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The Journal of Biological Chemistry May 2024Ebola virus (EBOV) is a filamentous negative-sense RNA virus, which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and...
Ebola virus (EBOV) is a filamentous negative-sense RNA virus, which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work, we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane-binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine and phosphatidylinositol 4,5-bisphosphate in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles. In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together, our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.
Topics: Humans; Amino Acid Substitution; Cell Membrane; Ebolavirus; HEK293 Cells; Hemorrhagic Fever, Ebola; Mutation; Nucleoproteins; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylserines; Protein Binding; Static Electricity; Viral Core Proteins; Viral Matrix Proteins; Virion; Virus Assembly; Virus Release
PubMed: 38522519
DOI: 10.1016/j.jbc.2024.107213 -
Scientific Reports Mar 2024Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface...
Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF EVs and should be carefully controlled.
Topics: Thromboplastin; Fluorescent Dyes; Blood Coagulation; Extracellular Vesicles
PubMed: 38494537
DOI: 10.1038/s41598-024-56841-5 -
JHEP Reports : Innovation in Hepatology Apr 2024Hepatocellular necrosis is common in both acute and chronic liver injury and may evolve to fibrosis and liver failure. Injury leads to accumulation of necrotic cell...
BACKGROUND & AIMS
Hepatocellular necrosis is common in both acute and chronic liver injury and may evolve to fibrosis and liver failure. Injury leads to accumulation of necrotic cell debris in the liver, which drives persistent inflammation and poor recovery. This study investigated the role of natural antibodies (NAbs) in the clearance of necrotic cells in the injured liver, their impact on tissue regeneration and their potential as a therapy for acute liver injury.
METHODS
We used murine models of drug-induced liver injury and focal thermal injury in immunocompetent and antibody-deficient mice ( and IgMi). Intravital microscopy was used to investigate the role of NAbs in the phagocytosis of necrotic cells in the liver . Immunostainings were used to quantify the extent of liver necrosis (fibrin), antibody deposition (IgM and IgG) and cellular proliferation (Ki67).
RESULTS
Both IgM and IgG NAbs bound necrotic liver areas and opsonized multiple debris molecules released during hepatocellular necrosis such as DNA, histones, actin, phosphoinositides and mitochondrial cardiolipin, but not phosphatidylserine. and IgMi mice presented impaired recovery from liver injury, which was correlated to the sustained presence of necrotic debris in the tissue, prolonged inflammation and reduced hepatocellular proliferation. These defects were rescued by treating mice with NAbs after the induction of injury. Mechanistically, and , phagocytosis of necrotic debris was dependent on NAbs via Fcγ receptors and CD11b. Moreover, NAb-mediated phagocytosis of necrotic cell debris occurs in two waves, firstly driven by neutrophils and then by recruited monocytes. Importantly, supplementation of immunocompetent mice with NAbs also improved liver regeneration significantly, demonstrating the therapeutic potential of natural IgM and IgG.
CONCLUSION
NAbs drive the phagocytosis of necrotic cells in liver injury and promote liver regeneration and recovery.
IMPACT AND IMPLICATIONS
Treatment with natural antibodies after acute liver injury improved recovery by increasing the clearance of necrotic debris and by improving cellular proliferation in the liver. This preclinical study provides a basis for the development of an immunotherapy for patients with early-stage, reversible, liver injury that aims to prevent disease chronification into fibrosis and liver failure.
PubMed: 38481390
DOI: 10.1016/j.jhepr.2024.101013 -
International Journal of Molecular... Mar 2024Adrenaline has recently been found to trigger phosphatidylserine (PS) exposure on blood platelets, resulting in amplification of the coagulation process, but the...
