Experimental Endotoxemia Research Articles

Brain Oxidative Stress During Experimental Sepsis Is Attenuated by Simvastatin Administration.

During sepsis, brain damage is associated with oxidative stress due to overproduction of reactive oxygen species (ROS). Although there are recent reports about the benefits of statins in experimental sepsis and endotoxemia in peripheral organs, little is known about their effects in the CNS. Here, we investigated the antioxidant properties of simvastatin and its possible neuroprotective role during experimental sepsis. Male Wistar rats (250-300g) were submitted to cecal ligation and puncture (CLP, n=34) or remained as non-manipulated (naive, n=34). Both groups were treated by gavage with simvastatin (20mg/kg) or an equivalent volume of saline. The animals submitted to CLP were treated 4days before and 48h after surgery. One animal group was decapitated and the blood and brain were collected to quantify plasma levels of cytokines and assess astrogliosis and apoptosis in the prefrontal cortex and hippocampus. Another group was perfused with PBS (0.01M), and the same brain structures were dissected to analyze oxidative damage. The CLP rats treated with simvastatin showed a reduction in nitric oxide (P<0.05), IL1-β (P<0.001), IL-6 (P<0.01), and TBARS levels (P<0.001) and an increase in catalase activity (P<0.01), citrate synthase enzyme (P<0.05), and normalized GSH/GSSG ratio. In addition, the histopathological analysis showed a reduction (P<0.001) in reactive astrocytes and caspase 3-positive apoptotic cells. The results suggest a possible neuroprotective effect of simvastatin in structures responsible for spatial learning and memory and indicate the need for behavioral studies evaluating the impact on cognitive damage, as frequently seen in patients surviving sepsis.

The influence of intravenous solution «Neoreodez» on the course of experimental endotoxemia and evaluation of the regenerative properties of drug in the conditions of application.

In the experiment it was shown that experimental endotoxemia induced by introduction of carbon tetrachloride and bacterial lipopolysaccharide is characterized by activation of free radical oxidation and failure of antioxidant defense system. Intravenous injection of solution «Neoreodez» under these experimental conditions has a positive impact on the course of endogenous intoxication, which is manifested by decrease in the concentration of MDA and DC in the liver on average by 1½ times (p≤0.05), decrease in the level of AlAT and AsAT serum by 2 times (p≤0.05) and to restore the balanced functioning of antiradical protection – increased activity of SOD by 1.6 times (p&lt;0.05) in comparison with the performance of the active control. It is established that two-week application «Neoreodez» of solution potentiate the processes of natural reparation of damaged skin.

Sumoylation of IkB attenuates NF-kB-induced nitrosative stress at rostral ventrolateral medulla and cardiovascular depression in experimental brain death

BackgroundSmall ubiquitin-related modifier (SUMO) is a group of proteins that participates in post-translational modifications. One known SUMO target is the transcription factor nuclear factor-kB (NF-kB) that plays a pivotal role in many disease processes; sumoylation inactivates NF-kB by conjugation with inhibitors of NF-kB (IkB). Our laboratory demonstrated previously that transcriptional upregulation of nitric oxide synthase II (NOS II) by NF-kB, leading to nitrosative stress by the formation of peroxynitrite in the rostral ventrolateral medulla (RVLM), underpins the defunct brain stem cardiovascular regulation that precedes brain death. Based on an experimental endotoxemia model, this study evaluated the hypothesis that sumoylation plays a pro-life role in brain death by interacting with the NF-kB/NOS II/peroxynitrite signaling pathway in the RVLM.ResultsIn Sprague–Dawley rats, intravenous administration of Escherichia coli lipopolysaccharide (LPS; 10 mg kg−1) elicited an augmentation of SUMO-1 and ubiquitin-conjugase 9 (Ubc9) mRNA or protein levels, alongside SUMO-1-conjugated proteins in the RVLM. Immunoneutralization of SUMO-1 or Ubc9 in the RVLM significantly potentiated the already diminished sumoylation of IkBα and intensified NF-kB activation and NOS II/peroxynitrite expression in this brain stem substrate, together with exacerbated fatality, cardiovascular depression and reduction of an experimental index of a life-and-death signal detected from arterial pressure that disappears in comatose patients signifying failure of brain stem cardiovascular regulation before brain death.ConclusionWe conclude that sumoylation of IkB in the RVLM ameliorates the defunct brain stem cardiovascular regulation that underpins brain death in our experimental endotoxemia modal by reducing nitrosative stress via inhibition of IkB degradation that diminishes the induction of the NF-kB/NOS II/peroxynitrite signaling cascade.

