Factor XI Inhibition for the Prevention of Catheter-Associated Thrombosis in Patients With Cancer Undergoing Central Line Placement: A Phase 2 Clinical Trial.

BACKGROUND: Despite the ubiquitous utilization of central venous catheters in clinical practice, their use commonly provokes thromboembolism. No prophylactic strategy has shown sufficient efficacy to justify routine use. Coagulation factors FXI (factor XI) and FXII (factor XII) represent novel targets for device-associated thrombosis, which may mitigate bleeding risk. Our objective was to evaluate the safety and efficacy of an anti-FXI mAb (monoclonal antibody), gruticibart (AB023), in a prospective, single-arm study of patients with cancer receiving central line placement. METHODS: We enrolled ambulatory cancer patients undergoing central line placement to receive a single dose of gruticibart (2 mg/kg) administered through the venous catheter within 24 hours of placement and a follow-up surveillance ultrasound at day 14 for evaluation of catheter thrombosis. A parallel, noninterventional study was used as a comparator. RESULTS: In total, 22 subjects (n=11 per study) were enrolled. The overall incidence of catheter-associated thrombosis was 12.5% in the interventional study and 40.0% in the control study. The anti-FXI mAb, gruticibart, significantly prolonged the activated partial thromboplastin time in all subjects on day 14 compared with baseline (P<0.001). Gruticibart was well tolerated and without infusion reactions, drug-related adverse events, or clinically relevant bleeding. Platelet flow cytometry demonstrated no difference in platelet activation following administration of gruticibart. T (thrombin)-AT (antithrombin) and activated FXI-AT complexes increased following central line placement in the control study, which was not demonstrated in our intervention study. CRP (C-reactive protein) did not significantly increase on day 14 in those who received gruticibart, but it did significantly increase in the noninterventional study. CONCLUSIONS: FXI inhibition with gruticibart was well tolerated without any significant adverse or bleeding-related events and resulted in a lower incidence of catheter-associated thrombosis on surveillance ultrasound compared with the published literature and our internal control study. These findings suggest that targeting FXI could represent a safe intervention to prevent catheter thrombosis. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04465760.

Factor XII plays a pathogenic role in organ failure and death in baboons challenged with Staphylococcus aureus.

Activation of coagulation factor (F) XI promotes multiorgan failure in rodent models of sepsis and in a baboon model of lethal systemic inflammation induced by infusion of heat-inactivated Staphylococcus aureus. Here we used the anticoagulant FXII-neutralizing antibody 5C12 to verify the mechanistic role of FXII in this baboon model. Compared with untreated control animals, repeated 5C12 administration before and at 8 and 24 hours after bacterial challenge prevented the dramatic increase in circulating complexes of contact system enzymes FXIIa, FXIa, and kallikrein with antithrombin or C1 inhibitor, and prevented cleavage and consumption of high-molecular-weight kininogen. Activation of several coagulation factors and fibrinolytic enzymes was also prevented. D-dimer levels exhibited a profound increase in the untreated animals but not in the treated animals. The antibody also blocked the increase in plasma biomarkers of inflammation and cell damage, including tumor necrosis factor, interleukin (IL)-1ß, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulating factor, nucleosomes, and myeloperoxidase. Based on clinical presentation and circulating biomarkers, inhibition of FXII prevented fever, terminal hypotension, respiratory distress, and multiorgan failure. All animals receiving 5C12 had milder and transient clinical symptoms and were asymptomatic at day 7, whereas untreated control animals suffered irreversible multiorgan failure and had to be euthanized within 2 days after the bacterial challenge. This study confirms and extends our previous finding that at least 2 enzymes of the contact activation complex, FXIa and FXIIa, play critical roles in the development of an acute and terminal inflammatory response in baboons challenged with heat-inactivated S aureus.

