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Impaired wound healing poses a significant burden on the healthcare system and patients. Stem cell therapy has demonstrated promising potential in the treatment of wounds. However, its clinical application is hindered by the low efficiency of cell homing. In this study, we successfully integrated P-selectin glycoprotein ligand-1 (PSGL-1) into the genome of human adipose-derived mesenchymal stem cells (ADSCs) using a Cas9-AAV6-based genome editing tool platform. Our findings revealed that PSGL-1 knock-in enhanced the binding of ADSCs to platelets and their adhesion to the injured site. Moreover, the intravenous infusion of PSGL-1 -engineered ADSCs (KI-ADSCs) significantly improved the homing efficiency and residence rate at the site of skin lesions in mice. Mechanistically, PSGL-1 knock-in promotes the release of some therapeutic cytokines by activating the canonical WNT/β-catenin signaling pathway and accelerates the healing of wounds by promoting angiogenesis, re-epithelialization, and granulation tissue formation at the wound site. This study provides a novel strategy to simultaneously address the problem of poor migration and adhesion of mesenchymal stem cells (MSCs). [ABSTRACT FROM AUTHOR]

Abstract Enterovirus 71 (EV-71) has strong neurotropism, and it is the main pathogen causing severe hand, foot, and mouth disease (HFMD). In clinical observations, significant differences were observed in the severity and prognosis of HFMD among children who were also infected with EV-71. Genetic differences among individuals could be one of the important causes of differences in susceptibility to EV-71–induced HFMD. As P-selectin glycoprotein ligand-1 (PSGL-1) is an important receptor of EV-71, the correlation between single-nucleotide polymorphisms (SNPs) in PSGL-1 and the susceptibility to severe HFMD following EV-71 infection is worth studying. Given the role of PSGL-1 in immunity, the correlations between PSGL-1 SNPs and the immune status after EV-71 infection are also worth studying. Meanwhile, PSGL-1 variable number of tandem repeats (VNTR) represents a research hotspot in cardiovascular and cerebrovascular diseases, but PSGL-1 VNTR polymorphism has not been investigated in HFMD caused by EV-71 infection. In this study, specific gene fragments were amplified by polymerase chain reaction, and PSGL-1 VNTR sequences were genotyped using an automatic nucleic acid analyzer. The correlations of PSGL-1 VNTR polymorphism with the susceptibility to EV-71–associated severe HFMD and the post-infection immune status were analyzed. The PSGL-1 VNTR A allele was identified as a susceptible SNP for severe HFMD. The risk of severe HFMD was higher for AA + AB genotype carriers than for BB genotype carriers. The counts of peripheral blood lymphocyte subsets were lower in AA + AB genotype carries than in BB genotype carries. In conclusion, PSGL-1 VNTR polymorphism is associated with the susceptibility to EV-71–induced severe HFMD and the immune status after infection. PSGL-1 VNTR might play a certain role in the pathogenesis of severe cases.

Enterovirus 71 (EV-71) has strong neurotropism, and it is the main pathogen causing severe hand, foot, and mouth disease (HFMD). In clinical observations, significant differences were observed in the severity and prognosis of HFMD among children who were also infected with EV-71. Genetic differences among individuals could be one of the important causes of differences in susceptibility to EV-71–induced HFMD. As P-selectin glycoprotein ligand-1 (PSGL-1) is an important receptor of EV-71, the correlation between single-nucleotide polymorphisms (SNPs) in PSGL-1 and the susceptibility to severe HFMD following EV-71 infection is worth studying. Given the role of PSGL-1 in immunity, the correlations between PSGL-1 SNPs and the immune status after EV-71 infection are also worth studying. Meanwhile, PSGL-1 variable number of tandem repeats (VNTR) represents a research hotspot in cardiovascular and cerebrovascular diseases, but PSGL-1 VNTR polymorphism has not been investigated in HFMD caused by EV-71 infection. In this study, specific gene fragments were amplified by polymerase chain reaction, and PSGL-1 VNTR sequences were genotyped using an automatic nucleic acid analyzer. The correlations of PSGL-1 VNTR polymorphism with the susceptibility to EV-71–associated severe HFMD and the post-infection immune status were analyzed. The PSGL-1 VNTR A allele was identified as a susceptible SNP for severe HFMD. The risk of severe HFMD was higher for AA + AB genotype carriers than for BB genotype carriers. The counts of peripheral blood lymphocyte subsets were lower in AA + AB genotype carries than in BB genotype carries. In conclusion, PSGL-1 VNTR polymorphism is associated with the susceptibility to EV-71–induced severe HFMD and the immune status after infection. PSGL-1 VNTR might play a certain role in the pathogenesis of severe cases. [ABSTRACT FROM AUTHOR]

In recent years, substantial advancements have been made in understanding the pathophysiology of ischemic stroke. Despite these developments, therapeutic options for cerebral ischemia remain limited due to stringent time windows and various contraindications. Consequently, there has been a concentrated effort to elucidate the underlying mechanisms of cerebral ischemic injury. Emerging research indicates that neutrophil extracellular traps (NETs) exacerbate inflammation and damage in ischemic brain tissue, contributing to neuronal cell death. The inhibition of NETs has shown potential in preventing thrombosis and the infiltration of immune cells. Central to the formation of NETs are P-selectin and its ligand, P-selectin glycoprotein ligand-1 (PSGL-1), which represent promising therapeutic targets. This review explores the detrimental impact of P-selectin, PSGL-1, and NETs on cerebral ischemia. Additionally, it delineates the processes by which P-selectin and PSGL-1 stimulate NETs production and provides evidence that blocking these molecules reduces NETs formation. This novel insight highlights a potential therapeutic avenue that warrants further investigation by researchers in the field. [ABSTRACT FROM AUTHOR]

Introduction: In clear cell renal cell carcinoma (ccRCC), intravascular extension often results in neoplastic thrombus formation, impacting patient outcomes. Material and methods: We assessed P-selectin glycoprotein ligand-1 (PSGL-1) expression in the primary tumours and thrombi of 82 ccRCC patients. Results: Notably, PSGL-1 expression varied between tumour compartments, with higher prevalence in thrombus tumour cells (TC) and primary tumour-associated immune cells (TAIC). Positive TC PSGL-1 correlated with high-grade histology, while TAIC PSGL-1 was associated with tumour necrosis. Univariate survival analysis identified PSGL-1 positivity in TC of primary tumours and TAIC in thrombi as indicators of poorer overall survival. These findings suggest a potential role for PSGL-1 in the mediation of tumour thrombus formation, highlighting its relevance in biology of ccRCC. Conclusions: The increased expression of PSGL-1 in venous thrombi suggests its potential role in facilitating TC interactions with platelets and endothelium, potentially contributing to metastatic spread and worse outcomes. Understanding these expression patterns may aid in the development of novel therapeutic strategies and personalised treatment approaches for ccRCC patients. [ABSTRACT FROM AUTHOR]

This document summarizes a research study conducted in India on the differentiation of human hematopoietic stem cells (HSCs) into articular chondrocytes, the main cells in articular cartilage. The study found that HSCs can transform into chondrocytes when stimulated by external signals and identified specific genes involved in this process. The researchers also explored the role of different components in the differentiation medium in promoting chondrogenesis. The study suggests that HSCs have potential for regenerating injured articular cartilage. The document includes tables with details of the research methods and acknowledgements for the support and facilities provided by the institute. It also mentions the funding source and information about ethical approval and conflicts of interest. [Extracted from the article]

