Tumour immune microenvironment in resected thymic carcinomas as a predictor of clinical outcome.

BACKGROUND: The spatial distribution of tumour-infiltrating lymphocytes (TILs) is a novel descriptor characterising the tumour immune microenvironment (TIME). The aim of our study was to assess whether a specific TIME of surgically resected thymic carcinoma (TC) can predict tumour invasiveness, recurrence or survival. METHODS: Digital microscopy was performed on 39 TCs immunohistochemically stained to investigate the activation of the immune checkpoint pathway (PD-L1/PD-1), along with density and spatial distribution of TILs phenotypes (CD3+, CD4+, CD8+, FOXP3+, CD56+). The impact of PD-L1 and TIL density considering the intratumoural (iTILs) and stromal (sTILs) distribution on pathological characteristics and clinical outcomes were analysed. RESULTS: In early TC stages, we observed a higher total density of CD3+ (p = 0.05) and CD8+ (p = 0.02) TILs. PD-L1 was expressed in 71.8% of TCs. In advanced TC stages, we observed a lower density of CD3+ (p = 0.04) and CD8+ (p = 0.01) iTILs compared to early stages. Serum concentrations of PD-L1 were significantly higher in TCs compared to healthy controls: 134.43 ± 18.51 vs. 82.01 ± 6.34 pg/ml (p = 0.001), respectively. High densities of stromal CD4+ TILs (54 vs. 32%, p = 0.043) and CD8+ TILs (65 vs. 17%, p = 0.048) were associated with improved freedom from recurrence (FFR) and cause-specific survival (CSS). High density of FoxP3+ TILs were associated with improved FFR (p = 0.03) and CSS (p = 0.003). DISCUSSION: Mapping TIL subpopulations complement the armamentarium for prognostication of TC outcomes. The improved outcome in patients with high density of TILs supports the use of immune checkpoint inhibitors in TC patients.

Mechanical aortic valve prostheses offer a survival benefit in 50-65 year olds: AUTHEARTVISIT study.

BACKGROUND: The present population-based cohort study investigated long-term mortality after surgical aortic valve replacement (AVR) with bioprosthetic (B) or mechanical aortic valve prostheses (M) in a European social welfare state. METHODS: We analysed patient data from health insurance records covering 98% of the Austrian population between 2010 and 2018. Subsequent patient-level record linkage with national health data provided patient characteristics and clinical outcomes. Further reoperation, myocardial infarction, heart failure and stroke were evaluated as secondary outcomes. RESULTS: A total of 13,993 patients were analysed and the following age groups were examined separately: <50 years (727 patients: 57.77% M, 42.23% B), 50-65 years (2612 patients: 26.88% M, 73.12% B) and >65 years (10,654 patients: 1.26% M, 98.74% B). Multivariable Cox regression revealed that the use of B-AVR was significantly associated with higher mortality in patients aged 50-65 years compared to M-AVR (HR = 1.676 [1.289-2.181], p < 0.001). B-AVR also performed worse in a competing risk analysis regarding reoperation (HR = 3.483 [1.445-8.396], p = 0.005) and myocardial infarction (HR = 2.868 [1.255-6.555], p = 0.012). However, the risk of developing heart failure and stroke did not differ significantly after AVR in any age group. CONCLUSIONS: Patients aged 50-65 years who underwent M-AVR had better long-term survival, and a lower risk of reoperation and myocardial infarction. Even though anticoagulation is crucial in patients with M-AVR, we did not observe significantly increased stroke rates in patients with M-AVR. This evident survival benefit in recipients of mechanical aortic valve prostheses aged <65 years critically questions current guideline recommendations.

Deciphering the functional heterogeneity of skin fibroblasts using single-cell RNA sequencing.

Though skin fibroblasts (FB) are the main cell population within the dermis, the different skin FB subsets are not well characterized and the traditional classification into reticular and papillary FBs has little functional relevance. To fill the gap of knowledge on FB diversity in human skin, we performed single-cell RNA sequencing. Investigation of marker genes for the different skin cell subtypes revealed a heterogeneous picture of FBs. When mapping reticular and papillary FB markers, we could not detect cluster specificity, suggesting that these two populations show a higher transcriptional heterogeneity than expected. This finding was further confirmed by in situ hybridization, showing that DPP4 was expressed in both dermal layers. Our analysis identified six FB clusters with distinct transcriptional signatures. Importantly, we could demonstrate that in human skin DPP4+ FBs are the main producers of factors involved in extracellular matrix (ECM) assembly. In conclusion, we provide evidence that hitherto considered FB markers are not ideal to characterize skin FB subpopulations in single-cell sequencing analyses. The identification of DPP4+ FBs as the main ECM-producing cells in human skin will foster the development of anti-fibrotic treatments for the skin and other organs.

