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Plasma leakage and blood parameters revealed P-sel-mediated rescue. Experimental outline (A). To evaluate the amelioration of PA or LT (4.5 mg/kg) treatment-induced pathogenesis, the effects of P-sel-Fc pretreatment were compared with those of control Ig treatment. Lung retaining Evans blue (B), platelet counts (C), hematocrit count (D), and serum aspartate aminotransferase (AST) levels (E) of mice were recorded before (0 h) and after (48 and 72 h) treatment with PA and LT. Ã P < 0.05, ÃÃ P < 0.01, vs. respective PA C control Ig groups; # P < 0.05, significant amelioration vs. respective LT C control Ig groups (B-E). n D 8. The mouse drawing used in this report was originally published in Blood by Huang, HS et al. © the American Society of Hematology. Reproduced by permission of Hsin-Hou Chang. Permission to reuse must be obtained from the rightsholder. 72
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As one of the virulence factors of Bacillus anthracis, lethal toxin (LT) induces various pathogenic responses including the suppression of the coagulation system. In this study, we observed that LT markedly increased the circulating soluble P-selectin (sP-sel) levels and microparticle (MP) count in wild-type but not P-selectin (P-sel, Selp⁻⁻⁻) or P...
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Context 1
... abnormalities such as plasma leakage, thrombocytopenia ( Fig. 1 and Fig. S1), decreased hematocrit count, 11 and liver damage. 13 The mice were treated with P-sel-Fc 4 h before a lethal dose of LT was administered. The results revealed that the P-sel-Fc treatment markedly ameliorated all aforementioned LT-mediated pathogenic abnormalities ( Fig. 5A experimental outline, ...
Context 2
... plasma leakage in the mice was measured using described previously methods. 13,16 The mice were administered a single intravenous injection of Evans blue dye, 30 min before a lethal dose challenge of LT. 16,74 Experimental courses including 72 h (Fig. 1), and 0, 48, 72 h (Fig. 5) after the LT treatment (4.5 mg/kg), the mice were killed and their tissue samples were collected and minced before incubation with formamide-water (1:1) for 48 h at 37 C. The concentration of dye was determined using a standard curve of Evans blue in formamide and a spectrophotometer (Hitachi, Japan). 13,16 Clotting time ...
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... analyze blood parameters such as hematocrit and platelet counts and the liver function, the blood and plasma samples of mice were collected from the retroorbital venous plexus using methods described in the previous clotting time section. Experimental courses include 0, 48, 72 h (Fig. 5) after the LT treatment (4.5 mg/kg). The hematocrit and platelet counts of mice were measured using a hematology analyzer (KX-21N; Sysmex). 72 The liver function was analyzed through detecting circulating aspartate aminotransferase (AST, a liver cell specifically expressed enzyme) levels with a clinical biochemistry analysis system ...
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... antibody labeling and flow cytometry analysis. To determine the MP count in the plasma, 6-mm reference beads with a known MP count (polystyrene latex beads, Sigma-Aldrich) were mixed with respective plasma samples with an unknown MP count. The MP count of these plasma samples was analyzed using the ratio between beads (known) and MPs (unknown) (Fig. S5). To determine the relative levels of sP-sel in the MP-bound and non-MP-bound forms through ELISA, the MP pellets obtained after ultracentrifugation were resuspended in the calcium-free Tyrode buffer (in the same plasma volume used before ultracentrifugation). In addition, the plasma sample was diluted 1 / 2 -fold using the Tyrode ...
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Citations
... C57BL/6J mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). P-selectin deficiency mice (Selp −/− ; B6.129S-Selp tm1Bay /J) were obtained from Jackson Laboratory (Bar Harbor, ME) [46]. All animals were maintained in a specific pathogen-free environment in the experimental animal center of Tzu Chi University (Hualien, Taiwan). ...
