[Therapy of experimental endotoxin shock. II. Morphologic changes in kidneys and adrenals in endotoxin shock under treatment with prednisolone and redergam].
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Adrenal Glands↗
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Antibiotic induced endotoxin release and clinical sepsis: a review.
Sepsis and peritonitis have not lost much of their danger for patients. The mortality rate in peritonitis has only marginally decreased during the last 30 years despite aggressive surgical and sophisticated intensive care treatment. In intra-abdominal infection and peritonitis source control remains the mainstay of treatment, although general principles and denominators of successful source control need to be established. Endotoxin has been recognized as a major player in the pathogenesis of sepsis and its significance in clinical disease has been investigated in clinical studies for more than 20 years. Since the Sixties there is a growing interest in the effect of antibiotics and other compounds on the release of endotoxin. The effect of antibiotics on the release of endotoxin and inflammatory parameters, e.g., cytokines, remains to be clarified despite a growing body of in-vitro studies, animal studies and a few clinical studies. The purpose of this review is to evaluate the evidence of endotoxin release in clinical studies and the effect that antibiotic treatment may have in-vitro, in-vivo and in clinical studies on endotoxin and cytokine release. In-vitro antibiotic-induced endotoxin release may depend on antibiotic class, presence of serum, type of organism, site of antibiotic action and Gram-stain. Endotoxin release may be different in late or early lysis, proportional to the number of killed pathogens. Morphology of bacteria may have an impact on endotoxin release and phagocytosis. Antibiotic-treated animals may show higher endotoxin levels with a higher survival rate than untreated animals. Plasma endotoxin may increase despite decreasing bacteremia. There may be a similar killing rate by different antibiotics but a difference in endotoxin release. Intestinal endotoxin does not necessarily correlate to the level of gram-negative bacteria. However, the alteration of the gut content by pretreatment may be associated with reduced endotoxemia and increased survival. Antibiotic-induced endotoxin release may be different depending on the type of infection, the location of infection, the virulence of strains, Gram-stain, mode of application and dosage of antibiotic. Different antibiotics may induce the release of different forms of endotoxin which may be lethal for sensitized animals. The combination of antibiotics with inhibitors of endotoxin or the pro-inflammatory response may be responsible for increased survival by decrease of endotoxin release. The clinical significance of antibiotic-induced endotoxin release is documented only in a few clinical disorders, e.g., meningitis, urosepsis. The difference in endotoxin release by PBP 2-specific antibiotics, e.g., imipenem, and PBP 3-specific antibiotics, e.g., ceftazidime, may not be visible in each study. Patients with increased multi-organ failure (MOF) scores may profit from treatment with antibiotics known to decrease endotoxin. In conclusion, the clinical significance of antibiotic-induced endotoxin release remains to be clarified. Type of pathogen and its virulence may be more important than recently suggested. gram-positive pathogens were just recently recognized as an important factor for the development of the host response. In case of fever of unknown origin in intensive care patients either failure of treatment, e.g., failure of source control in intra-abdominal infection, or a side effect of antibiotic treatment, e.g., endotoxin release, should be considered as a cause of the fever.
Animals↗
Isolation of an endotoxin-MD-2 complex that produces Toll-like receptor 4-dependent cell activation at picomolar concentrations.
Host proinflammatory responses to minute amounts of endotoxins derived from many Gram-negative bacteria require the interaction of lipopolysaccharide-binding protein (LBP), CD14, Toll-like receptor 4 (TLR4) and MD-2. Optimal sensitivity to endotoxin requires an ordered series of endotoxin-protein and protein-protein interactions. At substoichiometric concentrations, LBP facilitates delivery of endotoxin aggregates to soluble CD14 (sCD14) to form monomeric endotoxin-sCD14 complexes. Subsequent interactions of endotoxin-sCD14 with TLR4 and/or MD-2 have not been specifically defined. This study reports the purification of a stable, monomeric, bioactive endotoxin-MD-2 complex generated by treatment of endotoxin-sCD14 with recombinant MD-2. Efficient generation of this complex occurred at picomolar concentrations of endotoxin and nanogram per milliliter doses of MD-2 and required presentation of endotoxin to MD-2 as a monomeric endotoxin-CD14 complex. TLR4-dependent delivery of endotoxin to human embryonic kidney (HEK) cells and cell activation at picomolar concentrations of endotoxin occurred with the purified endotoxin-MD-2 complex, but not with purified endotoxin aggregates with or without LBP and/or sCD14. The presence of excess MD-2 inhibited delivery of endotoxin-MD-2 to HEK/TLR4 cells and cell activation. These findings demonstrate that TLR4-dependent activation of host cells by picomolar concentrations of endotoxin occurs by sequential interaction and transfer of endotoxin to LBP, CD14, and MD-2 and simultaneous engagement of endotoxin and TLR4 by MD-2.
