Comparison of the diagnostic accuracy of the Pluslife Mini Dock RHAM technology with Abbott ID Now and Cepheid GenXpert: A retrospective evaluation study.

Rapid and sensitive detection of pathogens is critical in interrupting the transmission chain of infectious diseases. Currently, real-time (RT-)PCR represents the gold standard for the detection of SARS-CoV-2. RNase HII-assisted amplification (RHAM) is a promising technology, enabling reliable point-of-care (PoC) testing; however, its diagnostic accuracy has not yet been investigated. The present study compared the Pluslife Mini Dock (RHAM technology), with Abbott ID Now and Cepheid GeneXpert IV. The positive percent agreement (PPA) and negative percent agreement (NPA) were determined in 100 SARS-CoV-2 positive and 210 SARS-CoV-2 negative samples. Further, the reliability of the Pluslife Mini Dock was investigated in different SARS-CoV-2 variants (Delta and Omicron subvariants). The PPA was 99.00% for Pluslife, 100.00% for Abbott ID Now, and 99.00% for Cepheid GeneXpert, with an NPA of 100.00%, 98.90%, and 93.72%, respectively. Abbott ID Now demonstrated the highest rate of invalid results. All SARS-CoV-2 analysed variants were detected by the Pluslife device. Altogether, the Pluslife Mini Dock demonstrated a PPA of 99.16% (235/237) for CT < 36 and an NPA of 100.00% (313/313), respectively. In conclusion, the Pluslife Mini Dock demonstrated better analytical performance than Abbott ID Now and Cepheid GeneXpert IV, representing a highly accurate and rapid PoC alternative to RT-PCR.

The Role of NOX2-Derived Reactive Oxygen Species in the Induction of Endothelin-Converting Enzyme-1 by Angiotensin II.

Endothelin-1 is a key regulator of vascular tone and blood pressure in health and disease. We have recently found that ET-1 production in human microvascular endothelial cells (HMECs) can be promoted by angiotensin II (Ang II) through a novel mechanism involving octamer-binding transcription factor-1 (Oct-1), NADPH oxidase-2 (NOX2), and superoxide anions. As the formation of bioactive ET-1 also depends on endothelin-converting enzyme-1 (ECE-1), we investigated the transcriptional regulation of the ECE1 gene. We found that exposure of HMECs to Ang II resulted in a concentration- and time-dependent increase in ECE1 mRNA expression. Pharmacological inhibition of ECE-1 reduced Ang II-stimulated ET-1 release to baseline values. The effect of Ang II on ECE1 mRNA expression was associated with Oct-1 binding to the ECE1 promoter, resulting in its increased activity. Consequently, the Ang II-stimulated increase in ECE1 mRNA expression could be prevented by siRNA-mediated Oct-1 inhibition. It could also be abolished by silencing the NOX2 gene and neutralizing superoxide anions with superoxide dismutase. In mice fed a high-fat diet, cardiac expression of Ece1 mRNA increased in wild-type mice but not in Nox2-deficient animals. It can be concluded that Ang II engages Oct-1, NOX2, and superoxide anions to stimulate ECE1 expression in the endothelium.

Expanded Hemodialysis ameliorates uremia-induced impairment of vasculoprotective KLF2 and concomitant proinflammatory priming of endothelial cells through an ERK/AP1/cFOS-dependent mechanism.