Adrenaline has recently been found to trigger phosphatidylserine (PS) exposure on blood platelets, resulting in amplification of the coagulation process, but the mechanism is only fragmentarily established. Using a panel of platelet receptors' antagonists and modulators of signaling pathways, we evaluated the importance of these in adrenaline-evoked PS exposure by flow cytometry. Calcium and sodium ion influx into platelet cytosol, after adrenaline treatment, was examined by fluorimetric measurements. We found a strong reduction in PS exposure after blocking of sodium and calcium ion influx via Na/H exchanger (NHE) and Na/Ca exchanger (NCX), respectively. ADP receptor antagonists produced a moderate inhibitory effect. Substantial limitation of PS exposure was observed in the presence of GPIIb/IIIa antagonist, phosphoinositide-3 kinase (PI3-K) inhibitors, or prostaglandin E, a cyclic adenosine monophosphate (cAMP)-elevating agent. We demonstrated that adrenaline may develop a procoagulant response in human platelets with the substantial role of ion exchangers (NHE and NCX), secreted ADP, GPIIb/IIIa-dependent outside-in signaling, and PI3-K. Inhibition of the above mechanisms and increasing cytosolic cAMP seem to be the most efficient procedures to control adrenaline-evoked PS exposure in human platelets.
Topics: Humans; Blood Platelets; Platelet Activation; Calcium; Epinephrine; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Sodium; Thrombin
PubMed: 38474244
DOI: 10.3390/ijms25052997 -
International Journal of Molecular... Mar 2024As an important functional protein molecule in the human body, human annexin A5 (hAnxA5) is widely found in human cells and body fluids. hAnxA5, the smallest type of... (Review)
Review
As an important functional protein molecule in the human body, human annexin A5 (hAnxA5) is widely found in human cells and body fluids. hAnxA5, the smallest type of annexin, performs a variety of biological functions by reversibly and specifically binding phosphatidylserine (PS) in a calcium-dependent manner and plays an important role in many human physiological and pathological processes. The free state hAnxA5 exists in the form of monomers and usually forms a polymer in a specific self-assembly manner when exerting biological activity. This review systematically discusses the current knowledge and understanding of hAnxA5 from three perspectives: physiopathological relevance, diagnostic value, and therapeutic utility. AnxA5 affects the occurrence and development of many physiopathological processes. Moreover, hAnxA5 can be used independently or in combination as a biomarker of physiopathological phenomena for the diagnosis of certain diseases. Importantly, based on the properties of hAnxA5, many novel drug candidates have been designed and prepared for application in actual medical practice. However, there are also some gaps and shortcomings in AnxA5 research. This in-depth study will not only expand the understanding of structural and functional relationships but also promote the application of hAnxA5 in the field of biomedicine.
Topics: Humans; Annexin A5; Apoptosis; Calcium; Calcium, Dietary; Phosphatidylserines
PubMed: 38474114
DOI: 10.3390/ijms25052865 -
Annals of Laboratory Medicine Sep 2024Coronavirus disease (COVID-19) induces inflammation, coagulopathy following platelet and monocyte activation, and fibrinolysis, resulting in elevated D-dimer levels....
BACKGROUND
Coronavirus disease (COVID-19) induces inflammation, coagulopathy following platelet and monocyte activation, and fibrinolysis, resulting in elevated D-dimer levels. Activated platelets and monocytes produce microvesicles (MVs). We analyzed the differences in platelet and monocyte MV counts in mild, moderate, and severe COVID-19, as well as their correlation with D-dimer levels.
METHODS
In this cross-sectional study, blood specimens were collected from 90 COVID-19 patients and analyzed for D-dimers using SYSMEX CS-2500. Platelet MVs (PMVs; PMVCD42b and PMVCD41a), monocyte MVs (MMVs; MMVCD14), and phosphatidylserine-binding annexin V (PS, AnnV) were analyzed using a BD FACSCalibur instrument.
RESULTS
PMV and MMV counts were significantly increased in COVID-19 patients. AnnV PMVCD42b and AnnV PMVCD41a cell counts were higher in patients with severe COVID-19 than in those with moderate clinical symptoms. The median (range) of AnnV PMVCD42b (MV/μL) in mild, moderate, and severe COVID-19 was 1,118.3 (328.1-1,910.5), 937.4 (311.4-2,909.5), and 1,298.8 (458.2-9,703.5), respectively ( =0.009). The median (range) for AnnV PMVCD41a (MV/μL) in mild, moderate, and severe disease was 885.5 (346.3-1,682.7), 663.5 (233.8-2,081.5), and 1,146.3 (333.3-10,296.6), respectively ( =0.007). D-dimer levels (ng/mL) weak correlated with AnnV PMVCD41a ( =0.047, r=0.258).
