3′-Nucleotidase/Nuclease Activity Allows Leishmania Parasites To Escape Killing by Neutrophil Extracellular Traps

ABSTRACTLeishmaniasis is a widespread neglected tropical disease caused by parasites of theLeishmaniagenus. These parasites express the enzyme 3′-nucleotidase/nuclease (3′NT/NU), which has been described to be involved in parasite nutrition and infection. Bacteria that express nucleases escape the toxic effects of neutrophil extracellular traps (NETs). Hence, we investigated the role of 3′NT/NU inLeishmaniasurvival of NET-mediated killing. Promastigotes ofLeishmania infantumwere cultured in high-phosphate (HP) or low-phosphate (LP) medium to modulate nuclease activity. We compared the survival of the two different groups ofLeishmaniaduring interaction with human neutrophils, assessing the role of neutrophil extracellular traps. As previously reported, we detected higher nuclease activity in parasites cultured in LP medium. Both LP and HP promastigotes were capable of inducing the release of neutrophil extracellular traps from human neutrophils in a dose- and time-dependent manner. LP parasites had 2.4 times more survival than HP promastigotes. NET disruption was prevented by the treatment of the parasites with ammonium tetrathiomolybdate (TTM), a 3′NT/NU inhibitor. Inhibition of 3′NT/NU by 3′-AMP, 5′-GMP, or TTM decreased promastigote survival upon interaction with neutrophils. Our results show thatLeishmania infantuminduces NET release and that promastigotes can escape NET-mediated killing by 3′-nucleotidase/nuclease activity, thus ascribing a new function to this enzyme.

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Immunomodulatory Role of Clarithromycin in Acinetobacter baumannii Infection via Formation of Neutrophil Extracellular Traps

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02063-15 ◽

2015 ◽

Vol 60 (2) ◽

pp. 1040-1048 ◽

Cited By ~ 26

Author(s):

Theocharis Konstantinidis ◽

Konstantinos Kambas ◽

Alexandros Mitsios ◽

Maria Panopoulou ◽

Victoria Tsironidou ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Ex Vivo ◽

Neutrophil Extracellular Traps ◽

Macrolide Antibiotics ◽

Dependent Manner ◽

Biofilm Inhibition ◽

Content Type ◽

Extracellular Traps

ABSTRACTMacrolide antibiotics have been shown to act as immunomodulatory molecules in various immune cells. However, their effect on neutrophils has not been extensively investigated. In this study, we investigated the role of macrolide antibiotics in the generation of neutrophil extracellular traps (NETs). By assessingex vivoandin vivoNET formation, we demonstrated that clarithromycin is able to induce NET generation bothin vitroandin vivo. Clarithromycin utilizes autophagy in order to form NETs, and these NETs are decorated with antimicrobial peptide LL-37. Clarithromycin-induced NETs are able to inhibitAcinetobacter baumanniigrowth and biofilm formation in an LL-37-dependent manner. Additionally, LL-37 antimicrobial function depends on NET scaffold integrity. Collectively, these data expand the knowledge on the immunomodulatory role of macrolide antibiotics via the generation of LL-37-bearing NETs, which demonstrate LL-37-dependent antimicrobial activity and biofilm inhibition againstA. baumannii.

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Neutrophil Extracellular Traps Exhibit Antibacterial Activity against Burkholderia pseudomallei and Are Influenced by Bacterial and Host Factors

Infection and Immunity ◽

10.1128/iai.00806-12 ◽

2012 ◽

Vol 80 (11) ◽

pp. 3921-3929 ◽

Cited By ~ 51

Author(s):

Donporn Riyapa ◽

Surachat Buddhisa ◽

Sunee Korbsrisate ◽

Jon Cuccui ◽

Brendan W. Wren ◽

...