... Reports showed upregulated sP-sel levels in the circulation of patients with inflammatory conditions, such as hypertension [39], cardiovascular disease [40], rheumatic mitral stenosis [80], myocardial infarction [81], and sepsis [82]. Consistently, animal experiments suggested that the sP-sel levels were upregulated in mice with inflammatory burdens, such as under anthrax lethal toxin [46] and snake venom treatments [83]. These studies suggested that sP-sel can be a native factor for conducting anti-inflammatory and repairing effects. ...
... The authors' previous study found that HSC mobilization following G-CSF administration was linked to pro-inflammatory factors such as IL-22, tumor necrosis factor, and interferon-γ [5]. These results align with earlier research on infectious diseases, toxin treatments, and cardiovascular injuries [46,94,95], indicating an association between circulating levels of sP-sel and inflammatory conditions. The previously reported pro-survival and tissue-repair functions of sP-sel [46,83,96] could be partly attributed to HSC mobilization, highlighting a natural self-rescue feedback mechanism between sP-sel-mediated tissue repair and inflammation-related tissue damage. ...
Background
Granulocyte colony-stimulating factor (G-CSF)-mediated mobilization of hematopoietic stem cells (HSCs) is a well-established method to prepare HSCs for transplantation nowadays. A sufficient number of HSCs is critical for successful HSC transplantation. However, approximately 2–6% of healthy stem cell donors are G-CSF-poor mobilizers for unknown reasons; thus increasing the uncertainties of HSC transplantation. The mechanism underlining G-CSF-mediated HSC mobilization remains elusive, so detailed mechanisms and an enhanced HSC mobilization strategy are urgently needed. Evidence suggests that P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) are one of the cell–cell adhesion ligand–receptor pairs for HSCs to keep contacting bone marrow (BM) stromal cells before being mobilized into circulation. This study hypothesized that blockage of PSGL-1 and P-selectin may disrupt HSC-stromal cell interaction and facilitate HSC mobilization.
Methods
The plasma levels of soluble P-selectin (sP-sel) before and after G-CSF administration in humans and male C57BL/6J mice were analyzed using enzyme-linked immunosorbent assay. Male mice with P-selectin deficiency (Selp−/−) were further employed to investigate whether P-selectin is essential for G-CSF-induced HSC mobilization and determine which cell lineage is sP-sel derived from. Finally, wild-type mice were injected with either G-CSF or recombinant sP-sel to investigate whether sP-sel alone is sufficient for inducing HSC mobilization and whether it accomplishes this by binding to HSCs and disrupting their interaction with stromal cells in the BM.
Results
A significant increase in plasma sP-sel levels was observed in humans and mice following G-CSF administration. Treatments of G-CSF induced a decrease in the level of HSC mobilization in Selp−/− mice compared with the wild-type (Selp+/+) controls. Additionally, the transfer of platelets derived from wild-type mice can ameliorate the defected HSC mobilization in the Selp−/− recipients. G-CSF induces the release of sP-sel from platelets, which is sufficient to mobilize BM HSCs into the circulation of mice by disrupting the PSGL-1 and P-selectin interaction between HSCs and stromal cells. These results collectively suggested that P-selectin is a critical factor for G-CSF-induced HSC mobilization.
Conclusions
sP-sel was identified as a novel endogenous HSC-mobilizing agent. sP-sel injections achieved a relatively faster and more convenient regimen to mobilize HSCs in mice than G-CSF. These findings may serve as a reference for developing and optimizing human HSC mobilization in the future.
... Male wild-type C57BL/6J mice aged 8-12 weeks were purchased from the National Laboratory Animal Center (Taipei, Taiwan) [14][15][16][17]. Genetically deficient Selp −/− (B6; 129S2-Selp tm1Hyn /J; P-selectin KO) [47,[77][78][79] with a C57BL/6J background were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Selp −/− mice were backcrossed with WT C57Bl/6J mice over six generations. ...
