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PubMed1992

Endotoxin tolerance is associated with altered GTP-binding protein function.

Previous studies have suggested that guanine nucleotide regulatory (G) proteins modulate endotoxin-stimulated peritoneal macrophage arachidonic acid (AA) metabolism. Endotoxin-stimulated metabolism of AA by peritoneal macrophages is decreased in endotoxin tolerance (Rogers et al. Prostaglandins 31: 639-650, 1986). These observations led to a study of G protein function and AA metabolism by peritoneal macrophages in endotoxin tolerance. Endotoxin tolerance was induced by the administration of sublethal doses of endotoxin. AA metabolism was assessed by measurement of thromboxane B2 (TxB2), a cyclooxygenase metabolite. NaF (5 mM), an activator of G proteins, significantly stimulated TxB2 synthesis in control macrophages from 7.7 +/- 0.2 to 19.1 +/- 0.6 (SE) ng/ml (P less than 0.05) at 2 h and was partially inhibited by pertussis toxin, suggesting a G protein-dependent mechanism. Salmonella enteritidis endotoxin (50 micrograms/ml) stimulated a similar increase in TxB2 levels (23 +/- 0.4 ng/ml, P less than 0.05). In contrast to control macrophages, macrophages from endotoxin-tolerant rats stimulated with either NaF or S. enteritidis endotoxin had TxB2 levels that were only 30 and 2% of the respective stimulated control cells. Basal guanosine-triphosphatase (GTPase) activity (33 +/- 6 pmol.mg-1.min-1) in endotoxin-tolerant macrophage membranes was significantly lower (P less than 0.05) than control basal activity (158 +/- 5 pmol.mg-1.min-1). This suppression of macrophage GTPase activity was apparent 48 h after the first in vivo sublethal endotoxin injection (100 micrograms/kg ip). The reduced GTPase activity paralleled in vitro cellular hyporesponsiveness to endotoxin-stimulated TxB2 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Endotoxin inhibits the surge secretion of gonadotropin-releasing hormone via a prostaglandin-independent pathway.

Immune/inflammatory challenges, such as bacterial endotoxin, disrupt gonadotropin secretion and ovarian cyclicity. We previously determined that endotoxin can block the estradiol-induced LH surge in the ewe. Here, we investigated mechanisms underlying this suppression. First, we tested the hypothesis that endotoxin blocks the estradiol-induced LH surge centrally, by preventing the GnRH surge. Artificial follicular phases were created in ovariectomized ewes, and either endotoxin or vehicle was administered together with a surge-inducing estradiol stimulus. In each ewe in which endotoxin blocked the LH surge, the GnRH surge was also blocked. Given this evidence that endotoxin blocks the estradiol-induced LH surge at the hypothalamic level, we began to assess underlying central mechanisms. Specifically, in view of the prior demonstration that prostaglandins mediate endotoxin-induced suppression of pulsatile GnRH secretion in ewes, we tested the hypothesis that prostaglandins also mediate endotoxin-induced blockade of the surge. The prostaglandin synthesis inhibitor flurbiprofen was delivered together with endotoxin and the estradiol stimulus. Although flurbiprofen abolished endotoxin-induced fever, which is a centrally generated, prostaglandin-mediated response, it failed to reverse blockade of the LH surge. Collectively, these results indicate endotoxin blocks the LH surge centrally, suppressing GnRH secretion via a mechanism not requiring prostaglandins. This contrasts with the suppressive effect of endotoxin on GnRH pulses, which requires prostaglandins as intermediates.

Animals↗
PubMed1990

Discrimination between endotoxin and (1----3)-beta-D-glucan using turbidimetric kinetic assay with Limulus amebocyte lysate.