Renal ischemia-reperfusion injury (IRI) is one of the most important causes of acute kidney injury (AKI). Interleukin (IL)-37 has been suggested as a novel anti-inflammatory factor for the treatment of IRI, but its application is still limited by its low stability and delivery efficiency. In this study, we reported a novel engineered method to efficiently and easily prepare neutrophil membrane-derived vesicles (N-MVs), which could be utilized as a promising vehicle to deliver IL-37 and avoid the potential side effects of neutrophil-derived natural extracellular vesicles. N-MVs could enhance the stability of IL-37 and targetedly deliver IL-37 to damaged endothelial cells of IRI kidneys via P-selectin glycoprotein ligand-1 (PSGL-1). In vitro and in vivo evidence revealed that N-MVs encapsulated with IL-37 (N-MV@IL-37) could inhibit endothelial cell apoptosis, promote endothelial cell proliferation and angiogenesis, and decrease inflammatory factor production and leukocyte infiltration, thereby ameliorating renal IRI. Our study establishes a promising delivery vehicle for the treatment of renal IRI and other endothelial damage-related diseases. [Display omitted] • Engineered method yields abundant, pure neutrophil membrane nanovesicles efficiently. • N-MVs target injured endothelial cells, avoiding neutrophil vesicle potential side effects. • N-MV@IL-37 enable targeted kidney delivery of IL-37 to counteract IRI. • N-MV@IL-37 mitigate renal IRI by reducing endothelial apoptosis and inflammation. [ABSTRACT FROM AUTHOR]

Enterovirus A71 (EV-A71) infection involves a variety of receptors. Among them, two transmembrane protein receptors have been investigated in detail and shown to be critical for infection: P-selectin glycoprotein ligand-1 (PSGL-1) in lymphocytes (Jurkat cells), and scavenger receptor class B member 2 (SCARB2) in rhabdomyosarcoma (RD) cells. PSGL-1 and SCARB2 have been reported to be expressed on the surface of Jurkat and RD cells, respectively. In the work reported here, we investigated the roles of PSGL-1 and SCARB2 in the process of EV-A71 entry. We first examined the expression of SCARB2 in Jurkat cells, and detected it within the cytoplasm, but not on the cell surface. Further, using PSGL-1 and SCARB2 knockout cells, we found that although both PSGL-1 and SCARB2 are essential for virus infection of Jurkat cells, virus attachment to these cells requires only PSGL-1. These results led us to evaluate the cell surface expression and the roles of SCARB2 in other EV-A71–susceptible cell lines. Surprisingly, in contrast to the results of previous studies, we found that SCARB2 is absent from the surface of RD cells and other susceptible cell lines we examined, and that although SCARB2 is essential for infection of these cells, it is dispensable for virus attachment. These results indicate that a receptor other than SCARB2 is responsible for virus attachment to the cell and probably for internalization of virions, not only in Jurkat cells but also in RD cells and other EV-A71–susceptible cells. SCARB2 is highly concentrated in lysosomes and late endosomes, where it is likely to trigger acid-dependent uncoating of virions, the critical final step of the entry process. Our results suggest that the essential interactions between EV-A71 and SCARB2 occur, not at the cell surface, but within the cell. Author summary: Enterovirus A71 (EV-A71) is a major pathogen of hand-foot-and-mouth disease. Although a variety of receptor molecules have been proposed to mediate EV-A71 infection, one of them, SCARB2, is widely believed to serve as the primary attachment and entry receptor on many cell types. SCARB2—initially identified as a lysosomal integral membrane protein, LIMP II—is primarily expressed within the cytoplasm rather than on the cell surface. In this study, we examined how this largely cytoplasmic protein functions during EV-A71 attachment and infection. In contrast to earlier reports, we found that SCARB2 was not expressed at detectable levels on the surface of EV-A71–susceptible cell lines, and that its elimination with CRISPR/Cas9 had no effect on attachment of virions to the cell surface. Nonetheless, SCARB2 was readily detected on late endosomes and lysosomes, and is essential for productive infection. The results suggest that non-SCARB2 receptors are responsible for virus attachment and internalization, whereas SCARB2 within late endosomes and lysosomes is an intracellular receptor essential for a subsequent event in infection. [ABSTRACT FROM AUTHOR]

Latent chronic inflammation has been proposed as a key mediator of multiple derangements in metabolic syndrome (MetS), which are increasingly becoming recognized as risk factors for age-related cognitive decline. However, the question remains whether latent chronic inflammation indeed induces brain inflammation and cognitive decline. A mouse model of latent chronic inflammation was constructed by a chronic subcutaneous infusion of low dose lipopolysaccharide (LPS) for four weeks. A receptor for advanced glycation end products (RAGE) knockout mouse, a chimeric myeloid cell specific RAGE-deficient mouse established by bone marrow transplantation and a human endogenous secretory RAGE (esRAGE) overexpressing adenovirus system were utilized to examine the role of RAGE in vivo. The cognitive function was examined by a Y-maze test, and the expression level of genes was determined by quantitative RT-PCR, western blot, immunohistochemical staining, or ELISA assays. Latent chronic inflammation induced MetS features in C57BL/6J mice, which were associated with cognitive decline and brain inflammation characterized by microgliosis, monocyte infiltration and endothelial inflammation, without significant changes in circulating cytokines including TNF-α and IL-1β. These changes as well as cognitive impairment were rescued in RAGE knockout mice or chimeric mice lacking RAGE in bone marrow cells. P-selectin glycoprotein ligand-1 (PSGL-1), a critical adhesion molecule, was induced in circulating mononuclear cells in latent chronic inflammation in wild-type but not RAGE knockout mice. These inflammatory changes and cognitive decline induced in the wild-type mice were ameliorated by an adenoviral increase in circulating esRAGE. Meanwhile, chimeric RAGE knockout mice possessing RAGE in myeloid cells were still resistant to cognitive decline and brain inflammation. These findings indicate that RAGE in inflammatory cells is necessary to mediate stimuli of latent chronic inflammation that cause brain inflammation and cognitive decline, potentially by orchestrating monocyte activation via regulation of PSGL-1 expression. Our results also suggest esRAGE-mediated inflammatory regulation as a potential therapeutic option for cognitive dysfunction in MetS with latent chronic inflammation. [ABSTRACT FROM AUTHOR]

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a novel negative checkpoint receptor (NCR) primarily involved in maintaining immune tolerance. It has a role in the pathogenesis of autoimmune disorders and cancer and has shown promising results as a therapeutic target. However, there is still some ambiguity regarding the ligands of VISTA and their interactions with each other. While V-Set and Immunoglobulin domain containing 3 (VSIG-3) and P-selectin glycoprotein ligand-1(PSGL-1) have been extensively studied as ligands for VISTA, the others have received less attention. It seems that investigating VISTA ligands, reviewing their functions and roles, as well as outcomes related to their interactions, may allow an understanding of their full functionality and effects within the cell or the microenvironment. It could also help discover alternative approaches to target the VISTA pathway without causing related side effects. In this regard, we summarize current evidence about VISTA, its related ligands, their interactions and effects, as well as their preclinical and clinical targeting agents. [ABSTRACT FROM AUTHOR]

