Selective Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibition by the SCH772984 Compound Attenuates In Vitro and In Vivo Inflammatory Responses and Prolongs Survival in Murine Sepsis Models.

Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome.

Hepatic FGF21 preserves thermoregulation and cardiovascular function during bacterial inflammation.

Sickness behaviors, including anorexia, are evolutionarily conserved responses to acute infections. Inflammation-induced anorexia causes dramatic metabolic changes, of which components critical to survival are unique depending on the type of inflammation. Glucose supplementation during the anorectic period induced by bacterial inflammation suppresses adaptive fasting metabolic pathways, including fibroblast growth factor 21 (FGF21), and decreases survival. Consistent with this observation, FGF21-deficient mice are more susceptible to mortality from endotoxemia and polybacterial peritonitis. Here, we report that increased circulating FGF21 during bacterial inflammation is hepatic derived and required for survival through the maintenance of thermogenesis, energy expenditure, and cardiac function. FGF21 signaling downstream of its obligate coreceptor, ß-Klotho (KLB), is required in bacterial sepsis. However, FGF21 modulates thermogenesis and chronotropy independent of the adipose, forebrain, and hypothalamus, which are operative in cold adaptation, suggesting that in bacterial inflammation, either FGF21 signals through a novel, undescribed target tissue or concurrent signaling of multiple KLB-expressing tissues is required.

Endothelial SOCS3 maintains homeostasis and promotes survival in endotoxemic mice.

SOCS3 is the main inhibitor of the JAK/STAT3 pathway. This pathway is activated by interleukin 6 (IL-6), a major mediator of the cytokine storm during shock. To determine its role in the vascular response to shock, we challenged mice lacking SOCS3 in the adult endothelium (SOCS3iEKO) with a nonlethal dose of lipopolysaccharide (LPS). SOCS3iEKO mice died 16-24 hours postinjection after severe kidney failure. Loss of SOCS3 led to an LPS-induced type I IFN-like program and high expression of prothrombotic and proadhesive genes. Consistently, we observed intraluminal leukocyte adhesion and neutrophil extracellular trap-osis (NETosis), as well as retinal venular leukoembolization. Notably, heterozygous mice displayed an intermediate phenotype, suggesting a gene dose effect. In vitro studies were performed to study the role of SOCS3 protein levels in the regulation of the inflammatory response. In human umbilical vein endothelial cells, pulse-chase experiments showed that SOCS3 protein had a half-life less than 20 minutes. Inhibition of SOCS3 ubiquitination and proteasomal degradation led to protein accumulation and a stronger inhibition of IL-6 signaling and barrier function loss. Together, our data demonstrate that the regulation of SOCS3 protein levels is critical to inhibit IL-6-mediated endotheliopathy during shock and provide a promising therapeutic avenue to prevent multiorgan dysfunction through stabilization of endothelial SOCS3.

Polymyxin B hemoperfusion in coronavirus disease 2019 patients with endotoxic shock: Case series from EUPHAS2 registry.

The coronavirus disease 2019 (COVID-19) has been shown to involve the gastrointestinal tract, which implies bacterial translocation and endotoxemia. The aim of this study was to evaluate the role of extracorporeal endotoxin removal by Polymyxin B hemoperfusion (PMX-HP), in the treatment of patients with COVID-19 and secondary bacterial infection. We conducted a subgroup analysis of a multicenter, multinational, prospective, and observational web-based database (EUPHAS2 registry). We included 12 patients with severe acute respiratory syndrome coronavirus 2 infection confirmed by real-time reverse transcriptase-polymerase chain reaction from nasal/oral swab, admitted to the intensive care unit between February and May 2020, who were affected by septic shock and received PMX-HP as per clinical indication of the attending physician. Septic shock was diagnosed in nine patients (75%), with a median time between symptoms onset and PMX-HP treatment of 16 (14-22) days. We identified Gram-negative bacteria in most of the microbiological cultures (N = 17, 65%), followed by Gram-positive bacteria in (N = 4, 15%), fungi (N = 3, 12%) and no growth (N = 2, 8%). Sequential Organ Failure Assessment (SOFA) score progressively improved over the next 120 hours following PMX-HP and it was associated with median endotoxin activity assay (EAA) decrease from 0.78 [0.70-0.92] at T0 to 0.60 [0.44-0.72] at T120 (P = .245). A direct correlation was observed between SOFA score and EAA. Lung Injury Score decreased and was associated with hemodynamic improvement over the same period. No statistically significant difference was observed for RIFLE score at each time point. Nine out of 12 patients (75%) required continuous renal replacement therapy because of acute kidney injury. In a series of consecutive COVID-19 patients with endotoxic shock, PMX-HP was associated with organ function recovery, hemodynamic improvement, and contemporary EAA level reduction. No PMX-HP-related complications were observed.