Keyword(s):

Burkholderia Pseudomallei ◽

Capsular Polysaccharide ◽

Neutrophil Extracellular Traps ◽

Polymorphonuclear Neutrophils ◽

Dependent Manner ◽

Predisposing Factor ◽

Bacterial Killing ◽

Content Type ◽

Extracellular Traps ◽

Type Iii Protein Secretion

ABSTRACTBurkholderia pseudomalleiis the causative pathogen of melioidosis, of which a major predisposing factor is diabetes mellitus. Polymorphonuclear neutrophils (PMNs) kill microbes extracellularly by the release of neutrophil extracellular traps (NETs). PMNs play a key role in the control of melioidosis, but the involvement of NETs in killing ofB. pseudomalleiremains obscure. Here, we showed that bactericidal NETs were released from human PMNs in response toB. pseudomalleiin a dose- and time-dependent manner.B. pseudomallei-induced NET formation required NADPH oxidase activation but not phosphatidylinositol-3 kinase, mitogen-activated protein kinases, or Src family kinase signaling pathways.B. pseudomalleimutants defective in the virulence-associated Bsa type III protein secretion system (T3SS) or capsular polysaccharide I (CPS-I) induced elevated levels of NETs. NET induction by such mutants was associated with increased bacterial killing, phagocytosis, and oxidative burst by PMNs. Taken together the data imply that T3SS and the capsule may play a role in evading the induction of NETs. Importantly, PMNs from diabetic subjects released NETs at a lower level than PMNs from healthy subjects. Modulation of NET formation may therefore be associated with the pathogenesis and control of melioidosis.

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Nitric oxide and peroxynitrite trigger and enhance release of neutrophil extracellular traps

Cellular and Molecular Life Sciences ◽

10.1007/s00018-019-03331-x ◽

2019 ◽

Vol 77 (15) ◽

pp. 3059-3075 ◽

Cited By ~ 5

Author(s):

Aneta Manda-Handzlik ◽

Weronika Bystrzycka ◽

Adrianna Cieloch ◽

Eliza Glodkowska-Mrowka ◽

Ewa Jankowska-Steifer ◽

...

Keyword(s):

Nitric Oxide ◽

Nitrosative Stress ◽

Calcium Ionophore ◽

Neutrophil Extracellular Traps ◽

Calcium Ionophore A23187 ◽

Human Neutrophils ◽

Ionophore A23187 ◽

Inflammatory Reactions ◽

Extracellular Traps

Abstract Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of reactive nitrogen species (RNS), still remain unclear. Therefore, our aim was to investigate the mechanisms underlying RNS-induced formation of NETs and contribution of RNS to NETs release triggered by various physiological and synthetic stimuli. The involvement of RNS in NETs formation was studied in primary human neutrophils and differentiated human promyelocytic leukemia cells (HL-60 cells). RNS (peroxynitrite and nitric oxide) efficiently induced NETs release and potentiated NETs-inducing properties of platelet activating factor and lipopolysaccharide. RNS-induced NETs formation was independent of autophagy and histone citrullination, but dependent on the activity of phosphoinositide 3-kinases (PI3K) and myeloperoxidase, as well as selective degradation of histones H2A and H2B by neutrophil elastase. Additionally, NADPH oxidase activity was required to release NETs upon stimulation with NO, as shown in NADPH-deficient neutrophils isolated from patients with chronic granulomatous disease. The role of RNS was further supported by increased RNS synthesis upon stimulation of NETs release with phorbol 12-myristate 13-acetate and calcium ionophore A23187. Scavenging or inhibition of RNS formation diminished NETs release triggered by these stimuli while scavenging of peroxynitrite inhibited NO-induced NETs formation. Our data suggest that RNS may act as mediators and inducers of NETs release. These processes are PI3K-dependent and ROS-dependent. Since inflammatory reactions are often accompanied by nitrosative stress and NETs formation, our studies shed a new light on possible mechanisms engaged in various immune-mediated conditions.

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Neutrophil Extracellular Traps Induce Platelet Adhesion and Thrombus Formation.

Blood ◽

10.1182/blood.v114.22.1345.1345 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1345-1345 ◽

Cited By ~ 4

Author(s):

Tobias Fuchs ◽

Alexander Brill ◽

Daniel Dürschmied ◽

Daphne Schatzberg ◽

John H. Hartwig ◽

...