Neutrophil extracellular trap inhibition increases inflammation, bacteraemia and mortality in murine necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease affecting primarily premature infants. The disease is characterized by intestinal inflammation and leucocyte infiltration, often progressing to necrosis, perforation, systemic inflammatory response and death. Neutrophil extracellular traps (NETs), denoting nuclear DNA, histone and antimicrobial protein release, have been suggested to play a role in NEC. This study aimed to determine the role of NETs in NEC and explore the effect of chloramidine, a NET inhibitor, on a murine NEC-like intestinal injury model. Blood and intestinal tissues were collected from infants diagnosed with ≥ Stage II NEC, and levels of nucleosomes and NETs, respectively, were compared with those of case-matched controls. In mice, NEC was induced with dithizone/Klebsiella, and mice in the treatment group received 40 mg/kg chloramidine. Bacterial load, intestinal histology, plasma myeloperoxidase and cytokine levels, and immunofluorescent staining were compared with controls. Nucleosomes were significantly elevated in both human and mouse NEC plasma, whereas NET staining was only present in NEC tissue in both species. Chloramidine treatment increased systemic inflammation, bacterial load, organ injury and mortality in murine NEC. Taken together, our findings suggest that NETs are critical in the innate immune defence during NEC in preventing systemic bacteraemia.

Peptidoglycan induces disseminated intravascular coagulation in baboons through activation of both coagulation pathways.

Anthrax infections exhibit progressive coagulopathies that may contribute to the sepsis pathophysiology observed in fulminant disease. The hemostatic imbalance is recapitulated in primate models of late-stage disease but is uncommon in toxemic models, suggesting contribution of other bacterial pathogen-associated molecular patterns (PAMPs). Peptidoglycan (PGN) is a bacterial PAMP that engages cellular components at the cross talk between innate immunity and hemostasis. We hypothesized that PGN is critical for anthrax-induced coagulopathies and investigated the activation of blood coagulation in response to a sterile PGN infusion in primates. The PGN challenge, like the vegetative bacteria, induced a sepsis-like pathophysiology characterized by systemic inflammation, disseminated intravascular coagulation (DIC), organ dysfunction, and impaired survival. Importantly, the hemostatic impairment occurred early and in parallel with the inflammatory response, suggesting direct engagement of coagulation pathways. PGN infusion in baboons promoted early activation of contact factors evidenced by elevated protease-serpin complexes. Despite binding to contact factors, PGN did not directly activate either factor XII (FXII) or prekallikrein. PGN supported contact coagulation by enhancing enzymatic function of active FXII (FXIIa) and depressing its inhibition by antithrombin. In parallel, PGN induced de novo monocyte tissue factor expression in vitro and in vivo, promoting extrinsic clotting reactions at later stages. Activation of platelets further amplified the procoagulant state during PGN challenge, leading to DIC and subsequent ischemic damage of peripheral tissues. These data indicate that PGN may be a major cause for the pathophysiologic progression of Bacillus anthracis sepsis and is the primary PAMP behind the pathogen-induced coagulopathy in late-stage anthrax.

Endothelial PAI-1 (Plasminogen Activator Inhibitor-1) Blocks the Intrinsic Pathway of Coagulation, Inducing the Clearance and Degradation of FXIa (Activated Factor XI).

Objective- Activation of coagulation FXI (factor XI) by FXIIa (activated factor XII) is a prothrombotic process. The endothelium is known to play an antithrombotic role by limiting thrombin generation and platelet activation. It is unknown whether the antithrombotic role of the endothelium includes sequestration of FXIa (activated factor XI) activity. This study aims to determine the role of endothelial cells (ECs) in the regulation of the intrinsic pathway of coagulation. Approach and Results- Using a chromogenic assay, we observed that human umbilical veins ECs selectively blocked FXIa yet supported kallikrein and FXIIa activity. Western blotting and mass spectrometry analyses revealed that FXIa formed a complex with endothelial PAI-1 (plasminogen activator inhibitor-1). Blocking endothelial PAI-1 increased the cleavage of a chromogenic substrate by FXIa and the capacity of FXIa to promote fibrin formation in plasma. Western blot and immunofluorescence analyses showed that FXIa-PAI-1 complexes were either released into the media or trafficked to the early and late endosomes and lysosomes of ECs. When baboons were challenged with Staphylococcus aureus to induce a prothrombotic phenotype, an increase in circulating FXIa-PAI-1 complex levels was detected by ELISA within 2 to 8 hours postchallenge. Conclusions- PAI-1 forms a complex with FXIa on ECs, blocking its activity and inducing the clearance and degradation of FXIa. Circulating FXIa-PAI-1 complexes were detected in a baboon model of S. aureus sepsis. Although ECs support kallikrein and FXIIa activity, inhibition of FXIa by ECs may promote the clearance of intravascular FXIa. Visual Overview- An online visual overview is available for this article.