Animals↗
Effect of a body burn on endotoxin-induced lipid peroxidation: comparison with physiologic and histologic changes.
We determined the effect of a 15% total body surface (TBS), full-thickness burn on the physiologic, histologic, and oxidant-induced lipid peroxidation changes produced by endotoxin. The endotoxin-burn response was compared with that of endotoxin alone. Twenty-two adult sheep with chronic lung and flank lymph fistulas were studied. In 11 sheep a burn was produced under anesthesia and 3 days later they were given 2 micrograms/kg of endotoxin. Data were also compared with those of control sheep and those that were given burns alone. Circulating conjugated dienes increased with endotoxin alone but remained at baseline with endotoxin and burn injury. The lung lymph flow response was increased significantly in the endotoxin-burn group (sixfold) compared with that of endotoxin alone (fourfold). Histologic quantitation of lung neutrophil count was comparable in both groups 6 hours after injury, although mononuclear cells were much more evident in lungs in the endotoxin-burn group. Lipid peroxidation measured by malondialdehyde was significantly increased in the endotoxin group compared with the endotoxin-burn group, despite the greater increase in lymph flow and lung water in the burned group. Oxygen consumption (VO2) remained constant after endotoxin alone compared with baseline. However, VO2 increased twofold immediately after endotoxin in the endotoxin-burn group. This marked increase was followed by a significant decrease in VO2 from baseline. Flank soft-tissue nonburned increased lung lymph flow twofold to threefold with endotoxin and burn, indicating increased soft-tissue permeability, whereas it remained unchanged with endotoxin alone. Liver malondialdehyde increased from a control of 110 +/- 20 to 210 +/- 80 mmol/gm tissue with endotoxin alone and to 450 +/- 54 nmol/gm tissue with endotoxin and burn. We can conclude that burn injury accentuates both the pulmonary and systemic physiologic response to endotoxin, possibly as a result of mediators released from mononuclear cells already activated in the presence of the burn. The increased lung physiologic response does not appear to be caused by greater oxidant-induced lipid peroxidation, as was seen in the liver with the combined injury.
Animals↗
Evaluation of biologic activity of ferric chloride-treated endotoxin in mice.
Endotoxin (lipopolysaccharide, LPS) treated with ferric chloride was tested for its potential as a non-toxic agent for enhancement of non-specific host resistance. A 1 mg dose of untreated endotoxin, injected i.p. into mice, resulted in 100 per cent mortality, whereas the same amount of chemically-treated endotoxin resulted in less than 35 per cent lethality. The radio-protective potential of the treated endotoxin was similar to that of untreated endotoxin, as 70 per cent of each group of mice tested with either substance survived a dose of 850 rad x-ray. Irradiated mice, challenged 8 days after 850 rad x-irradiation, died when injected with 25 mug of either untreated or treated endotoxin. Antibiotic decontamination of the intestinal tract of host animals reduced the possibility of toxicity from endogenous endotoxin after challenge. This treatment resulted in 100 per cent survival from a 25 mug challenge at 8 days post-irradiation. The ferric chloride-treated proved to be a more effective B-lymphocyte mitogen. At a dose of 100 mug, treated endotoxin resulted in a 50 per cent greater mitogenic stimulation of B-lymphocytes as compared with that found after exposure to untreated endotoxin. Several lines of evidence support the contention that tolerance to untreated endotoxin was induced by repeated injections of either endotoxin preparation 1) 100 per cent of all endotoxin-tolerant mice survived a 1 mg challenge dose of untreated endotoxin, 2) there was a reduced mitotic response of splenic B-lymphocytes after re-exposure with untreated endotoxin as compared with that observed for cells derived from saline-treated mice, and 3) all antibiotic decontaminated mice engrafted with spleen cells from mice made tolerant to either endotoxin preparation survive graft-versus-host disease. In conclusion, based on survival data from normal mice, ferric chloride-treated endotoxin is safer to use than normal endotoxin. Also, treated endotoxin can elicit biologic responses similar in magnitude to those found after injection of mice with untreated endotoxin.
Animals↗
Respiratory effect of acute and subacute exposure to endotoxin-contaminated metal working fluid (MWF) aerosols on Sprague-Dawley rats.