Aims: Expanded hemodialysis (HDx) therapy with improved molecular cut-off dialyzers exerts beneficial effects on lowering uremia-associated chronic systemic microinflammation, a driver of endothelial dysfunction and cardiovascular disease (CVD) in hemodialysis (HD) patients with end-stage renal disease (ESRD). However, studies on the underlying molecular mechanisms are still at an early stage. Here, we identify the (endothelial) transcription factor Krüppel-like factor 2 (KLF2) and its associated molecular signalling pathways as key targets and regulators of uremia-induced endothelial micro-inflammation in the HD/ESRD setting, which is crucial for vascular homeostasis and controlling detrimental vascular inflammation. Methods and results: First, we found that human microvascular endothelial cells (HMECs) and other typical endothelial and kidney model cell lines (e.g. HUVECs, HREC, and HEK) exposed to uremic serum from patients treated with two different hemodialysis regimens in the Permeability Enhancement to Reduce Chronic Inflammation II (PERCI-II) crossover clinical trial - comparing High-Flux (HF) and Medium Cut-Off (MCO) membranes - exhibited strongly reduced expression of vasculoprotective KLF2 with HF dialyzers, while dialysis with MCO dialyzers led to the maintenance and restoration of physiological KLF2 levels in HMECs. Mechanistic follow-up revealed that the strong downmodulation of KLF2 in HMECs exposed to uremic serum was mediated by a dominant engagement of detrimental ERK instead of beneficial AKT signalling, with subsequent AP1-/c-FOS binding in the KLF2 promoter region, followed by the detrimental triggering of pleiotropic inflammatory mediators, while the introduction of a KLF2 overexpression plasmid could restore physiological KLF2 levels and downmodulate the detrimental vascular inflammation in a mechanistic rescue approach. Conclusion: Uremia downmodulates vasculoprotective KLF2 in endothelium, leading to detrimental vascular inflammation, while MCO dialysis with the novel improved HDx therapy approach can maintain physiological levels of vasculoprotective KLF2.

An Efficient and Scalable Method for the Production of Immunogenic SARS-CoV-2 Virus-like Particles (VLP) from a Mammalian Suspension Cell Line.

The rapid evolution of new SARS-CoV-2 variants poses a continuing threat to human health. Vaccination has become the primary therapeutic intervention. The goal of the current work was the construction of immunogenic virus-like particles (VLPs). Here, we describe a human cell line for cost-efficient and scalable production of immunogenic SARS-CoV-2 VLPs. The modular design of the VLP-production platform facilitates rapid adaptation to new variants. Methods: The N, M-, and E-protein genes were integrated into the genome of Expi293 cells (ExpiVLP_MEN). Subsequently, this cell line was further modified for the constitutive expression of the SARS-CoV-2 spike protein. The resulting cell line (ExpiVLP_SMEN) released SARS-CoV-2 VLP upon exposure to doxycycline. ExpiVLP_SMEN cells were readily adapted for VLP production in a 5 L bioreactor. Purified VLPs were quantified by Western blot, ELISA, and nanoparticle tracking analysis and visualized by electron microscopy. Immunogenicity was tested in mice. Results: The generated VLPs contained all four structural proteins, are within the size range of authentic SARS-CoV-2 virus particles, and reacted strongly and specifically with immunoserum from naturally infected individuals. The VLPs were stable in suspension at 4 °C for at least 10 weeks. Mice immunized with VLPs developed neutralizing antibodies against lentiviruses pseudotyped with the SARS-CoV-2 spike protein. The flexibility of the VLP-production platform was demonstrated by the rapid switch of the spike protein to a new variant of concern (BA.1/Omicron). The present study describes an efficient, scalable, and adaptable production method of immunogenic SARS-CoV-2 VLPs with therapeutic potential.

Ang II Promotes ET-1 Production by Regulating NOX2 Activity Through Transcription Factor Oct-1.

BACKGROUND: Increasing evidence suggests that superoxide ions produced by NOX (nicotinamide adenine dinucleotide phosphate oxidases) mediate vascular effects of Ang II (angiotensin II) evoked by atherogenic diets. Here, we analyzed the mechanism by which NOX2 contributes to Ang II-induced ET-1 (endothelin 1) production in human microvascular endothelial cells. METHODS: The effects of high-fat diet were compared between WT (wild type) and Nox2 (mouse NOX2 gene)-deficient mice. ET-1 production and NOX2 expression by human microvascular endothelial cells in vitro were analyzed by ELISA, reverse transcription quantitative polymerase chain reaction, electrophoretic mobility shift assay, promoter deletions, RNA interference, and pharmacological inhibition. Production of superoxide anions was visualized by fluorescent cell labeling. RESULTS: Feeding mice high-fat diet for 10 weeks increased cardiac expression and plasma levels of Ang II and ET-1 in WT but not in Nox2-deficient animals. Exposure of human microvascular endothelial cells to Ang II resulted in increased ET-1 production, which could be blocked by silencing NOX2 (human NOX2 gene). Ang II promoted NOX2 expression through induction of the Oct-1 (human/mouse octamer binding transcription factor 1 protein) and activation of the NOX2 promoter region containing Oct-1-binding sites. Stimulation of NOX2 expression by Ang II was associated with increased production of superoxide anions. Inhibition of Oct-1 by small interfering RNA reduced Ang II-induced NOX2 expression and superoxide anion production, and neutralization of superoxide by SOD (superoxide dismutase) abolished Ang II-stimulated ET1 (human ET-1 gene) promoter activity, ET1 mRNA expression, and ET-1 release. CONCLUSIONS: Ang II may promote ET-1 production in the endothelium in response to atherogenic diets through a mechanism that involves the transcription factor Oct-1 and the increased formation of superoxide anions by NOX2.