Abstract # 1793 Sex differences in cytokine responses to endotoxin in humans unfold only in vivo

Both clinical observations and experimental findings in humans demonstrate that humoral and cell-mediated immune responses markedly differ between sexes. In line with this, we have recently shown that women mount stronger pro-inflammatory and weaker anti-inflammatory cytokine responses after in vivo challenge with bacterial endotoxin compared to men, but the underlying mechanisms remain elusive. Given the important role of neuroendocrine mediators such as cortisol and noradrenaline in the feedback regulation of the inflammatory response, we analyzed in this follow-up study whether peripheral blood immune cells from men and women differ in their sensitivity to glucocorticoid and adrenergic receptor agonists. Lipopolysaccharide-stimulated whole blood samples from healthy male ( n = 20) and female donors ( n = 20) were cultured in absence and presence of dexamethasone (DEX; 10-9 to 10-5 M) or terbutaline (TERB; 10-9 to 10-5 M), and the production of TNF-alpha, IL-6, and IL-10 was quantified by ELISA. In contrast to our in vivo findings, ex vivo cytokine production did not differ between men and women, suggesting that the previously observed sex differences in plasma cytokines during experimental endotoxemia must have resulted from differences in the systemic response. More importantly, we found no evidence for sex differences in the ex vivo sensitivity of peripheral immune cells to DEX and TERB. This suggests that inflammatory cells from women do not exhibit impaired sensitivity to neuroendocrine anti-inflammatory mediators compared to men.

Abstract # 1713 Predictors of bodily sickness symptoms during experimental endotoxemia

Patients with inflammatory conditions commonly suffer from non-specific bodily symptoms such as fatigue, widespread pain, and malaise. However, it remains unclear if these symptoms are driven by cytokines and/or psychological factors. Experimental administration of endotoxin (LPS) is an established model to induce a wide spectrum of behavioral and physical sickness symptoms. The aim of the present study was to identify predictors of LPS-induced physical sickness symptoms using multivariate regression analyses. Healthy volunteers (N = 128) were randomized to LPS (0.4 or 0.8 ng/kg) or placebo in published studies with identical protocols. Psychological state (state anxiety, positive mood) and trait variables (depression symptoms, trait anxiety, neuroticism, extraversion, coping skills) were measured along with plasma concentrations of IL-6, TNF-alpha, and cortisol. Non-specific sickness symptoms experienced during the acute (0–6 h post-injection) and late inflammatory (6–24 h post-injection) phases were assessed with the validated Generic Assessment of Side Effects (GASE). Results showed that LPS-treated subjects reported significantly more physical sickness symptoms. Symptoms during the early phase were predicted by decreases in positive mood and increases in IL-6 plasma levels, explaining 28.5% of variance in GASE scores. During the late phase, increases in cortisol, IL-6 and baseline depression were significant predictors, explaining 38.5% of variance. Hence, mood alterations may constitute a risk factor for physical sickness symptoms in inflammatory conditions.

Glucagon‐like peptide‐1 receptor signalling reduces microvascular thrombosis, nitro‐oxidative stress and platelet activation in endotoxaemic mice

Excessive inflammation in sepsis causes microvascular thrombosis and thrombocytopenia associated with organ dysfunction and high mortality. The present studies aimed to investigate whether inhibition of dipeptidyl peptidase-4 (DPP-4) and supplementation with glucagon-like peptide-1 (GLP-1) receptor agonists improved endotoxaemia-associated microvascular thrombosis via immunomodulatory effects. Endotoxaemia was induced in C57BL/6J mice by a single injection of LPS (17.5 mg kg-1 for survival and 10 mg kg-1 for all other studies). For survival studies, treatment was started 6 h after LPS injection. For all other studies, drugs were injected 48 h before LPS treatment. Mice treated with LPS alone showed severe thrombocytopenia, microvascular thrombosis in the pulmonary circulation (fluorescence imaging), increased LDH activity, endothelial dysfunction and increased markers of inflammation in aorta and whole blood (leukocyte-dependent oxidative burst, nitrosyl-iron haemoglobin, a marker of nitrosative stress, and expression of inducible NOS). Treatment with the DPP-4 inhibitor linagliptin or the GLP-1 receptor agonist liraglutide, as well as genetic deletion of DPP-4 (DPP4-/- mice) improved all these parameters. In GLP-1 receptor-deficient mice, both linagliptin and liraglutide lost their beneficial effects and improvement of prognosis. Incubation of platelets and cultured monocytes (containing GLP-1 receptor protein) with GLP-1 receptor agonists inhibited the monocytic oxidative burst and platelet activation, with a GLP-1 receptor-dependent elevation of cAMP levels and PKA activation. GLP-1 receptor activation in platelets by linagliptin and liraglutide strongly attenuated endotoxaemia-induced microvascular thrombosis and mortality by a cAMP/PKA-dependent mechanism, preventing systemic inflammation, vascular dysfunction and end organ damage. This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc.