Male Sprague-Dawley rats were exposed to a water-soluble metal working fluid (MWF) (5% v/v) contaminated with endotoxins (10,000 eu/ml or 100,000 eu/ml) at 10 mg/m3 for six hours per day for three days (acute exposure) or two weeks (subacute exposure). The geometric mean diameter of the MWF aerosols was 1.56 microm, and the airborne endotoxin concentrations ranged from 1,231 to 2,173 eu/m3 (10,000 eu/ml in the bulk MWF) for the low dose and 19,263-27,386 eu/m3 (100,000 eu/ml in the bulk MWF) for the high dose. Minimal effects were observed after exposure to 10 mg/m3 of the MWF without endotoxins for three days or two weeks. However, an increase in the number of polymorphonuclear cells (PMNs) and the level of protein was noted in the bronchoalveolar lavage (BAL) fluid from the rats acutely exposed to the MWF with endotoxins. The acute exposure produced a greater increase in the number of PMNs and total cell number in the BAL fluid than the subacute exposure. The number of white blood cells in the peripheral blood and the weight of the lungs both increased after the subacute exposure to the MWF aerosol with endotoxins, indicating increased vascular permeability in response to the endotoxin exposure. The levels of cyotokines such as IL-4, INF-gamma, and IL-1beta in the BAL fluid from the rats exposed to the MWF with or without endotoxins remained unchanged. Although the level of nitric oxide (NO(x)) in the BAL supernatant did not show any change, the induction of NO(x) from the alveolar macrophages increased in the rats acutely or subacutely exposed to the MWF contaminated with endotoxins. The ConA-induced proliferation response showed no change, yet the LPS-induced proliferation response was significantly increased in the splenocytes from the rats subacutely exposed to the MWF with and without endotoxins. The level of TNF-alpha in the spleen cell culture obtained from the rats exposed to the MWF with or without endotoxins increased without changing the levels of IL-1beta, IL-4, and INF-gamma. The level of endotoxin-specific IgE in the serum obtained from the rats exposed to the MWF with endotoxins increased dose-dependently, while the levels of total immunoglobulins (IgG(1), IgG(2a) and IgE) and endotoxin-specific IgG(1) and IgG(2a) remained unchanged. Accordingly, the current results indicate that lung inflammation can be immediately induced by acute or subacute exposure to an MWF contaminated with endotoxins, and macrophages would appear to play a role in the induction of inflammation along with B-cell functions rather than T-cell functions, after subacute exposure to an MWF with endotoxins. In addition, endotoxin-specific IgE is an early marker for endotoxin exposure in the workplace.
Aerosols↗
Enhancement by lipoprotein-free plasma of the inhibitory effect of endotoxin on endocytotic catabolism of low density lipoproteins in Hep G2 cells.
We previously showed that the presence of microgram levels of endotoxin inhibited low-density lipoprotein (LDL) uptake and degradation in Hep G2 cells. We also showed that both the polysaccharide and lipid A parts of endotoxins are needed for the inhibitory effects of endotoxins on cellular LDL uptake. The current study was carried out by inclusion of lipoprotein-free plasma (LFP) in tissue culture medium to observe the modulatory influence of non-lipoprotein factor(s) on endotoxin-induced inhibition of endocytotic catabolism of LDL in Hep G2 cells. We found that LFP dramatically promotes the inhibitory effect of endotoxins with a complete polysaccharide, but has no influence on the effect of the Re mutant endotoxin (from S. minnesota Re595), which lacks polysaccharide. By using gel-filtration chromatography, agarose electrophoresis and agarose isoelectric focusing, we further showed that in the presence of LFP, both the endotoxins with a complete polysaccharide and the Re mutant endotoxin complex with and anionize LDL, while in the absence of LFP, these endotoxins poorly interact with LDL. Thus, endotoxin inhibits cellular endocytotic catabolism of LDL by forming LDL-endotoxin complexes, and LFP enhances endotoxin-induced inhibition of endocytotic catabolism of LDL by promoting the interaction between endotoxin and LDL. In addition, our finding that the Re mutant endotoxin also interacts with LDL to form LDL-endotoxin complexes, but has no significant effect on LDL uptake and degradation, further supports the notion that both the polysaccharide and lipid A parts of endotoxins are needed for the inhibitory effects of endotoxins on cellular LDL uptake.
Carcinoma, Hepatocellular↗
Origin of endotoxemia influences the metabolic response to endotoxin in dogs.