Keyword(s):

Whole Blood ◽

Platelet Adhesion ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Neutrophil Extracellular Traps ◽

Thrombin Inhibitor ◽

Human Neutrophils ◽

Dependent Manner ◽

Extracellular Traps ◽

Deep Vein

Abstract Abstract 1345 Introduction Thrombus stability is provided by very large polymers adhering to platelets and anchoring the thrombus to the vessel wall. The best described polymers are fibrin and von Willebrand Factor (VWF). Activated neutrophils and other leukocytes can form an extracellular fibrous network which is composed of DNA, histones, and granular proteins. These neutrophil extracellular traps (NETs) are present in various inflammatory diseases. In deep vein thrombosis (DVT) inflammation closely cooperates with thrombosis. Here we examine whether NETs provide a new means to support the adhesion and recruitment of platelets and whether NETs are present in DVT. Methods and Results: To study the interaction of platelets with NETs, we isolated human neutrophils, induced NET formation and perfused over the NETs human platelets in plasma or whole blood anticoagulated with the thrombin inhibitor PPACK. Microscopic analysis revealed that under flow platelets adhere avidly to NETs. Perfusion of whole blood at physiological shear resulted in formation of thrombi on NETs in a time dependent manner. Addition of DNase1 degraded NETs and removed all platelets and thrombi demonstrating their adhesion to NETs. Thrombus formation on NETs was absent if blood was supplemented with EDTA indicating the requirement for divalent cations. Perfusion of NETs with heparinized blood dismantled NETs and prevented thrombus formation. Incubation of NETs with heparin alone released histones from NETs, indicating that heparin destroys the chromatin backbone of NETs. Furthermore, immunocytochemistry revealed that NETs were able to bind platelet adhesion molecules VWF and fibronectin from human plasma. Immunohistochemical analysis of a baboon deep vein thrombus showed abundant extracellular chromatin which co-localized with fibronectin and VWF. Conclusions: We show that extracellular traps are able to promote thrombosis in vitro and are abundant in vivo in DVT. We propose that extracellular chromatin provides a new type of scaffold that promotes platelet adhesion, activation, and aggregation and may be important for thrombus initiation or stability. Disclosures No relevant conflicts of interest to declare.

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Angiopoietin 1 release from human neutrophils is independent from neutrophil extracellular traps (NETs)

BMC Immunology ◽

10.1186/s12865-021-00442-8 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Elcha Charles ◽

Benjamin L. Dumont ◽

Steven Bonneau ◽

Paul-Eduard Neagoe ◽

Louis Villeneuve ◽

...

Keyword(s):

Nuclear Dna ◽

Neutrophil Extracellular Traps ◽

Innate Immune ◽

Human Neutrophils ◽

Extracellular Traps ◽

Pathological Conditions ◽

Healthy Control ◽

Angiopoietin 1

Abstract Background Neutrophils induce the synthesis and release of angiopoietin 1 (Ang1), a cytosolic growth factor involved in angiogenesis and capable of inducing several pro-inflammatory activities in neutrophils. Neutrophils also synthesize and release neutrophil extracellular traps (NETs), comprised from decondensed nuclear DNA filaments carrying proteins such as neutrophil elastase (NE), myeloperoxidase (MPO), proteinase 3 (PR3) and calprotectin (S100A8/S100A9), which together, contribute to the innate immune response against pathogens (e.g., bacteria). NETs are involved in various pathological conditions through pro-inflammatory, pro-thrombotic and endothelial dysfunction effects and have recently been found in heart failure (HF) and type 2 diabetes (T2DM) patients. The aim of the present study was to investigate the role of NETs on the synthesis and release of Ang1 by the neutrophils in patients with T2DM and HF with preserved ejection fraction (HFpEF) (stable or acute decompensated; ADHFpEF) with or without T2DM. Results Our data show that at basal level (PBS) and upon treatment with LPS, levels of NETs are slightly increased in patients suffering from T2DM, HFpEF ± T2DM and ADHF without (w/o) T2DM, whereas this increase was significant in ADHFpEF + T2DM patients compared to healthy control (HC) volunteers and ADHFpEF w/o T2DM. We also observed that treatments with PMA or A23187 increase the synthesis of Ang1 (from 150 to 250%) in HC and this effect is amplified in T2DM and in all cohorts of HF patients. Ang1 is completely released (100%) by neutrophils of all groups and does not bind to NETs as opposed to calprotectin. Conclusions Our study suggests that severely ill patients with HFpEF and diabetes synthesize and release a greater abundance of NETs while Ang1 exocytosis is independent of NETs synthesis.

