Abstract
The protease-activated receptor-1 (PAR-1) is critically involved in the co-activation of coagulation and inflammatory responses. Vorapaxar is a reversible, orally active, low molecular ...weight, competitive antagonist of PAR-1.We investigated the effects of PAR-1 inhibition by vorapaxar on the inflammatory response, the activation of coagulation, fibrinolysis and endothelium during experimental endotoxemia. In this randomized, double blind, crossover trial, 16 healthy volunteers received a bolus infusion of 2 ng/kg lipopolysaccharide (LPS) ± placebo/vorapaxar with a washout period of 8 weeks. Vorapaxar dosing was guided by thrombin receptor-activating peptide-6-induced whole blood aggregometry. Participants received 10 mg vorapaxar or placebo as an initial dose and, depending on the aggregometry, potentially an additional 10 mg. Goal was > 80% inhibition of aggregation compared with baseline. Vorapaxar significantly reduced the LPS-induced increase in pro-thrombin fragments F1 + 2 by a median of 27% (quartiles: 11–49%), thrombin–anti-thrombin concentrations by 22% (–3 to 46%) and plasmin–anti-plasmin levels by 38% (23–53%). PAR-1 inhibition dampened peak concentrations of tumour necrosis factor -α, interleukin-6 and consequently C-reactive protein by 66% (−11–71%), 50% (15–79%) and 23% (16–38%), respectively. Vorapaxar decreased maximum von Willebrand factor levels by 29% (26–51%) and soluble E-selectin concentrations by 30% (25–38%) after LPS infusion. PAR-1 inhibition did not affect thrombomodulin, soluble P-selectin and platelet factor-4 concentrations.
PAR-1 inhibition significantly reduced the activation of coagulation, fibrinolysis, the inflammatory response and endothelial activation during experimental human endotoxemia.
Macrophages are versatile cells that can be polarized by the tissue environment to fulfill required needs. Proinflammatory polarization is associated with increased tissue degradation and propagation ...of inflammation whereas alternative polarization within a Th2 cytokine environment is associated with wound healing and angiogenesis. To understand if polarization of macrophages can lead to a procoagulant macrophage subset we polarized human monocyte derived macrophages to a proinflammatory and an alternative activation state. Alternative polarization with interleukin-4 and IL-13 led to a macrophage phenotype characterized by increased tissue factor (TF) production and release and by an increase in extracellular vesicle production. In addition, also TF activity was enhanced in extracellular vesicles of alternatively polarized macrophages. This TF induction was dependent on signal transducer and activator of transcription-6 signaling and poly ADP ribose polymerase activity. In contrast to monocytes, human macrophages did not show increased tissue factor expression upon stimulation with lipopolysaccharide and interferon-γ. Previous polarization to either a proinflammatory or an alternative activation subset does not change the subsequent stimulation of TF. The inability of proinflammatory activated macrophages to respond to lipopolysaccharide and interferon-γ with an increase in TF production seems to be due to an increase in TF promoter methylation and was reversible when treating these macrophages with a demethylation agent. In conclusion, we provide evidence that proinflammatory polarization of macrophages does not lead to enhanced procoagulatory function, whereas alternative polarization of macrophages leads to an increased expression of TF and increased production of TF bearing extracellular vesicles by these cells suggesting a procoagulatory phenotype of alternatively polarized macrophages.
Macrophages are versatile immune cells capable of polarizing into functional subsets depending on environmental stimulation. In atherosclerotic lesions, proinflammatory polarized macrophages are ...associated with symptomatic plaques, whereas Th2 (T-helper cell type 2) cytokine-polarized macrophages are inversely related with disease progression. To establish a functional cause for these observations, we analyzed extracellular matrix degradation phenotypes in polarized macrophages.
We provide evidence that proinflammatory polarized macrophages rely on membrane-bound proteases including MMP-14 (matrix metalloproteinase-14) and the serine protease uPA (urokinase plasminogen activator) together with its receptor uPAR for extracellular matrix degradation. In contrast, Th2 cytokine alternatively primed macrophages do not show different proteolytic activity in comparison to unpolarized macrophages and lack increased localization of MMP-14 and uPA receptor to the cell membrane. Nonetheless, they express the highest amount of the serine protease uPA. However, uPA activity is blocked by similarly increased expression of its inhibitor PAI-1 (plasminogen activator inhibitor 1). When inhibiting PAI-1 or when analyzing macrophages deficient in PAI-1, Th2 cytokine-polarized macrophages display the same matrix degradation capability as proinflammatory-primed macrophages. Within atherosclerotic lesions, macrophages positive for the alternative activation marker CD206 express high levels of PAI-1. In addition, to test changed tissue remodeling capacities of alternatively activated macrophages, we used a bleomycin lung injury model in mice reconstituted with PAI-1
bone marrow. These results supported an enhanced remodeling phenotype displayed by increased fibrosis and elevated MMP activity in the lung after PAI-1 loss.
We were able to demonstrate matrix degradation dependent on membrane-bound proteases in proinflammatory stimulated macrophages and a forced proteolytical quiescence in alternatively polarized macrophages by the expression of PAI-1.
Colorectal cancer (CRC) is one of the most common cancers worldwide and demands more effective treatments. We sought to identify tumor selective CRC antigens and their therapeutic potential for ...cytotoxic T-cell targeting by transcriptomic and immunohistochemical analysis. LY6G6D was identified as a tumor selectively expressed CRC antigen, mainly in the microsatellite stable (MSS) subtype. A specific anti LY6G6D/CD3 T cell engager (TcE) was generated and demonstrated potent tumor cell killing and T cell activation
in vitro
. Ex vivo treatment of primary patient-derived CRC tumor slice cultures with the LY6G6D/CD3 TcE led to IFNγ secretion in LY6G6D positive tumor samples.
In vivo
, LY6G6D/CD3 TcE monotherapy demonstrated tumor regressions in pre-clinical mouse models of engrafted human CRC tumor cells and PBMCs. Lastly, 2D and 3D cocultures of LY6G6D positive and negative cells were used to explore the bystander killing of LY6G6D negative cells after specific activation of T cells by LY6G6D positive cells. LY6G6D/CD3 TcE treatment was shown to lyse target negative cells in the vicinity of target positive cells through a combined effect of IFNγ, TNFα and Fas/FasL. In summary, LY6G6D was identified as a selectively expressed CRC antigen that can be utilized to potently re-direct and activate cytotoxic T-cells to lyse LY6G6D expressing CRC using a TcE. This effect can be spread to target negative neighboring tumor cells, potentially leading to improved therapeutic efficacy.
Circulating extracellular vesicles are small particles enclosed by a phospholipid bilayer. Vesicles deriving directly from the cellular membrane by an active budding process retain cell origin ...specific proteins and RNA. These vesicles carry pathophysiological information from their parental cell and hold the potential to allow analysis of organs without the need for a biopsy. We included in our study 27 patients undergoing bariatric surgery. Hepatic extracellular vesicles were determined by flow cytometry. mRNA specific for hepatic cellular origin was determined in the extracellular vesicle fraction using qPCR. Surgery led to a massive reduction of weight and overall hepatic stress as determined by alanine transaminase (ALT), aspartate transaminase (AST) and γ-glutamyltransferase (GGT). Total extracellular vesicle numbers were reduced after bariatric surgery. Liver specific vesicles identified by HepPar1 or asialoglycoprotein receptor (ASGPR) were significantly reduced after bariatric surgery in both AnnexinV
and AnnexinV
subgroups. When analyzing circulating liver-specific mRNAs, we found reduced levels of these mRNAs after surgery even though total circulating RNA remained unchanged. We conclude that circulating hepatic extracellular vesicles are detectable in samples from patients undergoing gastric bypass surgery. These vesicles are reduced after a reduction of hepatic stress also observed with classic liver enzyme measurements. We conclude that ASGPR or HepPar positive vesicles hold the potential to serve as liver specific vesicle markers.
Abstract Background Monocytes form an important part of the human innate immune system by taking part in inflammatory reactions. With time, monocytes have gained interest in the role they may play ...during the event of myocardial infarction (MI). The current paradigm suggests that monocytes consist of three subdivisions which differ in phenotypic and dynamic patterns after an MI. In the inflammation that ensues, the different subsets have been shown to have an impact on reparative processes and patient recovery. Methods & results We searched Medline and Embase until April 5, 2016, for observational studies or clinical trials regarding monocyte functions and dynamics in MI. Apart from studies in humans, extensive work has been done in mice in an effort to understand the complex nature of monocyte dynamics. Animal models might add useful information on mapping these processes. Conclusion The question still remains whether animal data can, to a certain degree, be extrapolated to monocyte functions during human MI. This review aims to summarize current available evidence on both mice and men with particular focus on the understanding of monocyte subsets dynamics and effects in human MI.
Small molecule IAP antagonists - SMAC mimetics (SM) - are being developed as an anticancer therapy. SM therapy was demonstrated not only to sensitize tumor cells to TNFα-mediated cell death but also ...to exert immunostimulatory properties. Their good safety and tolerability profile, plus promising preclinical data, warrants further investigation into their various effects within the tumor microenvironment. Using in vitro models of human tumor cells and fibroblast spheroids co-cultured with primary immune cells, we investigated the effects of SM on immune cell activation. SM treatment induces the maturation of human PBMC- and patient-derived dendritic cells (DC), and modulates cancer-associated fibroblasts towards an immune interacting phenotype. Finally, SM-induced tumor necroptosis further enhances DC activation, leading also to higher T-cell activation and infiltration into the tumor site. These results highlight the relevance of using heterotypic in vitro models to investigate the effects of targeted therapies on different components of the tumor microenvironment.
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•Heterotypic 3D models enable the analysis of targeted therapy effects on the TME•SMAC mimetics (SM) activate PBMC and patient dendritic cells, T cells, and NK cells•SM modulate cancer-associated fibroblasts into an inflammatory status•SM-induced necroptosis enhances DC activation, increasing T cell infiltration
Biological sciences; Systems biology; Cancer systems biology; Cancer
Mitopus morio is a widespread harvestman species occurring in most of Europe and in moderate and cold‐moderate zones of Asia and North America. The species is characterized by extreme variability in ...body size and leg length. As leg length is correlated with habitat temperature, M. morio has been considered as an example of Allen's rule. Recently, observations for a single location in Tyrol, Austria, indicated the absence of mating between short‐ and long‐legged individuals. This study examines for signs of putative cryptic species in M. morio using an integrative approach that combines mating trials, amplified fragment length polymorphism whole‐genome scans, mitochondrial sequences and morphometrics. The mating trials did not corroborate the initial hypothesis of a reproductive barrier associated with leg size. Both types of genetic data revealed the existence of three distinct groups, in line with the mating results but largely unrelated to leg morphology and geographical origin of specimens. Morphometric characters supporting the findings of the other disciplines were identified using a supervised approach. We infer from all data together the existence of strongly diverged cryptic lineages among the analysed individuals, cautiously interpret them as three sympatric species and conclude that in these harvestmen Allen's rule applies at different levels. Due to the unexpected amount of differentiation found within a geographical scale very small compared with the distribution of M. morio, we suggest a thorough revision of the genus prior to formal taxonomic changes. Our case study underlines the general applicability of the integrative taxonomic protocol used and highlights the relevance of several rationales implemented in the protocol.
Abstract
Background
Postoperative atrial fibrillation (POAF) is assumed as a complex and multifactorial interaction of different pathogenic factors. Data suggests an inflammatory process as the main ...trigger of this specific type of atrial fibrillation. CD8
+
T lymphocytes that lack the surface protein CD28 were found to be crucially involved in chronic inflammatory processes within the cardiovascular system. Of utmost interest, these so-called CD8
+
CD28
null
T cells are known to present with autoaggressive behavior and deleterious cytotoxic effects on human tissue.
Methods
A total of 129 patients undergoing elective cardiac valve and/or coronary artery bypass graft surgery were enrolled. Fluorescence-activated cell sorting was performed to investigate lymphocyte subsets. Patients were stratified in two subgroups according to patients developing POAF (
n
= 60) and individuals free of POAF (
n
= 69).
Results
Comparing patients developing POAF to individuals free of POAF, the fraction of CD8
+
lymphocytes was significantly higher in individuals developing POAF (30.5% POAF vs. 25.7% no-POAF;
p
= 0.021). Interestingly, also the fraction of CD8
+
CD28
null
T lymphocytes was significantly higher in the POAF subgroup (66.7% POAF vs. 61.6% non-POAF;
p
= 0.043). Multivariate logistic regression proved that the fraction of CD8
+
CD28
null
cells is a strong and independent prognosticator for the development of POAF with an adjusted odds ratio per 1 standard deviation of 3.21 (95% confidence interval 1.01–10.18;
p
= 0.048).
Conclusion
We found that cytotoxic CD8
+
CD28
null
T lymphocytes proved to be a strong and independent predictor for the development of POAF after elective cardiac surgery. Our results potentially indicate an autoimmune impact of this preexisting, highly cytotoxic T cell subset in the pathogenesis of POAF.
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