A procedure for the Limulus amebocyte lysate (LAL) test discriminating between endotoxin and (1----3)-beta-D-glucan based on the turbidimetric kinetic method was proposed. Endotoxin and (1----3)-beta-D-glucan, which are elicitors of the activation of LAL, showed different reaction courses with this lysate. To analyze the difference in the reactions, two parameters, the maximum differential coefficient of the reaction (Dmax) and the reaction time required to obtain Dmax (Tp) were defined. The logarithmic plottings of Tp versus Dmax (Tp-Dmax plot) discriminated between endotoxin and (1----3)-beta-D-glucan. Endotoxin was measured with a standard curve plotting logarithmic endotoxin concentration versus Dmax (ET-Dmax plot). The endotoxin calculated from Dmax was less influenced by (1----3)-beta-D-glucan than that calculated from the usual gelation time. A small amount of endotoxin in a sample could be concealed by the addition of polymyxin B, which inhibited the activation of LAL by endotoxin. (1----3)-beta-D-glucan was measured without being affected by the presence of a small amount of endotoxin using LAL with polymyxin B. The following procedure is proposed as a LAL test to discriminate between endotoxin and (1----3)-beta-D-glucan. (1) Identify the main substance (endotoxin or (1----3)-beta-D-glucan) triggering the activation of LAL using the Tp-Dmax plot. (2) Use the appropriate method to measure the main substance: the ET-Dmax plot for endotoxin or the LAL with polymyxin B for (1----3)-beta-D-glucan.

Endotoxins↗
PubMed2005

Within-home versus between-home variability of house dust endotoxin in a birth cohort.

Endotoxin exposure has been proposed as an environmental determinant of allergen responses in children. To better understand the implications of using a single measurement of house dust endotoxin to characterize exposure in the first year of life, we evaluated room-specific within-home and between-home variability in dust endotoxin obtained from 470 households in Boston, Massachusetts. Homes were sampled up to two times over 5-11 months. We analyzed 1,287 dust samples from the kitchen, family room, and baby's bedroom for endotoxin. We fit a mixed-effects model to estimate mean levels and the variation of endotoxin between homes, between rooms, and between sampling times. Endotoxin ranged from 2 to 1,945 units per milligram of dust. Levels were highest during summer and lowest in the winter. Mean endotoxin levels varied significantly from room to room. Cross-sectionally, endotoxin was moderately correlated between family room and bedroom floor (r = 0.30), between family room and kitchen (r = 0.32), and between kitchen and bedroom (r = 0.42). Adjusting for season, the correlation of endotoxin levels within homes over time was 0.65 for both the bedroom and kitchen and 0.54 for the family room. The temporal within-home variance of endotoxin was lowest for bedroom floor samples and highest for kitchen samples. Between-home variance was lowest in the family room and highest for kitchen samples. Adjusting for season, within-home variation was less than between-home variation for all three rooms. These results suggest that room-to-room and home-to-home differences in endotoxin influence the total variability more than factors affecting endotoxin levels within a room over time.

Air Pollutants↗
PubMed1993

Turbidimetric-kinetic assay of endotoxin in rumen fluid or serum of cattle fed rations containing various levels of rolled barley.

A new, automated turbidimetric-kinetic (ATK) assay was used to quantitate bacterial endotoxin in rumen fluid or in serum of Holstein steers. The ATK method used Limulus amebocyte lysate (LAL) reagent with added beta-glucan (LAL-ES) which improved specific sensitivity to endotoxin. Design of the feeding trial permitted comparison of endotoxin levels found during consumption of a basal ration with those higher levels detected at various times following the introduction of increasing percentages of rolled barley to that basal ration. Both serum and ruminal endotoxin levels were significantly higher in steers on the higher concentrate rations. Peak endotoxin levels were detected 20 days following the change to the highest concentrate ration which contained 60% barley. Endotoxin levels from both sources subsequently decreased. Ruminal endotoxin stabilized at about 10 times the level, and serum endotoxin stabilized at 2 to 4 times the level, of that previously found during feeding of the basal ration. Test protocol included sample dilution and heating in order to avoid the effects of endotoxin inhibitors. Recovery rates for added endotoxin to either serum or rumen fluid supernates ranged from 120 to 136%. Coefficient of variation for endotoxin concentration in serum was lower than 10%, and in rumen fluid only slightly higher. There was significant correlation between ruminal concentration as measured by the ATK method and an alternative chromogenic substrate assay procedure. Changes in endotoxin level in experimental steers were those predictable from experience with naturally occurring incidents of grain engorgement. The ATK assay appears to be an accurate and rather simple technique which will prove useful for experimental and clinical studies in the future.