P-selectin glycoprotein ligand-1 (PSGL-1) has been established to be a cell adhesion molecule that is involved in the cellular rolling mechanism and the extravasation cascade, enabling the recruitment of immune cells to sites of inflammation. In recent years, researchers have established that PSGL-1 also functions as an HIV restriction factor. PSGL-1 has been shown to inhibit the HIV reverse transcription process and inhibit the infectivity of HIV virions produced by cells expressing PSGL-1. Cumulative evidence gleaned from contemporary literature suggests that PSGL-1 expression negatively affects the functions of immune cells, particularly T-cells, which are critical participants in the defense against HIV infection. Indeed, some researchers have observed that PSGL-1 expression and signaling provokes T-cell exhaustion. Additionally, it has been established that PSGL-1 may also mediate virus capture and subsequent transfer to permissive cells. We therefore believe that, in addition to its beneficial roles, such as its function as a proinflammatory molecule and an HIV restriction factor, PSGL-1 expression during HIV infection may be disadvantageous and may potentially predict HIV disease progression. In this hypothesis review, we provide substantial discussions with respect to the possibility of using PSGL-1 to predict the potential development of particular pathological conditions commonly seen during HIV infection. Specifically, we speculate that PSGL-1 may possibly be a reliable biomarker for immunological status, inflammation/translocation, cell exhaustion, and the development of HIV-related cancers. Future investigations directed towards our hypotheses may help to evolve innovative strategies for the monitoring and/or treatment of HIV-infected individuals. [ABSTRACT FROM AUTHOR]

Acute pancreatitis (AP) is a devastating disease characterized by an inflammatory disorder of the pancreas. P-selectin glycoprotein ligand-1 (PSGL-1) plays a crucial role in the initial steps of the adhesive at process to inflammatory sites, blockade of PSGL-1 might confer potent anti-inflammatory effects. In this study, we generated two non-human primate derived monoclonal antibodies capable of efficiently targeting human PSGL-1, RH001-6 and RH001-22, which were screened from immunized rhesus macaques. We found that RH001-6, can effectively block the binding of P-selectin to PSGL-1, and abolish the adhesion of leukocytes to endothelial cells in vitro. In vivo , we verified that RH001-6 relieved inflammatory responses and pancreatic injury in both caerulein and l -arginine induced AP models. We also evaluated the safety profile after RH001-6 treatment in mice, and verified that RH001-6 did not cause any significant pathological damages in vivo. Taken together, we developed a novel non-human primate derived PSGL-1 blocking antibody with high-specificity, named RH001-6, which can interrupt the binding of PSGL-1 and P-selectin and attenuate inflammatory responses during AP. Therefore, RH001-6 is highly potential to be further developed into therapeutics against acute inflammatory diseases, such as AP. A non-human primate derived monoclonal anti-human P-selectin glycoprotein ligand-1 antibody (RH001-6) was generated, which can effectively attenuate acute pancreatitis by reducing the inflammatory responses and pancreatic injury in mice, and may be the promising therapeutics. [Display omitted] [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Excessive neutrophil extracellular traps (NETs) is involved in the progression of acute pancreatitis (AP) but the mechanisms controlling NETs formation in AP are not fully understood. Therefore, our study sought to investigate the mechanism of the highly expressed P-selectin stimulating the formation of NETs in AP. Methods: NETs formation was detected by flow cytometry, immunofluorescence staining, and cf-DNA and MPO-DNA complexes were measured as biomarkers of NETs formation. Neutrophils treated with P-selectin and pharmacological inhibitors were examined by western blot, immunofluorescence staining and flow cytometry. Mouse model of AP was established by caerulein and the effect of inhibiting P-selectin by PSI-697 on the level of NETs and PAD4 in pancreatic tissue was observed. The severity of AP was evaluated by histopathological score and the detection of serum amylase and lipase. Results: Patients with AP had elevated levels of NETs and P-selectin compared with healthy volunteers. Stimulation of P-selectin up-regulated the expression of PSGL-1, increased the phosphorylation of Syk, mediated intracellular calcium signal and led to the activation and expression of PAD4, which modulated NETs formation in neutrophils. Pretreament with PSI-697 blunted NETs formation and PAD4 expression in the pancreatic tissue, and ameliorated the severity of AP in mice. Conclusion: Taken together, these results suggest that P-selectin induces NETs through PSGL-1 and its downstream Syk/Ca2+/PAD4 signaling pathway, and that targeting this pathway might be a promising strategy for the treatment of AP. [ABSTRACT FROM AUTHOR]

BackgroundExcessive neutrophil extracellular traps (NETs) is involved in the progression of acute pancreatitis (AP) but the mechanisms controlling NETs formation in AP are not fully understood. Therefore, our study sought to investigate the mechanism of the highly expressed P-selectin stimulating the formation of NETs in AP.MethodsNETs formation was detected by flow cytometry, immunofluorescence staining, and cf-DNA and MPO-DNA complexes were measured as biomarkers of NETs formation. Neutrophils treated with P-selectin and pharmacological inhibitors were examined by western blot, immunofluorescence staining and flow cytometry. Mouse model of AP was established by caerulein and the effect of inhibiting P-selectin by PSI-697 on the level of NETs and PAD4 in pancreatic tissue was observed. The severity of AP was evaluated by histopathological score and the detection of serum amylase and lipase.ResultsPatients with AP had elevated levels of NETs and P-selectin compared with healthy volunteers. Stimulation of P-selectin up-regulated the expression of PSGL-1, increased the phosphorylation of Syk, mediated intracellular calcium signal and led to the activation and expression of PAD4, which modulated NETs formation in neutrophils. Pretreament with PSI-697 blunted NETs formation and PAD4 expression in the pancreatic tissue, and ameliorated the severity of AP in mice.ConclusionTaken together, these results suggest that P-selectin induces NETs through PSGL-1 and its downstream Syk/Ca2+/PAD4 signaling pathway, and that targeting this pathway might be a promising strategy for the treatment of AP.

Background and Purpose: Abacavir, an antiretroviral drug used in HIV therapy associated with myocardial infarction, promotes thrombosis through P2X7 receptors. The role of platelets as pro‐thrombotic cells is acknowledged whereas that of neutrophils—due to their secretory capacity—is gaining recognition. This study analyses the role of neutrophils—specifically the secretome of abacavir‐treated neutrophils (SNABC)—in platelet activation that precedes thrombosis. Experimental Approach: Effects of abacavir or SNABC on platelet activation and platelet–leukocyte interactions and expression of lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) were analysed by flow cytometry. The secretome was analysed by proteomics. The role of leukocytes in the actions of abacavir was evaluated in a mouse model of thrombosis. Key Results: Abacavir induced platelet–leukocyte interactions, not directly via effects of abacavir on platelets, but via activation of neutrophils, which triggered interactions between platelet P‐selectin and neutrophil P‐selectin glycoprotein ligand‐1 (PSGL‐1). SNABC stimulated platelet activation and platelet–leukocyte interactions through a process that was dependent on LOX‐1, neutrophil P2X7 and platelet P2Y1, P2Y12 and P2X1 receptors. Abacavir induced the expression of LOX‐1 on neutrophils and of the soluble form of LOX‐1 (sLOX‐1) in SNABC. Neutrophils, LOX‐1, P2X7, P2Y1, P2Y12 and P2X1 receptors were required for the pro‐thrombotic actions of abacavir in vivo. Conclusion and Implications: Neutrophils are target cells in abacavir‐induced thrombosis. Abacavir released sLOX‐1 from neutrophils via activation of their P2X7 receptors, which in turn activated platelets. Hence, sLOX‐1 could be the missing link in the cardiovascular risk associated with abacavir. [ABSTRACT FROM AUTHOR]