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Neisseria gonorrhoeae Evades Calprotectin-Mediated Nutritional Immunity and Survives Neutrophil Extracellular Traps by Production of TdfH

Infection and Immunity ◽

10.1128/iai.00319-16 ◽

2016 ◽

Vol 84 (10) ◽

pp. 2982-2994 ◽

Cited By ~ 26

Author(s):

Sophonie Jean ◽

Richard A. Juneau ◽

Alison K. Criss ◽

Cynthia N. Cornelissen

Keyword(s):

Neisseria Gonorrhoeae ◽

Defense Mechanism ◽

Outer Membrane Proteins ◽

Neutrophil Extracellular Traps ◽

Host Protein ◽

Dependent Manner ◽

Bacterial Survival ◽

Content Type ◽

Extracellular Traps ◽

Nutritional Immunity

Neisseria gonorrhoeaesuccessfully overcomes host strategies to limit essential nutrients, termed nutritional immunity, by production of TonB-dependent transporters (TdTs)—outer membrane proteins that facilitate nutrient transport in an energy-dependent manner. Four gonococcal TdTs facilitate utilization of iron or iron chelates from host-derived proteins, including transferrin (TbpA), lactoferrin (LbpA), and hemoglobin (HpuB), in addition to xenosiderophores from other bacteria (FetA). The roles of the remaining four uncharacterized TdTs (TdfF, TdfG, TdfH, and TdfJ) remain elusive. Regulatory data demonstrating that production of gonococcal TdfH and TdfJ are unresponsive to or upregulated under iron-replete conditions led us to evaluate the role of these TdTs in the acquisition of nutrients other than iron. In this study, we found that production of gonococcal TdfH is both Zn and Zur repressed. We also found that TdfH confers resistance to calprotectin, an immune effector protein highly produced in neutrophils that has antimicrobial activity due to its ability to sequester Zn and Mn. We found that TdfH directly binds calprotectin, which enables gonococcal Zn accumulation in a TdfH-dependent manner and enhances bacterial survival after exposure to neutrophil extracellular traps (NETs). These studies highlight Zn sequestration by calprotectin as a key functional arm of NET-mediated killing of gonococci. We demonstrate for the first time thatN. gonorrhoeaeexploits this host strategy in a novel defense mechanism, in which TdfH production hijacks and directly utilizes the host protein calprotectin as a zinc source and thereby evades nutritional immunity.

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Neutrophil extracellular traps mediate joint hyperalgesia induced by immune inflammation

Rheumatology ◽

10.1093/rheumatology/keaa794 ◽

2020 ◽

Author(s):

Ayda Henriques Schneider ◽

Caio Cavalcante Machado ◽

Flávio Protásio Veras ◽

Alexandre Gomes de Macedo Maganin ◽

Flávio Falcão Lima de Souza ◽

...

Keyword(s):