CONCLUSIONS
PMV PMVCD42b and PMVCD41a counts were significantly increased in patients with severe clinical symptoms, and PMVCD41a counts correlated with D-dimer levels. Therefore, MV counts can be used as a potential biomarker of COVID-19 severity.
Topics: Humans; COVID-19; Cross-Sectional Studies; Monocytes; Female; Male; Fibrin Fibrinogen Degradation Products; Middle Aged; Biomarkers; Blood Platelets; SARS-CoV-2; Aged; Adult; Cell-Derived Microparticles; Severity of Illness Index; Pandemics; Pneumonia, Viral; Coronavirus Infections; Betacoronavirus; Aged, 80 and over
PubMed: 38469637
DOI: 10.3343/alm.2023.0395 -
Nature Communications Mar 2024Immune checkpoint inhibitors targeting PD-1/L1 have modest efficacy in hepatocellular carcinoma as single agents. Targeting membranous phosphatidylserine may induce...
Immune checkpoint inhibitors targeting PD-1/L1 have modest efficacy in hepatocellular carcinoma as single agents. Targeting membranous phosphatidylserine may induce pro-inflammatory and -immune stimulating effects that enhance immunotherapy activity. This hypothesis was tested in a single-arm phase 2 trial evaluating frontline bavituximab, a phosphatidylserine targeting antibody, plus pembrolizumab (anti-PD-1) in patients with unresectable hepatocellular carcinoma (NCT03519997). The primary endpoint was investigator-assessed objective response rate among evaluable patients, and secondary end points included progression-free survival, incidence of adverse events, overall survival, and duration of response. Among 28 evaluable patients, the confirmed response rate was 32.1%, which met the pre-specified endpoint, and the median progression-free survival was 6.3 months (95% CI, 1.3-11.3 months). Treatment related-adverse events of any grade occurred in 45.7% of patients, with grade 3 or greater adverse events in 14.3% of patients. Adverse events of any cause were observed in 33 patients (94.3%), with grade 3 or greater adverse events in 11 patients (31.4%). Prespecified exploratory analyses of baseline tumor specimens showed that a depletion of B cells, and the presence of fibrotic tissue and expression of immune checkpoints in stroma was associated with tumor response. These results suggest that targeting phosphatidylserine may lead to synergistic effects with PD-1 blockade without increasing toxicity rates, and future studies on this therapeutic strategy may be guided by biomarkers characterizing the pre-treatment tumor microenvironment.
Topics: Humans; Carcinoma, Hepatocellular; Phosphatidylserines; Programmed Cell Death 1 Receptor; Liver Neoplasms; Antineoplastic Combined Chemotherapy Protocols; Tumor Microenvironment; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized
PubMed: 38467639
DOI: 10.1038/s41467-024-46542-y -
Advanced Science (Weinheim,... May 2024Inflammation-responsive hydrogels loaded with therapeutic factors are effective biomaterials for bone tissue engineering and regenerative medicine. In this study, a...
Inflammation-responsive hydrogels loaded with therapeutic factors are effective biomaterials for bone tissue engineering and regenerative medicine. In this study, a matrix metalloproteinase (MMP)-responsive injectable hydrogel is constructed by integrating an MMP-cleavable peptide (pp) into a covalent tetra-armed poly-(ethylene glycol) (PEG) network for precise drug release upon inflammation stimulation. To establish a pro-regenerative environment, phosphatidylserine (PS) is encapsulated into a scaffold to form the PEG-pp-PS network, which could be triggered by MMP to release a large amount of PS during the early stage of inflammation and retain drug release persistently until the later stage of bone repair. The hydrogel is found to be mechanically and biologically adaptable to the complex bone defect area. In vivo and in vitro studies further demonstrated the ability of PEG-pp-PS to transform macrophages into the anti-inflammatory M2 phenotype and promote osteogenic differentiation, thus, resulting in new bone regeneration. Therefore, this study provides a facile, safe, and promising cell-free strategy on simultaneous immunoregulation and osteoinduction in bone engineering.