Different routes of endotoxin administration have been used to mimic inflammatory and metabolic responses observed during sepsis. Because the origin of endotoxemia may affect the reactions to endotoxin, we compared the induction of tumor necrosis factor (TNF), interleukin-6 (IL-6), hormones, and glucose production after endotoxin (1.0 microg/kg Escherichia coli 0111:B4) administration into a peripheral (n = 8) versus the portal (n = 8) vein in anesthetized dogs. Prior to endotoxin, a laparotomy was performed for cannulation of hepatic vessels. To evaluate the effects of surgery and anesthesia, we also studied the effects of peripheral endotoxin administration in six awake dogs. The rate of appearance of glucose was measured by primed continuous infusion of [6,6-2H2]glucose. In anesthetized dogs, arterial concentrations of TNF and IL-6 increased after endotoxin administration (P < 0.01 vs basal; NS between groups). Net hepatic TNF production was increased after endotoxin administration (peripheral vs portal endotoxin administration: 533 +/- 177 vs 2135 +/- 1127 ng/min, both P < 0.05 vs basal; NS between groups). Net hepatic IL-6 production was stimulated only after portal endotoxin delivery (from 86 +/- 129 to 4740 +/- 1899 ng/min, P < 0.05; NS between groups). Although there were no differences in neuroendocrine activation, portal endotoxin administration resulted in decreased glucose production compared with peripheral administration (13.6 +/- 0.9 vs 16.8 +/- 1.2 micromol/kg.min, P < 0. 05). In contrast to anesthetized dogs, endotoxin increased glucose production considerably in awake dogs from 13.8 +/- 1.2 to 24.2 +/- 3.2 micromol/kg.min (P < 0.05; P < 0.05 vs anesthetized dogs). The contribution of anesthesia and surgery increased the endotoxin-induced IL-6 response by approximately 350% compared with the effect of endotoxin in awake dogs (P < 0.01). In conclusion, there are no major differences in the responses to endotoxin between peripherally treated and portally treated dogs, except for differences in glucose production. Portal delivery compared with systemic delivery of endotoxin alters hepatic metabolism through nonendocrine mechanisms, reflected in decreased glucose production. The inflammatory, endocrine, and metabolic effects of endotoxin are altered by the combination of surgery and anesthesia.
Anesthesia↗
Progressive magnesium deficiency increases mortality from endotoxin challenge: protective effects of acute magnesium replacement therapy.
OBJECTIVES: To study the effects of endotoxin on magnesium homeostasis; to determine if progressive magnesium deficiency alters outcome from endotoxin challenge; and to evaluate the efficacy of magnesium therapy in reducing endotoxin-induced mortality. DESIGN: Prospective, placebo-controlled, randomized, multiexperiment studies. SETTING: Research laboratory of a university hospital. SUBJECTS: Male Sprague Dawley rats (n = 299). INTERVENTIONS: Experiment 1 was designed to test if endotoxin alters magnesium homeostasis. Circulating total and ionized magnesium (estimated by ultrafilterable values) concentrations were determined in blood samples collected from animals after the randomized administration of placebo or 0.3, 3.0, or 30 mg/kg of endotoxin. A baseline blood sample was collected and then a second blood sample was obtained at 5, 15, 30, 60, 120, or 180 mins after endotoxin or placebo administration. In experiment 2, animals were randomized to receive magnesium-sufficient diets or magnesium-deficient diets for 6 wks. After 6 wks, the effects of the randomized administration of 3.0 mg/kg endotoxin or placebo were evaluated on mortality and analyte values (pH and blood gases, sodium, potassium, chloride, glucose, ionized calcium, hematocrit, total and ultrafilterable magnesium concentrations) in the three study groups (magnesium-sufficient, 3-wk magnesium-deficient, or 6-wk magnesium-deficient). In experiment 3, magnesium-deficient animals were randomized to receive 50 mmol/kg magnesium chloride or placebo, before or after the administration of 3.0 mg/kg of endotoxin. Baseline and 24-hr analyte determinations were performed and outcome was analyzed. MEASUREMENTS AND MAIN RESULTS: Experiment 1: Significant increases (p < .05) in circulating total magnesium concentrations were found in animals that received 30 mg/kg of endotoxin, at 120 mins (0.79 +/- 0.10 vs. 0.60 +/- 0.05 mmol/L), and 180 mins (0.74 +/- 0.04 vs. 0.56 +/- 0.04 mmol/L) compared with baseline values. Similarly, significant increases (p < .05) in ionized magnesium concentrations were observed 120 and 180 mins after 3.0 and 30 mg/kg of endotoxin compared with baseline values. Experiment 2: Magnesium deficiency was strongly (p < .02) associated with increased mortality from endotoxin challenge. Endotoxin administration (3.0 mg/kg) was lethal in 10 (43%) of 23 magnesium-sufficient animals, 15 (65%) of 23 3-wk magnesium-deficient animals, and 20 (83%) of 24 6-wk magnesium-deficient animals. Experiment 3: In magnesium-deficient animals, rats treated with magnesium replacement therapy had significantly increased survival from endotoxin administration (15 [52%] of 29 vs. five [17%] of 29, p < .01) compared with placebo-treated animals. CONCLUSIONS: a) Endotoxin challenge causes significant increases in circulating total and ionized magnesium concentrations. b) Progressive magnesium deficiency is strongly associated with increased lethality, and magnesium replacement therapy provides significant protection from endotoxin challenge. c) These experimental results support the concept that cellular injury is probably associated with increases in circulating magnesium concentrations. Furthermore, these experimental findings suggest that magnesium deficiency predisposes to worse outcome from endotoxin challenge, and that replacement therapy in the setting of magnesium deficiency may be warranted, especially in critically ill subjects.