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the generation of anti-DNA autoantibodies due to exposure of immune cells to excessive amounts of extracellular DNA. Lack of P-selectin in mice induces the development of a lupus-like syndrome and patients with cutaneous lupus have reduced P-selectin expression in skin vessels. Using flow cytometry we analyzed in healthy donors and patients the expression of P-selectin Glycoprotein Ligand-1 (PSGL-1) in circulating neutrophils and the implication of PSGL-1/P-selectin interaction in neutrophil extracellular traps (NETs) generation. We found a statistical significance that neutrophils from active SLE patients have a reduced expression of PSGL-1 and low levels of PSGL-1 in neutrophils from SLE patients associated with the presence of anti-dsDNA antibodies, clinical lung involvement, Raynaud's phenomenon, and positive lupus anticoagulant. PSGL-1 is present along the DNA in the NET. In healthy donors, neutrophil interaction with immobilized P-selectin triggers Syk activation, increases the NETs percentage and reduces the amount of DNA extruded in the NETs. In active SLE patients, neutrophil interaction with P-selectin does not activate Syk or reduce the amount of DNA extruded in the NETs, that might contribute to increase the extracellular level of DNA and hence, to disease pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Macrophages secrete many cytokines and chemokines, which can provoke either an anti-tumor or pro-tumor immune response. P-selectin glycoprotein ligand-1 (PSGL-1) is expressed in macrophages and plays a vital role in synergizing for a more robust anti-tumor response. However, there are few studies about PSGL-1 expression status and clinical value of biological function in cervical cancer. Methods: In this study, 565 participants were enrolled. PSGL-1 mRNA was detected by real-time quantitative PCR (qPCR) with cervical cytology specimens. The relationship between PSGL-1 and cervical intraepithelial neoplasia in two grades and more (CIN2+) was analyzed, and the optimal cut-off values of PSGL-1 to predict CIN2+ were calculated. In addition, the clinical significance of PSGL-1 in cervical cancer was determined by Kaplan-Meier Cox regression based on the database. Results: The mean PSGL-1 increased significantly with cervical lesion development, especially compared with CIN2+ (p<0.05). Moreover, the expression of PSGL-1 increased significantly in HPV-16/18 positive and HPV-18 positive, but not in HPV-16 positive and other HR-HPV positive. And then, it demonstrated that the area under the receiver operating characteristic curve (AUC) of PSGL-1 was 0.820, and an optimal cut-off 0.245. Furthermore, the PSGL-1 had the highest odds ratio and highest OR (OR= 8.707; 95% CI (.371-19.321)) for the detection of CIN 2+. In addition, our result also indicated that higher PSGL-1 expression was significantly related to a better prognosis in cervical cancer due to immune cell infiltration. Conclusions: PSGL-1≥0.245 in cervical cytology specimens is a new auxiliary biomarker of CIN2+, and it may be a promising prognosis predictor and potential immunotherapy target linked with immune infiltration of cervical cancer. [ABSTRACT FROM AUTHOR]

Objective: Type 2 diabetes mellitus (T2D) is the result of a dysregulation of insulin production and signalling, leading to an increase in both glucose concentration and pro-inflammatory cytokines such as interleukin (IL)-6 and tumour necrosis factor (TNF)-α. Previous work showed that T2D patients exhibited immune dysfunction associated with increased adhesion molecule expression on endothelial cell surfaces, accompanied by decreased neutrophil rolling velocity on the endothelial cell surface. Changes in cell rolling adhesion have direct vascular and immune complications such as atherosclerosis and reduced healing time in T2D patients. While previous studies focused primarily on how endothelial cells affect neutrophil rolling under T2D conditions, little is known about changes to neutrophils that affect their rolling. In this study, we aim to show how the rolling behaviour of neutrophils is affected by T2D conditions on a controlled substrate. Results: We found that neutrophils cultured in T2D-serum mimicking media increased cell rolling velocity compared to neutrophils under normal conditions. Specifically, glucose alone is responsible for higher rolling velocity. While cytokines further increase the rolling velocity, they also reduce the cell size. Both glucose and cytokines likely reduce the function of P-selectin Glycoprotein Ligand-1 (PSGL-1) on neutrophils. [ABSTRACT FROM AUTHOR]

The risk factors for CVD are high blood pressure, high cholesterol levels, smoking, diabetes, obesity, and a family history of CVD. Atherosclerosis is considered the main precursor of CVD and is characterized by pathological remodeling of the vascular wall with the development of atheromatous plaques in the inner lining of medium and large arteries. The term CVD includes different pathologies affecting the heart and circulatory system, such as heart failure, coronary artery disease, stroke, hypertension, atherosclerosis, and aortic aneurysms. Cardiovascular diseases (CVD) are a major cause of morbidity and mortality worldwide, accounting for more than 17 million deaths each year. [Extracted from the article]

Activation of integrins is crucial for recruitment of flowing leukocytes to inflammatory or injured vascular sites, but their spatiotemporal characteristics are incompletely understood. We discovered that β2-integrin activation over the entire surface of neutrophils on immobilized P-selectin occurred via mitogen-activated protein kinase (MAPK) or non-MAPK signaling with a minute-level timescale in a force-dependent manner. In flow, MAPK signaling required intracellular Ca2+ release to activate integrin within 2 min. Integrin activation via non-MAPK signaling occurred first locally in the vicinity of ligated P-selectin glycoprotein ligand-1 (PSGL-1) within sub-seconds, and then over the entire cell surface within 1 min in an extracellular Ca2+ influxdependent manner. The transition from a local (but rapid) to global (but slow) activation mode was triggered by ligating the freshly activated integrin. Lipid rafts, moesin, actin, and talin were involved in non-MAPK signaling. Fluid loads had a slight effect on local integrin activation with a second-level timescale, but served as enhancers of global integrin activation. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) released from activated platelets contain microRNAs, the most abundant of which is hsa-miR-223-3p. Endogenous hsa-miR-223-3p suppresses the expression of tissue factor (TF), the initiator of the extrinsic coagulation pathway, in endothelial cells. Monocytes can be induced to express TF to enhance coagulation, but the role of hsa-miR-223-3p in regulating monocyte TF remains unknown. This study examined whether hsa-miR-223-3p from platelet-derived EVs (pdEVs) affects TF expression in monocytes. THP-1 cells, differentiated into a monocyte-like phenotype with 1α,25-dihydroxyvitaminD3, were transfected with hsa-miR-223-3p mimic or control microRNA. Alternatively, THP-1 cells were incubated with pdEVs from PAR1-agonist peptide activated-platelets, as platelet releasate, or pdEVs isolated by ultracentrifugation. Transfection with hsa-miR-223-3p mimic resulted in significant reductions in TF protein, determined by western blotting and flow cytometry and reduced procoagulant activity, measured by a TF-specific factor Xa generation assay, compared to cells transfected with control microRNA. This reduction was reversed by co-transfection with hsa-miR-223-3p inhibitor, AntagomiR-223. Incubation of THP-1 cells with pdEVs also decreased TF expression; however, this was not reversed by AntagomiR-223. Taken together, monocyte TF expression is downregulated by hsa-miR-223-3p, but when transferred via pdEVs the effect was not reversed with Antagomir-223, suggesting other pdEV components may contribute to TF regulation. Abbreviations: Tissue factor (TF), Factor VII (FVII), activated Factor VII (FVIIa), Factor X (FX), activated Factor X (FXa), extracellular vesicles (EVs), microvesicles (MVs), platelet-derived extracellular vesicles (pdEVs), protease-activated receptor 1 agonist peptide (PAR1-AP), lipopolysaccharide (LPS), P-selectin glycoprotein ligand-1 (PSGL-1), Tris-Buffered Saline Tween (TBST), room temperature (RT) [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae is an invasive pathogen with high morbidity and mortality in the immunocompromised children and elderly. NOD-like receptor family pyrin domain containing 6 (NLRP6) plays an important role in the host innate immune response against pathogen infections. Our previous studies have shown that NLRP6 plays a negative regulatory role in host defense against S. pneumoniae , but the underlying mechanism is still unclear. The further negative regulatory role of NLRP6 in the host was investigated in this study. Our results showed that NLRP6−/− mice in the lung had lower bacterial burdens after S. pneumoniae infection and expressed higher level of tight junction (TJ) protein occludin compared to WT mice, indicating the detrimental role of NLRP6 in the host defense against S. pneumoniae infection. Transcriptome analysis showed that genes related to leukocytes migration and recruitment were differentially expressed between wild-type (WT) and NLRP6 knockout (NLRP6−/−) mice during S. pneumoniae infection. Also, NLRP6−/− mice showed higher expression of chemokines including C-X-C motif chemokine ligand 1 (CXCL1) and 2 (CXCL2) and lower gene expression of complement C3a receptor 1 (C3aR1) and P-selectin glycoprotein ligand-1 (PSGL-1) which are the factors that inhibit the recruitment of neutrophils. Furthermore, NLRP6−/− neutrophils showed increased intracellular bactericidal ability and the formation of neutrophil extracellular traps (NETs) during S. pneumoniae infection. Taken together, our study suggests that NLRP6 is a negative regulator of neutrophil recruitment and function during S. pneumoniae infection. Our study provides a new insight to develop novel strategies to treat invasive pneumococcal infection. [ABSTRACT FROM AUTHOR]