Nailfold Videocapillaroscopy for Non-Invasive Assessment of Microcirculation and Prognostic Correlation with Endothelial Dysfunction, Cardiovascular Risk Factors, and Non-HLA Antibodies in Heart Transplant Recipients: A Pilot Study.

Early identification of allograft vasculopathy and the concomitant elimination of adverse risk factors is essential for improving the long-term prognosis of heart transplant (HTx) recipients with underlying cardiovascular disease (CVD). The major aim of this pilot study was to conduct a non-invasive imaging evaluation of the HTx patient microcirculation by employing nailfold video-capillaroscopy (NVC) in a well-characterized patient and control cohort, and to correlate these data with endothelial cell function, accompanied by studies of traditional cardiovascular risk factors and non-HLA antibodies in HTx recipients. Ten patients undergoing HTx (mean age of 38 ± 14 years) were recruited for the study and compared to a control group of 12 well-matched healthy volunteers (mean age 35 ± 5 years) with normal body mass index (BMI). Detailed medical records were collected from all individuals. NVC was performed using CapillaryScope 200 MEDL4N microscope. For functional readout and correlation analysis, endothelial cell network formation in conjunction with measurements of patient serum levels of vascular endothelial growth factor (VEGF) and non-HLA autoantibodies directed against the angiotensin II type-1-receptor (anti-AT1R-Ab), endothelin-1 type-A-receptor (anti-ETAR-Ab), protease-activated receptor-1 (anti-PAR-1-Ab), and VEGF-A (anti-VEGF-A-Ab) were studied. Our NVC analysis found that the average apical loop diameter of nailfold capillaries was significantly increased in HTx recipients (p = 0.001). In addition, HTx patients with more prominent changes in capillaroscopic patterns were characterized by the presence of traditional cardiovascular risk factors, and HTx patients had increased levels of anti-AT1R-ab, anti-ETAR-ab, and anti-VEGF-A-Ab (p = 0.017, p = 0.025, and p = 0.003, respectively). Capillary diameters most strongly correlated with elevated serum levels of troponin T and triglycerides (R = 0.69, p = 0.028 and R = 0.81, p = 0.004, respectively). In conclusion, we found that an abnormal NVC pattern in HTx patients is associated with traditional CVD risk factors and that NVC is a useful non-invasive tool to conveniently monitor changes in the microvasculature of HTx patients.

Autoimmune activation and hypersensitization of the AT1 and ETA receptors contributes to vascular injury in scleroderma renal crisis.

OBJECTIVES: Scleroderma renal crisis (SRC) is a rare vascular complication of systemic sclerosis with substantial risks for end-stage renal disease and premature death. Activating autoantibodies (Abs) targeting the angiotensin II type 1 (AT1R) and the endothelin-1 type A receptor (ETAR) have been identified as predictors for SRC. Here, we sought to determine their pathogenic significance for acute renal vascular injury potentially triggering kidney failure and malignant hypertension. METHODS: IgG from patients with SRC was studied for AT1R and ETAR dependent biologic effects on isolated rat renal interlobar arteries and vascular cells including contraction, signalling and mechanisms of receptor activation. RESULTS: In myography experiments, patient IgG exerted vasoconstriction sensitive to inhibition of AT1R and ETAR. This relied on MEK-ERK signalling indicating functional relevance of anti-AT1R and anti-ETAR Abs. The contractile response to angiotensin II and endothelin-1 was amplified by patient IgG containing anti-AT1R and anti-ETAR Abs with substantial crosstalk between both receptors implicating autoimmune receptor hypersensitization. Co-immunoprecipitation experiments indicated heterodimerization between both receptor types which may enable the observed functional interrelation by direct structural interactions. CONCLUSION: We provide experimental evidence that agonistic Abs may contribute to SRC. This effect is presumably related to direct receptor stimulation and additional allosteric effects, at least in heterodimeric receptor constellations. Novel therapies targeted at autoimmune hyperactivation of AT1R and ETAR might improve outcomes in severe cases of SRC.