... To analyze the platelet count, whole blood samples (100-120 µL) of the mice were collected with an anticoagulant, a citrate dextrose solution (38 mM citric acid, 75 mM sodium citrate, and 100 mM dextrose), in Eppendorf tubes. Subsequently, platelet counts were measured using a hematology analyzer (KX-21N, Sysmex, Kobe, Japan) at various time intervals [77,78,84]. To investigate the ameliorative effects of intravenous treatment with IVIg (high dose, 2 g/kg; vehicle: saline; injection at 0 h), cold (4 • C) exposure and restraint stress for 9 h was implemented. ...
Psychological stress is widely acknowledged as a major contributor to immunosuppression, rendering individuals more susceptible to various diseases. The complex interplay between the nervous, endocrine, and immune systems underlies stress-induced immunosuppression. However, the underlying mechanisms of psychological-stress-induced immunosuppression remain unclear. In this study, we utilized a restraint stress mouse model known for its suitability in investigating physiological regulations during psychological stress. Comparing it with cold exposure, we observed markedly elevated levels of stress hormones corticosterone and cortisol in the plasma of mice subjected to restraint stress. Furthermore, restraint-stress-induced immunosuppression differed from the intravenous immunoglobulin-like immunosuppression observed in cold exposure, with restraint stress leading to increased macrophage cell death in the spleen. Suppression of pyroptosis through treatments of inflammasome inhibitors markedly ameliorated restraint-stress-induced spleen infiltration and pyroptosis cell death of macrophages in mice. These findings suggest that the macrophage pyroptosis associated with restraint stress may contribute to its immunosuppressive effects. These insights have implications for the development of treatments targeting stress-induced immunosuppression, emphasizing the need for further investigation into the underlying mechanisms.
... Flow cytometry analysis of platelet surface P-selectin and CD41 (integrin αIIb) expression levels before and after stress was performed using FACScalibur (BD Biosciences, Franklin Lakes, NJ, USA) and Gallios (Beckman Coulter Life Sciences) flow cytometers [20,77,78]. For staining, anti-mouse phycoerythrin-conjugated P-selectin Ig (eBioscience, Thermo Fisher Scientific) [45] and Alexa Fluor 488-conjugated anti-mouse CD41 Ig (clone MWReg30 [79]; Biolegend, San Diego, CA, USA) were used to stain platelet-surface P-selectin and CD41, respectively. ...
Metformin is one of the most commonly used drugs for type 2 diabetes mellitus. In addition to its anti-diabetic property, evidence suggests more potential applications for metformin, such as antiaging, cellular protection, and anti-inflammation. Studies have reported that metformin activates pathways with anti-inflammatory effects, enhances the integrity of gut epithelial tight junctions, and promotes a healthy gut microbiome. These actions contribute to the protective effect of metformin against gastrointestinal (GI) tract injury. However, whether metformin plays a protective role in psychological-stress-associated GI tract injury remains elusive. We aim to elucidate the potential protective effect of metformin on the GI system and develop an effective intervention strategy to counteract GI injury induced by acute psychological stress. By monitoring the levels of GI-nonabsorbable Evans blue dye in the bloodstream, we assessed the progression of GI injury in live mice. Our findings demonstrate that the administration of metformin effectively mitigated GI leakage caused by psychological stress. The GI protective effect of metformin is more potent when used on wild-type mice than on activating-transcription-factor 3 (ATF3)-deficient (ATF3-/-) mice. As such, metformin-mediated rescue was conducted in an ATF3-dependent manner. In addition, metformin-mediated protection is associated with the induction of stress-induced GI mRNA expressions of the stress-induced genes ATF3 and AMP-activated protein kinase. Furthermore, metformin treatment-mediated protection of CD326+ GI epithelial cells against stress-induced apoptotic cell death was observed in wild-type but not in ATF3-/- mice. These results suggest that metformin plays a protective role in stress-induced GI injury and that ATF3 is an essential regulator for metformin-mediated rescue of stress-induced GI tract injury.