Background: P-selectin glycoprotein ligand-1 (PSGL-1/CD162) has been studied extensively for its role in mediating leukocyte rolling through interactions with its cognate receptor, P-selectin. Recently, PSGL-1 was identified as a novel HIV-1 host restriction factor, particularly when expressed at high levels in the HIV envelope. Importantly, while the potent antiviral activity of PSGL-1 has been clearly demonstrated in various complementary model systems, the breadth of PSGL-1 incorporation across genetically diverse viral isolates and clinical isolates has yet to be described. Additionally, the biological activity of virion-incorporated PSGL-1 has also yet to be shown. Results: Herein we assessed the levels of PSGL-1 on viruses produced through transfection with various amounts of PSGL-1 plasmid DNA (0–250 ng), compared to levels of PSGL-1 on viruses produced through infection of T cell lines and primary PBMC. We found that very low levels of PSGL-1 plasmid DNA (< 2.5 ng/well) were necessary to generate virus models that could closely mirror the phenotype of viruses produced via infection of T cells and PBMC. Unique to this study, we show that PSGL-1 is incorporated in a broad range of HIV-1 and SIV isolates and that virions with incorporated PSGL-1 are detectable in plasma from viremic HIV-1-infected individuals, corroborating the relevance of PSGL-1 in natural infection. Additionally, we show that PSGL-1 on viruses can bind its cognate selectin receptors, P-, E-, and L-selectins. Finally, we show viruses with endogenous levels of PSGL-1 can be captured by P-selectin and transferred to HIV-permissive bystander cells, highlighting a novel role for PSGL-1 in HIV-1 infection. Notably, viruses which contained high levels of PSGL-1 were noninfectious in our hands, in line with previous findings reporting the potent antiviral activity of PSGL-1. Conclusions: Our results indicate that levels of PSGL-1 incorporation into virions can vary widely among model systems tested, and that careful tailoring of plasmid levels is required to recapitulate physiological systems when using pseudovirus models. Taken together, our data suggest that PSGL-1 may play diverse roles in the physiology of HIV-1 infection, particularly due to the functionally active state of PSGL-1 on virion surfaces and the breadth of PSGL-1 incorporation among a wide range of viral isolates. [ABSTRACT FROM AUTHOR]

Simple Summary: Severe forms of coronavirus disease 2019 (COVID-19) are related to an alteration in the coagulation process determined by genetic factors. Variability on F5 and SERPINE1 genes has been previously reported as a risk factor for thrombosis associated with different diseases. In this study, we aimed to evaluate if two relevant variants in the F5 and SERPINE1 genes were related to the requirement of invasive mechanical ventilation in hospitalized patients with COVID-19 and/or the levels of coagulation-related proteins. We observed that patients presenting the studied variants in F5 and SERPINE1 exhibit different levels of coagulation-related proteins. Moreover, the levels of these proteins were higher among patients requiring invasive mechanical ventilation when compared to hospitalized patients with no ventilation support. This study contributes to the genetics insight regarding COVID-19 severity and the inter-individual susceptibility for a severe form of the infectious disease. An impaired coagulation process has been described in patients with severe or critical coronavirus disease (COVID-19). Nevertheless, the implication of coagulation-related genes has not been explored. We aimed to evaluate the impact of F5 rs6025 and SERPINE1 rs6092 on invasive mechanical ventilation (IMV) requirement and the levels of coagulation proteins among patients with severe COVID-19. Four-hundred fifty-five patients with severe COVID-19 were genotyped using TaqMan assays. Coagulation-related proteins (P-Selectin, D-dimer, P-selectin glycoprotein ligand-1, tissue plasminogen activator [tPA], plasminogen activator inhibitor-1, and Factor IX) were assessed by cytometric bead arrays in one- and two-time determinations. Accordingly, SERPINE1 rs6092, P-Selectin (GG 385 pg/mL vs. AG+AA 632 pg/mL, p = 0.0037), and tPA (GG 1858 pg/mL vs. AG+AA 2546 pg/mL, p = 0.0284) levels were different. Patients carrying the CT F5-rs6025 genotype exhibited lower levels of factor IX (CC 17,136 pg/mL vs. CT 10,247 pg/mL, p = 0.0355). Coagulation proteins were also different among IMV patients than non-IMV. PSGL-1 levels were significantly increased in the late stage of COVID-19 (>10 days). The frequencies of F5 rs6025 and SERPINE1 rs6092 variants were not different among IMV and non-IMV. The SERPINE1 rs6092 variant is related to the impaired coagulation process in patients with COVID-19 severe. [ABSTRACT FROM AUTHOR]

Background: Platelets are thought to be involved in the pathophysiology of asthma, presumably through direct adhesion to inflammatory cells, including group 2 innate lymphoid cells (ILC2s). Here, we tried to elucidate the effects of platelet adhesion to ILC2s in vitro and in vivo, as well as the mechanisms involved. Methods: Alternaria‐induced ILC2‐dependent airway inflammation models using wild‐type and c‐mpl−/− mice were evaluated. Both purified CD41+ and CD41−ILC2s were cultured with IL‐2 and IL‐33 to determine in vitro Type 2 (T2) cytokine production and cell proliferation. RNA‐seq data of flow‐cytometry‐sorted CD41+ and CD41−ILC2s were used to isolate ILC2‐specific genes. Flow cytometry was performed to determine the expression of CD41 and adhesion‐related molecules on ILC2s in both mouse and human tissues. Results: T2 inflammation and T2 cytokine production from ILC2s were significantly reduced in the c‐mpl−/− mice compared to wild‐type mice. Platelet‐adherent ILC2s underwent significant proliferation and showed enhanced T2 cytokine production when exposed to IL‐2 and IL‐33. The functions of ILC2‐specific genes were related to cell development and function. Upstream regulator analysis identified 15 molecules, that are thought to be involved in ILC2 activation. CD41 expression levels were higher in ILC2s from human PBMCs and mouse lung than in those from secondary lymphoid tissues, but they did not correlate with the P‐selectin glycoprotein ligand‐1 or CD24 expression level. Conclusion: Platelets spontaneously adhere to ILC2s, probably in the peripheral blood and airways, thereby potentiating ILC2s to enhance their responses to IL‐33. [ABSTRACT FROM AUTHOR]

