Packaging of supplemented urokinase into alpha-granules of in vitro-grown megakaryocytes for targeted nascent clot lysis.

Fibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding, but is clinically infeasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "anti-thrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+-megakaryocytes internalize and store in -granules single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs co-localized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+-megakaryocytes was mediated, in part, via LRP1 and IIb3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV, but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid-artery injury model in NOD-scid IL2rnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ib) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared to NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet -granules to sites of vascular injury.

Intention to leave, depersonalisation and job satisfaction in physicians and nurses: a cross-sectional study in Europe.

The European healthcare sector faces a significant shortage of healthcare workers. Assessing the prevalence of this issue and understanding its direct and indirect determinants are essential for formulating effective recruitment programs and enhancing job retention strategies for physicians and nurses. A multicentric cross-sectional study was conducted, involving 381 physicians and 1351 nurses recruited from eight European hospitals in Belgium, the Netherlands, Italy, and Poland. The study focused on assessing turnover intentions among healthcare workers based on the Job Demands-Resources model, using an online questionnaire. Structural equation models were employed to test the data collection questionnaires' construct validity and internal consistency. The turnover intention was assessed by agreement with the intention to leave either the hospital or the profession. Among physicians, 17% expressed an intention to leave the hospital, while 9% intended to leave the profession. For nurses, the figures were 8.9% and 13.6%, respectively. The internal consistency of the questionnaires exceeded 0.90 for both categories of health workers. Depersonalization and job dissatisfaction were identified as direct determinants of turnover intention, with work engagement being particularly relevant for nurses. We found a higher intention to leave the hospital among physicians, while nurses were more prone to leave their profession. To mitigate turnover intentions, it is recommended to focus on improving job satisfaction, work engagement and fostering a positive working climate, thereby addressing depersonalisation and promoting job retention.

Production of 129mXe and 131mXe via neutron activation of 128Xe and 130Xe at ILL-RHF and NCBJ-MARIA high-flux reactors.

The long-lived xenon isomers 129mXe and 131mXe are of interest for the GAMMA-MRI project, which aims at developing a novel imaging modality based on magnetic resonance of polarized unstable tracers. Here, we present the steps leading to and following the production of these two isomers via neutron irradiation of highly-enriched 128Xe and 130Xe gas samples at two high-flux reactors, the High-Flux Reactor (Réacteur à haut flux, RHF) at the Institut Laue-Langevin (ILL) and the MARIA reactor at the National Centre for Nuclear Research (NCBJ). We describe the experimental setups and procedures used to prepare the stable xenon samples, to open the irradiated samples, and to transfer xenon isomers into reusable transport vials. The activity of 129mXe and 131mXe was measured to be in the range of tens of MBq per sample of 0.8(1)mg, and was proportional to thermal neutron flux density. A small activity of unstable contaminants was also visible in the samples, but their level is not limiting for the GAMMA-MRI project's objectives. In addition, the minimum thermal neutron flux density required to produce 129mXe and 131mXe sufficient for the project could be also determined.

Packaging of supplemented urokinase into naked alpha-granules of in vitro -grown megakaryocytes for targeted therapeutic delivery.

Our prior finding that uPA endogenously expressed and stored in the platelets of transgenic mice prevented thrombus formation without causing bleeding, prompted us to develop a potentially clinically relevant means of generating anti-thrombotic human platelets in vitro from CD34 + hematopoietic cell-derived megakaryocytes. CD34 + -megakaryocytes internalize and store in α-granules single-chain uPA (scuPA) and a uPA variant modified to be plasmin-resistant, but thrombin-activatable, (uPAT). Both uPAs co-localized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3 (IFITM3), but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34 + -\megakaryocytes was mediated in part via LRP1 and αIIbß3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV, but not endogenous VWF, in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid-artery injury model in NOD-scid IL2rγnull (NSG) mice homozygous for VWF R1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein IbmlIX) to test whether platelets derived from scuPA-MKs or uPAT-Mks would prevent thrombus formation. NSG/VWF R1326H mice exhibited a lower thrombotic burden after carotid artery injury compared to NSG mice unless infused with human platelets or MKs, whereas intravenous injection of either uPA-containing megakaryocytes into NSG/VWF R1326H generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies suggest the potential to deliver uPA or potentially other ectopic proteins within platelet α-granules from in vitro- generated megakaryocytes. Key points: Unlike platelets, in vitro-grown megakaryocytes can store exogenous uPA in its α-granules.uPA uptake involves LRP1 and αIIbß3 receptors and is functionally available from activated platelets.