In vivo-generated thrombin and plasmin do not activate the complement system in baboons.

Sepsis concurrently activates both coagulation and complement systems. Although complement activation by bacteria is well documented, work in mice and in vitro suggests that coagulation proteases can directly cleave complement proteins. We aimed to determine whether generation of coagulation proteases in vivo can activate the complement cascade in 2 highly coagulopathic models. We compared temporal changes in activation biomarkers of coagulation (thrombin-antithrombin [TAT]), fibrinolysis (plasmin-antiplasmin [PAP]), and complement (C3b, C5a, C5b-9) in baboons infused with factor Xa (FXa) and phospholipids (FXa/phosphatidylcholine-phosphatidylserine [PCPS]) vs LD100 Escherichia coli We found that, albeit with different timing, both FXa/PCPS and E coli infusion led to robust thrombin and plasmin generation. Conversely, only E coli challenge activated the complement system, reaching a maximum at 2 hours postchallenge during the peaks of lipopolysaccharide and bacteremia but not of TAT and PAP. Despite inducing a strong burst of thrombin and plasmin, FXa/PCPS infusion did not produce measurable levels of complement activation in vivo. Similarly, ex vivo incubation of baboon serum with thrombin, plasmin, or FXa did not show noticeable complement cleavage unless supraphysiologic amounts of enzymes were used. Our results suggest that in vivo-generated thrombin and plasmin do not directly activate the complement in nonhuman primates.

Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo.

Activated protein C (APC) is a multifunctional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease-activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a nonhuman primate model of Escherichia coli-induced sepsis, pretreatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.

Serum Amyloid P and IgG Exhibit Differential Capabilities in the Activation of the Innate Immune System in Response to Bacillus anthracis Peptidoglycan.

We showed that human IgG supported the response by human innate immune cells to peptidoglycan (PGN) from Bacillus anthracis and PGN-induced complement activation. However, other serum constituents have been shown to interact with peptidoglycan, including the IgG-like soluble pattern recognition receptor serum amyloid P (SAP). Here, we compared the abilities of SAP and of IgG to support monocyte and complement responses to PGN. Utilizing in vitro methods, we demonstrate that SAP is superior to IgG in supporting monocyte production of cytokines in response to PGN. Like IgG, the response supported by SAP was enhanced by phagocytosis and signaling kinases, such as Syk, Src, and phosphatidylinositol 3-kinase, that are involved in various cellular processes, including Fc receptor signaling. Unlike IgG, SAP had no effect on the activation of complement in response to PGN. These data demonstrate an opsonophagocytic role for SAP in response to PGN that propagates a cellular response without propagating the formation of the terminal complement complex.

Inter-α inhibitor protein and its associated glycosaminoglycans protect against histone-induced injury.

Extracellular histones are mediators of tissue injury and organ dysfunction; therefore they constitute potential therapeutic targets in sepsis, inflammation, and thrombosis. Histone cytotoxicity in vitro decreases in the presence of plasma. Here, we demonstrate that plasma inter-α inhibitor protein (IAIP) neutralizes the cytotoxic effects of histones and decreases histone-induced platelet aggregation. These effects are mediated through the negatively charged glycosaminoglycans (GAGs) chondroitin sulfate and high-molecular-weight hyaluronan (HMW-HA) associated with IAIP. Cell surface anionic glycosaminoglycans heparan sulfate and HA protect the cells against histone-mediated damage in vitro. Surface plasmon resonance showed that both IAIP and HMW-HA directly bind to recombinant histone H4. In vivo neutralization of histones with IAIP and HMW-HA prevented histone-induced thrombocytopenia, bleeding, and lung microvascular thrombosis, decreased neutrophil activation, and averted histone-induced production of inflammatory cytokines and chemokines. IAIP and HMW-HA colocalized with histones in necrotic tissues and areas that displayed neutrophil extracellular traps. Increasing amounts of IAIP-histone complexes detected in the plasma of septic baboons correlated with increase in histones and/or nucleosomes and consumption of plasma IAIP. Our data suggest that IAIP, chondroitin sulfate, and HMW-HA are potential therapeutic agents to protect against histone-induced cytotoxicity, coagulopathy, systemic inflammation, and organ damage during inflammatory conditions such as sepsis and trauma.