Effect of pioglitazone on metabolic features in endotoxemia model in obese diabetic db/db mice.

Infectious diseases are more frequent in diabetic patients, leading to increased morbidity and mortality. Endotoxemia affects glucose metabolism and lipolytic capacity. The aims of the present study were to determine whether endotoxemia exacerbates metabolic features (adipose inflammation, adipogenesis, and insulin resistance [IR]) in an animal model of diabetes (i.e. db/db mice) after acute infection and the effects of pioglitazone. Female db/db mice treated with pioglitazone (3 and 30 mg/kg, p.o.) for 14 days were challenged with lipopolysaccharide (LPS; 200 μg/kg), followed by an oral glucose tolerance test (OGTT). Quantitative real-time polymerase chain reaction (PCR) was used to evaluate the expression of genes in white adipose tissue (WAT) involved in: (i) adipogenesis (lipoprotein lipase [Lpl], fatty acid binding protein-4 [Ap2] and adiponectin [Adipoq]); (ii) insulin signaling (peroxisome proliferator-activated receptor gamma [Pparg], suppressor of cytokine signaling 3 [Socs3], solute carrier family 2 [facilitated glucose transporter], member 4 [Slc2a4]); and (iii) inflammation (tumor necrosis factor [Tnf], interleukin-6 [Il6], monocyte chemoattractant protein-1 [Ccl2], cyclo-oxygenase-2 [prostaglandin-endoperoxide synthase 2; Ptgs2]). Experimental endotoxemia downregulated mRNA expression of Pparg, Slc2a4, Adipoq, Lpl, and Ap2, which coincided with upregulation of Il6, Tnf, Ccl2, Ptgs2, and Socs3 expression. Pioglitazone dose-dependently decreased Tnf, Il6, Ccl2, Ptgs2, and Socs3 expression in WAT, in association with upregulation of Lpl, Ap2, Slc2a4, and Adipoq expression, indicating improvement in endotoxin-induced IR. The findings suggest that LPS challenge exacerbates IR in db/db mice by altering the expression of genes in WAT involved in adipogenesis and inflammation, which is effectively controlled by pioglitazone treatment.

Differential in vivo activation of monocyte subsets during low-grade inflammation through experimental endotoxemia in humans

Human monocytes are a heterogeneous cell population, which can be divided into a classical (CD14++CD16−), a non-classical (CD14+CD16+), and an intermediate (CD14++CD16+) subset. We hypothesized that low-grade inflammation may differentially affect monocyte subsets. We used a human lipopolysaccharide (LPS) infusion model to mimic low-grade inflammation to identify, which monocyte subsets are preferentially activated under these conditions. Monocyte subsets were identified by staining for CD14 and CD16, activation status of monocytes was analyzed by staining for CD11b and a novel in situ mRNA hybridization approach to detect IL-6 and IL-8 specific mRNA at the single-cell level by flow cytometry. After LPS challenge, cell numbers of monocyte subsets dropped after 2 h with cell numbers recovering after 6 h. Distribution of monocyte subsets was skewed dramatically towards the intermediate subset after 24 h. Furthermore, intermediate monocytes displayed the largest increase of CD11b expression after 2 h. Finally, IL-6 and IL-8 mRNA levels increased in intermediate and non-classical monocytes after 6 h whereas these mRNA levels in classical monocytes changed only marginally. In conclusion, our data indicates that the main responding subset of monocytes to standardized low-grade inflammation induced by LPS in humans is the CD14++CD16+ intermediate subset followed by the CD14+CD16+ non-classical monocyte subset. Circulating classical monocytes showed comparably less reaction to LPS challenge in vivo.

Plasma Nuclear and Mitochondrial DNA Levels, and Markers of Inflammation, Shock, and Organ Damage in Patients with Septic Shock.