Platelet factor 4 limits neutrophil extracellular trap- and cell-free DNA-induced thrombogenicity and endothelial injury.

Plasma cell-free DNA (cfDNA), a marker of disease severity in sepsis, is a recognized driver of thromboinflammation and a potential therapeutic target. In sepsis, plasma cfDNA is mostly derived from neutrophil extracellular trap (NET) degradation. Proposed NET-directed therapeutic strategies include preventing NET formation or accelerating NET degradation. However, NET digestion liberates pathogens and releases cfDNA that promote thrombosis and endothelial cell injury. We propose an alternative strategy of cfDNA and NET stabilization with chemokine platelet factor 4 (PF4, CXCL4). We previously showed that human PF4 (hPF4) enhances NET-mediated microbial entrapment. We now show that hPF4 interferes with thrombogenicity of cfDNA and NETs by preventing their cleavage to short-fragment and single-stranded cfDNA that more effectively activates the contact pathway of coagulation. In vitro, hPF4 also inhibits cfDNA-induced endothelial tissue factor surface expression and von Willebrand factor release. In vivo, hPF4 expression reduced plasma thrombin-antithrombin (TAT) levels in animals infused with exogenous cfDNA. Following lipopolysaccharide challenge, Cxcl4-/- mice had significant elevation in plasma TAT, cfDNA, and cystatin C levels, effects prevented by hPF4 infusion. These results show that hPF4 interacts with cfDNA and NETs to limit thrombosis and endothelial injury, an observation of potential clinical benefit in the treatment of sepsis.

Evaluation of risk factors for treatment failure in canine patients undergoing photoactivated chromophore for keratitis - corneal cross-linking (PACK-CXL): a retrospective study using additive bayesian network analysis.

BACKGROUND: Infectious keratitis is a common ophthalmic condition in canine patients. Sequelae can include keratomalacia and corneal perforation, a vision threatening outcome. Photoactivated chromophore for keratitis - corneal cross-linking (PACK-CXL) is a non-surgical, adjunctive treatment method for infectious keratitis. The goal of this retrospective, multicenter study was to determine risk factors for treatment failure following PACK-CXL in canine patients suffering from suspected infectious keratitis. Medical records from four veterinary ophthalmology services were reviewed, and information related to patient demographics, ophthalmic findings, the PACK-CXL protocol used, and epithelialization time was collected and analyzed. Due to the potential for intervariable relationships, an additive Bayesian network (ABN) analysis was performed to evaluate these complex relationships. RESULTS: Records for 671 eyes (668 dogs) were included in the analysis. Based on the ABN, in the population included here, patients who underwent an accelerated PACK-CXL protocol were less likely to experience treatment failure versus patients treated with a slow protocol. Mutual dependencies between exposure variables were identified by ABN, which would have been overlooked using classical regression. Corneal re-epithelialization time was shortened following PACK-CXL combined with topical medical therapy compared to PACK-CXL alone. CONCLUSIONS: No risk factors associated with treatment failure were identified in the population included in the present study. Canine patients may benefit from the use of accelerated PACK-CXL protocols, especially when combined with topical antibiotics and anti-collagenolytic therapy. The reasons for this apparent positive impact on treatment outcome remain unclear.

Platelet-derived microparticles stimulate the invasiveness of colorectal cancer cells via the p38MAPK-MMP-2/MMP-9 axis.