A C-terminal fragment of adhesion protein Fibulin7 regulates neutrophil migration and functions and improves survival in LPS induced systemic inflammation.

Accumulation of hyperactive neutrophils in the visceral organs was shown to be associated with sepsis-induced multi-organ failure. Recently, a C-terminal fragment of secreted glycoprotein Fibulin7 (Fbln7-C) was shown to inhibit angiogenesis and regulate monocyte functions in inflammatory conditions. However, its effects on neutrophil functions and systemic inflammation induced lethality remain unknown. In this study, we show that human peripheral blood neutrophils adhered to Fbln7-C in a dose-dependent manner via integrin ß1. Moreover, the presence of Fbln7-C inhibited spreading, and fMLP mediated random migration of neutrophils on fibronectin. Significant reduction in ROS and inflammatory cytokine production (i.e., IL-6, IL-1ß) was observed, including a reduction in ERK1/2 phosphorylation in neutrophils stimulated with LPS and fMLP in the presence of Fbln7-C compared to untreated controls. In an in vivo model of endotoxemia, the administration of Fbln7-C (10 µg/dose) significantly improved survival and reduced the infiltration of neutrophils to the site of inflammation. Additionally, neutrophils infiltrating into the inflamed peritoneum of Fbln7-C administered animals expressed lower levels CD11b marker, IL-6, and produced lower levels of ROS upon stimulation with PMA compared to untreated controls. In conclusion, our results show that Fbln7-C could bind to the integrin ß1 on the neutrophil surface and regulate their inflammatory functions.

TRPM7 mediates kidney injury, endothelial hyperpermeability and mortality during endotoxemia.

Sepsis is the main cause of mortality in patients admitted to intensive care units. During sepsis, endothelial permeability is severely augmented, contributing to renal dysfunction and patient mortality. Ca2+ influx and the subsequent increase in intracellular [Ca2+]i in endothelial cells (ECs) are key steps in the establishment of endothelial hyperpermeability. Transient receptor potential melastatin 7 (TRPM7) ion channels are permeable to Ca2+ and are expressed in a broad range of cell types and tissues, including ECs and kidneys. However, the role of TRPM7 on endothelial hyperpermeability during sepsis has remained elusive. Therefore, we investigated the participation of TRPM7 in renal vascular hyperpermeability, renal dysfunction, and enhanced mortality induced by endotoxemia. Our results showed that endotoxin increases endothelial hyperpermeability and Ca2+ overload through the TLR4/NOX-2/ROS/NF-κB pathway. Moreover, endotoxin exposure was shown to downregulate the expression of VE-cadherin, compromising monolayer integrity and enhancing vascular hyperpermeability. Notably, endotoxin-induced endothelial hyperpermeability was substantially inhibited by pharmacological inhibition and specific suppression of TRPM7 expression. The endotoxin was shown to upregulate the expression of TRPM7 via the TLR4/NOX-2/ROS/NF-κB pathway and induce a TRPM7-dependent EC Ca2+ overload. Remarkably, in vivo experiments performed in endotoxemic animals showed that pharmacological inhibition and specific suppression of TRPM7 expression inhibits renal vascular hyperpermeability, prevents kidney dysfunction, and improves survival in endotoxemic animals. Therefore, our results showed that TRPM7 mediates endotoxemia-induced endothelial hyperpermeability, renal dysfunction, and enhanced mortality, revealing a novel molecular target for treating renal vascular hyperpermeability and kidney dysfunction during endotoxemia, sepsis, and other inflammatory diseases.