Transcriptional Differences in Lipid-Metabolizing Enzymes in Murine Sebocytes Derived from Sebaceous Glands of the Skin and Preputial Glands.

Sebaceous glands are adnexal structures, which critically contribute to skin homeostasis and the establishment of a functional epidermal barrier. Sebocytes, the main cell population found within the sebaceous glands, are highly specialized lipid-producing cells. Sebaceous gland-resembling tissue structures are also found in male rodents in the form of preputial glands. Similar to sebaceous glands, they are composed of lipid-specialized sebocytes. Due to a lack of adequate organ culture models for skin sebaceous glands and the fact that preputial glands are much larger and easier to handle, previous studies used preputial glands as a model for skin sebaceous glands. Here, we compared both types of sebocytes, using a single-cell RNA sequencing approach, to unravel potential similarities and differences between the two sebocyte populations. In spite of common gene expression patterns due to general lipid-producing properties, we found significant differences in the expression levels of genes encoding enzymes involved in the biogenesis of specialized lipid classes. Specifically, genes critically involved in the mevalonate pathway, including squalene synthase, as well as the sphingolipid salvage pathway, such as ceramide synthase, (acid) sphingomyelinase or acid and alkaline ceramidases, were significantly less expressed by preputial gland sebocytes. Together, our data revealed tissue-specific sebocyte populations, indicating major developmental, functional as well as biosynthetic differences between both glands. The use of preputial glands as a surrogate model to study skin sebaceous glands is therefore limited, and major differences between both glands need to be carefully considered before planning an experiment.

TGF-ß in the Secretome of Irradiated Peripheral Blood Mononuclear Cells Supports In Vitro Osteoclastogenesis.

Osteoclastogenesis required for bone remodeling is also a key pathologic mechanism of inflammatory osteolysis being controlled by paracrine factors released from dying cells. The secretome of irradiated, dying peripheral blood mononuclear cells (PBMCs) has a major impact on the differentiation of myeloid cells into dendritic cells, and macrophage polarization. The impact on osteoclastogenesis, however, has not been reported. For this aim, we used murine bone marrow macrophages exposed to RANKL and M-CSF to initiate osteoclastogenesis, with and without the secretome obtained from γ-irradiated PBMCs. We reported that the secretome significantly enhanced in vitro osteoclastogenesis as determined by means of histochemical staining of the tartrate-resistant acid phosphatase (TRAP), as well as the expression of the respective target genes, including TRAP and cathepsin K. Considering that TGF-ß enhanced osteoclastogenesis, we confirmed the TGF-ß activity in the secretome with a bioassay that was based on the increased expression of IL11 in fibroblasts. Neutralizing TGF-ß by an antibody decreased the ability of the secretome to support osteoclastogenesis. These findings suggested that TGF-ß released by irradiated PBMCs could enhance the process of osteoclastogenesis in vitro.

Role for Lipids Secreted by Irradiated Peripheral Blood Mononuclear Cells in Inflammatory Resolution in Vitro.

Periodontal inflammation is associated with dying cells that potentially release metabolites helping to promote inflammatory resolution. We had shown earlier that the secretome of irradiated, dying peripheral blood mononuclear cells support in vitro angiogenesis. However, the ability of the secretome to promote inflammatory resolution remains unknown. Here, we determined the expression changes of inflammatory cytokines in murine bone marrow macrophages, RAW264.7 cells, and gingival fibroblasts exposed to the secretome obtained from γ-irradiated peripheral blood mononuclear cells in vitro by RT-PCR and immunoassays. Nuclear translocation of p65 was detected by immunofluorescence staining. Phosphorylation of p65 and degradation of IκB was determined by Western blot. The secretome of irradiated peripheral blood mononuclear cells significantly decreased the expression of IL1 and IL6 in primary macrophages and RAW264.7 cells when exposed to LPS or saliva, and of IL1, IL6, and IL8 in gingival fibroblasts when exposed to IL-1ß and TNFα. These changes were associated with decreased phosphorylation and nuclear translocation of p65 but not degradation of IκB in macrophages. We also show that the lipid fraction of the secretome lowered the inflammatory response of macrophages exposed to the inflammatory cues. These results demonstrate that the secretome of irradiated peripheral blood mononuclear cells can lower an in vitro simulated inflammatory response, supporting the overall concept that the secretome of dying cells promotes inflammatory resolution.