Clinical Investigation ◽

Neutrophil Extracellular Traps ◽

Arginine Deiminase ◽

Human Neutrophils ◽

Oedema Formation ◽

Extracellular Traps ◽

Mechanical Threshold ◽

Articular Pain ◽

Dose Dependent

Abstract Objective To evaluate the role of neutrophil extracellular traps (NETs) in the genesis of joint hyperalgesia using an experimental model of arthritis and transpose the findings to clinical investigation. Methods C57BL/6 mice were subjected to antigen-induced arthritis (AIA) and treated with Pulmozyme (PLZ) to degrade NETs or Cl-amidine to inhibit NET production. Oedema formation, the histopathological score and mechanical hyperalgesia were evaluated. NETs were injected intra-articularly in wild type (WT), Tlr4−/−, Tlr9−/−, Tnfr1−/− and Il1r−/− mice, and the levels of cytokines and Cox2 expression were quantified. NETs were also quantified from human neutrophils isolated from RA patients and individual controls. Results AIA mice had increased NET concentration in joints, accompanied by increased Padi4 gene expression in the joint cells. Treatment of AIA mice with a peptidyl arginine deiminase 4 inhibitor or with PLZ inhibited the joint hyperalgesia. Moreover, the injection of NETs into joints of naïve animals generated a dose-dependent reduction of mechanical threshold, an increase of articular oedema, inflammatory cytokine production and cyclooxygenase-2 expression. In mice deficient for Tnfr1, Il1r, Tlr4 and Tlr9, joint hyperalgesia induced by NETs was prevented. Last, we found that neutrophils from RA patients were more likely to release NETs, and the increase in synovial fluid NET concentration correlated with an increase in joint pain. Conclusion The findings indicate that NETs cause hyperalgesia possibly through Toll-like receptor (TLR)-4 and TLR-9. These data support the idea that NETs contribute to articular pain, and this pathway can be an alternative target for the treatment of pain in RA.

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Angiopoietin 1 release from Human Neutrophils is Independent from Neutrophil Extracellular Traps (NETs)

10.21203/rs.3.rs-147798/v1 ◽

2021 ◽

Author(s):

Elcha Charles ◽

Benjamin Dumont ◽

Steven Bonneau ◽

Paul-Eduard Neagoe ◽

Louis Villeneuve ◽

...

Keyword(s):

Nuclear Dna ◽

Neutrophil Extracellular Traps ◽

Innate Immune ◽

Human Neutrophils ◽

Extracellular Traps ◽

Pathological Conditions ◽

Healthy Control ◽

Angiopoietin 1

Abstract Background: Neutrophils induce the synthesis and release of angiopoietin 1 (Ang1), a cytosolic growth factor involved in angiogenesis and capable of inducing several pro-inflammatory activities in neutrophils. Neutrophils also synthesize and release neutrophil extracellular traps (NETs), comprised from decondensed nuclear DNA filaments carrying proteins such as neutrophil elastase (NE), myeloperoxidase (MPO), proteinase 3 (PR3) and calprotectin (S100A8/S100A9), which together, contribute to the innate immune response against pathogens (e.g., bacteria). NETs are involved in various pathological conditions through pro-inflammatory, pro-thrombotic and endothelial dysfunction effects and have recently been found in heart failure (HF) and type 2 diabetes (T2DM) patients. The aim of the present study was to investigate the role of NETs on the synthesis and release of Ang1 by the neutrophils in patients with T2DM and HF with preserved ejection fraction (HFpEF) (stable or acute decompensated; ADHFpEF) with or without T2DM. Results: Our data show that at basal level (PBS) and upon treatment with LPS, levels of NETs are slightly increased in patients suffering from T2DM, HFpEF ± T2DM and ADHF w/o T2DM, whereas this increase was significant in ADHFpEF + T2DM patients compared to healthy control (HC) volunteers and ADHFpEF without T2DM. We also observed that treatments with PMA or A23187 increase the synthesis of Ang1 (from 150 to 250%) in HC and this effect is amplified in T2DM and in all cohorts of HF patients. Ang1 is completely released (100%) by neutrophils of all groups and does not bind to NETs as opposed to calprotectin. Conclusions: Our study suggests that severely ill patients with HFpEF and diabetes synthesize and release a greater abundance of NETs while Ang1 exocytosis is independent of NETs synthesis.