The Prognostic Value of Endotoxemia and Intestinal Barrier Biomarker ZO-1 in Bacteremic Sepsis.

BACKGROUND: Intestinal barrier dysfunction exerts a pivotal pathophysiological role in the development of multiple organ dysfunction in sepsis. The present study was undertaken to investigate the potential role of serum intestinal fatty acid-binding protein (I-FABP) and zonula occludens-1 (ZO-1) levels as biomarkers of intestinal barrier dysfunction in bacteremic sepsis. METHODS: Seventy-five patients with bacteremic sepsis of abdominal origin (n = 34) or nonabdominal origin (n = 41) and 12 healthy controls were retrospectively studied. Blood samples collected upon sepsis diagnosis were analyzed for serum ZO-1, I-FABP and endotoxin levels. Prognostic scores Sequential Organ Failure Assessment (SOFA), quickSOFA and Acute Physiology and Chronic Health Evaluation (APACHE-II) were determined over the first 24 hours after sepsis diagnosis and patients' outcome in terms of 28-day mortality was recorded. RESULTS: Serum ZO-1 levels were significantly higher in bacteremic septic patients as compared to controls with no difference between patients with abdominal or extra-abdominal source of infection. Serum I-FABP levels were significantly lower in septic patients as compared to control and this reduction was more evident in patients with bacteremic abdominal sepsis. Serum ZO-1 and endotoxin concentrations were found significantly higher in patients who did not survive from sepsis. In receiver operating characteristic curve analysis, both endotoxin and ZO-1 predicted 28-day mortality. In addition, ZO-1 and endotoxin were correlated with the prognostic scores of qSOFA, SOFA and APACHE II. CONCLUSIONS: The results of this study indicate that serum ZO-1 might be a reliable biomarker of gut barrier dysfunction in sepsis, not affected by the abdominal or extra-abdominal site of infection. ZO-1, measured early at sepsis diagnosis, might represent a valuable additional prognostic tool for patients' outcome.

Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

Association between endotoxemia and enterocyte injury and clinical course in patients with gram-positive septic shock: A posthoc analysis of a prospective observational study.

Endotoxemia often occurs in patients with gram-positive infections. The possible mechanism is thought to be bacterial translocation after enterocyte hypoperfusion injury. However, the association between endotoxemia and enterocyte injury among patients with gram-positive septic shock has never been assessed. The aim of this study was to evaluate the association between endotoxemia and enterocyte injury in gram-positive septic shock patients and to evaluate the association among endotoxemia, subsequent clinical course, and other related factors.This was a posthoc analysis of a prospective observational study that evaluated the capability of intestinal fatty acid-binding protein (I-FABP), an indicator of enterocyte injury, to predict mortality. Among 57 patients in septic shock, those whose causative microorganisms were gram positive were included. The correlation between endotoxin activity (EA), which indicates endotoxemia, and I-FABP levels upon admission to the intensive care unit (ICU), the clinical course, and other related factors were evaluated.A total of 21 patients were examined. One-third of the patients presented with high EA levels at the time of ICU admission. However, there was no significant correlation between EA and I-FABP levels (Spearman ρ = 0.002, P = .993). Additionally, high EA levels were not associated with abdominal complications after ICU admission or mortality. Similarly, high EA levels were not associated with severity scores, inotropic scores, or lactate levels upon ICU admission, which were previously reported to be factors related to high EA levels.In this posthoc analysis, no correlation was observed between endotoxemia and enterocyte injury among patients in gram-positive septic shock. Additionally, high EA levels were not associated with the clinical course and reported factors related to endotoxemia. Although our results need to be validated in a large prospective cohort study, hypoperfusion enterocyte injury might not be a cause of endotoxemia in these patients. Thus, if there is no correlation between EA and I-FABP levels, other mechanisms that induce high EA levels among patients with gram-positive septic shock should be elucidated.