Animals↗
Endotoxin promotes adhesion of human erythrocytes to human vascular endothelial cells under conditions of flow.
OBJECTIVE: To investigate the effects of endotoxin on adhesion of human red blood cells to human vascular endothelial cells under conditions of flow. DESIGN: Prospective, randomized, controlled in vitro study. SETTINGS: University-affiliated cell biology laboratory. SUBJECTS: Human erythrocytes and human vascular endothelial cells. INTERVENTIONS: Fresh human erythrocytes and human vascular endothelial cells grown as monolayers were incubated with either saline or endotoxin. After incubation, endothelial monolayers were superfused with erythrocytes, and the number of erythrocytes adhering to the endothelial monolayer was quantified. MEASUREMENTS AND MAIN RESULTS: Adhesion of erythrocytes to vascular endothelium was measured under conditions of continuous flow in different settings: a) exposure of both endothelial cells and erythrocytes to saline; b) incubation of both erythrocytes and endothelial cells with endotoxin; c) exposure of erythrocytes only to endotoxin; d) incubation of endothelial cells only to endotoxin; and e) both the endothelial cells and erythrocytes incubated with different concentrations of endotoxin. Erythrocyte adhesion in the saline control group was 71 +/- 8 cells/mm2. Incubation of both components with endotoxin increased the number of adhesive erythrocytes to 172 +/- 9 cells/mm2 (p < .05). When only the endothelial cells were treated with endotoxin, 142 +/- 8 cells/mm2 adhered to the endothelial monolayer, whereas the incubation of the erythrocytes only to endotoxin resulted in adhesion of 102 +/- 3 cells/mm2. Decreasing concentrations of endotoxin reduced adhesion from 172 +/- 9 cells/mm2 (endotoxin, 75 microg/mL) to 165 +/- 9 cells/mm2 (endotoxin, 25 microg/mL), 153 +/- 4 cells/mm2 (endotoxin, 1 microg/mL), and 146 +/- 6.1 cells/mm2 (endotoxin, 5 ng/mL). CONCLUSIONS: Exposure of human erythrocytes and human venous vascular endothelial cells to an inflammatory stimulus such as endotoxin promotes a dose-dependent adhesion of erythrocytes to endothelium in a dynamic environment. These adhesive erythrocyte-endothelium interactions can be produced by exposure of either red blood cells or endothelial cells to endotoxin, with a higher degree of adhesion after activation of the endothelial cell component.
Cell Adhesion↗
Effect of time post mortem on the concentration of endotoxin in rat organs: implications for sudden infant death syndrome (SIDS).
The aim of the study was to test the following hypotheses: (i) that endotoxin injected 40 min prior to death can be detected in rat organs post mortem and (ii) that endotoxin levels do not change with increasing time post mortem. Rats were injected with or without endotoxin in buffered saline, 40 min prior to being killed. Endotoxin levels in rat organs were assessed using a Limulus amoebocyte assay. The effect of storage time post mortem was assessed by following various storage regimes at 25 degrees C and 8 degrees C. Significant differences (P = < 0.001) in endotoxin levels of all samples tested were found between rats injected with and without endotoxin. A significant increase in detectable endotoxin was observed between 0 h and 6 h post mortem in rats injected with or without endotoxin. No difference in detectable endotoxin levels in the kidney, liver and spleen was observed from 30 h to 102 h post mortem in rats injected with or without endotoxin. In rats injected with endotoxin, detectable endotoxin levels in the heart were raised between 0 h and 6 h, 6 h and 54 h, and 30 h and 78 h. Endotoxin injected into rats 40 min prior to death can be detected post mortem. For rats injected with saline or endotoxin prior to death levels in the kidney, liver and spleen were not affected by storage at 8 degrees C for 30-102 h, after initial storage at room temperature for 6 h. Levels of endotoxin detected in the hearts of rats injected with saline were not affected by storage up to 102 h. In rats injected with endotoxin prior to death, detectable levels in the heart were significantly affected by increasing time in storage.
Animals↗
Chylomicrons enhance endotoxin excretion in bile.