Plasma levels of the danger-associated molecular patterns (DAMPs) nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) have been shown to be related to sepsis mortality. However, the intermediate factors and/or mechanisms contributing to this relation are largely unknown. Our aim was to determine whether plasma levels of nDNA and mtDNA are related to the markers of inflammation, severity of shock, and organ damage in septic shock patients. Moreover, we investigated the relationship between plasma levels of nDNA/mtDNA and inflammatory cytokines during experimental human endotoxemia, a model of systemic inflammation in humans in vivo mimicking some of the hallmarks of early sepsis. Blood was sampled from the onset of septic shock until day 28 in 121 septic shock patients and from 1 h before endotoxin administration until 8 h afterward in 12 healthy volunteers. Plasma concentrations of five cytokines and circulating levels of nDNA and mtDNA were measured, and correlations with shock-related parameters and markers of organ damage were investigated. In septic shock patients plasma cytokine concentrations, as well as nDNA and mtDNA levels, were increased at the onset of septic shock and remained elevated. During the first 5 days of septic shock, nDNA levels consistently correlated with plasma cytokine concentrations as well as with the shock-related parameter norepinephrine infusion rate and markers of organ damage (total bilirubin and creatinine). Experimental human endotoxemia also resulted in increased levels of plasma nDNA and mtDNA, but to a lesser extent than in septic shock patients. Furthermore, nDNA levels correlated with pro-inflammatory cytokines during endotoxemia. Our findings indicate a relationship between plasma nDNA levels and the inflammatory response. Furthermore, nDNA levels are associated with markers of shock and organ damage in septic shock patients. Nevertheless, the correlations found are relatively weak and it remains to be determined whether nDNA is merely a marker or directly involved in the pathophysiology of septic shock.

Differential Role of Neurohypophysial Hormones in Hypotension and Nitric Oxide Production During Endotoxaemia.

Besides their well-established endocrine roles, vasopressin and oxytocin are also important regulators of immune function, participating in a complex neuroendocrine-immune network. In the present study, we investigated whether and how vasopressin and oxytocin could modulate lipopolysaccharide (LPS)-induced nitric oxide (NO) production in a well-established model of experimental endotoxaemia. Male Wistar rats were previously treated i.v. with vasopressin V1 or oxytocin receptor antagonists and then received either an i.v. LPS injection to induce endotoxaemia or a saline imjection as a control. The animals were divided into two groups: in the first group, blood was collected at 2, 4 and 6h after LPS injection; in the second group, mean arterial blood pressure (MABP) and heart rate (HR) were recorded over 6h. Plasma vasopressin and oxytocin values were higher in LPS- compared to saline-injected animals at 2 and 4h but returned to basal levels at 6h. NO levels exhibited an opposite pattern, showing a progressive increase over the entire period. The previous administration of a vasopressin V1 receptor antagonist significantly reduced NO plasma concentrations at 2 and 4h but not at 6h. By contrast, oxytocin receptor agonist pre-treatment had no effect on the NO plasma concentration. In relation to MABP, previous treatment with vasopressin V1 receptor antagonist reversed the LPS-induced hypotension at 4h, although this was not the case for oxytocin antagonist-treated animals. None of the antagonists affected HR. Our findings indicate that vasopressin (but not oxytocin) has effects on NO production during endotoxaemia in rats, although they do not lend support to the proposed anti-inflammatory actions of vasopressin during endotoxaemia.

Inflammation-associated changes in lipid composition and the organization of the erythrocyte membrane

BackgroundReduced erythrocyte survival and deformability may contribute to the so-called anemia of inflammation observed in septic patients. Erythrocyte structure and function are affected by both the membrane lipid composition and the organization. We therefore aimed to determine whether these parameters are affected during systemic inflammation. MethodsA sensitive matrix-assisted laser desorption and ionization time-of-flight mass spectrometric method was used to investigate the effect of plasma components of 10 patients with septic shock and of 10 healthy volunteers subjected to experimental endotoxemia on erythrocyte membrane lipid composition. ResultsIncubation of erythrocytes from healthy control donors with plasma from patients with septic shock resulted in membrane phosphatidylcholine hydrolysis into lysophosphatidylcholine (LPC). Plasma from volunteers undergoing experimental human endotoxemia did not induce LPC formation. The secretory phospholipase A2 IIA concentration was enhanced up to 200-fold in plasma of septic patients and plasma from endotoxin-treated subjects, but did not correlate with the ability of these plasmas to generate LPC. Erythrocyte phosphatidylserine exposure increased up to two-fold during experimental endotoxemia. ConclusionsErythrocyte membrane lipid remodeling as reflected by LPC formation and/or PS exposure occurs during systemic inflammation in a secretory phospholipase A2 IIA-independent manner. General significanceSepsis-associated inflammation induces a lipid remodeling of the erythrocyte membrane that is likely to affect erythrocyte function and survival, and that is not fully mimicked by experimental endotoxemia.