Emodin reactivated autophagy and alleviated inflammatory lung injury in mice with lethal endotoxemia.

An uncontrolled inflammation induced critical health problems with serious morbidity and death, which namely acute lung injury (ALI). Recently researchs have found the anti-inflammatory effects of emodin. Here, we investigated the potential effects of emodin on a mouse model with a lethal dose of the potential mechanisms and lipopolysaccharide (LPS)-induced inflammatory lung injury in mice. The pulmonary histological abnormalities, the Evans blue's leakage, the myeloperoxidase (MPO) activity, the grades of TNF-α, IL-6, nitric oxide (NO), lactic acid (LA) in lung tissues were determined 18 h post exposure of LPS. Based on the expression of LC3-II with BECN1 was determined using Western blotting. Besides, the LPS-exposed mice for survival rate was monitored. The results indicated that intervention with emodin was important for mitigating LPS-induced pulmonary histological change and LPS-induced leakage of Evans blue, which were associated with suppressed elevation of MPO activity and inhibited up-regulation of TNF-α, IL-6, NO with LA in lung tissues. Moreover, intervention with emodin enhanced the survival rate of LPS-exposed mice. Finally, therapy with emodin increased the LC3 and BECN1 in lungs of LPS-exposed mice. Treatment with 3-MA (the autophagy inhibitor) reversed the beneficial effects of emodin. In conclusion, emodin might provide pharmacological benefits in LPS-induced inflammatory lung injury, and the mechanisms might be related to the restoration of autophagy.

Acute phase protein, α - 1- acid glycoprotein (AGP-1), has differential effects on TLR-2 and TLR-4 mediated responses.

Alpha-1-acid glycoprotein (AGP-1) is a major positive acute phase glycoprotein with unknown functions that likely play a role in inflammation. We tested its involvement in a variety of inflammatory responses using human AGP-1 purified to apparent homogeneity and confirmed its identity by immunoblotting and mass spectrometry. AGP-1 alone upregulated MAPK signaling in murine peritoneal macrophages. However, when given in combination with TLR ligands, AGP-1 selectively augmented MAPK activation induced by ligands of TLR-2 (Braun lipoprotein) but not TLR-4 (lipopolysaccharide). In vivo treatment of AGP-1 in a murine model of sepsis with or without TLR-2 or TLR-4 ligands, selectively potentiated TLR-2-mediated mortality, but was without significant effect on TLR-4-mediated mortality. Furthermore, in vitro, AGP-1 selectively potentiated TLR-2 mediated adhesion of human primary immune cell, neutrophils. Hence, our studies highlight a new role for the acute phase protein AGP-1 in sepsis via its interaction with TLR-2 signaling mechanisms to selectively promote responsiveness to one of the two major gram-negative endotoxins, contributing to the complicated pathobiology of sepsis.

A sensitive scoring system for the longitudinal clinical evaluation and prediction of lethal disease outcomes in newborn mice.

Neonatal animal models are increasingly employed in order to unravel age-specific disease mechanisms. Appropriate tools objectifying the clinical condition of murine neonates are lacking. In this study, we tested a scoring system specifically designed for newborn mice that relies on clinical observation and examination. Both, in a neonatal sepsis model and an endotoxic shock model, the scoring results strongly correlated with disease-induced death rates. Full as well as observation-restricted scoring, reliably predicted fatality and the remaining time until death. Clinical scores even proved as more sensitive biomarker than 6 traditionally used plasma cytokine levels in detecting sepsis at an early disease stage. In conclusion, we propose a simple scoring system that detects health impairments of newborn mice in a non-invasive longitudinal and highly sensitive manner. Its usage will help to meet animal welfare requirements and might improve the understanding of neonatal disease mechanisms.