Chylomicrons prevent endotoxin toxicity and increase endotoxin uptake by hepatocytes. As a consequence, less endotoxin is available to activate macrophages, thereby reducing tumor necrosis factor secretion. To determine whether the chylomicron-mediated increase in hepatocellular uptake of endotoxin results in increased endotoxin excretion into bile, we examined bile after endotoxin administration. A sublethal dose (7 micrograms/kg) of 125I-endotoxin was incubated with either rat mesenteric lymph containing nascent chylomicrons (500 mg of chylomicron triglyceride per kg of body weight) or an equal volume of normal saline (controls) for 3 h and then infused into male Sprague-Dawley rats. Bile samples were collected via a common bile duct catheter for 24 h. Infusion of endotoxin incubated with chylomicrons increased biliary excretion of endotoxin by 67% at 3 h (P < or = 0.006) and by 20% at 24 h (P < or = 0.01) compared with infusion of endotoxin incubated in saline. Endotoxin activity, as measured by the Limulus assay, was not detected in the bile of test animals. However, endotoxin activity was detected after hot phenol-water extraction of bile, demonstrating that endotoxin is inactive in the presence of bile but retains bioactivity after hepatic processing. Since the majority of an intravenous endotoxin load has been shown to be cleared by the liver, acceleration of hepatocyte clearance and biliary excretion of endotoxin may represent a component of the mechanism by which chylomicrons protect against endotoxin-induced lethality.
Animals↗
Beneficial effect of a platelet-activating factor antagonist, WEB 2086, on endotoxin-induced lung injury.
We tested the hypothesis that platelet-activating factor plays an important role in promoting endotoxin-induced lung injury by studying the effect of WEB 2086, a specific platelet-activating factor receptor antagonist, on lung vascular leak in endotoxin-treated rats. Intraperitoneal injection of Salmonella enteritidis endotoxin (2 mg/kg) increased the extravascular leakage of 125I-labeled albumin in perfused lungs at 30 min, 2 h, 6 h, and 48 h. Treatment with WEB 2086 (10 mg/kg ip) either 20 min before or 30 min after endotoxin injection significantly reduced lung injury at 2 h after endotoxin (leak index: control 0.74 +/- 0.03, endotoxin 1.79 +/- 0.14, endotoxin + pretreated WEB 1.23 +/- 0.09, endotoxin + posttreated WEB 1.21 +/- 0.13). In addition, posttreatment with WEB 2086 starting at 90 min after endotoxin injection markedly reduced lung leak at 6 h (control 0.74 +/- 0.03, endotoxin 1.29 +/- 0.14, endotoxin + WEB 0.71 +/- 0.06). The protective effect of WEB 2086 was not the result of cyclooxygenase blockade because the release of thromboxane B2 by endotoxin-treated lungs was not affected by WEB 2086. Furthermore, neither pretreatment nor posttreatment with WEB 2086 significantly reduced the endotoxin-induced increase in plasma glutathione disulfide, a marker of in vivo oxidative stress. In rats given a lethal dose of endotoxin (20 mg/kg ip), posttreatment with WEB 2086, starting at 2 h after endotoxin, significantly improved survival compared with vehicle treatment. We conclude that WEB 2086 ameliorated endotoxin-induced lung injury without reducing oxidative stress in the rat and suggest that blockade of platelet-activating factor receptor may be an important therapeutic consideration in sepsis-induced acute lung vascular injury.
Animals↗
Effects of different types and combinations of antimicrobial agents on endotoxin release from gram-negative bacteria: an in-vitro and in-vivo study.
Total and free endotoxin release in time from cultures of Escherichia coli by different antibiotics was studied in vitro for 4 h in relation to the antibiotic effect on viable counts and morphological features of the test cultures. The most rapid fall in viable counts was seen after treatment with imipenem or the combination of imipenem with tobramycin, accompanied by an early, but minimal increase (1.8-fold) of the total (free plus cell-bound) endotoxin level at 1 h. Total endotoxin levels increased approximately 5-fold upon incubation with ceftazidime, tobramycin or the combination of tobramycin with cefuroxime, whereas incubation with cefuroxime or aztreonam alone caused a late 22-and 49-fold increase in total endotoxin, respectively, at 4 h. In chloramphenicol treated cultures there was still an increase in viable counts during therapy, resulting in an ultimately 78-fold increase of mean levels of total endotoxin. Free endotoxin levels increased approximately 6-fold within 1 h upon treatment with imipenem, alone or in combination with tobramycin, or ceftazidime as the result of rapid lysis of bacteria. Treatment with cefuroxime or aztreonam induced a relatively late but much higher release of free endotoxin (118-and 222-fold, respectively), which was due to the formation of long filamentous structures during the first 2 h of incubation and eventually cell lysis. Both tobramycin and the combination of tobramycin with cefuroxime caused a more gradual rise in free endotoxin, with a +/- 15-fold increase in free endotoxin at 4 h. In chloramphenicol treated cultures, as in the control cultures, the level of free endotoxin remained proportional to the amount of viable organisms. We also studied plasma endotoxin levels in 20 patients with septic shock. 10 out of these 20 patients had a detectable endotoxemia (greater than 5 ng/l) on admission. We describe the patterns of plasma endotoxin in these patients during the first 24 h of antibiotic treatment. We conclude that, in the in-vitro study, values of total endotoxin, free endotoxin, and the rate of release of endotoxin varies with the antibiotic used. We also demonstrate that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics.