... The survival rate of human endothelial HMEC-1 cells [7] was assessed post-treatment with various substances, including vehicle, GST, EIII (0.6 µM, a dose of EIII functionally equivalent to the viral-load range of circulating DENV viron-associated EIII in DHF patients, determined by activated partial thromboplastin clotting time assay [26]), and GST-SNPs and EIII-SNPs (with a similar dose of soluble protein). The WST-1 kit (Roche Life Science, Penzberg, Germany) was employed to determine cell viability following the manufacturer's instructions [8,49,50]. The succinate-tetrazolium reductase system is responsible for breaking down the tetrazolium salt WST-1 into a soluble formazan. ...
... IL-6, IL-10, and TNF-α (ELISA, e-Bioscience, Thermo Fisher Scientific, Waltham, MA, USA), and the levels of expression of anti-coagulant proteins anthithrombin III (chromogenic, Sekisui Diagnostica, Burlington, MA, USA) and protein C (chromogenic, Sekisui Diagnostica). For platelet count analysis, blood samples were collected and placed into polypropylene tubes containing an anticoagulant solution of acid-citrate-dextrose (38 mM citric acid, 75 mM sodium citrate, and 100 mM dextrose) [49,52]. To obtain platelet-poor plasma for the analysis of cytokines and anticoagulant proteins, the samples were subjected to centrifugation at 1500× g for 20 min, followed by a subsequent centrifugation at 15,000× g for 3 min to remove contaminant cells from the plasma. ...
Dengue hemorrhagic fever (DHF) is a severe form of dengue virus (DENV) infection that can lead to abnormal immune responses, endothelial vascular dysfunction, and hemorrhage pathogenesis. The virion-associated envelope protein domain III (EIII) is thought to play a role in the virulence of DENV by damaging endothelial cells. However, it is unclear whether EIII-coated nanoparticles simulating DENV virus particles could cause a more severe pathogenesis than soluble EIII alone. This study aimed to investigate whether EIII-coated silica nanoparticles (EIII-SNPs) could elicit greater cytotoxicity in endothelial cells and hemorrhage pathogenesis in mice compared to EIII or silica nanoparticles alone. The main methods included in vitro assays to assess cytotoxicity and in vivo experiments to examine hemorrhage pathogenesis in mice. EIII-SNPs induced greater endothelial cytotoxicity in vitro than EIII or silica nanoparticles alone. Two-hit combined treatment with EIII-SNPs and antiplatelet antibodies to simulate DHF hemorrhage pathogenesis during secondary DENV infections resulted in higher endothelial cytotoxicity than either treatment alone. In mouse experiments, two-hit combined treatment with EIII-SNPs and antiplatelet antibodies resulted in more severe hemorrhage pathogenesis compared to single treatments of EIII, EIII-SNPs, or antiplatelet antibodies alone. These findings suggest that EIII-coated nanoparticles are more cytotoxic than soluble EIII and could be used to develop a tentative dengue two-hit hemorrhage pathogenesis model in mice. Additionally, our results indicated that EIII-containing DENV particles could potentially exacerbate hemorrhage pathogenesis in DHF patients who have antiplatelet antibodies, highlighting the need for further research on the potential role of EIII in DHF pathogenesis.
... In the present study, we found that the expression levels of platelet surface P-selectin are markedly up-regulated after restraint stress. Given the evidence that P-selectin [19][20][21] and platelets [17] exert protective roles during injuries, we hypothesize that P-selectin expressing platelets may have a protective role in the suppression of stress-induced GI injury. Results indicated that P-selectin gene deficient (Selp −/− ) mice displayed more severe GI leakage than wild-type (Selp +/+ ) mice, suggesting that P-selectin ameliorates GI injuries. ...
... A growing body of evidence suggests that the soluble form P-selectin exerts anti-inflammatory and cell pro-survival roles [19][20][21]39]. Although membrane-form P-selectin is associated with inflammation progression [40], anti-inflammatory soluble P-selectin can be released from the surfaces of platelets and endothelial cells in the forms of soluble ectodomain and cell-extracellular vesicle associated protein [20,41]. ...