Sympathetic Vasoconstrictor Responsiveness of the Leg Vasculature During Experimental Endotoxemia and Hypoxia in Humans.

Sympathetic vasoconstriction regulates peripheral circulation and controls blood pressure, but sepsis is associated with hypotension. We evaluated whether apparent loss of sympathetic vasoconstrictor responsiveness relates to distended smooth muscles or to endotoxemia and/or hypoxia. Prospective descriptive study. Hospital research laboratory. Ten healthy young men (age [mean ± SD], 31 ± 8 yr; body weight, 83 ± 10 kg) participated in the study. Leg blood flow and mean arterial pressure were determined, whereas leg vascular conductance was calculated during 1) adenosine infusion (vasodilator control), 2) hypoxia (FIO2 = 10%), 3) endotoxemia, and 4) endotoxemia + hypoxia. Leg sympathetic vasoconstrictor responsiveness (reduction in leg vascular conductance) was evaluated by femoral artery tyramine infusion. Endotoxemia increased body temperature from 36.9 ± 0.4°C to 38.6 ± 0.5°C (p < 0.01) and plasma tumor necrosis factor-α from 6 pg/mL (3-8 pg/mL) to 391 pg/mL (128-2258 pg/mL) (p < 0.01; median [range]). Mean arterial pressure decreased similarly during endotoxemia (-11% ± 16%) and endotoxemia + hypoxia (-10% ± 15%; both p < 0.05). Leg blood flow and leg vascular conductance were not affected by endotoxemia, whereas both were elevated by adenosine infusion (leg blood flow, +94% ± 61%; leg vascular conductance, +97% ± 57%), hypoxia (leg blood flow: +93% ± 58%; leg vascular conductance, +100% ± 115%), and endotoxemia + hypoxia (leg blood flow, +67% ± 120%; leg vascular conductance, +65% ± 57%; p < 0.05). Endotoxemia lessened the tyramine-induced reduction in leg vascular conductance (-28% ± 13%) compared with the reduction during adenosine infusion (-47% ± 5%; p < 0.05). Also, endotoxemia + hypoxia (-17% ± 21%) attenuated the tyramine-induced reduction in leg vascular conductance compared with both adenosine infusion and hypoxia (-45% ± 13%; p < 0.05). Both endotoxemia and combined hypoxia and endotoxemia blunted sympathetic vasoconstrictor responsiveness. Furthermore, tyramine normalized the doubled leg vascular conductance during administration of adenosine, suggesting that distension of vascular smooth muscles does not explain blunted sympathetic vasoconstrictor responsiveness during endotoxemia.

Restoring Antiendotoxin Activities of Inactive Antimicrobial Peptides

Gram‐negative bacteria cell wall is composed of two layers: the inner phospholipid membrane and the outer membrane with a highly conserved unique lipid called lipopolysaccharide (LPS; endotoxin). LPS acts as a permeability barrier against a number of bactericidal agents. Furthermore, LPS is well known as a potent inducer of the immune system when it is released to blood and often causes septic shock syndromes in human. Some but not every antimicrobial peptide can neutralize LPS stimulated proinflammatory responses. We have developed an easy method to restore antiendotoxin activities of inactive antimicrobial peptides by linking two inactive α‐helical peptides with the AGP sequence. Antibacterial activities were determined by the standard broth micro‐dilution method of National Committee for Clinical Laboratory Standards. Toxicities of the peptides were determined from measuring cell death by the MTT assay against human fibroblast (HFW cells). Dynamic light scattering (DLS) was used to measure the size increase of LPS in the presence of the peptides. The ability of LPS neutralization in vitro was measured by Limulus Amebocyte Lysate (LAL) assay. LPS can induce nitrite oxide (NO) and TNF‐α production in macrophage cells. Experimental mouse endotoxemia model was used to test the antiendotoxin activities of the peptides. Significant antiendotoxin effects were observed in vitro and in vivo with the AGP linked variant of the short α‐helical peptides KR12 and WR6. The results will be used to help us design more potent antimicrobial peptides for clinical application in the future.Support or Funding InformationGrant Number 103‐2113‐M‐007‐009‐MY2, Ministry of Science and Technology, Taiwan

Blockade of Thrombopoietin Reduces Organ Damage in Experimental Endotoxemia and Polymicrobial Sepsis.

