Leukotriene B4: A potential mediator and biomarker for cardiac allograft vasculopathy.

BACKGROUND: Cardiac allograft vasculopathy (CAV) remains the leading cause of long-term graft failure and mortality after heart transplantation. Effective preventive and treatment options are not available to date, largely because underlying mechanisms remain poorly understood. We studied the potential role of leukotriene B4 (LTB4), an inflammatory lipid mediator, in the development of CAV. METHODS: We used an established preclinical rat CAV model to study the role of LTB4 in CAV. We performed syngeneic and allogeneic orthotopic aortic transplantation, after which neointimal proliferation was quantified. Animals were then treated with Bestatin, an inhibitor of LTB4 synthesis, or vehicle control for 30 days post-transplant, and evidence of graft CAV was determined by histology. We also measured serial LTB4 levels in a cohort of 28 human heart transplant recipients with CAV, 17 matched transplant controls without CAV, and 20 healthy nontransplant controls. RESULTS: We showed that infiltration of the arterial wall with macrophages leads to neointimal thickening and a rise in serum LTB4 levels in our rat model of CAV. Inhibition of LTB4 production with the drug Bestatin prevents development of neointimal hyperplasia, suggesting that Bestatin may be effective therapy for CAV prevention. In a parallel study of heart transplant recipients, we found nonsignificantly elevated plasma LTB4 levels in patients with CAV, compared to patients without CAV and healthy, nontransplant controls. CONCLUSIONS: This study provides key evidence supporting the role of the inflammatory cytokine LTB4 as an important mediator of CAV development and provides preliminary data suggesting the clinical benefit of Bestatin for CAV prevention.

IL-22 promotes liver regeneration after portal vein ligation.

Background: Insufficient remnant liver volume (RLV) after the resection of hepatic malignancy could lead to liver failure and mortality. Portal vein ligation (PVL) prior to hepatectomy is subsequently introduced to increase the remnant liver volume and improve the outcome of hepatic malignancy. IL-22 has previously been reported to promote liver regeneration, while facilitating tumor development in the liver via Steap4 upregulation. Here we performed PVL in mouse models to study the role of IL-22 in liver regeneration post-PVL. Methods: Liver weight and volume was measured via magnetic resonance imaging (MRI). Immunohistochemistry for Ki67 and hepatocyte growth factor (HGF) was performed. IL-22 was analyzed by flow cytometry and quantitative polymerase chain reaction (qPCR) was used for acquisition of Il-33, Steap4, Fga, Fgb and Cebpd. To analyze signaling pathways, mice with deletion of STAT3 and a neutralizing antibody for IL-22 were used. Results: The remnant liver weight and volume increased over time after PVL. Additionally, we found that liver regenerative molecules, including Ki67 and HGF, were significantly increased in remnant liver at day 3 post-PVL, as well as IL-22. Administration of IL-22 neutralizing antibody could reduce Ki67 expression after PVL. The upregulation of IL-22 after PVL was mainly derived from innate cells. IL-22 blockade resulted in lower levels of IL-33 and Steap4 in the remnant liver, which was also the case in mice with deletion of STAT3, the main downstream signaling molecule of IL-22, in hepatocytes. Conclusion: IL-22 promotes liver regeneration after PVL. Thus, a combination of IL-22 supplementation and Steap4 blockade could potentially be applied as a novel therapeutic approach to boost liver regeneration without facilitating tumor progression after PVL.

Algorithm for Rapid Exclusion of Clinically Relevant Plasma Levels of Direct Oral Anticoagulants in Patients Using the DOAC Dipstick: An Expert Consensus Paper.

