by reduction of enveloped viruses (BVDV: ≥2.42; HIV-1: 4.53; PRV: ≥4.61) and to some degree of non-enveloped viruses as seen for HAV with a minimum log10 reduction factor of 2.92. No significant reduction could be measured for the non-enveloped virus PPV (2.60). DISCUSSION: Three manufacturing steps of APOSEC(TM) were evaluated under Good Laboratory Practice conditions for their efficacy at reducing and inactivating potentially present viruses. It could be demonstrated that all three steps contribute to the viral safety of APOSEC(TM).

Past research on video game effects was often limited to explaining effects of game content and mode, leaving structural and contextual game elements scarcely investigated. The present research examined the yet unclear role of narration in video games, by adapting concepts and methodology from video game research based on self-determination theory as well as past research on the effects of literary fiction. Results provided evidence for the facilitation of immersion and an immersion-mediated enhancement of autonomy and relatedness need satisfaction through in-game storytelling, suggesting a mutual enhancement of immersion and need satisfaction. Moreover, in-game storytelling enhanced affective theory of mind. Perspectives on future research, connecting in-game storytelling and game content to complement current knowledge of video game effects on various real-world outcomes, are discussed.