Animal Feed↗
PubMedDate not supplied

Endotoxin levels in steam and reservoirs of table-top steam sterilizers.

PURPOSE: To document endotoxin levels in "Statim" cassette sterilizer reservoirs and in steam delivered to the cassette in the unwrapped instrument cycle. To document endotoxin levels in sterilizer reservoir water using different management protocols. METHODS: Endotoxin levels were determined using the Limulus Amebocyte Lysate test. Endotoxin preparations were from Escherichia coli and Ralstonia pickettii. All samples were collected in depyrogenated glassware and stored at -20 degrees C until assayed. RESULTS: The majority of water samples contained < 1.0 Endotoxin Unit (EU)/ml. The highest level found in sterilizers in clinical use was 5.3 EU/ml. Endotoxin was not detected in steam condensate within the limits of the assay. When the endotoxin level in the reservoir water was experimentally enhanced to 200 EU/ml, cassette steam condensate endotoxin levels were from 0.5% to 5% of the reservoir level. Daily and weekly emptying of the cassette reservoir consistently yielded low endotoxin levels as did monthly emptying, but with the latter there was a trend toward higher levels that favors weekly emptying as a precautionary measure. CONCLUSIONS: Endotoxin levels in the reservoirs of 23 sterilizers involving 240 samplings were never high enough to yield detectable endotoxin levels in steam in the sterilizer cassette. Regular weekly emptying of sterilizer reservoirs would eliminate the risk of endotoxin transfer during steam sterilization.

Ambulatory Care↗
PubMed2005

Molecular basis of reduced potency of underacylated endotoxins.

Potent TLR4-dependent cell activation by gram-negative bacterial endotoxins depends on sequential endotoxin-protein and protein-protein interactions with LPS-binding protein, CD14, myeloid differentiation protein 2 (MD-2), and TLR4. Previous studies have suggested that reduced agonist potency of underacylated endotoxins (i.e., tetra- or penta- vs hexa-acylated) is determined by post-CD14 interactions. To better define the molecular basis of the differences in agonist potency of endotoxins differing in fatty acid acylation, we compared endotoxins (lipooligosaccharides (LOS)) from hexa-acylated wild-type (wt), penta-acylated mutant msbB meningococcal strains as well as tetra-acylated LOS generated by treatment of wt LOS with the deacylating enzyme, acyloxyacylhydrolase. To facilitate assay of endotoxin:protein and endotoxin:cell interactions, the endotoxins were purified after metabolic labeling with [3H]- or [14C]acetate. All LOS species tested formed monomeric complexes with MD-2 in an LPS-binding protein- and CD14-dependent manner with similar efficiency. However, msbB LOS:MD-2 and acyloxyacylhydrolase-treated LOS:MD-2 were at least 10-fold less potent in inducing TLR4-dependent cell activation than wt LOS:MD-2 and partially antagonized the action of wt LOS:MD-2. These findings suggest that underacylated endotoxins produce decreased TLR4-dependent cell activation by altering the interaction of the endotoxin:MD-2 complex with TLR4 in a way that reduces receptor activation. Differences in potency among these endotoxin species is determined not by different aggregate properties, but by different properties of monomeric endotoxin:MD-2 complexes.

Acylation↗
PubMed1999

Comparative personal exposures to organic dusts and endotoxin.