BACKGROUND: With the widespread use of direct oral anticoagulants (DOACs), there is an urgent need for a rapid assay to exclude clinically relevant plasma levels. Accurate and rapid determination of DOAC levels would guide medical decision-making to (1) determine the potential contribution of the DOAC to spontaneous or trauma-induced hemorrhage; (2) identify appropriate candidates for reversal, or (3) optimize the timing of urgent surgery or intervention. METHODS AND RESULTS: The DOAC Dipstick test uses a disposable strip to identify factor Xa- or thrombin inhibitors in a urine sample. Based on the results of a systematic literature search followed by an analysis of a simple pooling of five retrieved clinical studies, the test strip has a high sensitivity and an acceptably high negative predictive value when compared with levels measured with liquid chromatography tandem mass spectrometry or calibrated chromogenic assays to reliably exclude plasma DOAC concentrations ≥30 ng/mL. CONCLUSION: Based on these data, a simple algorithm is proposed to enhance medical decision-making in acute care indications useful primarily in hospitals not having readily available quantitative tests and 24/7. This algorithm not only determines DOAC exposure but also differentiates between factor Xa and thrombin inhibitors to better guide clinical management.

Highly sensitive therapeutic drug monitoring of infliximab in serum by targeted mass spectrometry in comparison to ELISA data.

BACKGROUND: Presently, antibody concentration measurements for patients undergoing treatment are predominantly determined by ELISA, which still comes with known disadvantages. Therefore, our aim was to establish a targeted mass-spectrometric assay enabling the reproducible absolute quantification of peptides from the hypervariable and interaction regions of infliximab. METHODS: Peptides of infliximab were measured post-trypsin digestion and subsequent separation on a Vanquish Horizon UHPLC coupled to a TSQ Altis Triple-Quad mass spectrometer. Normalization and absolute quantification were conducted using stable isotope-synthesized peptides. Calibration curves covering a range of 0.25-50 µg/ml were employed for quantitation. RESULTS: We demonstrated the substantial influence of peptide selection, choice of hydrolase for digestion, and digestion time on absolute peptide yield (28-44% for peptide 1 and 64-97% for peptide 2). Furthermore, we showed that the generated calibration curves for absolute quantification were highly reproducible and robust (LLOQ1 0.72 µg/ml and LLOQ2 1.00 µg/ml) over several months. In comparison to ELISA values, the absolute values obtained by mass spectrometry often yielded lower results for both targeted peptides. CONCLUSIONS: In this study, a semi-automated workflow was employed and tested with 8 patients and corresponding replicates (n = 3-4). We demonstrated the robust implementation of calibration curves for the absolute quantification of infliximab in patient samples, with coefficients of variation ranging from 0.5 to 9%. Taken together, we have developed a platform enabling the rapid (2 days of sample preparation and 30 min of measurement time per sample) and robust quantification of Infliximab antibody concentration in patients. The use of mass spectrometry also facilitates the straightforward expansion of the method to include additional antibody peptides.

The History of Extracorporeal Membrane Oxygenation and the Development of Extracorporeal Membrane Oxygenation Anticoagulation.

Extracorporeal membrane oxygenation (ECMO) was first started for humans in early 1970s by Robert Bartlett. Since its inception, there have been numerous challenges with extracorporeal circulation, such as coagulation and platelet activation, followed by consumption of coagulation factors and platelets, and biocompatibility of tubing, pump, and oxygenator. Unfractionated heparin (heparin hereafter) has historically been the defacto anticoagulant until recently. Also, coagulation monitoring was mainly based on bedside activated clotting time and activated partial thromboplastin time. In the past 50 years, the technology of ECMO has advanced tremendously, and thus, the survival rate has improved significantly. The indication for ECMO has also expanded. Among these are clinical conditions such as postcardiopulmonary bypass, sepsis, ECMO cardiopulmonary resuscitation, and even severe coronavirus disease 2019 (COVID-19). Not surprisingly, the number of ECMO cases has increased according to the Extracorporeal Life Support Organization Registry and prolonged ECMO support has become more prevalent. It is not uncommon for patients with COVID-19 to be on ECMO support for more than 1 year until recovery or lung transplant. With that being said, complications of bleeding, thrombosis, clot formation in the circuit, and intravascular hemolysis still remain and continue to be major challenges. Here, several clinical ECMO experts, including the "Father of ECMO"-Dr. Robert Bartlett, describe the history and advances of ECMO.

Goal-directed colloid versus crystalloid therapy and microcirculatory blood flow following ischemia/reperfusion.