Background and PurposeThrombopoietin (TPO), a growth factor primarily involved in thrombopoiesis may also have a role in the pathophysiology of sepsis. In patients with sepsis, indeed, TPO levels are markedly increased, with disease severity being the major independent determinant of TPO concentrations. Moreover, TPO increases and correlates with ex vivo indices of platelet activation in patients with burn injury upon sepsis development, and may contribute to depress cardiac contractility in septic shock. Still, the role of TPO in sepsis pathophysiology remains controversial, given the protective role of TPO in other experimental disease models, for instance in doxorubicin-induced cardiotoxicity and myocardial ischemia/reperfusion injury. The aim of our study was to define the contribution of TPO in the development of organ damage induced by endotoxemia or sepsis, and to investigate the effects of inhibiting TPO in these conditions.MethodsWe synthesized a chimeric protein able to inhibit TPO, mTPOR-MBP, and studied its effect in two murine experimental models, acute endotoxemia and cecal ligation and puncture (CLP) model.ResultsIn both models, TPO levels markedly increased, from 289.80±27.87 pg/mL to 465.60±45.92 pg/mL at 3 hours in the LPS model (P<0.01), and from 265.00±26.02 pg/mL to 373.70±26.20 pg/mL in the CLP model (P<0.05), respectively. Paralleling TPO levels, also platelet-monocyte aggregates increased, from 32.86±2.48% to 46.13±1.39% at 3 hours in the LPS model (P<0.01), and from 43.68±1.69% to 56.52±4.66% in the CLP model (P<0.05). Blockade of TPO by mTPOR-MBP administration reduced histological damage in target organs, namely lung, liver, and gut. In particular, neutrophil infiltration and lung septal thickening were reduced from a score of 1.86±0.34 to 0.60±0.27 (P<0.01) and from 1.43±0.37 to 0.40±0.16 (P<0.05), respectively, in the LPS model at 3 hours, and from a score of 1.75±0.37 to 0.38±0.18 (P<0.01) and from 1.25±0.31 to 0.13±0.13 (P<0.001), respectively, in the CLP model. Similarly, the number of hepatic microabscesses was decreased from 14.14±1.41 to 3.64±0.56 in the LPS model at 3 hours (P<0.001), and from 1.71±0.29 to 0.13±0.13 in the CLP model (P<0.001). Finally, the diameter of intestinal villi decreased from 90.69±3.95 μm to 70.74±3.60 μm in the LPS model at 3 hours (P<0.01), and from 74.29±4.29 μm to 57.50±1.89 μm in the CLP model (P<0.01). This protective effect was associated with the blunting of the increase in platelet-monocyte adhesion, and, on the contrary, with increased platelet-neutrophil aggregates in the circulation, which may be related to decreased neutrophil sequestration into the inflamed tissues. Conversely, circulating cytokine levels were not significantly changed, in both models, by mTPOR-MBP administration.ConclusionOur results demonstrate that TPO participates in the development of organ damage induced by experimental endotoxemia or polymicrobial sepsis via a mechanism involving increased platelet-leukocyte adhesion, but not cytokine release, and suggest that blocking TPO may be useful in preventing organ damage in patients affected by systemic inflammatory response or sepsis.

Time-related changes in equine neutrophils after experimental endotoxemia: myeloperoxidase staining, size, and numbers.

Neutrophil myeloperoxidase content is determined by the Advia 2120 hematology system by staining characteristics. Changes in myeloperoxidase staining are shown by location of neutrophils on Advia peroxidase dot plots and as myeloperoxidase index (MPXI). Significant changes in MPXI have been reported during severe inflammation in horses, dogs, and people but conclusions were inconsistent. Infusion of endotoxin was used to initiate an inflammatory stimulus under controlled conditions and over a longer time period than in previous studies to document kinetics of changes in neutrophil numbers, morphology, and myeloperoxidase staining. Identification of consistent time-related changes may allow better interpretation of changes in neutrophil characteristics during inflammation. Five Standardbred trotting horses received an intravenous infusion over a 6-hour period with Escherichia coli endotoxin. Neutrophil count, MPXI, neutrophil characteristics in Advia 2120 Perox dot plots and neutrophil morphology in blood smears were monitored with repeated sampling for up to 10 days. Endotoxin infusion immediately caused severe neutropenia which converted to neutrophilia 14 hours after start of endotoxin infusion. Neutrophilia was still present 78 hours after start of infusion. Large "giant" neutrophils first appeared in blood smears and Advia Perox dot plots after 36-48 hours. A marked and consistent decrease in MPXI was seen in all horses 6 days (150 hours) after endotoxin exposure. Endotoxemia caused prominent, time-related changes in equine neutrophil characteristics including emergence of giant neutrophils and markedly decreased MPXI several days after endotoxin infusion.