BACKGROUND: Metastasis is the main cause of death in patients with colorectal cancer (CRC). Apart from platelets, platelet-derived microparticles (PMPs) are also considered important factors that can modify the activity of cancer cells. PMPs are incorporated by cancer cells and can also serve as intracellular signalling vesicles. PMPs are believed to affect cancer cells by upregulating their invasiveness. To date, there is no evidence that such a mechanism occurs in colorectal cancer. It has been shown that platelets can stimulate metalloproteases (MMPs) expression and activity via the p38MAPK pathway in CRC cells, leading to their elevated migratory potential. This study aimed to investigate the impact of PMPs on the invasive potential of CRC cells of various phenotypes via the MMP-2, MMP-9 and p38MAPK axis. METHODS: We used various CRC cell lines, including the epithelial-like HT29 and the mesenchymal-like SW480 and SW620. Confocal imaging was applied to study PMP incorporation into CRC cells. The presence of surface receptors on CRC cells after PMP uptake was evaluated by flow cytometry. Transwell and scratch wound-healing assays were used to evaluate cell migration. The level of C-X-C chemokine receptor type 4 (CXCR4), MMP-2, and MMP-9 and the phosphorylation of ERK1/2 and p38MAPK were measured by western blot. MMP activity was determined using gelatine-degradation assays, while MMP release was evaluated by ELISA. RESULTS: We found that CRC cells could incorporate PMPs in a time-dependent manner. Moreover, PMPs could transfer platelet-specific integrins and stimulate the expression of integrins already present on tested cell lines. While mesenchymal-like cells expressed less CXCR4 than epithelial-like CRC cells, PMP uptake did not increase its intensity. No significant changes in CXCR4 level either on the surface or inside CRC cells were noticed. Levels of cellular and released MMP-2 and MMP-9 were elevated in all tested CRC cell lines after PMP uptake. PMPs increased the phosphorylation of p38MAPK but not that of ERK1/2. Inhibition of p38MAPK phosphorylation reduced the PMP-induced elevated level and release of MMP-2 and MMP-9 as well as MMP-dependent cell migration in all cell lines. CONCLUSIONS: We conclude that PMPs can fuse into both epithelial-like and mesenchymal-like CRC cells and increase their invasive potential by inducing the expression and release of MMP-2 and MMP-9 via the p38MAPK pathway, whereas CXCR4-related cell motility or the ERK1/2 pathway appears to not be affected by PMPs. Video Abstract.

Neutrophil extracellular trap stabilization by platelet factor 4 reduces thrombogenicity and endothelial cell injury.

Neutrophil extracellular traps (NETs) are abundant in sepsis, and proposed NET-directed therapies in sepsis prevent their formation or accelerate degradation. Yet NETs are important for microbial entrapment, as NET digestion liberates pathogens and NET degradation products (NDPs) that deleteriously promote thrombosis and endothelial cell injury. We proposed an alternative strategy of NET-stabilization with the chemokine, platelet factor 4 (PF4, CXCL4), which we have shown enhances NET-mediated microbial entrapment. We now show that NET compaction by PF4 reduces their thrombogenicity. In vitro, we quantified plasma thrombin and fibrin generation by intact or degraded NETs and cell-free (cf) DNA fragments, and found that digested NETs and short DNA fragments were more thrombogenic than intact NETs and high molecular weight genomic DNA, respectively. PF4 reduced the thrombogenicity of digested NETs and DNA by interfering, in part, with contact pathway activation. In endothelial cell culture studies, short DNA fragments promoted von Willebrand factor release and tissue factor expression via a toll-like receptor 9-dependent mechanism. PF4 blocked these effects. Cxcl4-/- mice infused with cfDNA exhibited higher plasma thrombin anti-thrombin (TAT) levels compared to wild-type controls. Following challenge with bacterial lipopolysaccharide, Cxcl4-/- mice had similar elevations in plasma TAT and cfDNA, effects prevented by PF4 infusion. Thus, NET-stabilization by PF4 prevents the release of short fragments of cfDNA, limiting the activation of the contact coagulation pathway and reducing endothelial injury. These results support our hypothesis that NET-stabilization reduces pathologic sequelae in sepsis, an observation of potential clinical benefit.

Platelet factor 4 (CXCL4/PF4) upregulates matrix metalloproteinase-2 (MMP-2) in gingival fibroblasts.