RationaleCirculating monocytes play pivotal roles in chronic inflammatory diseases. Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid, are known to have anti-inflammatory effects and are hydrolyzed by soluble epoxide hydrolase (sEH).ObjectiveWe aimed to investigate the effect of sEH inhibition in atherogenesis.Methods and resultsMice with low-density lipoprotein receptor deficiency (Ldlr(-/-)) with or without sEH inhibitor, and Ldlr/sEH double-knockout (DK) mice were fed a Western-type diet (WTD) for 6weeks to induce arteriosclerosis. Both sEH inhibition and gene depletion decreased the WTD-induced hyperlipidemia, plaque area and macrophage infiltration in mice arterial wall. Ly6C(hi) infiltration of monocytes remained similar in blood, spleen and bone marrow of DK mice, but was decreased in aortic lesions. To further assess the role of sEH or EETs in monocyte/macrophage infiltration in atherogenesis, we transplanted DK bone marrow into Ldlr(-/-) recipients, and then fed mice the WTD. Aortic lesions and Ly6C(hi) monocyte infiltration were reduced in mice with transplanted bone marrow of DK mice without diminishing the cholesterol level. Furthermore, sEH inhibition or gene depletion increased the ratio of EETs/DHETs and diminished the expression of P-selectin glycoprotein ligand 1 (PSGL-1) in mice peripheral-blood mononuclear cells. Monocyte adhesion to P-selectin and to tumor necrosis factor α-activated endothelial cells was also diminished by sEH inhibition.ConclusionsEH inhibition and gene depletion attenuated atherosclerosis in mice by decreasing the infiltration of monocytes into the artery wall. EET and PSGL-1 may play pivotal roles in monocyte/macrophage infiltration and atherogenesis.

Chronic viruses and cancers thwart immune responses in humans by inducing T cell dysfunction. Using a murine chronic virus that models human infections, we investigated the function of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), that is upregulated on responding T cells. PSGL-1-deficient mice cleared the virus due to increased intrinsic survival of multifunctional effector T cells that had downregulated PD-1 as well as other inhibitory receptors. Notably, this response resulted in CD4(+)-T-cell-dependent immunopathology. Mechanistically, PSGL-1 ligation on exhausted CD8(+) T cells inhibited T cell receptor (TCR) and interleukin-2 (IL-2) signaling and upregulated PD-1, leading to diminished survival with TCR stimulation. In models of melanoma cancer in which T cell dysfunction occurs, PSGL-1 deficiency led to PD-1 downregulation, improved T cell responses, and tumor control. Thus, PSGL-1 plays a fundamental role in balancing viral control and immunopathology and also functions to regulate T cell responses in the tumor microenvironment.

ABSTRACTEnterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine, Salvia miltiorrhiza (Danshen), were screened for anti-EV-A71. We identified a natural product, rosmarinic acid (RA), as a potential inhibitor of EV-A71 by cell-based antiviral assay and in vivo mouse model. Results also show that RA may affect the early stage of viral infection and may target viral particles directly, thereby interfering with virus-P-selectin glycoprotein ligand-1 (PSGL1) and virus-heparan sulfate interactions without abolishing the interaction between the virus and scavenger receptor B2 (SCARB2). Sequencing of the plaque-purified RA-resistant viruses revealed a N104K mutation in the five-fold axis of the structural protein VP1, which contains positively charged amino acids reportedly associated with virus-PSGL1 and virus-heparan sulfate interactions via electrostatic attraction. The plasmid-derived recombinant virus harbouring this mutation was confirmed to be refractory to RA inhibition. Receptor pull-down showed that this non-positively charged VP1-N104 is critical for virus binding to heparan sulfate. As the VP1-N104 residue is conserved among different EV-A71 strains, RA may be useful for inhibiting EV-A71 infection, even for emergent virus variants. Our study provides insight into the molecular mechanism of virus-host interactions and identifies a promising new class of inhibitors based on its antiviral activity and broad spectrum effects against a range of EV-A71.

Objectives: Disease-specific biomarkers are an essential tool for the efficient management of pathological conditions, including susceptibility determination, diagnosis, and preventive monitoring. P-selectin (CD62p) is selectively expressed after platelet activation, and is involved in thrombus formation and immune response. The serum and plasma level of CD62p increases in conditions such as heart attack, stroke, some immune diseases, and cancer. The aim of this study was to develop a new, aptamer-based method for CD62p measurement. Methods: Aptamers can be used to target specific biomarkers based on their molecular shape. The systematic evolution of ligands by exponential enrichment (SELEX) process is a method to identify aptamers with a high affinity for a specific macromolecular target. This study explored using aptamers to measure CD62p. First, aptamers that specifically bind to CD62p were isolated using the SELEX method. The aptamers that demonstrated the highest binding affinity to the CD62p protein were used to coat 96-well plates. Next, the level of CD62p in human serum was measured using this aptamer and the test performance parameters of sensitivity, specificity, and precision were evaluated. Results: Among the aptamers used, Apt-1, Apt-2, and Apt-3, bound to CD62p protein with high affinity. Apt-2 had the greatest binding affinity to CD62, demonstrating a binding constant of -9.6 kcal/mol, and a dissociation constant (Kd) of 18.15±2.36 nM. Bovine serum albumin was used in the specificity test as a negative control. No binding between the selected aptamers and this protein was observed. The performance showed that of intra-assay coefficient of variation (CV) was <6.52% and inter-assay CV was <3.96%. The recovery values were between 95.06% and 107.95%, and the linearity values were between 99.49% and 113.17%. Sensitivity was calculated at 0.30 ng/mL of CD62p. Conclusion: The aptamer method to measure CD62p proved to be a sensitive, specific, time-saving, and low-cost option. [ABSTRACT FROM AUTHOR]

Background: Cancer immunotherapy has gained increasing popularity as a novel approach to treat cancer. A member of the B7 family, V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a novel immune checkpoint that regulates a broad spectrum of immune responses. VISTA is an acidic pH-selective ligand for P-selectin glycoprotein ligand-1(PSGL-1). CA-170, a first-in-class small-molecule dual antagonist of VISTA/PD-L1, was collaboratively developed by Aurigene Discovery Technologies Limited and Curis, Inc. It is currently in Phase I clinical trial. Results: In this study, we develop homology modeling for the VISTA 3D structure and subsequent virtual screening for VISTA small-molecule hit ligands. Visualization of the binding postures of docked ligands with the VISTA protein indicates that some small molecular compounds target VISTA. The ability of antagonist to disrupt immune checkpoint VISTA pathways was investigated though functional studies in vitro. Conclusions: Affinity active molecule for VISTA was obtained through virtual screening, and the antagonist compound activity to VISTA was assayed in cellular level. We reported a small molecule with high VISTA affinity as antagonist, providing ideas for development VISTA-targeted small molecule compound in cancer immunotherapy. [ABSTRACT FROM AUTHOR]