... Although membrane-form P-selectin is associated with inflammation progression [40], anti-inflammatory soluble P-selectin can be released from the surfaces of platelets and endothelial cells in the forms of soluble ectodomain and cell-extracellular vesicle associated protein [20,41]. Treatments of soluble P-selectin can mobilize anti-inflammatory CD34 + stem cells and block leukocyte infiltration -mediated inflammation [19][20][21]39], suggesting the anti-inflammatory properties of soluble P-selectin. Hence, P-selectin exerts dual roles in inflammatory regulation depending on expression type (soluble vs. cell-surface bound) and tissue environment. ...
Psychological stress is associated with increased risk of gastrointestinal (GI) tract diseases. Evidence indicated that platelets facilitate GI tissue repair in intestinal anastomosis models. However, whether platelets are involved in native mechanism of the rescue of stress-induced GI injury for maintaining the GI homeostasis remains elusive. Because P-selectin-deficient (Selp−/−) mice displayed higher stress-induced GI injury compared to the wild-type (Selp+/+) mice, and P-selectin is specifically expressed in platelets, we hypothesize that P-selectin-expressing platelets play a protective role in the rescue of stress-induced GI injury. Our goal is to clarify the putative protective role of platelets in a GI system, thereby develop a feasible intervention strategy, such as platelet transfer, to overcome stress-induced GI injury. Through monitoring the plasma levels of GI-nonabsorbable Evans blue dye to reveal the progression course of GI injury in live mice, we found that intravenous treatments of purified platelets ameliorated stress-induced GI leakage. The transfer of platelets from wild-type mice was more potent than from Selp−/− mice in the rescue of stress-induced-GI leakage in the recipients. As such, platelet transfer-mediated rescue was conducted in a P-selectin dependent manner. Additionally, platelet-mediated protection is associated with corrections of stress-induced aberrant GI mRNA expressions, including tight junctions claudin 3 and occludin, as well as stress-induced genes activating transcription factor 3 and AMP-activated protein kinase, after the transfer of wild-type platelets into wild-type and Selp−/− mice. Furthermore, the stress-induced apoptosis of CD326+ GI epithelial cells was rescued by the transfer of wild type, but not P-selectin-deficient platelets. These results suggest that platelet plays a protective role for maintaining the GI homeostasis during stress in vivo, and that P-selectin is a molecular target for managing stress-induced GI tract injury.
... Gene knockout mice with a C57BL/6J background, including Nlrp3 -/and Casp1 -/-(61), were kindly provided by the Centre National de Recherche Scientifique (Orleáns, France) (61)(62)(63). C57BL/6J male mice (8-12 wk old) deficient in P-selectin (B6; 129S2-Selp tm1Hyn /J) (Selp -/-) (19,64,65) were purchased from the Jackson Laboratory (Maine, USA). All animals were maintained in a specific-pathogen-free (SPF) facility in the Laboratory Animal Center of Tzu Chi University (Hualien, Taiwan). ...
... Collected mouse blood samples were transferred into polypropylene tubes containing anticoagulant acid-citratedextrose solution (38 mM citric acid, 75 mM sodium citrate, and 100 mM dextrose) (64,65). Washed platelets were prepared as previously described (19,38). ...
... Washed platelets were prepared as previously described (19,38). Platelet counts of mice were measured using a hematology analyzer (KX-21N; Sysmex, Kobe, Japan) (64)(65)(66). ...