OBJECTIVE: Ischemia/reperfusion can impair microcirculatory blood flow. It remains unknown whether colloids are superior to crystalloids for restoration of microcirculatory blood flow during ischemia/reperfusion injury. We tested the hypothesis that goal-directed colloid - compared to crystalloid - therapy improves small intestinal, renal, and hepatic microcirculatory blood flow in pigs with ischemia/reperfusion injury. METHODS: This was a randomized trial in 32 pigs. We induced ischemia/reperfusion by supra-celiac aortic-cross-clamping. Pigs were randomized to receive either goal-directed isooncotic hydroxyethyl-starch colloid or balanced isotonic crystalloid therapy. Microcirculatory blood flow was measured using Laser-Speckle-Contrast-Imaging. The primary outcome was small intestinal, renal, and hepatic microcirculatory blood flow 4.5 h after ischemia/reperfusion. Secondary outcomes included small intestinal, renal, and hepatic histopathological damage, macrohemodynamic and metabolic variables, as well as specific biomarkers of tissue injury, renal, and hepatic function and injury, and endothelial barrier function. RESULTS: Small intestinal microcirculatory blood flow was higher in pigs assigned to isooncotic hydroxyethyl-starch colloid therapy than in pigs assigned to balanced isotonic crystalloid therapy (768.7 (677.2-860.1) vs. 595.6 (496.3-694.8) arbitrary units, p = .007). There were no important differences in renal (509.7 (427.2-592.1) vs. 442.1 (361.2-523.0) arbitrary units, p = .286) and hepatic (604.7 (507.7-701.8) vs. 548.7 (444.0-653.3) arbitrary units, p = .376) microcirculatory blood flow between groups. Pigs assigned to colloid - compared to crystalloid - therapy also had less small intestinal, but not renal and hepatic, histopathological damage. CONCLUSIONS: Goal-directed isooncotic hydroxyethyl-starch colloid - compared to balanced isotonic crystalloid - therapy improved small intestinal, but not renal and hepatic, microcirculatory blood flow in pigs with ischemia/reperfusion injury. Whether colloid therapy improves small intestinal microcirculatory blood flow in patients with ischemia/reperfusion needs to be investigated in clinical trials.

Glycolytic reprogramming fuels myeloid cell-driven hypercoagulability.

BACKGROUND: Myeloid cell metabolic reprogramming is a hallmark of inflammatory disease; however, its role in inflammation-induced hypercoagulability is poorly understood. OBJECTIVES: We aimed to evaluate the role of inflammation-associated metabolic reprogramming in regulating blood coagulation. METHODS: We used novel myeloid cell-based global hemostasis assays and murine models of immunometabolic disease. RESULTS: Glycolysis was essential for enhanced activated myeloid cell tissue factor expression and decryption, driving increased cell-dependent thrombin generation in response to inflammatory challenge. Similarly, inhibition of glycolysis enhanced activated macrophage fibrinolytic activity through reduced plasminogen activator inhibitor 1 activity. Macrophage polarization or activation markedly increased endothelial protein C receptor (EPCR) expression on monocytes and macrophages, leading to increased myeloid cell-dependent protein C activation. Importantly, inflammation-dependent EPCR expression on tissue-resident macrophages was also observed in vivo. Adipose tissue macrophages from obese mice fed a high-fat diet exhibited significantly enhanced EPCR expression and activated protein C generation compared with macrophages isolated from the adipose tissue of healthy mice. Similarly, the induction of colitis in mice prompted infiltration of EPCR+ innate myeloid cells within inflamed colonic tissue that were absent from the intestinal tissue of healthy mice. CONCLUSION: Collectively, this study identifies immunometabolic regulation of myeloid cell hypercoagulability, opening new therapeutic possibilities for targeted mitigation of thromboinflammatory disease.

The phosphodiesterase 2A controls lymphatic junctional maturation via cGMP-dependent notch signaling.

