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Interleukin-4 (IL-4) and its receptors (IL-4R) promote the proliferation and polarization of macrophages. However, it is unknown if IL-4R also influences monocyte homeostasis and if steady state IL-4 levels are sufficient to affect monocytes. Employing full IL-4 receptor alpha knockout mice (IL-4Rα

Background Monocyte–platelet aggregates (MPAs) are a sensitive marker of in vivo platelet activation in acute coronary syndrome (ACS) and associated with clinical outcomes. MicroRNAs (miRs) play an important role in the regulation of platelet activation, and may influence MPA formation. Both, miRs and MPA, could be influenced by the type of P2Y12 inhibitor. Aim To study the association of platelet-related miRs with MPA formation in ACS patients on dual antiplatelet therapy (DAPT), and to compare miRs and MPA levels between prasugrel- and ticagrelor-treated patients. Methods and Results We analyzed 10 circulating platelet-related miRs in 160 consecutive ACS patients on DAPT with low-dose aspirin and either prasugrel (n = 80) or ticagrelor (n = 80). MPA formation was measured by flow cytometry without addition of platelet agonists and after simulation with the toll-like receptor (TLR)-1/2 agonist Pam3CSK4, adenosine diphosphate (ADP), or arachidonic acid (AA). In multivariate regression analyses, we identified miR-21 (β = 9.50, 95% confidence interval [CI]: 1.60–17.40, p = 0.019) and miR-126 (β = 7.50, 95% CI: 0.55–14.44, p = 0.035) as independent predictors of increased MPA formation in vivo and after TLR-1/2 stimulation. In contrast, none of the investigated miRs was independently associated with MPA formation after stimulation with ADP or AA. Platelet-related miR expression and MPA formation did not differ significantly between prasugrel- and ticagrelor-treated patients. Conclusion Platelet-related miR-21 and miR-126 are associated with MPA formation in ACS patients on DAPT. miRs and MPA levels were similar in prasugrel- and ticagrelor-treated patients.

Atherosclerosis is a chronic, inflammatory disease of the vessel wall where triggered immune cells bind to inflamed endothelium, extravasate and sustain local inflammation. Leukocyte adhesion and extravasation are mediated by adhesion molecules expressed by activated endothelial cells, like intercellular adhesion molecule 1 (ICAM-1). Extracellular adherence protein (Eap) from Staphylococcus aureus binds to a plethora of extracellular matrix proteins, including ICAM-1 and its ligands macrophage-1 antigen (Mac-1, α

BACKGROUND: Plg-R(KT), a unique transmembrane plasminogen receptor, enhances the activation of plasminogen to plasmin, and localizes the proteolytic activity of plasmin on the cell surface. OBJECTIVES: We investigated the role of Plg-R(KT) in adipose function, metabolic homeostasis and obesity. METHODS: We used adipose tissue (AT) sections from bariatric surgery patients and from high fat diet (HFD)-induced obese mice together with immunofluorescence and real-time PCR to study adipose expression of Plg-R(KT). Mice genetically deficient in Plg-R(KT) and littermate controls fed a HFD or control low fat diet (LFD) were used to determine the role of Plg-R(KT) in insulin resistance, glucose tolerance, T2D, and associated mechanisms including adipose inflammation, fibrosis, and ectopic lipid storage. The role of Plg-R(KT) in adipogenesis was determined using 3T3-L1 preadipocytes and primary cultures established from Plg-R(KT) deficient and littermate control mice. RESULTS: Plg-R(KT) was highly expressed in both human and mouse adipose tissue AT, and its levels dramatically increased during adipogenesis. Plg-R(KT) deficient mice, when fed a HFD, gained more weight, developed more hepatic steatosis and were more insulin resistant/glucose intolerant than HFD-fed wild type littermates. Mechanistically, these metabolic defects were linked with increased AT inflammation, AT macrophage and T cell accumulation, adipose and hepatic fibrosis, and decreased insulin signaling in the AT and liver. Moreover, Plg-R(KT) regulated the expression of PPARγ and other adipogenic molecules, suggesting a novel role for Plg-R(KT) in the adipogenic program. CONCLUSIONS: Plg-R(KT) coordinately regulates multiple aspects of adipose function that are important to maintain efficient metabolic homeostasis.