Complement inhibition decreases early fibrogenic events in the lung of septic baboons.

Acute respiratory distress syndrome (ARDS) induced by severe sepsis can trigger persistent inflammation and fibrosis. We have shown that experimental sepsis in baboons recapitulates ARDS progression in humans, including chronic inflammation and long-lasting fibrosis in the lung. Complement activation products may contribute to the fibroproliferative response, suggesting that complement inhibitors are potential therapeutic agents. We have been suggested that treatment of septic baboons with compstatin, a C3 convertase inhibitor protects against ARDS-induced fibroproliferation. Baboons challenged with 10(9) cfu/kg (LD50) live E. coli by intravenous infusion were treated or not with compstatin at the time of challenge or 5 hrs thereafter. Changes in the fibroproliferative response at 24 hrs post-challenge were analysed at both transcript and protein levels. Gene expression analysis showed that sepsis induced fibrotic responses in the lung as early as 24 hrs post-bacterial challenge. Immunochemical and biochemical analysis revealed enhanced collagen synthesis, induction of profibrotic factors and increased cell recruitment and proliferation. Specific inhibition of complement with compstatin down-regulated sepsis-induced fibrosis genes, including transforming growth factor-beta (TGF-ß), connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinase 1 (TIMP1), various collagens and chemokines responsible for fibrocyte recruitment (e.g. chemokine (C-C motif) ligand 2 (CCL2) and 12 (CCL12)). Compstatin decreased the accumulation of myofibroblasts and proliferating cells, reduced the production of fibrosis mediators (TGF-ß, phospho-Smad-2 and CTGF) and inhibited collagen deposition. Our data demonstrate that complement inhibition effectively attenuates collagen deposition and fibrotic responses in the lung after severe sepsis. Inhibiting complement could prove an attractive strategy for preventing sepsis-induced fibrosis of the lung.

Bacillus anthracis peptidoglycan activates human platelets through FcγRII and complement.

Platelet activation frequently accompanies sepsis and contributes to the sepsis-associated vascular leakage and coagulation dysfunction. Our previous work has implicated peptidoglycan (PGN) as an agent causing systemic inflammation in gram-positive sepsis. We used flow cytometry and fluorescent microscopy to define the effects of PGN on the activation of human platelets. PGN induced platelet aggregation, expression of the activated form of integrin αIIbß3, and exposure of phosphatidylserine (PS). These changes were dependent on immunoglobulin G and were attenuated by the Fcγ receptor IIa-blocking antibody IV.3, suggesting they are mediated by PGN-anti-PGN immune complexes signaling through Fcγ receptor IIa. PS exposure was not blocked by IV.3 but was sensitive to inhibitors of complement activation. PGN was a potent activator of the complement cascade in human plasma and caused deposition of C5b-9 on the platelet surface. Platelets with exposed PS had greatly accelerated prothrombinase activity. We conclude that PGN derived from gram-positive bacteria is a potent platelet agonist when complexed with anti-PGN antibody and could contribute to the coagulation dysfunction accompanying gram-positive infections.

Acute lung injury and fibrosis in a baboon model of Escherichia coli sepsis.

Sepsis-induced inflammation of the lung leads to acute respiratory distress syndrome (ARDS), which may trigger persistent fibrosis. The pathology of ARDS is complex and poorly understood, and the therapeutic approaches are limited. We used a baboon model of Escherichia coli sepsis that mimics the complexity of human disease to study the pathophysiology of ARDS. We performed extensive biochemical, histological, and functional analyses to characterize the disease progression and the long-term effects of sepsis on the lung structure and function. Similar to humans, sepsis-induced ARDS in baboons displays an early inflammatory exudative phase, with extensive necrosis. This is followed by a regenerative phase dominated by proliferation of type 2 epithelial cells, expression of epithelial-to-mesenchymal transition markers, myofibroblast migration and proliferation, and collagen synthesis. Baboons that survived sepsis showed persistent inflammation and collagen deposition 6-27 months after the acute episodes. Long-term survivors had almost double the amount of collagen in the lung as compared with age-matched control animals. Immunostaining for procollagens showed persistent active collagen synthesis within the fibroblastic foci and interalveolar septa. Fibroblasts expressed markers of transforming growth factor-ß and platelet-derived growth factor signaling, suggesting their potential role as mediators of myofibroblast migration and proliferation, and collagen deposition. In parallel, up-regulation of the inhibitors of extracellular proteases supports a deregulated matrix remodeling that may contribute to fibrosis. The primate model of sepsis-induced ARDS mimics the disease progression in humans, including chronic inflammation and long-lasting fibrosis. This model helps our understanding of the pathophysiology of fibrosis and the testing of new therapies.