Periodontitis is a chronic inflammatory disease characterized by the release of matrix metalloproteinases (MMPs) from resident connective tissue cells in tooth-supporting tissues (periodontium). Platelet activation, and the attendant release of pro-inflammatory chemokines such as platelet factor 4 (CXCL4/PF4), are associated with periodontitis although the associated biochemical pathways remain undefined. Here we report that recombinant PF4 is internalized by cultured human gingival fibroblasts (hGFs), resulting in significant (p < 0.05) upregulation in both the production and release of MMP-2 (gelatinase A). This finding was corroborated by elevated circulating levels of MMP-2 (p < 0.05) in PF4-overexpressing transgenic mice, relative to controls. We also determined that PF4 induces the phosphorylation of NF-κB; notably, the suppression of NF-κB signaling by the inhibitor BAY 11-7082 abrogated PF4-induced MMP-2 upregulation. Moreover, the inhibition of surface glycosaminoglycans (GAGs) blocked both PF4 binding and NF-κB phosphorylation. Partial blockade of PF4 binding to the cells was achieved by treatment with either chondroitinase ABC or heparinase III, suggesting that both chondroitin sulfate and heparan sulfate mediate PF4 signaling. These results identify a novel pathway in which PF4 upregulates MMP-2 release from fibroblasts in an NF-κB- and GAG-dependent manner, and further our comprehension of the role of platelet signaling in periodontal tissue homeostasis.

Dose Escalation Trial of Desulfated Heparin (ODSH) in Septic Peritonitis.

Objective: Septic peritonitis is associated with significant morbidity and mortality. As a potential therapeutic agent in the treatment of sepsis, 2-O, 3-O desulfated heparin (ODSH) reduces histones and platelet factor 4 (PF4) in mouse sepsis models. This pilot clinical trial evaluated the safety and effect of ODSH in client-owned dogs with septic peritonitis. Interventions: In an IACUC-approved, open-label, prospective, dose-escalation clinical trial in 6 dogs with spontaneous septic peritonitis, ODSH administration was initiated following surgical explore to achieve source control. Acute patient physiology and laboratory evaluation (APPLEfast and APPLEfull) scores on admission, source of septic peritonitis, requirement for vasopressors, the administration of blood products, and survival to discharge were recorded. Platelet count, cell free DNA (cfDNA) concentration, and platelet factor 4 (PF4) concentrations were measured at the time of each ODSH dosage. A dose of ODSH was administered every 8 hs for a total of 4 doses (maximum total dosage 75 mg/kg) based on a pre-determined escalation protocol. Patients were monitored in the ICU following administration for evidence of clinical hemorrhage. Main Results: The mean APPLEfast and APPLEfull scores on admission were 22 +/- 6 and 32 +/-10, respectively. Four dogs received 4 total dosages of ODSH and 2 dogs received 3 total dosages of ODSH intravenously. The mean total dosage of ODSH administered during the study period was 48.3 +/- 21.6 mg/kg. No dog required dose de-escalation or had any evidence of bleeding. Four dogs survived to discharge. Conclusions: No adverse effects of ODSH administration were documented in dogs with septic peritonitis. A randomized controlled trial is necessary to evaluate ODSH as a novel therapeutic in the treatment of septic peritonitis.

CXCL4 drives fibrosis by promoting several key cellular and molecular processes.

Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy.

Neutral endopeptidase depletion decreases colon cancer cell proliferation and TGF-ß1 synthesis in indirect co-cultures with normal colon fibroblasts.

PURPOSE: Although recent studies have suggested that neutral endopeptidase (NEP) is implicated in the regulation of colon cancer (CC) cell growth and metastasis, the influence of the tumor microenvironment on this role of NEP has not been investigated so far. Normal colon fibroblasts (NCFs) constitute a component of the stroma surrounding a tumor in an early stage of its development. NCFs can influence transformed cells via different paracrine factors, including TGF-ß1. This in vitro study was undertaken to evaluate the role of NEP in CC promotion in conditions of indirect co-culture of CC cells (LS180 and SW620) with NCFs (CCD-18Co) or their conditioned medium (CM-18Co). METHODS: We examined cell proliferation (with the BrdU assay) and invasiveness (using BME-coated inserts, 8 µm) of NEP-expressing, NEP-silenced (siRNA), and NEP-inhibited (with thiorphan, i.e. a NEP specific inhibitor) CC cells cultured alone or co-cultured with CCD-18Co or with their conditioned medium. The Western blot and ELISA methods were used to assess the level of TGF-ß1. RESULTS: The results showed that the co-culture of the NEP-depleted CC cells with NCFs or their conditioned medium resulted in a significant decrease in cell proliferation in comparison with the proliferative potential of NEP-silenced/inhibited CC cells cultivated alone. In contrast, the NEP depletion did not influence the invasiveness of CC cells in the co-cultures. The co-culture of CC cells with CCD-18Co or CM-C18Co resulted in increased synthesis of TGF-ß1, while the NEP downregulation decreased the synthesis of TGF-ß1 in CC cells and abolished the stimulatory effect of the co-cultures on TGF-ß1 production. CONCLUSIONS: The results suggest that the expression of NEP by colon cancer cells is essential for their proliferation and TGF-ß1 synthesis during paracrine interactions with NCFs.