Training of the innate immune system with orally ingested bacterial extracts was demonstrated to have beneficial effects on infection clearance and disease outcome. The aim of our study was to identify cellular and molecular processes responsible for these immunological benefits. We used a murine coronavirus (MCoV) A59 mouse model treated with the immune activating bacterial extract Broncho-Vaxom (BV) OM-85. Tissue samples were analysed with qPCR, RNA sequencing, histology, and flow cytometry. After BV OM-85 treatment, interstitial macrophages accumulated in lung tissue leading to a faster response of type I interferon (IFN) signalling after MCoV infection resulting in overall lung tissue protection. Moreover, RNA sequencing showed that lung tissue from mice receiving BV OM-85 resembled an intermediate stage between healthy and viral infected lung tissue at day 4, indicating a faster return to normal tissue homoeostasis. The pharmacologic effect was mimicked by adoptively transferring naive lung macrophages into lungs from recipient mice before virus infection. The beneficial effect of BV OM-85 was abolished when inhibiting initial type I IFN signalling. Overall, our data suggest that BV OM-85 enhances lung macrophages allowing for a faster IFN response towards a viral challenge as part of the oral-induced innate immune system training.

Apart from clinical data, mouse models for SARS have helped advance our knowledge in fighting against COVID-19. We emphasised on a physiological model of infection and host-compatibility and used the murine coronavirus mCoV-A59, which belongs to the same subgroup as SARS-CoV-2. Thus, we could mimic asymptomatic infections with an immune response that results in major virus clearance at day 10 post infection. Using histology, multi-colour flow cytometry and RNAseq of mCoV infected lungs, we were able to detect an intriguing temporal interplay of innate and adaptive immunity in virus resolution. Virus clearance was already evident after 4 days and RNAseq revealed the unexpectedly crucial role of innate immune cell regulation, being strongest regulated cellular pathway. Moreover, interferon type I signalling was also remarkably upregulated during this early phase; a hallmark of SARS-CoV infections and mainly produced by innate immune cells. Lung cell apoptosis was highest at day 10, at which we could also observe a distinct increase in cytotoxic CD8 T cells. This indicates that successful, rapid SARS-CoV clearance depends mainly on innate immunity rather than on adaptive responses, while at a later phase recruited CD8 T cells contribute to lung tissue damage. This early innate immune response might explain the observed decline of SARS-CoV-2 antibodies in asymptomatic or mild infections. Thus, we highlight the importance to strengthen the innate immune system at the beginning of a SARS-CoV infection with the requirement to dampen CD8 T cell function. Lastly, our model allows screening of compounds modulating the innate and adaptive immunity during disease.

Recent data suggest that the disease-associated microenvironment, known as the leukemic stem cell (LSC) niche, is substantially involved in drug resistance of LSC in BCR-ABL1(+) chronic myeloid leukemia (CML). Attacking the LSC niche in CML may thus be an effective approach to overcome drug resistance. We have recently shown that osteoblasts are a major site of niche-mediated LSC resistance against second- and third-generation tyrosine kinase inhibitors (TKI) in CML. In the present study, we screened for drugs that are capable of suppressing the growth and viability of osteoblasts and/or other niche cells and can thereby overcome TKI resistance of CML LSC. Proliferation was analyzed by determining (3)H-thymidine uptake in niche-related cells, and apoptosis was measured by Annexin-V/DAPI-staining and flow cytometry. We found that the dual PI3 kinase (PI3K) and mTOR inhibitor BEZ235 and the selective pan-PI3K inhibitor copanlisib suppress proliferation of primary osteoblasts (BEZ235 IC(50): 0.05 μM; copanlisib IC(50): 0.05 μM), the osteoblast cell line CAL-72 (BEZ235 IC(50): 0.5 μM; copanlisib IC(50): 1 μM), primary umbilical vein-derived endothelial cells (BEZ235 IC(50): 0.5 μM; copanlisib IC(50): 0.5 μM), and the vascular endothelial cell line HMEC-1 (BEZ235 IC(50): 1 μM; copanlisib IC(50): 1 μM), whereas no comparable effects were seen with the mTOR inhibitor rapamycin. Furthermore, we show that BEZ235 and copanlisib cooperate with nilotinib and ponatinib in suppressing proliferation and survival of osteoblasts and endothelial cells. Finally, BEZ235 and copanlisib were found to overcome osteoblast-mediated resistance against nilotinib and ponatinib in K562 cells, KU812 cells and primary CD34(+)/CD38(-) CML LSC. Together, targeting osteoblastic niche cells through PI3K inhibition may be a new effective approach to overcome niche-induced TKI resistance in CML. Whether this approach can be translated into clinical application and can counteract drug resistance of LSC in patients with CML remains to be determined in clinical trials.