The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we identify the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic but not of blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, whereas blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, whereas cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to the activation of p38 signaling and downregulation of NOTCH signaling. However, DLL4-induced NOTCH activation restores junctional maturation and contact inhibition in PDE2A-deficient human lymphatic endothelial cells. In postnatal mouse mesenteries, PDE2A is specifically enriched in collecting lymphatic valves, and loss of Pde2a results in the formation of abnormal valves. Our data demonstrate that PDE2A selectively finetunes a crosstalk of cGMP, p38, and NOTCH signaling during lymphatic vessel maturation.

Prognostic utility of a multi-biomarker panel in patients with suspected myocardial infarction.

BACKGROUND: The accurate identification of patients with high cardiovascular risk in suspected myocardial infarction (MI) is an unmet clinical need. Therefore, we sought to investigate the prognostic utility of a multi-biomarker panel with 29 different biomarkers in in 748 consecutive patients with symptoms indicative of MI using a machine learning-based approach. METHODS: Incident major cardiovascular events (MACE) were documented within 1 year after the index admission. The selection of the best multi-biomarker model was performed using the least absolute shrinkage and selection operator (LASSO). The independent and additive utility of selected biomarkers was compared to a clinical reference model and the Global Registry of Acute Coronary Events (GRACE) Score, respectively. Findings were validated using internal cross-validation. RESULTS: Median age of the study population was 64 years. At 1 year of follow-up, 160 cases of incident MACE were documented. 16 of the investigated 29 biomarkers were significantly associated with 1-year MACE. Three biomarkers including NT-proBNP (HR per SD 1.24), Apolipoprotein A-I (Apo A-I; HR per SD 0.98) and kidney injury molecule-1 (KIM-1; HR per SD 1.06) were identified as independent predictors of 1-year MACE. Although the discriminative ability of the selected multi-biomarker model was rather moderate, the addition of these biomarkers to the clinical reference model and the GRACE score improved model performances markedly (∆C-index 0.047 and 0.04, respectively). CONCLUSION: NT-proBNP, Apo A-I and KIM-1 emerged as strongest independent predictors of 1-year MACE in patients with suspected MI. Their integration into clinical risk prediction models may improve personalized risk stratification. Prognostic utility of a multi-biomarker approach in suspected myocardial infarction. In a cohort of 748 patients with symptoms indicative of myocardial infarction (MI) to the emergency department, we measured a 29-biomarker panel and performed regressions, machine learning (ML)-based variable selection and discriminative/reclassification analyses. We identified three biomarkers as top predictors for 1-year major adverse cardiovascular events (MACE). Their integration into a clinical risk prediction model and the Global Registry of Acute Coronary Events (GRACE) Score allowed for marked improvement in discrimination and reclassification for 1-year MACE. Apo apolipoprotein; CRP C-reactive protein; CRS clinical risk score; ECG electrocardiogram; EN-RAGE extracellular newly identified receptor for advanced glycation end-products binding protein; FABP fatty acid-binding protein; GS Grace Score; hs-cTnI high-sensitivity cardiac troponin I; KIM-1 kidney injury molecule-1; LASSO least absolute shrinkage and selection operator; MACE major adverse cardiovascular events; MI myocardial infarction; NRI net reclassification improvement; NT-proBNP N-terminal prohormone of brain natriuretic peptide.

Polyphosphate-loaded silk fibroin membrane as hemostatic agent in oral surgery: a pilot study.

PURPOSE: Post-interventional hemorrhage can result in serious complications, especially in patients with hemostatic disorders. Identification of safe and efficient local hemostatic agents is important, particularly in the context of an ageing society and the emergence of new oral anticoagulants. The aim of this in vitro study was to investigate the potential of silk fibroin membranes coated with the inorganic polymer polyphosphate (polyP) as a novel hemostatic device in oral surgery. METHODS: Cocoons of the silkworm Bombyx mori were degummed and dissolved. Varying amounts of long-chain polyP (2-2000 µg/mm2) were adsorbed to the surface of silk fibroin membranes. Analysis of the procoagulant effect of polyP-coated silk membranes was performed using real-time thrombin generation assays in human plasma. Increasing concentrations of polyP (0.15-500 µg/ml) served as a positive control, while uncoated silk fibroin membranes were used as negative control. RESULTS: PolyP-coated silk fibroin membranes triggered coagulation when compared to plasma samples and pure silk fibroin membranes. A polyP-dose-dependent effect of thrombin generation could be found with a maximum (ETP = 1525.7 nM⋅min, peak thrombin = 310.1 nM, time to peak = 9.8 min, lag time = 7.6 min.) at 200 µg/mm2 of polymer loading on the silk fibroin membrane surface. CONCLUSIONS: In this study, it was demonstrated that silk fibroin membranes coated with polyP have the potential to act as a promising novel hemostatic device.