Nanodiamond (ND) has been developed as a carrier to conduct various in vivo diagnostic and therapeutic uses. Safety is one of the major considerations, while the hemocompatibility of ND is not clearly addressed. Here we found that, compared to the other sizes of ND with relatively inert properties, treatments of 50 nm ND induced stronger platelet aggregation, platelet pyroptosis, apoptosis and thrombocytopenia in mice. Blockage treatments of soluble P-selectin, reactive oxygen species (ROS), and Nlrp3 inflammasome inhibitors markedly suppressed such adverse effects, suggesting ND-induced platelet activation and pyroptosis involves surface P-selectin-mediated enhancement of mitochondrial superoxide levels and Nlrp3 inflammasome activation. In addition, challenges of NDs induced less platelet pyroptosis and displayed less thrombocytopenia in P-selectin (Selp-/- ), Nlrp3 (Nlrp3-/- ) and caspase-1 (Casp1-/- ) mutants, as compared to the wild type mice. Blockers of P-selectin, ROS, and Nlrp3 inflammasome pathways could be considered as antidotes for ND induced platelet activation and thrombocytopenia.
... After intravenous injection, Evans blue, an azo dye with a high affinity for serum albumin, can bind to the plasma protein albumin to form a 69-kDa tracer, a molecular-sized protein not normally able to penetrate viable cells and peripheral tissues unless vascular leakage or tissue injuries occur [73]. As a result, Evans blue dye is widely used to measure tissue injuries [35,42,[74][75][76]. This is likely the reason that Evans blue has previously only been used in analyses of gut leakage using proximal-distal-ligated intestine samples that were isolated from the sacrificed animal [77]; the leakage levels of dead, not live intestinal tissue were recorded. ...
Psychological stress increases the risk of gastrointestinal (GI) tract diseases, which involve bidirectional communication of the GI and nerves systems. Acute stress leads to GI ulcers; however, the mechanism of the native cellular protection pathway, which safeguards tissue integrality and maintains GI homeostasis, remains to be investigated. In a mouse model of this study, restraint stress induced GI leakage, abnormal tight junction protein expression, and cell death of gut epithelial cells. The expression of activating transcription factor 3 (ATF3), a stress-responsive transcription factor, is upregulated in the GI tissues of stressed animals. ATF3-deficient mice displayed an exacerbated phenotype of GI injuries. These results suggested that, in response to stress, ATF3 is part of the native cellular protective pathway in the GI system, which could be a molecular target for managing psychological stress-induced GI tract diseases.
... Immune system defenses against external stimulations and pathogen invasions [48][49][50][51][52][53][54][55][56][57], in which the inflammasome-IL-1 axis exerts critical role on the induction of inflammation in various conditions [58][59][60][61][62][63][64][65][66][67][68][69][70]. Itaconate was demonstrated to inhibit SDH, and subsequently succinate oxidation and thus blocking succinate-mediated inflammatory processes [10,14]. ...
Metabolic regulations play vital roles on maintaining the homeostasis of our body. Evidence have suggested that ATF3 and nuclear factor erythroid 2–related factor 2 (NRF2) are critical for maintaining cell function, metabolism, and inflammation/anti-inflammation regulations when cells are under stress, while the upstream regulators in the stressed cells remain elusive. Recent findings have shown that tricarboxylic acid cycle metabolites such as itaconate and succinate are not just mitochondrial metabolites, but rather important signaling mediators, involving in the regulations of metabolism, immune modulation. Itaconate exerts anti-inflammatory role through regulating ATF3 and NRF2 pathways under stressed conditions. In addition, itaconate inhibits succinate dehydrogenase, succinate oxidation and thus blocking succinate-mediated inflammatory processes. These findings suggest itaconate-ATF3 and itaconate-NRF2 axes are well-coordinated machineries that facilitate the rescue against cellular stress. Here, we review these fascinating discoveries, a research field may help the development of more effective therapeutic approach to manage stress-induced inflammation, tissue damage, and metabolic disorder.