Granzyme M and K release in human experimental endotoxemia

Granzymes are serine proteases involved in killing of tumor cells and virally infected cells. However, granzymes are also upregulated in blood under inflammatory conditions and contribute to cytokine release and processing. Here, we show that granzyme M (GrM) and to a lesser extent GrK are transiently elevated in the circulation following LPS administration in humans. GrM is released upon stimulation of whole blood with LPS or the gram-negative bacteria Escherichia coli BL21, Pseudomonas aeruginosa, and Neisseria meningitidis. GrK is only released upon stimulation with P. aeruginosa. Thus, GrM and GrK are differentially released in response to LPS and gram-negative bacteria.

Mood disturbance during experimental endotoxemia: Predictors of state anxiety as a psychological component of sickness behavior

Lipopolysaccharide (LPS) administration is a well-established model to assess afferent immune-to-brain communication and behavioral aspects of inflammation. Nevertheless, only few studies in comparatively small samples have assessed state anxiety as a psychological component of sickness behavior despite possible clinical implications for the pathophysiology of neuropsychiatric conditions. Thus, the goal of the present analyses carried out in a large, pooled dataset from two independent study sites was to analyze the state anxiety response to LPS administration and to investigate predictors (i.e., cytokine changes; pre-existing anxiety and depression symptoms assessed with the Hospital Anxiety and Depression Scale) of the LPS-induced state anxiety changes at different time points after LPS administration. Data from 186 healthy volunteers who participated in one of six randomized, placebo-controlled human studies involving intravenous administration of LPS at doses of 0.4–0.8ng/kg body weight were combined. State anxiety as well as circulating interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-10 concentrations were significantly increased 2h and 3h after LPS administration, with a peak at 2h, and returned to baseline 6h after administration. Greater changes in IL-6 from baseline to 3h after LPS administration significantly and independently predicted a more pronounced LPS-induced state anxiety response. In addition, higher pre-existing subclinical anxiety symptoms significantly predicted a lower increase in state anxiety 3h and 6h after LPS-administration, which was mediated by TNF-α changes. In conclusion, our findings give additional support for a putative role of inflammatory mechanisms in the pathophysiology of stress-related and anxiety disorders and give new insight on the potential role of pre-existing subclinical affective symptoms.

Endotoxemia Engages the RhoA Kinase Pathway to Impair Cardiac Function By Altering Cytoskeleton, Mitochondrial Fission, and Autophagy.

The RhoA/ROCK pathway controls crucial biological processes involved in cardiovascular pathophysiology, such as cytoskeleton dynamics, vascular smooth muscle contraction, and inflammation. In this work, we tested whether Rho kinase inhibition would beneficially impact cardiac cytoskeleton organization, bioenergetics, and autophagy in experimental endotoxemia induced by lipopolysaccharides (LPSs) in mice. Fasudil, a potent ROCK inhibitor, prevented LPS-induced cardiac inflammation, oxidative stress, cytoskeleton disarray, and mitochondrial injury. ROCK inhibition prevented phosphorylation of cofilin and dynamin-related protein-1, which promotes stabilization-polymerization of F-actin and mediates mitochondrial fission, respectively. Pyr1, which exclusively alters actin dynamics, prevented LPS-induced myocardial dysfunction, suggesting that beneficial impact of ROCK inhibition was not mainly related to pleiotropic effects of fasudil on cardiac inflammation and oxidative stress. Fasudil reduced mitochondrial fragmentation, stimulated initiation of autophagy, and elicited cardioprotection in LPS heart. Mdivi-1, a potent mitochondria fission inhibitor, converted cardioprotective autophagy to an inefficient form due to cargo loading failure in which autophagic vacuoles fail to trap cytosolic cargo, despite their formation at enhanced rates and lysosomal elimination. In experimental endotoxemia, cardioprotection by RhoA/ROCK inhibition may be related to changes in actin cytoskeleton reorganization and mitochondrial homeostasis. Improvement of LPS-induced mitochondrial dysfunction by fasudil was attributed to inhibition of ROCK-dependent Drp1 phosphorylation and activation of autophagic processes that can limit mitochondrial fragmentation and enhance degradation of damaged mitochondria, respectively. Fasudil prevented LPS-induced heart oxidative stress, abnormal F-actin distribution, and oxidative phosphorylation, which concur to improve cardiac contractile and bioenergetic function. We suggest that fasudil may represent a valuable therapy for patients with sepsis.