Reactive oxygen species-induced activation of ERK and p38 MAPK mediates PMA-induced NETs release from human neutrophils.

Neutrophils/polymorphonuclear leukocytes (PMNs), an important component of innate immune system, release extracellular traps (NETs) to eliminate invaded pathogens; however understanding of the role of signaling molecules/proteins need to be elucidated. In the present study role of p38 MAPK and extracellular signal regulated kinase (ERK) against phorbol 12-myristate 13-acetate (PMA) induced reactive oxygen species (ROS) generation and NETs formation has been investigated. Human neutrophils were treated with PMA to induce free radical generation and NETs release, which were monitored by NBT reduction and elastase/DNA release, respectively. PMA treatment led to the time dependent phosphorylation of p38 MAPK and ERK in PMNs. Pretreatment of PMNs with SB202190 or U0126 did not significantly reduce PMA induce free radical generation, but prevented NETs release. Pretreatment of PMNs with NADPH oxidase inhibitor (diphenyleneiodonium chloride) significantly reduced free radical generation, p38 MAPK and ERK phosphorylation as well as NETs release, suggesting that p38 MAPK and ERK activation was downstream to free radical generation. The present study thus demonstrates ROS dependent activation of ERK and p38 MAPK, which mediated PMA induced NETs release from human neutrophils.

Anti-miR-93-5p therapy prolongs sepsis survival by restoring the peripheral immune response.

Sepsis remains a leading cause of death for humans and currently has no pathogenesis-specific therapy. Hampered progress is partly due to a lack of insight into deep mechanistic processes. In the past decade, deciphering the functions of small noncoding miRNAs in sepsis pathogenesis became a dynamic research topic. To screen for new miRNA targets for sepsis therapeutics, we used samples for miRNA array analysis of PBMCs from patients with sepsis and control individuals, blood samples from 2 cohorts of patients with sepsis, and multiple animal models: mouse cecum ligation puncture-induced (CLP-induced) sepsis, mouse viral miRNA challenge, and baboon Gram+ and Gram- sepsis models. miR-93-5p met the criteria for a therapeutic target, as it was overexpressed in baboons that died early after induction of sepsis, was downregulated in patients who survived after sepsis, and correlated with negative clinical prognosticators for sepsis. Therapeutically, inhibition of miR-93-5p prolonged the overall survival of mice with CLP-induced sepsis, with a stronger effect in older mice. Mechanistically, anti-miR-93-5p therapy reduced inflammatory monocytes and increased circulating effector memory T cells, especially the CD4+ subset. AGO2 IP in miR-93-KO T cells identified important regulatory receptors, such as CD28, as direct miR-93-5p target genes. In conclusion, miR-93-5p is a potential therapeutic target in sepsis through the regulation of both innate and adaptive immunity, with possibly a greater benefit for elderly patients than for young patients.

Neutrophil extracellular traps contain mitochondrial as well as nuclear DNA and exhibit inflammatory potential.

Neutrophils expel extracellular traps (NETs) to entrap and exterminate the invaded micro-organisms. Acute/chronic inflammatory disorders are often observed with aberrantly enhanced NETs formation and high nitric oxide (NO) availability. Recent study from this laboratory demonstrated release of NETs from human neutrophils following treatment with SNP or SNAP. This study is an extension of our previous finding to explore the extracellular bacterial killing, source of DNA in the expelled NETs, their ability to induce proinflammatory cytokines release from platelets/THP-1 cells, and assessment of NO-mediated free radical formation by using a consistent NO donor, DETA-NONOate. NO-mediated NETs exhibited extracellular bacterial killing as determined by colony forming units. NO-mediated NETs formation was due to the activation of NADPH oxidase and myeloperoxidase. NO- or PMA-mediated NETs were positive for both nuclear and mitochondrial DNA as well as proteolytic enzymes. Incubation of NETs with human platelets enhanced the release of IL-1ß and IL-8, while with THP-1 cells, release of IL-1ß, IL-8, and TNFα was observed. This study demonstrates that NO by augmenting enzymatic free radical generation release NETs to promote extracellular bacterial killing. These NETs were made up of mitochondrial and nuclear DNA and potentiated release of proinflammatory cytokines.