Aged↗
Endotoxin suppresses mouse hepatic low-density lipoprotein-receptor expression via a pathway independent of the toll-like receptor 4.
Endotoxin provokes an inflammatory state in the infected host. C3H/HeJ mice are tolerant to endotoxin because of an Lps gene mutation. Recent studies have identified that this gene encodes the Toll-like receptor 4. Endotoxin also induces hyperlipidemia and suppresses hepatic low-density lipoprotein (LDL)-receptor expression. In the current study, we investigated whether a defective Lps gene would impair the hepatic LDL-receptor response to endotoxin in C3H/HeJ mice. Eighteen hours after an intraperitoneal injection of endotoxin, the hepatic LDL-receptor expression and the plasma lipoprotein pattern were analyzed. Endotoxin increased plasma triglyceride and apoE in very low-density lipoproteins (VLDL) and intermediate-density lipoproteins, and decreased apoAI in high-density lipoproteins (HDL) in the endotoxin-sensitive mice (C3H/HeN), but not in the endotoxin-resistant mice (C3H/HeJ). These data indicate that a defective Lps gene impairs the endotoxin signaling to alter these lipoproteins. However, the hepatic LDL-receptor response to endotoxin in the endotoxin-resistant mice was similar to that in the endotoxin-sensitive mice. Thus, at a dose of 5 microg/mouse, endotoxin reduced hepatic LDL-receptor expression by 35% in C3H/HeN mice and by 52% in C3H/HeJ mice. At a dose of 50 microg/mouse, endotoxin reduced hepatic LDL-receptor expression by 61% in C3H/HeN mice and by 63% in C3H/HeJ mice. It is concluded that endotoxin suppresses hepatic LDL-receptor expression in vivo via a pathway independent of the Toll-like receptor 4.
Animals↗
Decreased endotoxin-binding capacity of whole blood in patients with alcoholic liver disease.
BACKGROUND/AIMS: The proinflammatory effects of endotoxemia, which is often observed in alcohol-abusing patients with various degrees of liver disease, may be modulated by changes in the concentration of endotoxin binding factors. Therefore, the plasma endotoxin concentration and the overall endotoxin binding capacity of whole blood were measured in these patients. METHODS: Patients with minor (A1; n=27), more pronounced (A2; n=13), cirrhotic alcoholic liver disease (A3; n=18), and non-alcoholic cirrhosis (NC; n=6), and 15 healthy control persons (HC) were included in the study. Endotoxin plasma levels were determined using a standardized limulus assay. A modified assay was applied to additionally detect tightly bound endotoxin. To measure the endotoxin-binding capacity, aliquots of whole blood were incubated with serial dilutions of endotoxin, supernatants were obtained, and endotoxin retrieval was estimated by addition of limulus lysate, followed by photometric measurement of the maximal reaction velocity (dODmax). Endotoxin binding capacity equals the endotoxin concentration at which dODmax reaches a predefined threshold. RESULTS: All groups of alcohol abusers had significantly elevated endotoxin plasma levels with a considerable portion of 'bound' endotoxin. Conversely, the endotoxin binding capacity was markedly diminished, mainly in patients with more advanced liver disease (A1: 85.8% of the control value [non-significant vs. controls]; A2: 25.4% [p<0.05]; A3: 43.6% [p<0.02], NC: 43.2%). CONCLUSIONS: The endotoxin-binding capacity is diminished in patients with alcoholic and non-alcoholic cirrhosis, as well as in less advanced alcoholic liver disease. Reduced endotoxin binding may contribute to the adverse effects of endotoxemia.
Adult↗
Free radicals alter maximal diaphragmatic mitochondrial oxygen consumption in endotoxin-induced sepsis.