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The interrelationship between phagocytosis, autophagy and formation of neutrophil extracellular traps following infection of human neutrophils by Streptococcus pneumoniae

Innate Immunity ◽

10.1177/1753425917704299 ◽

2017 ◽

Vol 23 (5) ◽

pp. 413-423 ◽

Cited By ~ 24

Author(s):

Ihsan Ullah ◽

Neil D Ritchie ◽

Tom J Evans

Keyword(s):

Streptococcus Pneumoniae ◽

Pneumococcal Infection ◽

Neutrophil Extracellular Traps ◽

Inhibitory Effect ◽

Human Neutrophils ◽

Phosphatidylinositol 3 Kinase ◽

Cellular Processes ◽

Extracellular Traps ◽

Potent Inhibitory Effect

Neutrophils play an important role in the innate immune response to infection with Streptococcus pneumoniae, the pneumococcus. Pneumococci are phagocytosed by neutrophils and undergo killing after ingestion. Other cellular processes may also be induced, including autophagy and the formation of neutrophil extracellular traps (NETs), which may play a role in bacterial eradication. We set out to determine how these different processes interacted following pneumococcal infection of neutrophils, and the role of the major pneumococcal toxin pneumolysin in these various pathways. We found that pneumococci induced autophagy in neutrophils in a type III phosphatidylinositol-3 kinase dependent fashion that also required the autophagy gene Atg5. Pneumolysin did not affect this process. Phagocytosis was inhibited by pneumolysin but enhanced by autophagy, while killing was accelerated by pneumolysin but inhibited by autophagy. Pneumococci induced extensive NET formation in neutrophils that was not influenced by pneumolysin but was critically dependent on autophagy. While pneumolysin did not affect NET formation, it had a potent inhibitory effect on bacterial trapping within NETs. These findings show a complex interaction between phagocytosis, killing, autophagy and NET formation in neutrophils following pneumococcal infection that contribute to host defence against this pathogen.

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Emerging Fungal PathogenCandida aurisEvades Neutrophil Attack

mBio ◽

10.1128/mbio.01403-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 35

Author(s):

Chad J. Johnson ◽

J. Muse Davis ◽

Anna Huttenlocher ◽

John F. Kernien ◽

Jeniel E. Nett

Keyword(s):

Invasive Candidiasis ◽

Fungal Pathogen ◽

Fungal Infections ◽

Neutrophil Extracellular Traps ◽

Invasive Disease ◽

Human Neutrophils ◽

Candida Auris ◽

Zebrafish Model ◽

Content Type ◽

Extracellular Traps

ABSTRACTCandida aurishas recently emerged as the first fungal pathogen to cause a global public health threat. The reason this species is causing hospital-associated outbreaks of invasive candidiasis with high mortality is unknown. In this study, we examine the interaction ofC. auriswith neutrophils, leukocytes critical for control of invasive fungal infections. We show that human neutrophils do not effectively killC. auris. Compared toCandida albicans, neutrophils poorly recruited toC. aurisand failed to form neutrophil extracellular traps (NETs), which are structures of DNA, histones, and proteins with antimicrobial activity. In mixed cultures, neutrophils preferentially engaged and killedC. albicansoverC. auris. Imaging of neutrophils in a zebrafish larval model of invasive candidiasis revealed the recruitment of approximately 50% fewer neutrophils in response toC. auriscompared toC. albicans. Upon encounter withC. albicansin the zebrafish hindbrain, neutrophils produced clouds of histones, suggesting the formation of NETs. These structures were not observed inC. aurisinfection. Evasion of neutrophil attack and innate immunity offers an explanation for the virulence of this pathogen.IMPORTANCEThe emerging fungal pathogenCandida aurishas produced numerous outbreaks of invasive disease in hospitals worldwide. Why this species causes deadly disease is unknown. Our findings reveal a failure of neutrophils to killC. auriscompared to the most commonly encounteredCandidaspecies,C. albicans. While neutrophils produce neutrophil extracellular traps (NETs) upon encounter withC. albicans, these antimicrobial structures are not formed in response toC. auris. Using human neutrophils and a zebrafish model of invasive candidiasis, we show thatC. aurispoorly recruits neutrophils and evades immune attack. Identification of this impaired innate immune response toC. aurissheds light on the dismal outcomes for patients with invasive disease.

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