Early Antibiotic Exposure Alters Intestinal Development and Increases Susceptibility to Necrotizing Enterocolitis: A Mechanistic Study.

Increasing evidence suggests that prolonged antibiotic therapy in preterm infants is associated with increased mortality and morbidities, such as necrotizing enterocolitis (NEC), a devastating gastrointestinal pathology characterized by intestinal inflammation and necrosis. While a clinical correlation exists between antibiotic use and the development of NEC, the potential causality of antibiotics in NEC development has not yet been demonstrated. Here, we tested the effects of systemic standard-of-care antibiotic therapy for ten days on intestinal development in neonatal mice. Systemic antibiotic treatment impaired the intestinal development by reducing intestinal cell proliferation, villi height, crypt depth, and goblet and Paneth cell numbers. Oral bacterial challenge in pups who received antibiotics resulted in NEC-like intestinal injury in more than half the pups, likely due to a reduction in mucous-producing cells affecting microbial-epithelial interactions. These data support a novel mechanism that could explain why preterm infants exposed to prolonged antibiotics after birth have a higher incidence of NEC and other gastrointestinal disorders.

CD14 inhibition improves survival and attenuates thrombo-inflammation and cardiopulmonary dysfunction in a baboon model of Escherichia coli sepsis.

BACKGROUND: During sepsis, gram-negative bacteria induce robust inflammation primarily via lipopolysacharride (LPS) signaling through TLR4, a process that involves the glycosylphosphatidylinositol (GPI)-anchored receptor CD14 transferring LPS to the Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) complex. Sepsis also triggers the onset of disseminated intravascular coagulation and consumptive coagulopathy. OBJECTIVES: We investigated the effect of CD14 blockade on sepsis-induced coagulopathy, inflammation, organ dysfunction, and mortality. METHODS: We used a baboon model of lethal Escherichia (E) coli sepsis to study two experimental groups (n = 5): (a) E coli challenge; (b) E coli challenge plus anti-CD14 (23G4) inhibitory antibody administered as an intravenous bolus 30 minutes before the E coli. RESULTS: Following anti-CD14 treatment, two animals reached the 7-day end-point survivor criteria, while three animals had a significantly prolonged survival as compared to the non-treated animals that developed multiple organ failure and died within 30 hours. Anti-CD14 reduced the activation of coagulation through inhibition of tissue factor-dependent pathway, especially in the survivors, and enhanced the fibrinolysis due to strong inhibition of plasminogen activator inhibitor 1. The treatment prevented the robust complement activation induced by E coli, as shown by significantly decreased C3b, C5a, and sC5b-9. Vital signs, organ function biomarkers, bacteria clearance, and leukocyte and fibrinogen consumption were all improved at varying levels. Anti-CD14 reduced neutrophil activation, cell death, LPS levels, and pro-inflammatory cytokines (tumor necrosis factor, interleukin (IL)-6, IL-1ß, IL-8, interferon gamma, monocyte chemoattractant protein-1), more significantly in the survivors than non-surviving animals. CONCLUSIONS: Our results highlight the crosstalk between coagulation/fibrinolysis, inflammation, and complement systems and suggest a protective role of anti-CD14 treatment in E coli sepsis.

Acceleration of Small Intestine Development and Remodeling of the Microbiome Following Hyaluronan 35 kDa Treatment in Neonatal Mice.