Recent studies indicate that sepsis is associated with enhanced generation of several free radical species (nitric oxide, superoxide, hydrogen peroxide) in skeletal muscle. While studies suggest that free radical generation causes uncoupling of oxidative phosphorylation in sepsis, no previous report has examined the role of free radicals in modulating skeletal muscle oxygen consumption during State 3 respiration or inhibiting the electron transport chain in sepsis. The purpose of the present study was to examine the effects of endotoxin-induced sepsis on State 3 diaphragm mitochondrial oxygen utilization and to determine if inhibitors/scavengers of various free radical species would protect against these effects. We also examined mitochondrial protein electrophoretic patterns to determine if observed endotoxin-related physiological derangements were accompanied by overt alterations in protein composition. Studies were performed on: (a) control animals, (b) endotoxin-treated animals, (c) animals given endotoxin plus PEG-SOD, a superoxide scavenger, (d) animals given endotoxin plus L-NAME, a nitric oxide synthase inhibitor, (e) animals given only PEG-SOD or L-NAME, (f) animals given endotoxin plus D-NAME, and (g) animals given endotoxin plus denatured PEG-SOD. We found: (a) no alteration in maximal State 3 mitochondrial oxygen consumption rate at 24 h after endotoxin administration, but (b) a significant reduction in oxygen consumption rate at 48 h after endotoxin, (c) no effect of endotoxin to induce uncoupling of oxidative phosphorylation, (d) either PEG-SOD or L-NAME (but neither denatured PEG-SOD nor D-NAME) prevented endotoxin-mediated reductions in State 3 respiration rates, (e) some mitochondrial proteins underwent tyrosine nitrosylation at 24 h after endotoxin administration, and (f) SDS-page electrophoresis of mitochondria from endotoxin-treated animals revealed a selective depletion of several proteins at 48 h after endotoxin administration (but not at 24 h); (g) administration of L-NAME or PEG-SOD prevented this protein depletion. These data provide the first evidence that endotoxin-induced reductions in State 3 mitochondrial oxygen consumption are free radical-mediated.
Animals↗
In vivo quantification of endotoxin-induced nitric oxide production in pigs from Na15NO3-infusion.
1. In this investigation the NO production rate is quantified in the pig during normotensive endotoxin-induced shock with increased cardiac output and during subsequent treatment with the NO synthase inhibitor N omega-monomethy-L-arginine (L-NMMA). NO production rate was derived from the plasma isotope-enrichment of 15N-labelled nitrate (15NO3-). 2. Three groups of animals (control, n = 5; endotoxin, n = 6; endotoxin + L-NMMA, n = 6) were anaesthetized and instrumented for the measurement of systemic and pulmonary haemodynamics. Each animal received a primed-continuous infusion of stable, non-radioactively labelled Na15 NO3 (bolus 30 mg, infusion rate 2.1 mg h-1). Arterial blood samples were taken 5, 10, 15, 30, 60 and 90 min later and every 90 minutes until the end of the experiment. 3. Continuous i.v. infusion of endotoxin was incrementally adjusted until mean pulmonary artery pressure (PAP) reached 50 mmHg and subsequently titrated to keep mean PAP approximately 35 mmHg. Hydroxyethylstarch was administered as required to maintain mean arterial pressure (MAP) > 60 mmHg. Six hours after the start of the endotoxin continuous i.v. L-NMMA (1 mg kg-1 h-1) was administered to the endotoxin + L-NMMA group. Haemodynamic data were measured before as well as 9 h after the start of the endotoxin. 4. After conversion of NO3- to nitro-trimethoxybenzene and gas chromatography-mass spectrometry analysis the total NO3- pool, basal NO3- production rate and the increase per unit time in NO3- production rate were calculated from the time-course of the 15NO3- plasma isotope-enrichment. A two compartment model was assumed for the NO3- kinetics, one being an active pool in which newly generated NO3- appears and from which it is eliminated, the other being an inactive volume of distribution in which only passive exchange takes place with the active compartment. 5. Although MAP did not change during endotoxin infusion alone, cardiac output (CO) increased by 42 +/- 40% (P < 0.05 versus baseline) by the end of the experiment due to a significant (P < 0.05 versus baseline) fall in systemic vascular resistance (SVR) to 65 +/- 25% of the baseline value. L-NMMA given with endotoxin did not change MAP, and both CO and SVR were maintained close to the pre-shock levels. 6. Baseline plasma NO3- concentrations were 43 +/- 13 and 40 +/- 10 mumol l-1 in the control and endotoxin animals, respectively, and did not differ at the end of the experiment (39 +/- 8 and 44 +/- 15 mumol l-1, respectively). The mean NO3- pool and basal NO3- production rate were 1155 +/- 294 mumol and 140 +/- 32 mumol h-1, respectively, without any intergroup difference. Endotoxin significantly increased NO3- production rate (23 +/- 10 mumol h-2, P < 0.05 versus control (6 +/- 7 mumol h-2) and endotoxin + L-NMMA groups). L-NMMA given with endotoxin (-1 +/- 2 mumol h-2, P < 0.05 versus control and endotoxin groups) had no effect. 7. Analysis of the time course of the 15NO3- plasma isotope enrichment during primed-continuous infusion of Na15NO3 allowed us to quantify the endotoxin-induced increase in NO3- production rate independently of total NO3- plasma concentrations. Low-dose L-NMMA blunted the increase in NO3- production rate while maintaining basal NO3- formation.
Animals↗