Snail Overexpression Alters the microRNA Content of Extracellular Vesicles Released from HT29 Colorectal Cancer Cells and Activates Pro-Inflammatory State In Vivo.

During metastasis, cancer cells undergo phenotype changes in the epithelial-mesenchymal transition (EMT) process. Extracellular vesicles (EVs) released by cancer cells are the mediators of intercellular communication and play a role in metastatic process. Knowledge of factors that influence the modifications of the pre-metastatic niche for the migrating carcinoma cells is important for prevention of metastasis. We focus here on how cancer progression is affected by EVs released from either epithelial-like HT29-cells or from cells that are in early EMT stage triggered by Snail transcription factor (HT29-Snail). We found that EVs released from HT29-Snail, as compared to HT29-pcDNA cells, have a different microRNA profile. We observed the presence of interstitial pneumonias in the lungs of mice injected with HT29-Snail cells and the percent of mice with lung inflammation was higher after injection of HT29-Snail-EVs. Incorporation of EVs released from HT29-pcDNA, but not released from HT29-Snail, leads to the increased secretion of IL-8 from macrophages. We conclude that Snail modifications of CRC cells towards more invasive phenotype also alter the microRNA cargo of released EVs. The content of cell-released EVs may serve as a biomarker that denotes the stage of CRC and EVs-specific microRNAs may be a target to prevent cancer progression.

Population based frequency of naturally occurring loss-of-function variants in genes associated with platelet disorders.

Essentials The frequency of predicted loss-of-function (pLoF) variants in platelet-associated genes is unknown in the general population. Datasets like Genome Aggregation Database allow us to analyze pLoF variants with increased resolution. Expected prevalence of significant pLoF variants in platelet-associated genes in 0.329% in the general population. Platelet-associated genes that cause phenotypes due to haploinsufficiency are significantly depleted for deleterious variation. ABSTRACT: Background Inherited platelet disorders are being recognized more frequently as advanced sequencing technologies become more commonplace in clinical scenarios. The prevalence of each inherited platelet disorder and the disorders in aggregate are not known. This deficit in the field makes it difficult for clinicians to discuss results of sequencing assays and provide appropriate anticipatory guidance. Objectives In this study, we aim to calculate the prevalence of predicted loss-of-function variants in platelet-associated genes in the general population. Methods Here, we leverage the aggregation of exomes from the general population in the form of Genome Aggregation Database to assess 58 platelet-associated genes with phenotypic correlates. We use the loss-of-function transcript effect estimator (LOFTEE) to identify predicted loss-of-function mutations in these platelet-associated genes. These variants are curated and we then quantify the frequency of predicted loss-of-function variants in each gene. Results Our data show that 0.329% of the general population have a clinically meaningful predicted loss-of-function variant in a platelet-associated gene. Thus, these individuals are at risk for bleeding disorders that can range from mild to severe. Conclusions These data provide a novel lens through which clinicians can analyze sequencing results in their patients as well as an additional method to curate newly discovered platelet-associated genes in the future.

Human mutational constraint as a tool to understand biology of rare and emerging bone marrow failure syndromes.