EpCAM-positive circulating tumor cells and serum AFP levels predict outcome after curative resection of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has high recurrence rates exceeding 50% despite curative resection. The serum biomarker alpha-fetoprotein (AFP) is a well-known prognostic marker for HCC. EpCAM-positive circulating tumor cells (CTC) have a high predictive value for early HCC recurrence after curatively intended resection, most likely indicating micro-metastases at the time of resection. However, sensitivity remains low. The objective of this study was to evaluate a composite test comprising both CTC and AFP to identify patients at high risk for early HCC recurrence. We prospectively enrolled 58 patients undergoing curative intended resection for HCC at a tertiary referral center. Blood specimens were obtained prior to resection and analyzed for EpCAM-positive CTC and serum AFP levels. A positive result was defined as either detection of CTC or AFP levels ≥ 400 ng/ml. Eight patients tested positive for CTC, seven for AFP, and two for both markers. A positive composite test was significantly associated with shorter early recurrence-free survival (5 vs. 16 months, p = 0.005), time to recurrence (5 vs. 16 months, p = 0.011), and overall survival (37 vs. not reached, p = 0.034). Combining CTC and AFP identified patients with poor outcome after surgical resection, for whom adjuvant or neoadjuvant therapies may be particularly desirable.

Cardiovascular Functions of Ena/VASP Proteins: Past, Present and Beyond.

Actin binding proteins are of crucial importance for the spatiotemporal regulation of actin cytoskeletal dynamics, thereby mediating a tremendous range of cellular processes. Since their initial discovery more than 30 years ago, the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family has evolved as one of the most fascinating and versatile family of actin regulating proteins. The proteins directly enhance actin filament assembly, but they also organize higher order actin networks and link kinase signaling pathways to actin filament assembly. Thereby, Ena/VASP proteins regulate dynamic cellular processes ranging from membrane protrusions and trafficking, and cell-cell and cell-matrix adhesions, to the generation of mechanical tension and contractile force. Important insights have been gained into the physiological functions of Ena/VASP proteins in platelets, leukocytes, endothelial cells, smooth muscle cells and cardiomyocytes. In this review, we summarize the unique and redundant functions of Ena/VASP proteins in cardiovascular cells and discuss the underlying molecular mechanisms.

In-vivo functions and regulation of polyphosphate in the vascular system.

PURPOSE OF REVIEW: Polyphosphate, an inorganic polymer consisting of linearly linked phosphate subunits, is ubiquitously found in living organisms. Functions and regulation of the polymer have been analyzed in plants, bacteria and yeast; however, the roles of polyphosphate in mammals are still emerging. RECENT FINDINGS: In contrast to synthetic polyphosphate that has been extensively utilized in ex-vivo studies, natural polyphosphate is complexed with bivalent cations (mostly Ca 2+ ) and regardless of chain length, forms microparticles that are retained on the surface of procoagulant platelets, platelet-derived microparticles and cancer extracellular vesicles. On cell surfaces, these Ca 2+ /polyphosphate aggregates initiate the factor XII-driven contact system, triggering proinflammatory and procoagulant reactions through the kallikrein kinin system and intrinsic pathway of coagulation, respectively. Polyphosphate inhibitors interfere with thrombosis while sparing hemostasis, replicating the effect of factor XII neutralizing agents. Furthermore, polyphosphate binds to platelet factor 4, which has implications for autoimmune thrombotic diseases, such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT), potentially contributing to their pathogenesis. The metabolism and organ-specific distribution of the polymer remain incompletely defined and is the topic of ongoing research. SUMMARY: Polyphosphate acts as a procoagulant and proinflammatory mediator. Neutralizing polyphosphate provides well tolerated thromboprotection, mimicking the effects of factor XII deficiency.