[Display omitted] Pancreatic ductal adenocarcinoma is one of the most lethal malignant tumors, its drug resistance, immunosuppression and metastasis makes the traditional chemotherapy and immunotherapy inefficient. Here we confirmed a 3-aminophenylboronic acid-modified low molecular weight heparin-D-α-tocopheryl succinate micellar nanoparticle (PBA-LMWH-TOS NP, PLT NP) could inhibit orthotopic pancreatic tumor and its spontaneous metastases. The small particle size and high affinity of PBA to sialic acid residue (SA) made PLT/PTX NPs significantly targeted and accumulated in both pancreatic tumor tissues and metastases. The immunosuppressive microenvironment of pancreatic tumor was most caused by the infiltration of immunosuppressive cells, mainly myeloid-derived suppressor cells (MDSCs). We first reported that P-selectin glycoprotein ligand-1 (PSGL-1) was expressed on the surfaces of MDSCs in pancreatic tumor tissues. Meanwhile, we found that LMWH could inhibit the early stage of adhesion cascade between vascular endothelial cells (VECs) and MDSCs by interfering with P-selectin/PSGL-1 binding, thus inhibiting MDSC recruitment to pancreatic tumor tissues. The therapeutic results indicated that PLT/PTX NPs could significantly improve the immune microenvironment of pancreatic tumor and inhibit spontaneous metastases. This nanosystem provides a new immune microenvironment regulation mechanism based on carrier materials in pancreatic tumor, and has high clinical application potential. [ABSTRACT FROM AUTHOR]

An unknown, but surely substantial, proportion of the vast number of patients included in various COVID-19 GWAS will have presented with ARDS, but the relevance of this to non-COVID-19 ARDS remains uncertain. Genome-wide association studies (GWAS) have demonstrated the power of genomics in helping us better understand complex diseases. As an illustration, in Type II Diabetes Mellitus, GWAS and gene-resequencing studies have identified a protective loss-of-function gene mutation that encodes a zinc transporter in pancreatic islet cells that in turn has led to an interest in developing therapies that inhibit this zinc-transporter [[1]]. [Extracted from the article]

Differently expressed genes of atherosclerotic sample analysis are helpful to sort the prominent genes that influence the plaque formation and progression. Scientific evidence-based protein–protein interaction network (PPIN) studies were used to find hub proteins in complex disease conditions. Druggable capacity is one of the important parameters to confirm as a successful drug target. Construction of protein interaction network and principal node analysis (PNA) on atherosclerotic data sets lead to screen the hub proteins. Furthermore, druggable property of protein pocket confirms the targetability of susceptible target candidates and for target selection. Differentially expressed genes are identified through GEO2R analyzer on data sets of various atherosclerotic samples. STRING database and Cytoscape are employed to construct PPIN. Targets were identified by PNA such as centrality measures and clustering algorithm. Gene Ontology enrichment also used as one of the screening parameters to filter the candidates related to atherosclerotic terms. Topological evaluation of target protein was successfully done by ITASSER and GROMACS, respectively. Grid-based principle of DoGSiteScorer is utilized for druggability analysis. Six proteins such as integrin alpha L (ITGAL), metallothionein 1F (MT1F), metallothionein 1X (MT1X), P-selectin glycoprotein ligand-1 (SELPLG), solute carrier family 30 A, zinc transporter protein (SLC30A1), and TNFSF13B are screened as potential biomarkers through network-based analysis. Among the six, ITGAL, SELPLG, SLC30A1, and TNSF13B are identified as better prioritized atherosclerotic targets through druggability efficiency. [ABSTRACT FROM AUTHOR]

P-selectin as a treatment for lupus: Dysfunctional regulatory T (Treg) cells are known to be involved in the pathogenesis of systemic lupus erythematosus (SLE), but the mechanisms mediating Treg cell dysfunction are not clear. Here, Scherlinger et al. demonstrated that platelets can impair Treg cell function after forming aggregates mediated by platelet (P)–selectin expression on platelets and P-selectin glycoprotein ligand-1 (PSGL-1) expression on Treg cells. Treg cells aggregated more frequently with platelets from individuals with active SLE as compared to healthy donors. In addition, blocking the interaction between P-selectin and PSGL-1 reduced disease severity in a mouse model of SLE. Thus, the P-selectin/PSGL-1 axis may represent a therapeutic target for SLE. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of tolerance toward self-nucleic acids, autoantibody production, interferon expression and signaling, and a defect in the regulatory T (Treg) cell compartment. In this work, we identified that platelets from patients with active SLE preferentially interacted with Treg cells via the P-selectin/P-selectin glycoprotein ligand-1 (PSGL-1) axis. Selectin interaction with PSGL-1 blocked the regulatory and suppressive properties of Treg cells and particularly follicular Treg cells by triggering Syk phosphorylation and an increase in intracytosolic calcium. Mechanistically, P-selectin engagement on Treg cells induced a down-regulation of the transforming growth factor–β axis, altering the phenotype of Treg cells and limiting their immunosuppressive responses. In patients with SLE, we found an up-regulation of P- and E-selectin both on microparticles and in their soluble forms that correlated with disease activity. Last, blocking P-selectin in a mouse model of SLE improved cardinal features of the disease, such as anti-dsDNA antibody concentrations and kidney pathology. Overall, our results identify a P-selectin–dependent pathway that is active in patients with SLE and validate it as a potential therapeutic avenue. [ABSTRACT FROM AUTHOR]

Aim of the study: P-selectin (CD62P) is a platelet activation marker that was claimed to mediate the accumulation of platelets induced by cholestasis. The nature of platelet dysfunction and hemostasis abnormalities in cholestatic liver disease needs to be more explored. The aim of this study was to assess platelet CD62P expression in cirrhotic patients with and without cholestasis, and to evaluate its relationship with a bleeding tendency. Material and methods: 150 patients were included in this case-control study. Participants were divided into 84 patients with liver cirrhosis (group I), 44 of whom had cholestasis (Group Ia) and 40 patients were without cholestasis (group Ib); 36 patients who were cholestatic without liver cirrhosis (group II); and 30 healthy subjects who formed the control group (group III). Platelet CD62P expression was assessed by a flow cytometer. Results: Platelets expressing CD62P were significantly increased in all patient groups compared to controls (p < 0.001). Platelets expressing CD62P were significantly increased in gastrointestinal (GIT) bleeders compared to non-bleeders in cirrhotic and cholestatic groups (p < 0.001 each). Among group I patients at cut-off > 12.4, up-regulation of platelet CD62P yielded 72% sensitivity and 44.1% specificity to discriminate bleeders from non-bleeders (p = 0.01), while among group II at cut-off > 12.9, it yielded 90% sensitivity and 80.8% specificity (p < 0.001). In cirrhotic patients, platelet CD62P expression was significantly increased in patients with an advanced Child-Pugh class (p < 0.001). Platelet expressing CD62P was shown as an independent risk factor for bleeding among cirrhotic cases with an odds ratio of 1.07 and CI 0.99-1.15. Conclusions: Up-regulation of platelet CD62P expression can serve as a GIT bleeding predictor in liver cirrhosis. [ABSTRACT FROM AUTHOR]

Glycan biosynthesis on cell surface proteins and lipids is orchestrated by different classes of enzymes and proteins including the following: i. glycosyltransferases that add saccharides; ii. glycosidases that trim glycans; iii. conserved oligomeric golgi complex members that regulate intracellular transport; iv. enzymes aiding the biosynthesis of sugar–nucleotides; and v. sulfotransferases. This manuscript describes a pooled "glycoGene CRISPR" lentiviral library that targets 347 human genes involved in the above processes. Approximately 10 single-guide RNA (sgRNA) are included against each glycogene, with the putative editing site spanning the length of the target. A data analysis scheme is presented in order to determine glycosylation pathways regulating biological processes. As proof of principle, forward genetic screen results are presented to identify penetrating glycogenes that regulate the binding of P-/E-selectin, anti-sialyl Lewis-X monoclonal antibody HECA-452 and selected lectins (phaseolus vulgaris leucoagglutinin, vicia villosa lectin, peanut agglutinin) to HL-60 promyelocytic cells. Besides validating previously established biology, the study identifies three enzymes, PAPSS1, SLC35B2 and TPST2, as key molecules regulating sulfation of the major P-selectin glycoprotein ligand-1 in leukocytes. Approximately 80–90% of the sgRNA used in this study displayed high editing efficiency, and the CRISPR library picked up entire gene sets regulating specific biosynthetic pathways rather than only isolated genes. These data suggest that the glycoGene CRISPR library contains high-efficiency sgRNA. Further, this resource could be useful for the rapid screening of glycosylation-related genes and pathways that control lectin recognition in a variety of contexts. [ABSTRACT FROM AUTHOR]