SARS-CoV-2 Virus-like Particles (VLPs) Specifically Detect Humoral Immune Reactions in an ELISA-Based Platform.

A key in controlling the SARS-CoV-2 pandemic is the assessment of the immune status of the population. We explored the utility of SARS-CoV-2 virus-like particles (VLPs) as antigens to detect specific humoral immune reactions in an enzyme-linked immunosorbent assay (ELISA). For this purpose, SARS-CoV-2 VLPs were produced from an engineered cell line and characterized by Western blot, ELISA, and nanoparticle tracking analysis. Subsequently, we collected 42 serum samples from before the pandemic (2014), 89 samples from healthy subjects, and 38 samples from vaccinated subjects. Seventeen samples were collected less than three weeks after infection, and forty-four samples more than three weeks after infection. All serum samples were characterized for their reactivity with VLPs and the SARS-CoV-2 N- and S-protein. Finally, we compared the performance of the VLP-based ELISA with a certified in vitro diagnostic device (IVD). In the applied set of samples, we determined a sensitivity of 95.5% and a specificity of 100% for the certified IVD. There were seven samples with an uncertain outcome. Our VLP-ELISA demonstrated a superior performance, with a sensitivity of 97.5%, a specificity of 100%, and only three uncertain outcomes. This result warrants further research to develop a certified IVD based on SARS-CoV-2 VLPs as an antigen.

SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications.

Introduction: The ongoing COVID-19 pandemic situation caused by SARS-CoV-2 and variants of concern such as B.1.617.2 (Delta) and recently, B.1.1.529 (Omicron) is posing multiple challenges to humanity. The rapid evolution of the virus requires adaptation of diagnostic and therapeutic applications. Objectives: In this study, we describe camelid heavy-chain-only antibodies (hcAb) as useful tools for novel in vitro diagnostic assays and for therapeutic applications due to their neutralizing capacity. Methods: Five antibody candidates were selected out of a naïve camelid library by phage display and expressed as full length IgG2 antibodies. The antibodies were characterized by Western blot, enzyme-linked immunosorbent assays, surface plasmon resonance with regard to their specificity to the recombinant SARS-CoV-2 Spike protein and to SARS-CoV-2 virus-like particles. Neutralization assays were performed with authentic SARS-CoV-2 and pseudotyped viruses (wildtype and Omicron). Results: All antibodies efficiently detect recombinant SARS-CoV-2 Spike protein and SARS-CoV-2 virus-like particles in different ELISA setups. The best combination was shown with hcAb B10 as catcher antibody and HRP-conjugated hcAb A7.2 as the detection antibody. Further, four out of five antibodies potently neutralized authentic wildtype SARS-CoV-2 and particles pseudotyped with the SARS-CoV-2 Spike proteins of the wildtype and Omicron variant, sublineage BA.1 at concentrations between 0.1 and 0.35 ng/mL (ND50). Conclusion: Collectively, we report novel camelid hcAbs suitable for diagnostics and potential therapy.

Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation.

The angiotensin II (Ang II) type 1 receptor (AT1R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT1R can also be activated by auto-antibodies (AT1R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT1R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT1R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT1R signaling. AT1R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects Gq/11 and Gi activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT1R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT1R is impeded by binding of AT1R-Abs. Secondly, exclusive AT1R-Abs-induced Gq/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT1R activation of Gi signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of Gi basal activity potentiates proliferation triggered by AT1R-Abs. Finally, although AT1R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT1R and Abs. This current study thus provides extended insights into the molecular action of AT1R-Abs and associated mechanisms of interrelated pathogenesis.