miR-155 Contributes to Normal Keratinocyte Differentiation and Is Upregulated in the Epidermis of Psoriatic Skin Lesions.

The role of microRNAs (miRNAs) during keratinocyte (KC) differentiation and in skin diseases with epidermal phenotypes has attracted strong interest over the past few years. However, combined mRNA and miRNA expression analyses to elucidate the intricate mRNA-miRNA networks of KCs at different stages of differentiation have not been performed yet. In the present study, we investigated the dynamics of miRNA and mRNA expression during KC differentiation in vitro and in normal and psoriatic epidermis. While we identified comparable numbers of up- and downregulated mRNAs (49% and 51%, respectively), miRNAs were predominantly upregulated (76% vs 24%) during KC differentiation. Further bioinformatics analyses suggested an important inhibitory role for miR-155 in KC differentiation, as it was repressed during KC differentiation in normal skin but strongly upregulated in the epidermis of psoriatic skin lesions. Mimicking the inflammatory milieu of psoriatic skin in vitro, we could show that the pro-inflammatory cytokines IL17, IL1ß and INFγ synergistically upregulated miR-155 expression in KCs. Forced over-expression of miR-155 in human in vitro skin models specifically reduced the expression of loricrin (LOR) in KCs, indicating that miR-155 interferes with the establishment of a normal epidermal barrier. Together, our data indicate that downregulation of miR-155 during KC differentiation is a crucial step for epidermal barrier formation. Furthermore, its strong upregulation in psoriatic lesions suggests a contributing role of miR-155 in the altered keratinocyte differentiation observed in psoriasis. Therefore, miR-155 represents as a potential target for treating psoriatic skin lesions.

Antithymocyte Globulin Inhibits CD8+ T Cell Effector Functions via the Paracrine Induction of PDL-1 on Monocytes.

BACKGROUND: Antithymocyte globulins (ATG) are T cell-depleting antibodies used in solid organ transplantation for induction therapy in sensitized patients with a high risk of graft rejection. Previously described effects besides the depletion of T cells have suggested additional modes of action and identified further cellular targets. METHODS: We examined the transcriptional changes arising in immune cells from human blood after ex vivo stimulation with ATG at the single-cell level to uncover additional mechanisms by which ATG regulates T cell activity and effector functions. FINDINGS: Analysis of the paracrine factors present in the plasma of ATG-treated whole blood revealed high levels of chemokines and cytokines, including interferon-γ (IFN-γ). Furthermore, we identified an increase in the surface expression of the programmed death ligand 1 (PDL-1) on monocytes mediated by the released paracrine factors. In addition, we showed that this induction is dependent on the activation of JAK/STAT signaling via the binding of IFN-γ to interferon-γ receptor 1 (IFN-γR1). Lastly, we demonstrated 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 the proliferation and granzyme B release of activated T cells. INTERPRETATION: Together, our findings represent a novel mode of action by which ATG exerts its immunosuppressive effects.

Identification of Treatment Protocols for Effective Cross-Linking of the Peripheral Cornea: An Experimental Study.