The beneficial effects of human milk suppressing the development of intestinal pathologies such as necrotizing enterocolitis in preterm infants are widely known. Human milk (HM) is rich in a multitude of bioactive factors that play major roles in promoting postnatal maturation, differentiation, and the development of the microbiome. Previous studies showed that HM is rich in hyaluronan (HA) especially in colostrum and early milk. This study aims to determine the role of HA 35 KDa, a HM HA mimic, on intestinal proliferation, differentiation, and the development of the intestinal microbiome. We show that oral HA 35 KDa supplementation for 7 days in mouse pups leads to increased villus length and crypt depth, and increased goblet and Paneth cells, compared to controls. We also show that HA 35 KDa leads to an increased predominance of Clostridiales Ruminococcaceae, Lactobacillales Lactobacillaceae, and Clostridiales Lachnospiraceae. In seeking the mechanisms involved in the changes, bulk RNA seq was performed on samples from the terminal ileum and identified upregulation in several genes essential for cellular growth, proliferation, and survival. Taken together, this study shows that HA 35 KDa supplemented to mouse pups promotes intestinal epithelial cell proliferation, as well as the development of Paneth cells and goblet cell subsets. HA 35 KDa also impacted the intestinal microbiota; the implications of these responses need to be determined.

C3 Opsonization of Anthrax Bacterium and Peptidoglycan Supports Recognition and Activation of Neutrophils.

Neutrophils are the most abundant innate cell population and a key immune player against invading pathogens. Neutrophils can kill both bacterium and spores of Bacillus anthracis, the causative anthrax pathogen. Unlike interactions with professional phagocytes, the molecular recognition of anthrax by neutrophils is largely unknown. In this study, we investigated the role of complement C3 deposition on anthrax particles for neutrophil recognition of bacterium and/or its cell wall peptidoglycan, an abundant pathogen-associated molecular pattern that supports anthrax sepsis. C3 opsonization and recognition by complement receptors accounted for 70-80% of the affinity interactions between neutrophils and anthrax particles at subphysiologic temperatures. In contrast, C3 supported up to 50% of the anthrax particle ingestion under thermophysiologic conditions. Opsonin-dependent low affinity interactions and, to a lower extent, opsonin-independent mechanisms, provide alternative entry routes. Similarly, C3 supported 58% of peptidoglycan-induced degranulation and, to a lower extent, 23% of bacterium-induced degranulation. Interestingly, an opsonin independent mechanism mediated by complement C5, likely through C5a anaphylatoxin, primes azurophilic granules in response to anthrax particles. Overall, we show that C3 deposition supports anthrax recognition by neutrophils but is dispensable for pathogen ingestion and neutrophil degranulation, highlighting immune recognition redundancies that minimize the risk of pathogen evasion.

Fondaparinux pentasaccharide reduces sepsis coagulopathy and promotes survival in the baboon model of Escherichia coli sepsis.

BACKGROUND: Sepsis triggers dysfunction of coagulation and fibrinolytic systems leading to disseminated intravascular coagulation (DIC) that contributes to organ failure and death. Fondaparinux (FPX) is a synthetic pentasaccharide that binds to antithrombin (AT) and selectively inhibits factor (F) Xa and other upstream coagulation proteases but not thrombin (T). OBJECTIVES: We used a baboon model of lethal Escherichia coli sepsis to investigate the effects of FPX treatment on DIC, organ function, and outcome. METHODS: Two experimental groups were studied: (a) E. coli challenge (n = 4); and (b) E coli plus FPX (n = 4). Bacteremia was modeled by intravenous infusion of pathogen (1-2 × 1010  CFU/kg). Fondaparinux (0.08 mg/kg) was administered subcutaneously, 3 h prior to and 8 h after bacteria infusion. RESULTS: Bacteremia rapidly increased plasma levels of inhibitory complexes of AT with coagulation proteases. Activation markers of both intrinsic (FXIa-AT), and extrinsic (FVIIa-AT) pathways were significantly reduced in FPX-treated animals. Factor Xa-AT and TAT complexes were maximal at 4 to 8 h post challenge and reduced >50% in FPX-treated animals. Fibrinogen consumption, fibrin generation and degradation, neutrophil and complement activation, and cytokine production were strongly induced by sepsis. All parameters were significantly reduced, while platelet count was unchanged by the treatment. Fondaparinux infusion attenuated organ dysfunction, prolonged survival, and saved two of four challenged animals (log-rank Mantel-Cox test, P = .0067). CONCLUSION: Our data indicate that FPX-mediated inhibition of coagulation prevents sepsis coagulopathy; protects against excessive complement activation, inflammation, and organ dysfunction; and provides survival benefit in E. coli sepsis.