... The zebrafish undergoing testing were exposed to TiO 2 NPs in well water for 21 days, and the mortality was recorded each day. The Kaplan Meier curves are plotted using the Online Application for the Survival Analysis of Lifespan Assay (http://sbi.postech.ac.kr/oasis) [41][42][43][44]. Fish from three of the 3-L tanks were selected for behavior analysis, in which the swimming speed of the zebrafish was analyzed using Kinovea software (version 0.8.24; available at http://www.kinovea.org/) in accordance with methods used in previous studies [45,46]. ...
... Statistical analysis. The mortality of zebrafish was calculated using Online Application for the Survival Analysis of Lifespan Assay (http://sbi.postech.ac.kr/oasis) [41][42][43][44]. A t test was used to assess the statistical significance of differences in antimicrobial effects. ...
The large amounts of engineered titanium dioxide nanoparticles (TiO2NPs) that have been manufactured have inevitably been released into the ecosystem. Reports have suggested that TiO2 is a relatively inert material that has low toxicity to animals. However, as various types of NPs increasingly accumulate in the ocean, their effects on aquatic life-forms remain unclear. In this study, a zebrafish model was used to investigate TiO2NP-induced injury and mortality. We found that the treatment dosages of TiO2NP are positively associated with increased motility of zebrafish and the bacterial counts in the water. Notably, gill but not dorsal fin and caudal fin of the zebrafish displayed considerably increased bacterial load. Metagenomic analysis further revealed that gut microflora, such as phyla Proteobacteria, Bacteroidetes, and Actinobacteria, involving more than 95% of total bacteria counts in the NP-injured zebrafish gill samples. These results collectively suggest that opportunistic bacterial infections are associated with TiO2NP-induced mortality in zebrafish. Infections secondary to TiO2NP-induced injury could be a neglected factor determining the detrimental effects of TiO2NPs on wild fish.
... Mouse blood samples were collected through the retro-orbital venous plexus technique by using plain capillary tubes (Thermo Fisher Scientific, Waltham, MA, USA) and were transferred into polypropylene tubes containing anticoagulant acid-citrate-dextrose solution (38 mM citric acid, 75 mM sodium citrate, and 100 mM dextrose) (29,30). Washed platelets were prepared as previously described (31,32). ...
... The fluorescence intensities of rEIII-bound platelets were analyzed using a fluorescent microplate reader (Varioskan ™ Flash Multimode reader; Thermo Fisher Scientific) (26,29) at Excitation/Emission spectrum = 480/530 nm. A flow cytometer (FACScalibur, BD Biosciences) (29,30) was used for analyzing platelet surface P-selectin expression and biotin-rEIII binding by using anti-mouse P-selectin Ig-phycoerythrin (PE; eBioscience, Thermo Fisher Scientific) and biotin-conjugated rEIII, followed by avidin-PE/Cy5 (Biolegend) labeling. Scanning electron microscopy analysis for platelet morphology was conducted as previously described (31). ...
In tropical and subtropical regions, mosquito-borne dengue virus (DENV) infections can lead to severe dengue, also known as dengue hemorrhage fever, which causes bleeding, thrombocytopenia, and blood plasma leakage and increases mortality. Although DENV-induced platelet cell death was linked to disease severity, the role of responsible viral factors and the elicitation mechanism of abnormal platelet activation and cell death remain unclear. DENV and virion-surface envelope protein domain III (EIII), a cellular binding moiety of the virus particle, highly increase during the viremia stage. Our previous report suggested that exposure to such viremia EIII levels can lead to cell death of endothelial cells, neutrophils, and megakaryocytes. Here we found that both DENV and EIII could induce abnormal platelet activation and predominantly necrotic cell death pyroptosis. Blockages of EIII-induced platelet signaling using the competitive inhibitor chondroitin sulfate B or selective Nlrp3 inflammasome inhibitors OLT1177 and Z-WHED-FMK markedly ameliorated DENV- and EIII-induced thrombocytopenia, platelet activation, and cell death. These results suggest that EIII could be considered as a virulence factor of DENV, and that Nlrp3 inflammasome is a feasible target for developing therapeutic approaches against dengue-induced platelet defects.