Endotoxemia-induced endothelial fibrosis inhibition improves hypotension, tachycardia, multiple organ dysfunction syndrome, cytokine response, oxidative stress, and survival.

Sepsis syndrome is the leading cause of mortality in critically ill patients admitted to intensive care. However, current therapies for sepsis treatment are unsatisfactory, and the mortality rate is still high. The main pathological characteristics observed during sepsis syndrome and endotoxemia include hypotension, tachycardia, multiple organ dysfunction syndrome (MODS), tissue damage, and cytokine and oxidative bursts. These conditions severely decrease the survival rates of endotoxemic patients. As a consequence of endotoxemia, large amounts of endotoxin circulate in the bloodstream throughout the vascular system and interact directly with endothelial cells that cover the inner wall of blood vessels. Endothelial cells exposed to lipopolysaccharides exhibit conversion to activated fibroblasts. By means of endotoxin-induced endothelial fibrosis, endothelial cells downregulate the expression of endothelial proteins and express fibrotic and ECM markers throughout endothelial protein expression reprogramming. Although endotoxin-induced endothelial fibrosis should, in theory, be detrimental to endothelial vascular function, the role of endothelial fibrosis in sepsis syndrome or endotoxemia is not known. Therefore, we employed a rat model to investigate whether the inhibition of endotoxin-induced endothelial fibrosis protects against endotoxemia and whether this inhibition increases survival. Our results show that the inhibition of endotoxin-induced endothelial fibrosis reduced both hypotension and tachycardia. Endotoxemia-induced MODS was also decreased when endothelial fibrosis was inhibited; treated rats showed normal kidney and liver function, inhibition of muscle mass wasting and normal glycemia. Liver and kidney histology was preserved, and organ fibrosis and fibrotic protein expression were reduced. Furthermore, pro-inflammatory cytokine secretion and NOX-2-mediated oxidative stress bursts were decreased when endothelial fibrosis was inhibited. Remarkably, the risk of death associated with sepsis syndrome at early and late time points was decreased when endotoxemia-induced endothelial fibrosis was inhibited, and a significant increase in survival was observed. These results reveal a potential novel treatment strategy to protect against sepsis syndrome and endotoxemia.

Polymyxin B hemoperfusion in endotoxemic septic shock patients without extreme endotoxemia: a post hoc analysis of the EUPHRATES trial.

PURPOSE: The EUPHRATES trial examined the impact of polymyxin B hemoperfusion (PMX) on mortality in patients with septic shock and endotoxemia, defined as EAA ≥ 0.60. No difference was found in 28-day all-cause mortality. However, the trial showed that in some patients with septic shock the burden of endotoxin activity was extreme (EAA ≥ 0.9). In a post hoc analysis, we evaluated the impact of PMX use in patients with septic shock and endotoxin activity measured between 0.6-0.89. METHODS: Post-hoc analysis of the EUPHRATES trial for the 194 patients with EAA ≥ 0.6-0.89 who completed two treatments (PMX or sham). The primary end point was mortality at 28 days adjusted for APACHE II score and baseline mean arterial pressure (MAP). Additional end points included changes in MAP, cumulative vasopressor index (CVI), median EAA reduction, ventilator-free days (VFD), dialysis-free days (DFD) and hospital length of stay. Subpopulations analyzed were site and type of infection and those with norepinephrine dose > 0.1 mcg/kg/min at baseline. RESULTS: At 28 days, 23 patients of 88 (26.1%) in the PMX group died versus 39 of 106 (36.8%) in the sham group [risk difference 10.7%, OR 0.52, 95% CI (0.27, 0.99), P = 0.047]. When unadjusted for baseline variables, P = 0.11. The 28-day survival time in the PMX group was longer than for the sham group [HR 0.56 (95% CI 0.33, 0.95) P = 0.03]. PMX treatment compared with sham showed greater change in MAP [median (IQR) 8 mmHg (- 0.5, 19.5) vs. 4 mmHg (- 4.0, 11) P = 0.04] and VFD [median (IQR) 20 days (0.5, 23.5) vs. 6 days (0, 20), P = 0.004]. There were no significant differences in other end points. There was a significant difference in mortality in PMX-treated patients with no bacterial growth on culture [PMX, 6/30 (20%) vs. sham, 13/31 (41.9%), P = 0.005]. The median EAA change in the population was - 12.9% (range: increase 49.2%-reduction 86.3%). The mortality in the above median EAA change group was PMX: 6/38 (15.7%) vs. sham 15/49 (30.6%), P = 0.08. CONCLUSIONS: These hypothesis-generating results, based on an exploratory post hoc analysis of the EUPHRATES trial, suggest measurable responses in patients with septic shock and an EAA ≥ 0.6 to 0.89 on changes in mean arterial pressure, ventilator-free days and mortality. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT01046669. Funding Spectral Medical Incorporated.