Introduction: Background: açaí is the fruit of the palm tree Euterpe oleracea Martius, which is native to the Amazon region. This fruit has been extensively studied due to its potential effects on human health. Studies have also evaluated the potential effect of açaí on the inflammatory response, but there are still few studies that have assessed this property in humans. Objective: in this study we aimed to evaluate the effects of 200 g of açaí pulp consumption per day during four weeks on a rich panel of inflammatory biomarkers. Methods: a prospective nutritional intervention study was conducted on forty apparently healthy women who consumed 200 g of açaí pulp per day for four weeks. A panel of serum inflammatory markers were evaluated before and after the nutritional intervention, namely, cell adhesion molecules (ICAM-1, IVAM-1, P-selectin, MCP-1, and fractalkine), interleukins (IL-1β, IL-6, IL-8, IL-10, and IL-17) and adipokines (adiponectin, leptin, visfatin, and adipsin). The data were analyzed using paired Student's t-test to evaluate the effect of the intervention using PASW Statistics, version 17.0, and a p-value of < 0.05 was considered significant. Results: four weeks of açaí pulp consumption decreased p-selectin, leptin, and visfatin concentrations in the serum of the participating women. Conclusion: these results show that consumption of açaí pulp was able to modulate important biomarkers of the inflammatory process in apparently healthy women. [ABSTRACT FROM AUTHOR]

Gout is a painful arthritic inflammatory disease caused by buildup of monosodium urate (MSU) crystals in the joints. Colchicine, a microtubule-depolymerizing agent that is used in prophylaxis and treatment of acute gout flare, alleviates the painful inflammatory response to MSU crystals. Using i.p. and intra-articular mouse models of gout-like inflammation, we found that GEF-H1/GEF-H1/AHRGEF2, a microtubule-associated Rho-GEF, was necessary for the inhibitory effect of colchicine on neutrophil recruitment. GEF-H1 was required for neutrophil polarization in response to colchicine, characterized by uropod formation, accumulation of F-actin and myosin L chain at the leading edge, and accumulation of phosphorylated myosin L chain, flotillin-2, and P-selectin glycoprotein ligand-1 (PSGL-1) in the uropod. Wild-type neutrophils that were pre-exposed to colchicine failed to roll or accumulate on activated endothelial monolayers, whereas GEF-H1 knockout (GEF-H1-/-) neutrophils were unaffected by treatment with colchicine. In vivo, colchicine blocked MSU-induced recruitment of neutrophils to the peritoneum and the synovium in wild-type mice, but not in GEF-H1-/- mice. Inhibition of macrophage IL-1b production by colchicine was independent of GEF-H1, supporting a neutrophil-intrinsic mode of action. Our results suggest that the anti-inflammatory effects of colchicine in acute gout-like inflammation can be accounted for by inhibition of neutrophil-rolling interactions with the inflamed vasculature and occurs through GEF-H1-dependent neutrophil stimulation by colchicine. These results contribute to our understanding of the therapeutic action of colchicine, and could inform the application of this drug in other conditions. [ABSTRACT FROM AUTHOR]

Acute inflammation begins with leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) binding to P-selectin on inflamed endothelium and platelets. In pathologic conditions, this process may contribute to secondary organ damage, like sepsis-induced liver injury. Therefore, developing novel therapies to attenuate inflammation may be beneficial. We previously reported that recombinant human vimentin (rhVim) binds P-selectin to block leukocyte adhesion to endothelium and platelets. In this study, we used SPOT-peptide arrays to identify the rod domain as the active region within rhVim that interacts with P-selectin. Indeed, recombinant human rod domain of vimentin (rhRod) binds to P-selectin with high affinity, with in silico modeling suggesting that rhRod binds P-selectin at or near the PSGL-1 binding site. Using bio-layer interferometry, rhRod decreases PSGL-1 binding to immobilized P-selectin, corroborating the in silico data. Under parallel-plate flow, rhRod blocks leukocyte adhesion to fibrin(ogen)-captured platelets, P-selectin/Fc-coated channels, and IL-1β/IL-4-co-stimulated human umbilical vein endothelial cells. Finally, using intravital microscopy in endotoxemic C57Bl/6 mice, rhRod co-localizes with P-selectin in the hepatic sinusoids and decreases neutrophil adhesion to hepatic sinusoids. These data suggest a potential role for rhRod in attenuating inflammation through directly blocking P-selectin-PSGL-1 interactions. [ABSTRACT FROM AUTHOR]

PSGL-1 has recently been identified as an HIV restriction factor that inhibits HIV DNA synthesis and more potently, virion infectivity. But the underlying mechanisms of these inhibitions are unknown. Here we show that PSGL-1 directly binds to cellular actin filaments (F-actin) to restrict actin dynamics, which leads to inhibition of HIV DNA synthesis. PSGL-1 is incorporated into nascent virions and restricts actin dynamics in the virions, which partially accounts for the inhibition of virion infectivity. More potently, PSGL-1 inhibits incorporation of Env proteins into nascent virions, causing a loss of envelope spikes on the virions as shown by Cryo-electron microscopy and super-resolution imaging. This loss is associated with a profound defect in viral entry. Mechanistically, PSGL-1 binds gp41 and sequesters gp41 at the plasma membrane, explaining the inhibition of Env incorporation in nascent virions. PSGL-1's dual anti-HIV mechanisms represent novel strategies of human cells to defend against HIV infection. [ABSTRACT FROM AUTHOR]

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins. PSGL-1 is expressed primarily on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of endothelium for migration into inflamed tissues. Although it has been reported that PSGL-1 expression inhibits HIV-1 replication, the mechanism of PSGL-1– mediated anti-HIV activity remains to be elucidated. Here we report that PSGL-1 in virions blocks the infectivity of HIV-1 particles by preventing the binding of particles to target cells. This inhibitory activity is independent of the viral glycoprotein present on the virus particle; the binding of particles bearing the HIV-1 envelope glycoprotein or vesicular stomatitis virus G glycoprotein or even lacking a viral glycoprotein is impaired by PSGL-1. Mapping studies show that the extracellular N-terminal domain of PSGL-1 is necessary for its anti–HIV-1 activity, and that the PSGL-1 cytoplasmic tail contributes to inhibition. In addition, we demonstrate that the PSGL-1–related monomeric E-selectin–binding glycoprotein CD43 also effectively blocks HIV-1 infectivity. HIV-1 infection, or expression of either Vpu or Nef, down-regulates PSGL-1 from the cell surface; expression of Vpu appears to be primarily responsible for enabling the virus to partially escape PSGL-1–mediated restriction. Finally, we show that PSGL-1 inhibits the infectivity of other viruses, such as murine leukemia virus and influenza A virus. These findings demonstrate that PSGL-1 is a broad-spectrum antiviral host factor with a unique mechanism of action. [ABSTRACT FROM AUTHOR]

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin-ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin-ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains. [ABSTRACT FROM AUTHOR]