Monocytes are activated in inflammatory conditions via a variety of cytokine receptors as well as in a procoagulatory setting through thrombin, acting upon protease-activated receptors (PARs). This study investigated the expression pattern of PAR1 and PAR3 on human monocyte subsets. Furthermore, a possible regulation of the expression of PAR1 and PAR3 in these cells by inflammatory activation were studied. CD16+ monocytes showed significantly higher levels of PAR1 and PAR3 as compared with CD16− monocytes. Ex vivo treatment of whole blood with lipopolysaccharide (LPS) increased PAR1 and PAR3 messenger ribonucleic acid (mRNA) in human monocytes. In addition, increase of PAR1 was seen in all three subsets upon LPS treatment, whereas PAR3 increased significantly only in CD16− monocytes and nonclassical CD16+ monocytes. Protein levels of PAR1 and PAR3 significantly increased on monocytes in vivo in human endotoxemia 1 hour after LPS infusion. PAR1 increased significantly in CD16− monocytes and nonclassical CD16+ monocytes. In this in vivo model, PAR3 was also significantly elevated in CD16− monocytes and increased slightly albeit not significantly in CD16+ monocytes. Endotoxemia increased plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression in monocytes in humans. Pretreatment of healthy volunteers with the PAR1 antagonist vorapaxar blocked the increase in PAI-1 but not the increase in TF. We here provide new evidence for a critical role for monocytes as cellular mediators that contribute to the activation of coagulation in diseases characterized by an inflammatory state.

The Kopernikus project “FlexASU” investigates an enhancement of the load flexibility of air separation units with the goal to synchronize their power consumption with the availability of renewable energy sources. The main heat exchanger of these plants was identified as a key component for a dynamic plant operation because startup, shutdown and load change procedures can lead to thermal stress and an increased lifetime consumption of the apparatus. Aluminium brazed plate fin heat exchangers (PFHE) are used in these plants because of their high process integration, low production cost and compact design. Finding the optimum operating scheme for the rectification columns depends on detailed knowledge of their effect on the lifetime of the PFHE.The transient behavior of temperature profiles within a PFHE can be calculated and a FEM-based model is available to investigate the thermal stress induced by dynamic temperature changes in the apparatus. Hence, the reduction of lifetime due to certain dynamic plant operating scenarios can be estimated and critical operating modes can be identified. For further development of the model, a PFHE test rig is designed which is representative for extreme operating conditions in air separation units. A test scenario matching the conditions of plant startup after extended downtime in cold condition has been designed. To reach a level of thermal stress comparable to industrial sized plants, the PFHE weighing around 1.5 tons is heated up to 50 °C before rapidly being cooled down by a stream of gaseous nitrogen at -173 °C in a cyclic operation scheme. This test scenario leads to thermal stress that is expected to damage the PFHE within a few weeks of testing and the location of maximum thermal stress in the FEM model matches typical cracks observed in PFHE of air separation units. The test rig is designed to gain a clearer understanding of the underlying damage mechanism and to evaluate measures for lowering thermal stress in further test series. The knowledge gained from the experimental investigations is crucial for maximizing the operation flexibility of an ASU plant.

Background: Fabry disease is a hereditary genetic defect resulting in reduced activity of the enzyme α-galactosidase-A and the accumulation of globotriaosylceramide (Gb3) in body fluids and cells. Gb3 accumulation was especially reported for the vascular endothelium in several organs. Methods: Three Fabry disease patients were screened using a micro-RNA screen. An in vitro approach in human endothelial cells was used to determine miRNA regulation by Gb3. Results: In a micro-RNA screen of three Fabry patients undergoing enzyme replacement therapy, we found that miRNAs let-7a and let-7d were significantly increased after therapy. We demonstrate in vitro in endothelial cells that Gb3 induced activation of NF-κB and activated downstream targets. In addition, NF-κB activity directly reduced let-7a and let-7d miRNA expression as inhibiting NF-kB nuclear entry abolished the Gb3 effects. Conclusion: We suggest that let-7a and let-7d are potential markers for enzyme activity and inflammation in Fabry disease patients.