Angiogenic Role of Mesothelium-Derived Chemokine CXCL1 During Unfavorable Peritoneal Tissue Remodeling in Patients Receiving Peritoneal Dialysis as Renal Replacement Therapy.

Peritoneal dialysis (PD) is a valuable 'home treatment' option, even more so during the ongoing Coronavirus pandemic. However, the long-term use of PD is limited by unfavourable tissue remodelling in the peritoneal membrane, which is associated with inflammation-induced angiogenesis. This appears to be driven primarily through vascular endothelial growth factor (VEGF), while the involvement of other angiogenic signaling pathways is still poorly understood. Here, we have identified the crucial contribution of mesothelial cell-derived angiogenic CXC chemokine ligand 1 (CXCL1) to peritoneal angiogenesis in PD. CXCL1 expression and peritoneal microvessel density were analysed in biopsies obtained by the International Peritoneal Biobank (NCT01893710 at www.clinicaltrials.gov), comparing 13 children with end-stage kidney disease before initiating PD to 43 children on chronic PD. The angiogenic potential of mesothelial cell-derived CXCL1 was assessed in vitro by measuring endothelial tube formation of human microvascular endothelial cells (HMECs) treated with conditioned medium from human peritoneal mesothelial cells (HPMCs) stimulated to release CXCL1 by treatment with either recombinant IL-17 or PD effluent. We found that the capillary density in the human peritoneum correlated with local CXCL1 expression. Both CXCL1 expression and microvessel density were higher in PD patients than in the age-matched patients prior to initiation of PD. Exposure of HMECs to recombinant CXCL1 or conditioned medium from IL-17-stimulated HPMCs resulted in increased endothelial tube formation, while selective inhibition of mesothelial CXCL1 production by specific antibodies or through silencing of relevant transcription factors abolished the proangiogenic effect of HPMC-conditioned medium. In conclusion, peritoneal mesothelium-derived CXCL1 promotes endothelial tube formation in vitro and associates with peritoneal microvessel density in uremic patients undergoing PD, thus providing novel targets for therapeutic intervention to prolong PD therapy.

Native and Oxidized Low-Density Lipoproteins Increase the Expression of the LDL Receptor and the LOX-1 Receptor, Respectively, in Arterial Endothelial Cells.

Atherosclerotic artery disease is the major cause of death and an immense burden on healthcare systems worldwide. The formation of atherosclerotic plaques is promoted by high levels of low-density lipoproteins (LDL) in the blood, especially in the oxidized form. Circulating LDL is taken up by conventional and non-classical endothelial cell receptors and deposited in the vessel wall. The exact mechanism of LDL interaction with vascular endothelial cells is not fully understood. Moreover, it appears to depend on the type and location of the vessel affected and the receptor involved. Here, we analyze how native LDL (nLDL) and oxidized LDL (oxLDL) modulate the expression of their receptors-classical LDLR and alternative LOX-1-in endothelial cells derived from human umbilical artery (HUAECs), used as an example of a medium-sized vessel, which is typically affected by atherosclerosis. Exposure of HUAECs to nLDL resulted in moderate nLDL uptake and gradual increase in LDLR, but not LOX-1, expression over 24 h. Conversely, exposure of HUAECs to oxLDL, led to significant accumulation of oxLDL and rapid induction of LOX-1, but not LDLR, within 7 h. These activation processes were associated with phosphorylation of protein kinases ERK1/2 and p38, followed by activation of the transcription factor AP-1 and its binding to the promoters of the respective receptor genes. Both nLDL-induced LDLR mRNA expression and oxLDL-induced LOX-1 mRNA expression were abolished by blocking ERK1/2, p-38 or AP-1. In addition, oxLDL, but not nLDL, was capable of inducing LOX-1 through the NF-κB-controlled pathway. These observations indicate that in arterial endothelial cells nLDL and oxLDL signal mainly via LDLR and LOX-1 receptors, respectively, and engage ERK1/2 and p38 kinases, and AP-1, as well as NF-κB transcription factors to exert feed-forward regulation and increase the expression of these receptors, which may perpetuate endothelial dysfunction in atherosclerosis.