INTRODUCTION: This study aimed to test and evaluate modified corneal cross-linking (CXL) protocols regarding improved treatment effects on the peripheral cornea in terms of tissue stability and cellular response. METHODS: Peripheral CXL (pCXL) was performed within a ring of 9-11 mm of 36 human donor corneas with variations in applied energy (5.4, 7.2, and 10 J/cm2) at 9 mW/cm2 irradiance. Each energy level was additionally modulated regarding the oxygen level surrounding the cornea during treatment (21%; 100%). Stress-strain tests with endpoints at 12% strain and collagenase A-assisted digestions to complete digestion were performed to evaluate the rigidity and resistance of treated and control tissue. Further, corneas were processed histologically via TUNEL assay and H&E staining to demonstrate the effects on stromal cells during treatment under varying CXL conditions. RESULTS: Increases in energy dosage achieved significant increases in resistance to stress in all variations except when comparing protocols A and B under normoxic conditions. Supplemental oxygen significantly increased rigidity in protocols B (p < 0.01) and C (p = 0.018). Hyperoxic conditions significantly increased resistance to digestion in all protocols. The number of DNA strand breaks in TUNEL assay staining showed significant increases in all increases in energy as well as with oxygen supplementation. CONCLUSIONS: Increases in energy and supplemental oxygen improved the effect of CXL, though endothelial safety could not be verified with confidence in high-fluence CXL with supplemental oxygen. Results suggest that CXL protocols using 7.2 J/cm2 with 100% O2 or 10 J/cm2 without supplemental oxygen prove most effective without anticipated risk of endothelial damage.

The transcriptional profile of keloidal Schwann cells.

Recently, a specific Schwann cell type with profibrotic and tissue regenerative properties that contributes to keloid formation has been identified. In the present study, we reanalyzed published single-cell RNA sequencing (scRNA-seq) studies of keloids, healthy skin, and normal scars to reliably determine the specific gene expression profile of keloid-specific Schwann cell types in more detail. We were able to confirm the presence of the repair-like, profibrotic 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 not increased, nor was their gene expression profile distinctly different from that of Schwann cells of normal skin. In addition, our bioinformatics analysis provided evidence for a role of transcription factors of the AP1, STAT, and KLF families, and members of the IER genes in the dedifferentiation process of keloidal Schwann cells. Together, our analysis strengthens the role of the profibrotic Schwann cell type in the formation of keloids. Knowledge of the exact gene expression profile of these Schwann cells will facilitate their identification in other organs and diseases.

Paracrine Factors of Stressed Peripheral Blood Mononuclear Cells Activate Proangiogenic and Anti-Proteolytic Processes in Whole Blood Cells and Protect the Endothelial Barrier.

Tissue-regenerative properties have been attributed to secreted paracrine factors derived from stem cells and other cell types. In particular, the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCsec) has been shown to possess high tissue-regenerative and proangiogenic capacities in a variety of preclinical studies. In light of future therapeutic intravenous applications of PBMCsec, we investigated the possible effects of PBMCsec on white blood cells and endothelial cells lining the vasculature. To identify changes in the transcriptional profile, whole blood was drawn from healthy individuals and stimulated with PBMCsec for 8 h ex vivo before further processing for single-cell RNA sequencing. PBMCsec significantly altered the gene signature of granulocytes (17 genes), T-cells (45 genes), B-cells (72 genes), and, most prominently, monocytes (322 genes). We detected a strong upregulation of several tissue-regenerative and proangiogenic cyto- and chemokines in monocytes, including VEGFA, CXCL1, and CXCL5. Intriguingly, inhibitors of endopeptidase activity, such as SERPINB2, were also strongly induced. Measurement of the trans-endothelial electrical resistance of primary human microvascular endothelial cells revealed a strong barrier-protective effect of PBMCsec after barrier disruption. Together, we show that PBMCsec induces angiogenic and proteolytic processes in the blood and is able to attenuate endothelial barrier damage. These regenerative properties suggest that systemic application of PBMCsec might be a promising novel strategy to restore damaged organs.