Inherited bone marrow failure (IBMF) syndromes are rare blood disorders characterized by hematopoietic cell dysfunction and predisposition to hematologic malignancies. Despite advances in the understanding of molecular pathogenesis of these heterogeneous diseases, genetic variant interpretation, genotype-phenotype correlation, and outcome prognostication remain difficult. As new IBMF and other myelodysplastic syndrome (MDS) predisposition genes continue to be discovered (frequently in small kindred studies), there is an increasing need for a systematic framework to evaluate penetrance and prevalence of mutations in genes associated with IBMF phenotypes. To address this need, we analyzed population-based genomic data from >125 000 individuals in the Genome Aggregation Database for loss-of-function (LoF) variants in 100 genes associated with IBMF. LoF variants in genes associated with IBMF/MDS were present in 0.426% of individuals. Heterozygous LoF variants in genes in which haploinsufficiency is associated with IBMF/MDS were identified in 0.422% of the population; homozygous LoF variants associated with autosomal recessive IBMF/MDS diseases were identified in only .004% of the cohort. Using age distribution of LoF variants and 2 measures of mutational constraint, LOEUF ("loss-of-function observed/expected upper bound fraction") and pLI ("probability of being loss-of-function intolerance"), we evaluated the pathogenicity, tolerance, and age-related penetrance of LoF mutations in specific genes associated with IBMF syndromes. This analysis led to insights into rare IBMF diseases, including syndromes associated with DHX34, MDM4, RAD51, SRP54, and WIPF1. Our results provide an important population-based framework for the interpretation of LoF variant pathogenicity in rare and emerging IBMF syndromes.

Glypican-1 Level Is Elevated in Extracellular Vesicles Released from MC38 Colon Adenocarcinoma Cells Overexpressing Snail.

The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer cells with invasive properties during tumor progression. Extracellular vesicles (EVs) released from cancer cells at various stages of cancer progression are known to influence the tumor pre-metastatic niche and metastatic potential. The aim of this study was to analyze the effect of Snail on murine colon adenocarcinoma cells (MC38 line) and on the characteristics of their EVs. Stable clones of Snail-overexpressing MC38 cells were investigated in vitro versus Mock cells. Increased expression of matrix metalloproteinase MMP-14 and augmented activity of MMP-9 and -14 were observed in Snail-MC38 cells. There was no change in the transcriptomic profile of proteoglycans in Snail-MC38 cells; however, the protein level of Glypican-1 (GPC1) was enhanced in EVs released from those cells. Our finding that GPC1 protein level was enhanced in EVs released from MC38 cells that overexpressed Snail and were in an early EMT stage might explain the specificity of the GPC1 biomarker in colon cancer diagnosis. Further, our data suggest that Snail, by changing the level of GPC1 on EVs released by colon cancer cells, may affect the generation of a distant premetastatic niche and metastatic organotropism in colon adenocarcinoma.

FcRn augments induction of tissue factor activity by IgG-containing immune complexes.

Thromboembolism complicates disorders caused by immunoglobulin G (IgG)-containing immune complexes (ICs), but the underlying mechanisms are incompletely understood. Prior evidence indicates that induction of tissue factor (TF) on monocytes, a pivotal step in the initiation, localization, and propagation of coagulation by ICs, is mediated through Fcγ receptor IIa (FcγRIIa); however, the involvement of other receptors has not been investigated in detail. The neonatal Fc receptor (FcRn) that mediates IgG and albumin recycling also participates in cellular responses to IgG-containing ICs. Here we asked whether FcRn is also involved in the induction of TF-dependent factor Xa (FXa) activity by IgG-containing ICs by THP-1 monocytic cells and human monocytes. Induction of FXa activity by ICs containing IgG antibodies to platelet factor 4 (PF4) involved in heparin-induced thrombocytopenia (HIT), ß-2-glycoprotein-1 implicated in antiphospholipid syndrome, or red blood cells coated with anti-(α)-Rh(D) antibodies that mediate hemolysis in vivo was inhibited by a humanized monoclonal antibody (mAb) that blocks IgG binding to human FcRn. IgG-containing ICs that bind to FcγR and FcRn induced FXa activity, whereas IgG-containing ICs with an Fc engineered to be unable to engage FcRn did not. Infusion of an α-FcRn mAb prevented fibrin deposition after microvascular injury in a murine model of HIT in which human FcγRIIa was expressed as a transgene. These data implicate FcRn in TF-dependent FXa activity induced by soluble and cell-associated IgG-containing ICs. Antibodies to FcRn, now in clinical trials in warm autoimmune hemolytic anemia to lower IgG antibodies and IgG containing ICs may also reduce the risk of venous thromboembolism.