Expanded Hemodialysis Therapy Ameliorates Uremia-Induced Systemic Microinflammation and Endothelial Dysfunction by Modulating VEGF, TNF-α and AP-1 Signaling.

Abstract: Systemic chronic microinflammation and altered cytokine signaling, with adjunct cardiovascular disease (CVD), endothelial maladaptation and dysfunction is common in dialysis patients suffering from end-stage renal disease and associated with increased morbidity and mortality. New hemodialysis filters might offer improvements. We here studied the impact of novel improved molecular cut-off hemodialysis filters on systemic microinflammation, uremia and endothelial dysfunction. Human endothelial cells (ECs) were incubated with uremic serum obtained from patients treated with two different hemodialysis regimens in the Permeability Enhancement to Reduce Chronic Inflammation (PERCI-II) crossover clinical trial, comparing High-Flux (HF) and Medium Cut-Off (MCO) membranes, and then assessed for their vascular endothelial growth factor (VEGF) production and angiogenesis. Compared to HF membranes, dialysis with MCO membranes lead to a reduction in proinflammatory mediators and reduced endothelial VEGF production and angiogenesis. Cytokine multiplex screening identified tumor necrosis factor (TNF) superfamily members as promising targets. The influence of TNF-α and its soluble receptors (sTNF-R1 and sTNF-R2) on endothelial VEGF promoter activation, protein release, and the involved signaling pathways was analyzed, revealing that this detrimental signaling was indeed induced by TNF-α and mediated by AP-1/c-FOS signaling. In conclusion, uremic toxins, in particular TNF-signaling, promote endothelial maladaptation, VEGF expression and aberrant angiogenesis, which can be positively modulated by dialysis with novel MCO membranes. Translational Perspective and Graphical Abstract: Systemic microinflammation, altered cytokine signaling, cardiovascular disease, and endothelial maladaptation/dysfunction are common clinical complications in dialysis patients suffering from end-stage renal disease. We studied the impact of novel improved medium-cut-off hemodialysis filters on uremia and endothelial dysfunction. We can show that uremic toxins, especially TNF-signaling, promote endothelial maladaptation, VEGF expression and aberrant angiogenesis, which can be positively modulated by dialysis with novel improved medium-cut-off membranes.

Viscoelastic testing reveals normalization of the coagulation profile 12 weeks after severe COVID-19.

COVID 19 is associated with a hypercoagulable state and frequent thromboembolic complications. For how long this acquired abnormality lasts potentially requiring preventive measures, such as anticoagulation remains to be delineated. We used viscoelastic rotational thrombelastometry (ROTEM) in a single center cohort of 13 critical ill patients and performed follow up examinations three months after discharge from ICU. We found clear signs of a hypercoagulable state due to severe hypofibrinolysis and a high rate of thromboembolic complications during the phase of acute illness. Three month follow up revealed normalization of the initial coagulation abnormality and no evidence of venous thrombosis in all thirteen patients. In our cohort the coagulation profile was completely normalized three months after COVID-19. Based on these findings, discontinuation of anticoagulation can be discussed in patients with complete venous reperfusion.

Transcriptional Regulation of Thrombin-Induced Endothelial VEGF Induction and Proangiogenic Response.

Thrombin, the ligand of the protease-activated receptor 1 (PAR1), is a well-known stimulator of proangiogenic responses in vascular endothelial cells (ECs), which are mediated through the induction of vascular endothelial growth factor (VEGF). However, the transcriptional events underlying this thrombin-induced VEGF induction and angiogenic response are less well understood at present. As reported here, we conducted detailed promotor activation and signal transduction pathway studies in human microvascular ECs, to decipher the transcription factors and the intracellular signaling events underlying the thrombin and PAR-1-induced endothelial VEGF induction. We found that c-FOS is a key transcription factor controlling thrombin-induced EC VEGF synthesis and angiogenesis. Upon the binding and internalization of its G-protein-coupled PAR-1 receptor, thrombin triggers ERK1/2 signaling and activation of the nuclear AP-1/c-FOS transcription factor complex, which then leads to VEGF transcription, extracellular secretion, and concomitant proangiogenic responses of ECs. In conclusion, exposure of human microvascular ECs to thrombin triggers signaling through the PAR-1-ERK1/2-AP-1/c-FOS axis to control VEGF gene transcription and VEGF-induced angiogenesis. These observations offer a greater understanding of endothelial responses to thromboinflammation, which may help to interpret the results of clinical trials tackling the conditions associated with endothelial injury and thrombosis.