Background Inflammation is a key process during atherosclerotic lesion development and propagation. Recent evidence showed clearly that especially the inhibition of interleukin (IL)-1β reduced atherosclerotic adverse events in human patients. Fatty acid oxidation (FAO) was previously demonstrated to interact with the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) pathway which is required for mature IL-1β secretion. To understand possible anti-inflammatory properties of FAO inhibition, we tested the effect of pharmacological FAO inhibition using the inhibitor for long-chain 3-ketoacyl coenzyme A thiolase trimetazidine on atherosclerotic plaque development and inflammation. Experimental Approach The effect of FAO inhibition was determined in LDL-R−/− male mice on a C57/BL6 background. In vitro effects of trimetazidine treatment were analyzed in human umbilical vein endothelial cells and human monocyte derived macrophages. Key Results We were able to demonstrate that inhibition of FAO reduced atherosclerotic plaque growth. We did not find direct anti-inflammatory properties of trimetazidine in endothelial cells or macrophages in vitro. However, we found that the activation of the NLRP3 system and the secretion of IL-1β were significantly reduced in macrophages after FAO inhibition. These results were confirmed in atherosclerotic lesions of mice treated with trimetazidine as they showed a significant reduction of IL-1β and cleaved caspase-1 in the atherosclerotic lesion as well as of IL-1β and IL-18 in the circulation. Conclusion Overall, we therefore suggest that the main mechanism of reducing inflammation of trimetazidine and FAO inhibition is the reduction of the NLRP-3 activation leading to reduced levels of the proinflammatory cytokine IL-1β.

Nicorandil, a balanced vasodilator, is used in the second-line therapy of angina pectoris. In this study, we aimed to illuminate the effects of nicorandil on inflammation, apoptosis, and atherosclerotic plaque progression. Twenty-five LDL-R -/- mice were fed a high-fat diet for 14 weeks. After 6 weeks mice were randomly allocated to treatment with nicorandil (10 mg/kg/day) or tap water. Nicorandil treatment led to a more stable plaque phenotype, displaying an increased thickness of the fibrous cap (p = 0.014), a significant reduction in cholesterol clefts (p = 0.045), and enhanced smooth muscle cell content (p = 0.009). In endothelial cells nicorandil did not reduce the induction of adhesion molecules or proinflammatory cytokines. In H2O2 challenged endothelial cells, pretreatment with nicorandil significantly reduced the percentage of late apoptotic/necrotic cells (p = 0.016) and the ratio of apoptotic to living cells (p = 0.036). Atherosclerotic lesions of animals treated with nicorandil exhibited a significantly decreased content of cleaved caspase-3 (p = 0.034), lower numbers of apoptotic nuclei (p = 0.040), and reduced 8-oxogunanine staining (p = 0.039), demonstrating a stabilizing effect of nicorandil in established atherosclerotic lesions. We suggest that nicorandil has a positive effect on atherosclerotic plaque stabilization by reducing apoptosis.

Supplemental Digital Content is available in the text., Objective: Macrophages are immune cells, capable to remodel the extracellular matrix, which can harbor extracellular DNA incorporated into neutrophil extracellular traps (NETs). To study the breakdown of NETs we studied the capability of macrophage subsets to degrade these structures in vitro and in vivo in a murine thrombosis model. Furthermore, we analyzed human abdominal aortic aneurysm samples in support of our in vitro and in vivo results. Approach and Results: Macrophages were seeded onto blood clots or isolated NETs and polarized. All macrophages were capable to degrade NETs. For initial breakdown, macrophages relied on extracellular deoxyribonucleases. Proinflammatory polarization enhanced NET degradation. The boost in degradation was because of increased macropinocytosis, as inhibition by imipramine diminished their NET breakdown. Inhibition of macropinocytosis in a murine thrombosis model led to increased NET burden and reduced thrombus resolution in vivo. When analyzing abdominal aortic aneurysm samples, macrophage density furthermore corresponded negatively with the amount of local NETs in the intraluminal thrombi as well as in the vessel wall, as increased macrophage density was associated with a reduction in NET burden. Conclusions: We provide evidence that macrophages degrade NETs by extracellular predigestion and subsequent uptake. Furthermore, we show that proinflammatory macrophages increase NET degradation through enhanced macropinocytosis, priming them for NET engulfment. Based on our findings, that inhibition of macropinocytosis in mice corresponded to increased NET amounts in thrombi and that local macrophage density in human abdominal aortic aneurysm is negatively associated with surrounding NETs, we hypothesize, that macrophages are able to degrade NETs in vivo.