EGR1 Is Implicated in Right Ventricular Cardiac Remodeling Associated with Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a vasoconstrictive disease characterized by elevated mean pulmonary arterial pressure (mPAP) at rest. Idiopathic pulmonary arterial hypertension (iPAH) and chronic thromboembolic pulmonary hypertension (CTEPH) represent two distinct subtypes of PH. Persisting PH leads to right ventricular (RV) hypertrophy, heart failure, and death. RV performance predicts survival and surgical interventions re-establishing physiological mPAP reverse cardiac remodeling. Nonetheless, a considerable number of PH patients are deemed inoperable. The underlying mechanism(s) governing cardiac regeneration, however, remain largely elusive. METHODS: In a longitudinal approach, we profiled the transcriptional landscapes of hypertrophic RVs and recovered hearts 3 months after surgery of iPAH and CTEPH patients. RESULTS: Genes associated with cellular responses to inflammatory stimuli and metal ions were downregulated, and cardiac muscle tissue development was induced in iPAH after recovery. In CTEPH patients, genes related to muscle cell development were decreased, and genes governing cardiac conduction were upregulated in RVs following regeneration. Intriguingly, early growth response 1 (EGR1), a profibrotic regulator, was identified as a major transcription factor of hypertrophic RVs in iPAH and CTEPH. A histological assessment confirmed our biocomputational results, and suggested a pivotal role for EGR1 in RV vasculopathy. CONCLUSION: Our findings improved our understanding of the molecular events driving reverse cardiac remodeling following surgery. EGR1 might represent a promising candidate for targeted therapy of PH patients not eligible for surgical treatment.

The secretome of irradiated peripheral blood mononuclear cells attenuates activation of mast cells and basophils.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: 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).

The Roles of S100A4 and the EGF/EGFR Signaling Axis in Pulmonary Hypertension with Right Ventricular Hypertrophy.

Pulmonary hypertension (PH) is characterized by increased pulmonary arterial pressure caused by the accumulation of mesenchymal-like cells in the pulmonary vasculature. PH can lead to right ventricular hypertrophy (RVH) and, ultimately, heart failure and death. In PH etiology, endothelial-to-mesenchymal transition (EndMT) has emerged as a critical process governing the conversion of endothelial cells into mesenchymal cells, and S100A4, EGF, and EGFR are implicated in EndMT. However, a potential role of S100A4, EGF, and EGFR in PH has to date not been elucidated. We therefore quantified S100A4, EGF, and EGFR in patients suffering from chronic thromboembolic pulmonary hypertension (CTEPH) and idiopathic pulmonary arterial hypertension (iPAH). To determine specificity for unilateral heart disease, the EndMT biomarker signature was further compared between PH patients presenting with RVH and patients suffering from aortic valve stenosis (AVS) with left ventricular hypertrophy. Reduced S100A4 concentrations were found in CTEPH and iPAH patients with RVH. Systemic EGF was increased in CTEPH but not in iPAH, while AVS patients displayed slightly diminished EGF levels. EGFR was downregulated in all patient groups when compared to healthy controls. Longitudinal data analysis revealed no effect of surgical therapies on EndMT markers. Pulmonary thrombo-endarterectomized samples were devoid of S100A4, while S100A4 tissue expression positively correlated with higher grades of Heath-Edwards histopathological lesions of iPAH-derived lung tissue. Histologically, EGFR was not detectable in CTEPH lungs or in iPAH lesions. Together, our data suggest an intricate role for S100A4 and EGF/EGFR in PH with right heart pathology.

Secretome of Stressed Peripheral Blood Mononuclear Cells Alters Transcriptome Signature in Heart, Liver, and Spleen after an Experimental Acute Myocardial Infarction: An In Silico Analysis.

Acute myocardial infarction (AMI) is a result of cardiac non-perfusion and leads to cardiomyocyte necrosis, inflammation, and compromised cardiac performance. Here, we showed that the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCsec) improved heart function in a porcine AMI model and displayed beneficial long- and short-term effects. As an AMI is known to strongly affect gene regulation of the ischemia non-affected heart muscle and distal organs, we employed a transcriptomics approach to further study the immediate molecular events orchestrated using the PBMCsec in myocardium, liver, and spleen 24 h post ischemia. In the infarcted area, the PBMCsec mainly induced genes that were essential for cardiomyocyte function and simultaneously downregulated pro-inflammatory genes. Interestingly, genes associated with pro-inflammatory processes were activated in the transition zone, while being downregulated in the remote zone. In the liver, we observed a pronounced inhibition of immune responses using the PBMCsec, while genes involved in urea and tricarboxylic cycles were induced. The spleen displayed elevated lipid metabolism and reduced immunological processes. Together, our study suggested several types of pharmacodynamics by which the PBMCsec conferred immediate cardioprotection. Furthermore, our data supported the assumption that an AMI significantly affects distal organs, suggesting that a holistic treatment of an AMI, as achieved by PBMCsec, might be highly beneficial.

Severity of thermal burn injury is associated with systemic neutrophil activation.