Topics: Bone Regeneration; Hydrogels; Animals; Phosphatidylserines; Immunomodulation; Matrix Metalloproteinases; Tissue Engineering; Mice; Osteogenesis; Polyethylene Glycols; Disease Models, Animal; Tissue Scaffolds; Biocompatible Materials; Models, Animal
PubMed: 38460178
DOI: 10.1002/advs.202306924 -
Journal of Autoimmunity May 2024Type 1 diabetes (T1D) results from a breakdown in immunological tolerance, with pivotal involvement of antigen-presenting cells. In this context, antigen-specific...
Type 1 diabetes (T1D) results from a breakdown in immunological tolerance, with pivotal involvement of antigen-presenting cells. In this context, antigen-specific immunotherapies have been developed to arrest autoimmunity, such as phosphatidylserine (PS)-liposomes. However, the role of certain antigen-presenting cells in immunotherapy, particularly human macrophages (Mφ) in T1D remains elusive. The aim of this study was to determine the role of Mφ in antigen-specific immune tolerance and T1D. To that end, we evaluated Mφ ability to capture apoptotic-body mimicking PS-liposomes in mice and conducted a phenotypic and functional characterisation of four human monocyte-derived Mφ (MoMφ) subpopulations (M0, M1, M2a and M2c) after PS-liposomes uptake. Our findings in mice identified Mφ as the most phagocytic cell subset in the spleen and liver. In humans, while phagocytosis rates were comparable between T1D and control individuals, PS-liposome capture dynamics differed among Mφ subtypes, favouring inflammatory (M1) and deactivated (M2c) Mφ. Notably, high nanoparticle concentrations did not affect macrophage viability. PS-liposome uptake by Mφ induced alterations in membrane molecule expression related to immunoregulation, reduced secretion of IL-6 and IL-12, and diminished autologous T-cell proliferation in the context of autoantigen stimulation. These results underscore the tolerogenic effects of PS-liposomes and emphasize their potential to target human Mφ, providing valuable insights into the mechanism of action of this preclinical immunotherapy.
Topics: Diabetes Mellitus, Type 1; Liposomes; Animals; Humans; Phosphatidylserines; Mice; Immunotherapy; Macrophages; Autoantigens; Female; Immune Tolerance; Phagocytosis; Male; Mice, Inbred NOD; Autoimmunity; Adult
PubMed: 38458075
DOI: 10.1016/j.jaut.2024.103196 -
Nature Cell Biology Apr 2024β-Propeller protein-associated neurodegeneration (BPAN) is a rare X-linked dominant disease, one of several conditions that manifest with neurodegeneration and brain...
β-Propeller protein-associated neurodegeneration (BPAN) is a rare X-linked dominant disease, one of several conditions that manifest with neurodegeneration and brain iron accumulation. Mutations in the WD repeat domain 45 (WDR45) gene encoding WIPI4 lead to loss of function in BPAN but the cellular mechanisms of how these trigger pathology are unclear. The prevailing view in the literature is that BPAN is simply the consequence of autophagy deficiency given that WIPI4 functions in this degradation pathway. However, our data indicate that WIPI4 depletion causes ferroptosis-a type of cell death induced by lipid peroxidation-via an autophagy-independent mechanism, as demonstrated both in cell culture and in zebrafish. WIPI4 depletion increases ATG2A localization at endoplasmic reticulum-mitochondrial contact sites, which enhances phosphatidylserine import into mitochondria. This results in increased mitochondrial synthesis of phosphatidylethanolamine, a major lipid prone to peroxidation, thus enabling ferroptosis. This mechanism has minimal overlap with classical ferroptosis stimuli but provides insights into the causes of neurodegeneration in BPAN and may provide clues for therapeutic strategies.
Topics: Animals; Ferroptosis; Zebrafish; Carrier Proteins; Autophagy; Mutation
PubMed: 38454050
DOI: 10.1038/s41556-024-01373-3