Autoantibodies Targeting AT1- and ETA-Receptors Link Endothelial Proliferation and Coagulation via Ets-1 Transcription Factor.

Scleroderma renal crisis (SRC) is an acute life-threatening manifestation of systemic sclerosis (SSc) caused by obliterative vasculopathy and thrombotic microangiopathy. Evidence suggests a pathogenic role of immunoglobulin G (IgG) targeting G-protein coupled receptors (GPCR). We therefore dissected SRC-associated vascular obliteration and investigated the specific effects of patient-derived IgG directed against angiotensin II type 1 (AT1R) and endothelin-1 type A receptors (ETAR) on downstream signaling events and endothelial cell proliferation. SRC-IgG triggered endothelial cell proliferation via activation of the mitogen-activated protein kinase (MAPK) pathway and subsequent activation of the E26 transformation-specific-1 transcription factor (Ets-1). Either AT1R or ETAR receptor inhibitors/shRNA abrogated endothelial proliferation, confirming receptor activation and Ets-1 signaling involvement. Binding of Ets-1 to the tissue factor (TF) promoter exclusively induced TF. In addition, TF inhibition prevented endothelial cell proliferation. Thus, our data revealed a thus far unknown link between SRC-IgG-induced intracellular signaling, endothelial cell proliferation and active coagulation in the context of obliterative vasculopathy and SRC. Patients' autoantibodies and their molecular effectors represent new therapeutic targets to address severe vascular complications in SSc.

Thromboembolic complications in critically ill COVID-19 patients are associated with impaired fibrinolysis.

BACKGROUND: There is emerging evidence for enhanced blood coagulation in coronavirus 2019 (COVID-19) patients, with thromboembolic complications contributing to morbidity and mortality. The mechanisms underlying this prothrombotic state remain enigmatic. Further data to guide anticoagulation strategies are urgently required. METHODS: We used viscoelastic rotational thromboelastometry (ROTEM) in a single-center cohort of 40 critically ill COVID-19 patients. RESULTS: Clear signs of a hypercoagulable state due to severe hypofibrinolysis were found. Maximum lysis, especially following stimulation of the extrinsic coagulation system, was inversely associated with an enhanced risk of thromboembolic complications. Combining values for maximum lysis with D-dimer concentrations revealed high sensitivity and specificity of thromboembolic risk prediction. CONCLUSIONS: The study identifies a reduction in fibrinolysis as an important mechanism in COVID-19-associated coagulopathy. The combination of ROTEM and D-dimer concentrations may prove valuable in identifying patients requiring higher intensity anticoagulation.

MSC Therapies for COVID-19: Importance of Patient Coagulopathy, Thromboprophylaxis, Cell Product Quality and Mode of Delivery for Treatment Safety and Efficacy.

Numerous clinical trials of mesenchymal stromal/stem cells (MSCs) as a new treatment for coronavirus-induced disease (COVID-19) have been registered recently, most of them based on intravenous (IV) infusion. There is no approved effective therapy for COVID-19, but MSC therapies have shown first promise in the treatment of acute respiratory distress syndrome (ARDS) pneumonia, inflammation, and sepsis, which are among the leading causes of mortality in COVID-19 patients. Many of the critically ill COVID-19 patients are in a hypercoagulable procoagulant state and at high risk for disseminated intravascular coagulation, thromboembolism, and thrombotic multi-organ failure, another cause of high fatality. It is not yet clear whether IV infusion is a safe and effective route of MSC delivery in COVID-19, since MSC-based products express variable levels of highly procoagulant tissue factor (TF/CD142), compromising the cells' hemocompatibility and safety profile. Of concern, IV infusions of poorly characterized MSC products with unchecked (high) TF/CD142 expression could trigger blood clotting in COVID-19 and other vulnerable patient populations and further promote the risk for thromboembolism. In contrast, well-characterized products with robust manufacturing procedures and optimized modes of clinical delivery hold great promise for ameliorating COVID-19 by exerting their beneficial immunomodulatory effects, inducing tissue repair and organ protection. While the need for MSC therapy in COVID-19 is apparent, integrating both innate and adaptive immune compatibility testing into the current guidelines for cell, tissue, and organ transplantation is critical for safe and effective therapies. It is paramount to only use well-characterized, safe MSCs even in the most urgent and experimental treatments. We here propose three steps to mitigate the risk for these vulnerable patients: (1) updated clinical guidelines for cell and tissue transplantation, (2) updated minimal criteria for characterization of cellular therapeutics, and (3) updated cell therapy routines reflecting specific patient needs.