Inhibition of peptidylarginine deiminase alleviates LPS-induced pulmonary dysfunction and improves survival in a mouse model of lethal endotoxemia.

Immune cell death caused by neutrophil extracellular traps (NETs), referred to as NETosis, can contribute to the pathogenesis of endotoxemia and organ damage. Although the mechanisms by which infection induces NETosis and how that leads to organ dysfunction remain largely unknown, NET formation is often found following citrullination of histone H3 (CitH3) by peptidylarginine deiminase (PAD). We hypothesized that lipopolysaccharide (LPS)-induced activation of PAD and subsequent CitH3-mediated NET formation increases endothelial permeability and pulmonary dysfunction and, therefore, that inhibition of PAD can mitigate damage and improve survival in lethal endotoxemia. Here, we showed that treatment with YW3-56, a PAD2/PAD4 inhibitor, significantly diminished PAD activation, blocked LPS-induced pulmonary vascular leakage, alleviated acute lung injury, and improved survival in a mouse model of lethal LPS-induced endotoxemia. We found CitH3 in the bloodstream 30 min after intraperitoneal injection of LPS (35 mg/kg) into mice. Additionally, CitH3 production was induced in cultured neutrophils exposed to LPS, and NETs derived from these LPS-treated neutrophils increased the permeability of endothelial cells. However, YW3-56 reduced CitH3 production and NET formation by neutrophils following LPS exposure. Moreover, treatment with YW3-56 decreased the levels of circulating CitH3 and abolished neutrophil activation and NET formation in the lungs of mice with endotoxemia. These data suggest a novel mechanism by which PAD-NET-CitH3 can play a pivotal role in pulmonary vascular dysfunction and the pathogenesis of lethal endotoxemia.

Protective effect of hemopexin on systemic inflammation and acute lung injury in an endotoxemia model.

BACKGROUND: Hemopexin (HPX) has been identified as an anti-inflammatory agent, but its role in endotoxemia is unclear. The purpose of this study was to determine whether HPX suppresses systemic and lung inflammation in a mouse model of endotoxemia. MATERIALS AND METHODS: At 30 min of intraperitoneal administration of lipopolysaccharide (LPS; 10 mg/kg), either distilled water (LPS-only treated animals) or HPX (5 mg/kg) was injected into mice via the tail vein, and the survival rates were analyzed after 36 h. Furthermore, the serum levels of tumor necrosis factor-α, interleukin-6 (IL-6), and HPX were determined at 0, 3, and 6 h, and the expression levels and DNA binding activities of phosphorylated cytoplasmic inhibitor κB-α, nuclear factor-κB (NF-κB), and the p65 subunit of NF-κB were evaluated and compared with the rates of histologic lung injury after 6 h. RESULTS: Serum tumor necrosis factor-α and interleukin-6 levels were decreased in HPX-treated animals at 3 and 6 h (P < 0.05). HPX suppressed the NF-κB pathway (P < 0.05) and reduced acute lung injury at 6 h, and 36 h after initial treatment, the survival rate was higher in HPX-treated animals than that in LPS-treated animals (P < 0.05). CONCLUSIONS: HPX downregulated proinflammatory cytokine production and acute lung injury as well as improved survival rates in a mouse model of endotoxemia. These effects were associated with HPX-mediated suppression of the NF-κB pathway.

Neutrophil myeloperoxidase diminishes the toxic effects and mortality induced by lipopolysaccharide.

Neutrophils have crucial antimicrobial functions but are also thought to contribute to tissue injury upon exposure to bacterial products, such as lipopolysaccharide (LPS). To study the role of neutrophils in LPS-induced endotoxemia, we developed a new mouse model, PMNDTR mice, in which injection of diphtheria toxin induces selective neutrophil ablation. Using this model, we found, surprisingly, that neutrophils serve to protect the host from LPS-induced lethal inflammation. This protective role was observed in conventional and germ-free animal facilities, indicating that it does not depend on a particular microbiological environment. Blockade or genetic deletion of myeloperoxidase (MPO), a key neutrophil enzyme, significantly increased mortality after LPS challenge, and adoptive transfer experiments confirmed that neutrophil-derived MPO contributes importantly to protection from endotoxemia. Our findings imply that, in addition to their well-established antimicrobial properties, neutrophils can contribute to optimal host protection by limiting the extent of endotoxin-induced inflammation in an MPO-dependent manner.

Serum calcium as an indicator of persistent organ failure in acute pancreatitis.

AIM: Decreased level of serum calcium was commonly seen in critical illness. Hypocalcemia was significantly more frequent in patients with severe form of acute pancreatitis (AP), and a negative correlation was observed between endotoxemia and serum calcium in AP. AP patients with persistent organ failure (POF) show an extremely high mortality. The association underlying calcium and POF in AP has not been characterized. METHODS: We conducted a retrospective cohort study of adult patients who presented within 72hours from symptom onset of AP at our center between January 2014 and May 2015. Demographic parameters on admission, organ failure assessment, laboratory data and in-hospital mortality were compared between patients with and without POF. Uni-and multi-variate logistic regression analyses were utilized to evaluated the predictive ability of serum calcium. RESULTS: A total of 128 consecutive AP patients, including 29 with POF, were included. Compared to patients without POF, patients with POF showed a significantly lower value of serum calcium on admission (2.11±0.46 vs. 1.55±0.36mmol/L, P<0.001). After multivariate logistic analysis, serum calcium remained an independent risk factor for POF (Hazard ratio 0.21, 95% confident interval: 0.08-0.58; P=0.002). A calcium value of 1.97mmol/L predicted POF with an area under the curve (AUC) of 0.888, a sensitivity with 89.7% and specificity with 74.8%, respectively. CONCLUSION: Our results indicate that serum calcium on admission is independently associated with POF in AP and may serve as a potential prognostic factor.

Gelsolin Inhibits the Inflammatory Process Induced by LPS.

BACKGROUND/AIMS: Endotoxemia is a life-threatening situation that signifies a key challenge in the field of intensive care medicine. Proinflammatory mediators produced by macrophages play a key role in endotoxemia. Gelsolin (GSN) is involved in the process of inflammation. METHODS: IL-6 and TNF-α in the supernatant were measured with an ELISA kit. NO production was assessed by measurement of nitrite concentration with the Griess assay. si-RNA directed against GSN (si-GSN) was transfected by Lipofectamine. RESULTS: LPS decreased the levels of GSN. Recombinant GSN inhibited the cytokines induced by LPS and rescued mice from LPS-induced death, and si-GSN increased death in the LPS-pretreated mice. CONCLUSION: GSN exhibited a protective role in endotoxemia.