Burn 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. Serum 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. Serum 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). Taken 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.

The Effect of Paracrine Factors Released by Irradiated Peripheral Blood Mononuclear Cells on Neutrophil Extracellular Trap Formation.

Neutrophil extracellular trap (NET)-formation represents an important defence mechanism for the rapid clearance of infections. However, exaggerated NET formation has been shown to negatively affect tissue-regeneration after injury. As our previous studies revealed the strong tissue-protective and regenerative properties of the secretome of stressed peripheral blood mononuclear cells (PBMCsec), we here investigated the influence of PBMCsec on the formation of NETs. The effect of PBMCsec on NET formation was assessed ex vivo in ionomycin stimulated neutrophils derived from healthy donors using flow cytometry, image stream analysis, and quantification of released extracellular DNA. The effect of PBMCsec on molecular mechanisms involved in NET formation, including Ca-flux, protein kinase C activity, reactive oxygen species production, and protein arginine deiminase 4 activity, were analysed. Our results showed that PBMCsec significantly inhibited NET formation. Investigation of the different biological substance classes found in PBMCsec revealed only a partial reduction in NET formation, suggesting a synergistic effect. Mechanistically, PBMCsec treatment did not interfere with calcium signalling and PKC-activation, but exerted anti-oxidant activity, as evidenced by reduced levels of reactive oxygen species and upregulation of heme oxygenase 1 and hypoxia inducible-factor 1 in PBMCsec-treated neutrophils. In addition, PBMCsec strongly inhibited the activation of protein arginine deiminase 4 (PAD4), ultimately leading to the inhibition of NET formation. As therapeutics antagonizing excessive NET formation are not currently available, our study provides a promising novel treatment option for a variety of conditions resulting from exaggerated NET formation.

Schwann cells contribute to keloid formation.

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.

Experimental Models for the Study of Hereditary Cornification Defects.

Ichthyoses comprise a broad spectrum of keratinization disorders due to hereditary defects of cornification. Until now, mutations in more than 50 genes, mostly coding for structural proteins involved in epidermal barrier formation, have been identified as causes for different types of these keratinization disorders. However, due to the high heterogeneity and difficulties in the establishment of valid experimental models, research in this field remains challenging and translation of novel findings to clinical practice is difficult. In this review, we provide an overview of existing models to study hereditary cornification defects with focus on ichthyoses and palmoplantar keratodermas.

Inflammatory immune response in recipients of transcatheter aortic valves.

Objective: Transcatheter aortic valve implantation (TAVI) is rapidly replacing cardiac surgery due to its minimal invasiveness and practicality. Midterm immunological studies on the biocompatibility of galactose-alpha-1,3-galactose (α-Gal)-carrying bioprosthetic heart valves for TAVI are not available. In this study we investigated whether bioprosthetic heart valves employed for TAVI augment an α-Gal-specific antibody-dependent and antibody-independent immune response 3 months after TAVI implantation. Methods: This prospective observational study included 27 patients with severe aortic valve stenosis undergoing TAVI and 10 patients with severe mitral valve regurgitation treated with a transcatheter MitraClip (Abbott Laboratories, Abbott Park, Ill) procedure. Blood samples were drawn before and 90 days after treatment at a routine checkup. Serum samples were analyzed using enzyme-linked immunosorbent assay. Serum concentrations of α-Gal-specific immunoglobulin (Ig) G, IgG subclasses and IgE, complement factor 3a, NETosis-specific citrullinated H3, and the systemic inflammation markers soluble suppression of tumorigenicity and interleukin 33 were evaluated. Results: Three months after TAVI, we found significantly increased serum concentrations of α-Gal-specific IgG3, complement factor complement factor 3a, citrullinated H3 levels, and soluble suppression of tumorigenicity (P = .002, P = .001, P = .025, and P = .039, respectively). Sensitization of α-Gal-specific IgE antibodies occurred in 55% of all patients after TAVI. Conclusions: Our results indicate that TAVI elicits a midterm, specific humoral immune response against α-Gal and causes an unspecific humoral inflammation compared with patients undergoing MitraClip implantation. This observation will lead to a better understanding of postintervention morbidity and the long-term durability of bioprostheses and indicates that caution is appropriate when designing implantation strategies for younger patients.