The comparative efficacy of tyloxapol versus pentoxifylline against induced acute phase response in an ovine experimental endotoxemia model

The effective treatments of endotoxemia are necessary to prevent high mortality rates. Hence, the present study was performed to clarify the antiendotoxic effects of tyloxapol and pentoxifylline in experimentally induced endotoxemia in sheep. Thirty clinically healthy 1-year-old Iranian fat-tailed ewes were randomly divided into six equal experimental (n=5) groups, comprising Negative and Positive control, Tyloxapol 1, Tyloxapol 2, Pentoxifylline 1 and Pentoxifylline 2. Phenol extracted lipopolysaccharide from Escherichia coli serotype O55:B5 was infused at 2µg/kg intravenously. Tyloxapol (200 and 400mg/kg) and pentoxifylline (30 and 60mg/kg) were injected to Tyloxapol and Pentoxifylline groups, respectively, at 90min after endotoxemia induction over 60min along with intravenous fluids. Blood samples were collected from all ewes prior and 1.5, 3, 4.5, 6, 24 and 48h after lipopolysaccharide injection and sera and plasmas were separated, subsequently. Haptoglobin, serum amyloid A, tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), superoxide dismutase and glutathione peroxidase were measured in all samples. Serum concentrations of haptoglobin, serum amyloid A, TNF-α and IFN-γ in Tyloxapol 1 and 2 and Pentoxifylline 1 and 2 groups were significantly lower than Positive control one after hour 3. There were no significant differences among Tyloxapol and Pentoxifylline groups (P>0.05). Superoxide dismutase and glutathione peroxidase activities in Tyloxapol 1 and 2 and Pentoxifylline 1 and 2 groups were significantly lower than Positive control one after hour 3. There were no significant differences among Tyloxapol 1 and 2 and Pentoxifylline 1 and 2 groups (P>0.05). Tyloxapol and pentoxifylline act as the anti-inflammatory mediators by decreasing pro-inflammatory cytokines and hepatic APPs and modulating oxidative enzymes activity after endotoxemia induction in sheep. Furthermore, their efficacies at different doses were significantly similar together and both drugs don't induce their effects by dose dependent manner and the anti- and pro-inflammatory effects of them were statistically similar.

Human metabolic response to systemic inflammation: assessment of the concordance between experimental endotoxemia and clinical cases of sepsis/SIRS.

IntroductionTwo recent, independent, studies conducted novel metabolomics analyses relevant to human sepsis progression; one was a human model of endotoxin (lipopolysaccharide (LPS)) challenge (experimental endotoxemia) and the other was community acquired pneumonia and sepsis outcome diagnostic study (CAPSOD). The purpose of the present study was to assess the concordance of metabolic responses to LPS and community-acquired sepsis.MethodsWe tested the hypothesis that the patterns of metabolic response elicited by endotoxin would agree with those in clinical sepsis. Alterations in the plasma metabolome of the subjects challenged with LPS were compared with those of sepsis patients who had been stratified into two groups: sepsis patients with confirmed infection and non-infected patients who exhibited systemic inflammatory response syndrome (SIRS) criteria. Common metabolites between endotoxemia and both these groups were individually identified, together with their direction of change and functional classifications.ResultsResponse to endotoxemia at the metabolome level elicited characteristics that agree well with those observed in sepsis patients despite the high degree of variability in the response of these patients. Moreover, some distinct features of SIRS have been identified. Upon stratification of sepsis patients based on 28-day survival, the direction of change in 21 of 23 metabolites was the same in endotoxemia and sepsis survival groups.ConclusionsThe observed concordance in plasma metabolomes of LPS-treated subjects and sepsis survivors strengthens the relevance of endotoxemia to clinical research as a physiological model of community-acquired sepsis, and gives valuable insights into the metabolic changes that constitute a homeostatic response. Furthermore, recapitulation of metabolic differences between sepsis non-survivors and survivors in LPS-treated subjects can enable further research on the development and assessment of rational clinical therapies to prevent sepsis mortality. Compared with earlier studies which focused exclusively on comparing transcriptional dynamics, the distinct metabolomic responses to systemic inflammation with or without confirmed infection, suggest that the metabolome is much better at differentiating these pathophysiologies. Finally, the metabolic changes in the recovering patients shift towards the LPS-induced response pattern strengthening the notion that the metabolic, as well as transcriptional responses, characteristic to the endotoxemia model represent necessary and “healthy” responses to infectious stimuli.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-0783-2) contains supplementary material, which is available to authorized users.