Quantitative and functional analysis of mononuclear leukocyte populations is an invaluable tool to understand the role of the immune system in the pathogenesis of a disease. Cryopreservation of mononuclear cells (MNCs) is routinely used to guarantee similar experimental conditions. Immune cells react differently to cryopreservation, and populations and functions of immune cells change during the process of freeze–thawing. To allow for a setup that preserves cell number and function optimally, we tested four different cryopreservation media. MNCs from 15 human individuals were analyzed. Before freezing and after thawing, the distribution of leukocytes was quantified by flow cytometry. Cultured cells were stimulated using lipopolysaccharide, and their immune response was quantified by flow cytometry, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). Ultimately, the performance of the cryopreservation media was ranked. Cell recovery and viability were different between the media. Cryopreservation led to changes in the relative number of monocytes, T cells, B cells, and their subsets. The inflammatory response of MNCs was altered by cryopreservation, enhancing the basal production of inflammatory cytokines. Different cryopreservation media induce biases, which needs to be considered when designing a study relying on cryopreservation. Here, we provide an overview of four different cryopreservation media for choosing the optimal medium for a specific task.

The influence of inlet placement and distributor geometry on the steady-state efficiency and pressure drop of cryogenic plate-fin heat exchangers (PFHEs) is studied using a three-dimensional simulation model. The simulation model employs a porous media approach to combine computational fluid dynamics with well-established correlations for heat transfer and pressure drop in fin materials. An isothermal study is conducted for comparison of six different distributor geometries. The selection of a favorable design depends on a trade-off between heat transfer and allowable pressure drop of a specific task but the presented results can serve as a guideline. Furthermore, two different configurations of a 6-stream PFHE are compared to an idealized, pseudo one-dimensional design. Both feasible designs lead to a significant increase in pressure drop and the thermal efficiency of the feasible designs differs by 5 percentage points. This highlights the necessity of considering the inlet- and distributor configuration in the design of a cryogenic PFHE.

This work proposes a novel, three-dimensional model for transient simulation of cryogenic plate-fin heat exchangers (PFHE). This model is based on the combination of computational fluid dynamics with well-established correlations for calculating heat transfer and pressure drop occurring during fluid flow through various fin types. This reduces the complex geometry of a PFHE to a geometrically simple mesh, allowing for efficient simulation of large-scale industrial PFHE. Successful validation of the model is carried out using both data available in the literature and results calculated using commercial software. The necessity for a three-dimensional model is further demonstrated using a transient case study in which the design of inlet distributors is shown to cause unfavorable distribution of fluid flow and hence severe temperature maldistribution in the PFHE. These results show that the proposed model can be useful in both detailed engineering and lifetime investigations of flexible PFHE.

In this paper, an advanced adsorption cooling cum desalination system comprising four adsorption beds and two evaporators is presented. The proposed system continuously produces desalinated water in the condenser and chilled water at two different temperature levels. It employs silica gel as adsorbent and water as adsorbate pair. One evaporator is operated at low pressure for low temperature cooling (≈10 °C) while the pressure of the other evaporator is set higher for sufficient desalination and cooling water production at high temperature (≈20 °C). The proposed system is simulated with two different configurations. In the first configuration each evaporator is connected to only two beds, whereas in the second configuration, all beds are connected to both evaporators for consecutive low pressure and high pressure adsorption. Both configurations are compared with conventional system in regards of specific cooling power for the two temperature levels (SCP-L and SCP-H), specific daily water production (SDWP), performance ratio (PR), coefficient of performance (COP) and overall conversion ratio (OCR). It is found that the proposed system with the second configuration outperforms the conventional 4 bed system by 45% and 40% in terms of SDWP and OCR. Further, the flexibility of the system is proven by the variation of timing schemes, leading to an adjustment in SCP and SDWP depending on the fresh water and cooling energy demands. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore)