The blood coagulation test is an indispensable test for monitoring the blood coagulation and fibrinolysis functions. Currently, activated partial thromboplastin time (APTT) is the most widely used approach to coagulation testing. However, APTT reagents need to be optimized due to the fact that they are unstable, highly variable, and cannot be easily controlled. In this study, we created apoptotic cell-inspired methacryloyloxyethyl phosphorylserine (MPS) particles for blood coagulation as an alternative to conventional APTT reagents. Particle size could be controlled by changing the concentration of the polymer. The blood coagulation ability of particles was stable at different environmental temperatures. Moreover, the procoagulant activity could be enhanced by increasing the concentration to 0.06 mg/mL and reducing the size of the particles to around 900 nm. Fibrin clotted by particles showed no significant difference from that formed by APTT regent Actin FSL. We propose that MPS particles are a potential alternative to Actin FS for the application of blood coagulation tests.

Spinal cord injury (SCI) is a severe neurological complication following spinal fracture, which has long posed a challenge for clinicians. Microglia play a dual role in the pathophysiological process after SCI, both beneficial and detrimental. The underlying mechanisms of microglial actions following SCI require further exploration. The present study combined three different machine learning algorithms, namely weighted gene co-expression network analysis, random forest analysis and least absolute shrinkage and selection operator analysis, to screen for differentially expressed genes in the GSE96055 microglia dataset after SCI. It then used protein-protein interaction networks and gene set enrichment analysis with single genes to investigate the key genes and signaling pathways involved in microglial function following SCI. The results indicated that microglia not only participate in neuroinflammation but also serve a significant role in the clearance mechanism of apoptotic cells following SCI. Notably, bioinformatics analysis and lipopolysaccharide + UNC569 (a MerTK-specific inhibitor) stimulation of BV2 cell experiments showed that the expression levels of Anxa2, Myo1e and Spp1 in microglia were significantly upregulated following SCI, thus potentially involved in regulating the clearance mechanism of apoptotic cells. The present study suggested that Anxa2, Myo1e and Spp1 may serve as potential targets for the future treatment of SCI and provided a theoretical basis for the development of new methods and drugs for treating SCI.

The process of dead cell clearance by phagocytic cells, called efferocytosis, prevents inflammatory cell necrosis and promotes resolution and repair. Defective efferocytosis contributes to the progression of numerous diseases in which cell death is prominent, including liver disease. Many gaps remain in our understanding of how hepatic macrophages carry out efferocytosis and how this process goes awry in various types of liver diseases. Thus far, studies have suggested that, upon liver injury, liver-resident Kupffer cells and infiltrating monocyte-derived macrophages clear dead cells, limit inflammation, and, through macrophage reprogramming, repair liver damage. However, in unusual settings, efferocytosis can promote liver disease. In this review, we will focus on efferocytosis in various types of acute and chronic liver diseases, including metabolic dysfunction-associated steatohepatitis. Understanding the mechanisms and consequences of efferocytosis by hepatic macrophages has the potential to shed new light on liver disease pathophysiology and to guide new treatment strategies to prevent disease progression.

Clearance of dying cells, named efferocytosis, is a pivotal function of professional phagocytes that impedes the accumulation of cell debris. Efferocytosis can be experimentally assessed by differentially tagging the target cells and professional phagocytes and analyzing by cell imaging or flow cytometry. Here, we describe an assay to evaluate the uptake of apoptotic cells (ACs) by human macrophages in vitro by labeling the different cells with commercially available dyes and analysis by flow cytometry. We detail the methods to prepare and label human macrophages and apoptotic lymphocytes and the in vitro approach to determine AC uptake. This protocol is based on previously published literature and allows for in vitro modeling of the efficiency of AC engulfment during continual efferocytosis process. Also, it can be modified to evaluate the clearance of different cell types by diverse professional phagocytes.

Hyper-inflammatory immune response, a hallmark of severe COVID-19, is associated with increased mortality. Acute respiratory distress syndrome (ARDS) is a common manifestation. We undertook two phase I/II studies in five and then 16 subjects with severe/critical COVID-19 to assess the safety and preliminary efficacy of apoptotic cells (Allocetra™-OTS, Enlivex Therapeutics), a cellular immunomodulatory therapy that reprograms macrophages to reduce hyper-inflammatory response severity.
Eligible patients presenting to the Emergency Room with severe COVID-19 and respiratory dysfunction received one intravenous administration of Allocetra™-OTS and were monitored for adverse events (AEs) for 28 days. The primary aim was to determine the safety profile of treatment; secondary aims were recovery from ARDS, intensive care unit (ICU) and hospital length-of-stay, and mortality. Immune modulator markers were measured to elucidate the mechanism of action of Allocetra™-OTS.
21 patients with severe-critical COVID-19 of Gamma, Alpha and Delta variants, were treated with a single dose of apoptotic cells. 19/21 patients had mild-to-severe ARDS at presentation. Median age was 53 years, 16/21 were males, 16/21 were overweight/obese. No serious related adverse events (SAEs) were reported. All 21 study subjects survived to day 28 (end of study); 19/21 recovered completely. Comparable mortality rates at the hospital were 3.8%-8.9% for age- and gender-matched patients, and 39%-55% for critical patients. Recovering patients exhibited rapid ARDS resolution and parallel resolution of inflammation markers and elevated cytokines/chemokines.
In patients with severe/critical COVID-19 associated with ARDS, Allocetra™-OTS was safe, well-tolerated, and showed promising results for resolution of respiratory failure and inflammation.
https://clinicaltrials.gov/ct2/show/study/NCT04513470, https://clinicaltrials.gov/ct2/show/study/NCT04590053, Identifiers NCT04513470, NCT04590053.

Membranous CD14 is crucial in the phagocytic activity of neutrophils. However, the role of CD14(+) microparticles (MPs) derived from apoptotic neutrophils (apo-MP) during the phagocytic process is not clear. All trans-retinoic acid (ATRA) induces acute promyelocytic leukemic NB4 cells along granulocytic differentiation. In this study, we investigated the role of CD14(+)apo-MP in the cell-cell interaction during the phagocytic process of apoptotic cells by viable ATRA-NB4 cells. We firstly demonstrate that CD14 expression and phagocytic activity of NB4 cells were upregulated simultaneously after ATRA treatment in a time-dependent manner, and both were significantly enhanced via concurrent lipopolysaccharide treatment. The phagocytic activity of ATRA-NB4 cells and lipopolysaccharide-treated ATRA-NB4 cells were both significantly attenuated by pre-treating cells with an antibody specific to either CD14 or TLR4. Further flow cytometric analysis demonstrates that apoptotic ATRA-NB4 cells release CD14(+)apo-MP in an idarubicin dosage-dependent manner. Both CD14 expression and the phagocytic activity of viable ATRA-NB4 cells were significantly enhanced after incubation with apo-MP harvested from apoptotic ATRA-NB4 cells, and the apo-MP-enhanced phagocytic activity was significantly attenuated by pre-treating apo-MP with an anti-CD14 antibody before incubation with viable cells. We conclude that CD14(+)apo-MP derived from apoptotic ATRA-NB4 cells promotes the phagocytic activity of viable ATRA-NB4 cells in engulfing apoptotic cells.

Phosphatidylserine (PS) is a lipid component of the plasma membrane. It is asymmetrically distributed to the inner leaflet in live cells. In cells undergoing apoptosis, phosphatidylserine is exposed to the outer surfaces. The exposed phosphatidylserine acts as an evolutionarily conserved \"eat-me\" signal that attracts neighboring engulfing cells in metazoan organisms, including the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and mammals. During apoptosis, the exposure of phosphatidylserine to the outer surface of a cell is driven by the membrane scramblases and flippases, the activities of which are regulated by caspases. Cells undergoing necrosis, a kind of cell death frequently associated with cellular injuries and morphologically distinct from apoptosis, were initially believed to allow passive exposure of phosphatidylserine through membrane rupture. Later studies revealed that necrotic cells actively expose phosphatidylserine before any rupture occurs. A recent study in C. elegans further reported that the calcium ion (Ca2+) plays an essential role in promoting the exposure of phosphatidylserine on the surfaces of necrotic cells. These findings indicate that necrotic and apoptotic cells, which die through different molecular mechanisms, use common and unique mechanisms for promoting the exposure of the same \"eat me\" signal. This article will review the mechanisms regulating the exposure of phosphatidylserine on the surfaces of necrotic and apoptotic cells and highlight their similarities and differences.

This study investigated the potential effects of China dust (CD) exposure on cyclophosphamide (CP)-induced testicular toxicity in mice, focusing on spermatogenesis and oxidative damage. CP treatment reduced testicular and epididymal weight and sperm motility and enhanced sperm abnormality. Histopathological examination presented various morphological alterations in the testis, including increased exfoliation of spermatogenic cells, degeneration of early spermatogenic cells, vacuolation of Sertoli cells, a decreased number of spermatogonia/spermatocytes/spermatids, along with a high number of apoptotic cells. In addition, the testis exhibited reduced glutathione (GSH) levels and glutathione reductase (GR) activity and enhanced malondialdehyde (MDA) concentration. Meanwhile, CD exposure exacerbated testicular histopathological alterations induced by CP. CD exposure also aggravated oxidative damage by increasing the lipid peroxidative product MDA and decreasing GSH levels and antioxidant enzyme activities in the testis. These results suggest that CD exposure exacerbates CP-induced testicular toxicity in mice, which might be attributed to the induction of lipid peroxidation and reduced antioxidant activity.

Eosinophils are multifunctional, evolutionary conserved leukocytes that are involved in a plethora of responses ranging from regulation of tissue homeostasis, host defense and cancer. Although eosinophils have been studied mostly in the context of Type 2 inflammatory responses, it is now evident that they participate in Type 1 inflammatory responses and can respond to Type 1 cytokines such as IFN-γ. Notably, both Type 1- and Type 2 inflammatory environments are characterized by tissue damage and cell death. Collectively, this raises the possibility that eosinophils can interact with apoptotic cells, which can alter eosinophil activation in the inflammatory milieu. Herein, we demonstrate that eosinophils can bind and engulf apoptotic cells. We further show that exposure of eosinophils to apoptotic cells induces marked transcriptional changes in eosinophils, which polarize eosinophils towards an anti-inflammatory phenotype that is associated with wound healing and cell migration. Using an unbiased RNA sequencing approach, we demonstrate that apoptotic cells suppress the inflammatory responses of eosinophils that were activated with IFN-γ + E. coli (e.g., Type 1 eosinophils) and augment IL-4-induced eosinophil activation (e.g., Type 2 eosinophils). These data contribute to the growing understanding regarding the heterogeneity of eosinophil activation patterns and highlight apoptotic cells as potential regulators of eosinophil polarization.

Several chronic inflammatory diseases are associated with non-resolving inflammation. Conventional anti-inflammatory drugs fail to completely cure these diseases. Resolution pharmacology is a new therapeutic approach based on the use of pro-resolving mediators that accelerate the resolution phase of inflammation by targeting the productive phase of inflammation. Indeed, pro-resolving mediators prevent leukocyte recruitment and induce apoptosis of accumulated leukocytes. This approach is now called resolution therapy with the introduction of complex biological drugs and cell-based therapies. The main objective of resolution therapy is to specifically reduce the duration of the resolution phase to accelerate the return to homeostasis. Under physiological conditions, macrophages play a critical role in the resolution of inflammation. Indeed, after the removal of apoptotic cells (a process called efferocytosis), macrophages display anti-inflammatory reprogramming and subsequently secrete multiple pro-resolving factors. These factors can be used as resolution therapy. Here, we review the different mechanisms leading to anti-inflammatory reprogramming of macrophages after efferocytosis and the pro-resolving factors released by these efferocytic macrophages. We classify these mechanisms in three different categories: macrophage reprogramming induced by apoptotic cell-derived factors, by molecules expressed by apoptotic cells (i.e., \"eat-me\" signals), and induced by the digestion of apoptotic cell-derived materials. We also evoke that macrophage reprogramming may result from cooperative mechanisms, for instance, implicating the apoptotic cell-induced microenvironment (including cellular metabolites, specific cytokines or immune cells). Then, we describe a new drug candidate belonging to this resolution therapy. This candidate, called SuperMApo, corresponds to the secretome of efferocytic macrophages. We discuss its production, the pro-resolving factors present in this drug, as well as the results obtained in experimental models of chronic (e.g., arthritis, colitis) and acute (e.g., peritonitis or xenogeneic graft-versus-host disease) inflammatory diseases.