Regulation of coagulation activation in newly diagnosed AML by the heme enzyme myeloperoxidase.

INTRODUCTION: Patients with acute myeloid leukemia (AML) are at increased risk of thrombohemorrhagic complications. Overexpressed tissue factor (TF) on AML blasts contributes to systemic coagulation activation. We have recently shown that the heme enzyme myeloperoxidase (MPO) negatively regulates TF procoagulant activity (PCA) on myelomonocytic cells in vitro. We now aimed to further characterize the functional interaction of MPO and TF in AML in vivo. METHODS: We prospectively recruited 66 patients with newly diagnosed AML. TF PCA of isolated peripheral blood mononuclear cells (PBMC) was assessed by single-stage clotting assay in the presence or absence of inhibitors against MPO catalytic activity (ABAH) or against MPO-binding integrins (anti-CD18). MPO in plasma and in AML blasts was measured by ELISA, and plasma D-dimers and prothrombin fragment F1+2 were quantified by automated immunoturbidimetric and chemiluminescence assays, respectively. RESULTS: Patients with AML had significantly higher MPO plasma levels compared to healthy controls and exhibited increased levels of D-dimers and F1+2. In vivo thrombin generation was mediated by TF PCA on circulating PBMC. Ex vivo incubation of isolated PBMC with ABAH or anti-CD18 antibody resulted in either increased or decreased TF PCA. The strong and robust correlation of F1+2 with TF PCA of circulating PBMC was abrogated at MPO plasma levels higher than 150 ng/mL, indicating a modulatory role for MPO on TF-mediated in vivo thrombin generation above this threshold. CONCLUSION: Our study indicates that catalytically active MPO released by circulating myeloblasts regulates TF-dependent coagulation in patients with newly diagnosed AML in a CD18-dependent manner.

Targeting NETs using dual-active DNase1 variants.

Background: Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Methods: Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. Results: We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Conclusion: Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states.

Repetitive Antigen Responses of LDL-Reactive CD4+ T Cells Induce Tr1 Cell-Mediated Immune Tolerance.

BACKGROUND: Inflammation triggered by the deposition of LDL (low-density lipoprotein) in the arterial wall leads to the development of atherosclerosis. Regulatory T (Treg) cells inhibit vascular inflammation through the induction of immune tolerance toward LDL-related antigens. However, tolerogenic mechanisms that promote the generation of LDL-specific Treg cells in vivo remain unclear. METHODS: We identified LDL-specific T cells by activation-induced marker expression and analyzed expression profiles and suppressive functions of TCR (T-cell antigen receptor)-transgenic T cells upon repetitive transfer into antigen-transgenic mice via flow cytometry. RESULTS: We investigated the naturally occurring Treg-cell response against human LDL in standard chow diet-fed mice that are transgenic for human ApoB100 (apolipoprotein B100). We found that IL (interleukin)-10 expression in LDL-specific T cells from spleen increases with age, albeit LDL-specific populations do not enlarge in older mice. To investigate the generation of IL-10-producing LDL-specific T cells, we transferred naive CD4+ T cells recognizing human ApoB100 from TCR-transgenic mice into human ApoB100-transgenic mice. Adoptive transfer of human ApoB100-specific T cells induced immune tolerance in recipient mice and effectively inhibited activation of subsequently transferred naive T cells of the same specificity in vivo. Moreover, repetitive transfers increased the population of Treg type 1 cells that suppress ApoB100-specific responses via IL-10. In a translational approach, LDL-specific Treg type 1 cells from blood of healthy donors suppressed the activation of monocytic THP-1 cells in an IL-10-dependent manner. CONCLUSIONS: We show that repetitive transfer of naive ApoB100-specific T cells and recurrent LDL-specific T-cell stimulation induces Treg type 1 cell-mediated immune tolerance against LDL in vivo. Our results provide insight into the generation of autoantigen-specific anti-inflammatory T cells under tolerogenic conditions.

Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism.

Venous thromboembolism (VTE) is a common, multi-causal disease with potentially serious short- and long-term complications. In clinical practice, there is a need for improved plasma biomarker-based tools for VTE diagnosis and risk prediction. Here we show, using proteomics profiling to screen plasma from patients with suspected acute VTE, and several case-control studies for VTE, how Complement Factor H Related 5 protein (CFHR5), a regulator of the alternative pathway of complement activation, is a VTE-associated plasma biomarker. In plasma, higher CFHR5 levels are associated with increased thrombin generation potential and recombinant CFHR5 enhanced platelet activation in vitro. GWAS analysis of ~52,000 participants identifies six loci associated with CFHR5 plasma levels, but Mendelian randomization do not demonstrate causality between CFHR5 and VTE. Our results indicate an important role for the regulation of the alternative pathway of complement activation in VTE and that CFHR5 represents a potential diagnostic and/or risk predictive plasma biomarker.

Seroprevalence of arthropod-borne bacterial infections in homeless individuals in Hamburg in 2020.

PURPOSE: The number of homeless people in Germany is steadily increasing. Due to their often precarious living conditions, this specific population may be increasingly exposed to ectoparasites that can transmit various pathogens. To assess the prevalence and thus the risk of such infections, we analyzed the seropositivity of rickettsiosis, Q fever, tularemia and bartonellosis in homeless individuals. METHODS: A total of 147 homeless adults from nine shelters in Hamburg, Germany, were included. The individuals underwent questionnaire-based interviewing, physical examination, and venous blood was drawn between May and June 2020. Blood samples were analyzed for antibodies against rickettsiae (Rickettsia typhi and R. conorii), Coxiella burnetii, Francisella tularensis and bartonellae. RESULTS AND CONCLUSION: A very low seroprevalence of R. typhi and F. tularensis infection was found (0-1%), while antibodies against R. conorii and C. burnetii were more common (7% each), followed by a relatively high seroprevalence of 14% for bartonellosis. Q fever seroprevalence was associated with the country of origin, whereas bartonellosis seroprevalence was associated with the duration of homelessness. Preventive measures targeting ectoparasites, especially body lice, should be put in place continuously.

Human platelets release amyloid peptides ß1-40 and ß1-42 in response to haemostatic, immune, and hypoxic stimuli.

Background: Platelets contain high levels of amyloid ß (Aß) peptides and have been suggested to participate in the deposition of amyloid plaques in Alzheimer's Disease (AD). Objectives: This study aimed to determine whether human platelets release pathogenic Aß peptides Aß1-42 and Aß1-40 and to characterise the mechanisms regulating this phenomenon. Methods and Results: Enzyme-linked immunosorbent assays (ELISAs) revealed that the haemostatic stimulus thrombin and the pro-inflammatory molecule lipopolysaccharide (LPS) induce platelet release of both Aß1-42 and Aß1-40. Notably, LPS preferentially induced the release of Aß1-42, which was potentiated by the reduction of oxygen from atmospheric levels to physiological hypoxia. The selective ß secretase (BACE) inhibitor LY2886721 showed no effect on the release of either Aß1-40 or Aß1-42 in our ELISA experiments. This suggested a store-and-release mechanism that was confirmed in immunostaining experiments showing co-localisation of cleaved Aß peptides with platelet alpha granules. Conclusions: Taken together, our data suggest that human platelets release pathogenic Aß peptides as a result of a store-and-release mechanism rather than a de novo proteolytic event. Although further studies are required to fully characterise this phenomenon, we suggest the possibility of a role for platelets in the deposition of Aß peptides and the formation of amyloid plaques. Interestingly, the combination of hypoxia and inflammation that we simulated in vitro with reduced oxygen tension and LPS may increase the release of fibrillogenic Aß1-42 and, consequently, exacerbate amyloid plaque deposition in the brain of AD patients.