Addressing multiple gastroenterological aspects of coronavirus disease 2019.

The outbreak of the coronavirus disease 2019 (COVID­19) pandemic has become the biggest challenge for the whole human community since many years. It seems that the proper identification of all people infected with severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) is the best strategy to limit the transmission. However, in a significant proportion of patients, there are no clinical manifestations of the disease, and symptoms may be very mild or atypical. There is a growing body of evidence that digestive manifestations of COVID­19 are frequently reported and may precede typical respiratory symptoms. Moreover, SARS­CoV­2 particles were found in the gastrointestinal epithelial cells, and viral RNA was detected in the feces of patients with COVID­19. These data suggest that gastrointestinal symptoms in COVID­19 are not accidental findings and they may result from direct digestive involvement. Patients with new­onset diarrhea, abdominal pain, nausea, and vomiting without any other evident etiological factors should be tested for SARS­CoV­2 infection. Gastroenterologists and members of other medical specialties should also remember that the current epidemiological situation has changed diagnostic and therapeutic algorithms in the management of several gastrointestinal and liver disorders. This review article summarizes the currently available data on multiple gastroenterological aspects of COVID­19 and provides information on practical recommendations and position statements of the most prominent associations in the field of gastroenterology, which appeared in response to the emergence of the pandemic.

Control of neutrophil influx during peritonitis by transcriptional cross-regulation of chemokine CXCL1 by IL-17 and IFN-γ.

Neutrophil infiltration is a hallmark of peritoneal inflammation, but mechanisms regulating neutrophil recruitment in patients with peritoneal dialysis (PD)-related peritonitis are not fully defined. We examined 104 samples of PD effluent collected during acute peritonitis for correspondence between a broad range of soluble parameters and neutrophil counts. We observed an association between peritoneal IL-17 and neutrophil levels. This relationship was evident in effluent samples with low but not high IFN-γ levels, suggesting a differential effect of IFN-γ concentration on neutrophil infiltration. Surprisingly, there was no association of neutrophil numbers with the level of CXCL1, a key IL-17-induced neutrophil chemoattractant. We investigated therefore the production of CXCL1 by human peritoneal mesothelial cells (HPMCs) under in vitro conditions mimicking clinical peritonitis. Stimulation of HPMCs with IL-17 increased CXCL1 production through induction of transcription factor SP1 and activation of the SP1-binding region of the CXCL1 promoter. These effects were amplified by TNFα. In contrast, IFN-γ dose-dependently suppressed IL-17-induced SP1 activation and CXCL1 production through a transcriptional mechanism involving STAT1. The SP1-mediated induction of CXCL1 was also observed in HPMCs exposed to PD effluent collected during peritonitis and containing IL-17 and TNFα, but not IFN-γ. Supplementation of the effluent with IFN-γ led to a dose-dependent activation of STAT1 and a resultant inhibition of SP1-induced CXCL1 expression. Transmesothelial migration of neutrophils in vitro increased upon stimulation of HPMCs with IL-17 and was reduced by IFN-γ. In addition, HPMCs were capable of binding CXCL1 at their apical cell surface. These observations indicate that changes in relative peritoneal concentrations of IL-17 and IFN-γ can differently engage SP1-STAT1, impacting on mesothelial cell transcription of CXCL1, whose release and binding to HPMC surface may determine optimal neutrophil recruitment and retention during peritonitis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.