Fc-modified HIT-like monoclonal antibody as a novel treatment for sepsis.

Sepsis is characterized by multiorgan system dysfunction that occurs because of infection. It is associated with high morbidity and mortality and is in need of improved therapeutic interventions. Neutrophils play a crucial role in sepsis, releasing neutrophil extracellular traps (NETs) composed of DNA complexed with histones and toxic antimicrobial proteins that ensnare pathogens, but also damage host tissues. At presentation, patients often have a significant NET burden contributing to the multiorgan damage. Therefore, interventions that inhibit NET release would likely be ineffective at preventing NET-based injury. Treatments that enhance NET degradation may liberate captured bacteria and toxic NET degradation products (NDPs) and likely be of limited therapeutic benefit as well. We propose that interventions that stabilize NETs and sequester NDPs may be protective in sepsis. We showed that platelet factor 4 (PF4), a platelet-associated chemokine, binds and compacts NETs, increasing their resistance to DNase I. We now show that PF4 increases NET-mediated bacterial capture, reduces the release of NDPs, and improves outcome in murine models of sepsis. A monoclonal antibody KKO which binds to PF4-NET complexes, further enhances DNase resistance. However, the Fc portion of this antibody activates the immune response and increases thrombotic risk, negating any protective effects in sepsis. Therefore, we developed an Fc-modified KKO that does not induce these negative outcomes. Treatment with this antibody augmented the effects of PF4, decreasing NDP release and bacterial dissemination and increasing survival in murine sepsis models, supporting a novel NET-targeting approach to improve outcomes in sepsis.

TUBB4B Downregulation Is Critical for Increasing Migration of Metastatic Colon Cancer Cells.

Tumor metastasis, the major problem for clinical oncology in colon cancer treatment, is linked with an epithelial-mesenchymal transition (EMT). The observed cellular transformation in this process is manifested by cell elongation, enhanced cell migration and invasion ability, coordinated by cytoskeleton reorganization. In the present study, we examined the role of tubulin-ß4 (TUBB4B) downregulation that occurs during EMT in colon cancer cells, in the modulation of the function of microtubules. Based on biochemical and behavioral analysis (transmigration) we posit that the decrease of the TUBB4B level is critical for microtubule-vimentin interaction and contributes to the maintenance of polarity in migrating cells. The microscopic studies revealed that TUBB4B decrease is accompanied by cell elongation and increased number of matured focal adhesion sites, which is a characteristic of the cell metastatic stage. We also demonstrated faster polymerization of microtubules in cells with a lower level of TUBB4B. Simultaneous TUBB3 upregulation, reported during EMT, acts additively in this process. Our studies suggest that the protein level of TUBB4B could be used as a marker for detection of the preinvasive stages of the colon cancer cells. We also concluded that chemotherapy enriched to increase TUBB4B level and/or to stabilize microtubule polymerization might more effectively prevent metastasis in colon cancer development.

Laser Spectroscopy of Neutron-Rich Tin Isotopes: A Discontinuity in Charge Radii across the N=82 Shell Closure.

The change in mean-square nuclear charge radii δ⟨r^{2}⟩ along the even-A tin isotopic chain ^{108-134}Sn has been investigated by means of collinear laser spectroscopy at ISOLDE/CERN using the atomic transitions 5p^{2} ^{1}S_{0}→5p6 s^{1}P_{1} and 5p^{2} ^{3}P_{0}→5p6s ^{3}P_{1}. With the determination of the charge radius of ^{134}Sn and corrected values for some of the neutron-rich isotopes, the evolution of the charge radii across the N=82 shell closure is established. A clear kink at the doubly magic ^{132}Sn is revealed, similar to what has been observed at N=82 in other isotopic chains with larger proton numbers, and at the N=126 shell closure in doubly magic ^{208}Pb. While most standard nuclear density functional calculations struggle with a consistent explanation of these discontinuities, we demonstrate that a recently developed Fayans energy density functional provides a coherent description of the kinks at both doubly magic nuclei, ^{132}Sn and ^{208}Pb, without sacrificing the overall performance. A multiple correlation analysis leads to the conclusion that both kinks are related to pairing and surface effects.