The aims of the study were to provide valid comparative data for personal exposures to dust and endotoxins for different occupations and to calculate comparative data for the contamination of organic dusts with endotoxin. Nine different occupational settings were studied, drawn from the textile, agricultural and animal handling industries. Samples were collected by personal sampling techniques, using the Institute of Occupational Medicine (IOM) sampling head, glass fibre filters and rechargeable sampling pumps. The dust exposures were calculated by gravimetric analysis and using the calculated volume of air sampled were expressed as mg/m3. Endotoxin exposures were measured using a simple water extraction from the collected dusts, followed by a quantitative turbidimetric assay. Results were expressed as ng/m3, using the calculated volume of air sampled. In addition, the levels of the contamination of dusts with endotoxin for individual industries were expressed as ng/mg of collected dust. Two hundred and fifty-nine samples, collected from 9 different industries and across 36 different sites were analysed. This represented a sampling rate of 25% for the total work force. The average sampling time was 4.62 h. For all the dusts collected, a significant correlation between the collected dust and endotoxin was seen (r = 0.7 and p < 0.001). The highest dust exposures occurred during cleaning activities (grain handling: 72.5 mg/m3). The individuals exposed to the highest median level of dust and endotoxin were the animal handlers (poultry handlers, dust: 11.53 mg/m3, endotoxin: 71,995 ng/m3). Weaving and mushroom cultivation had the lowest exposures for dust and endotoxins. The mostly highly contaminated dusts (median values expressed as ng of endotoxin per mg of collected dust) were found in the animal handling (poultry: 1,030 ng/mg, swine: 152 ng/mg) and cotton spinning (522 ng/mg) industries. Processing of cotton and wool fibres was found to reduce the levels of contamination of dusts with endotoxin. In the study, valid comparative data for personal exposures to organic dusts and endotoxins have been presented. The highest exposures were found amongst animal handlers and during cleaning activities. The results highlight that dust exposures are greater in a number of industries than the set exposure standards. In addition, endotoxin exposures are found to be greater than levels at which harmful effects have been demonstrated.

Agriculture↗
PubMedDate not supplied

Endotoxin-independent white cell cytokine production in haemodynamically stable patients with idiopathic dilated cardiomyopathy.

INTRODUCTION: In heart failure, increased circulating white cell tumour necrosis factor-alpha production could be attributed to elevated plasma endotoxin concentrations or an increase in white cell sensitivity to endotoxin. AIMS: To ascertain whether, in patients with IDC, circulating white cell TNF-alpha production is also mediated through endotoxin-independent mechanisms. METHODS: Whole blood production of TNF-alpha, both with and without the presence of an endotoxin stimulus, was evaluated in 89 controls and in 60 patients with IDC in New York Heart Association functional class I, II or III heart failure and without evidence of oedema, reduced peripheral perfusion or elevated plasma endotoxin concentrations. Circulating concentrations of selected pro and anti-inflammatory factors were also measured. RESULTS: In patients compared to controls, IgG (p < 0.01) (IgG1 and IgG3), but not IgM concentrations were elevated, and plasma TNF-alpha and TGF-beta concentrations were raised (p < 0.001, p < 0.02 respectively). In addition, endotoxin-free cultured whole blood TNF-alpha production (p < 0.0005) was increased. Against a role for endotoxin-mediated pre-activation of white cells in patients, the sensitivity of white cells to endotoxin, as determined from the excitatory endotoxin concentration producing 50% maximal TNF-alpha production was unchanged. Moreover, in favour of non-endotoxin-mediated white cell activation, the calcineurin inhibitor, cyclosporin-A, which did not alter endotoxin-induced TNF-alpha production, decreased TNF-alpha produced by unstimulated cultured cells in patients to values not significantly greater than those in controls. CONCLUSIONS: We concluded that circulating white cell cytokine over-production can occur through both endotoxin- dependent and -independent mechanisms in IDC.

Cardiomyopathy, Dilated↗
PubMed1976

Effects of endotoxin on carbohydrate metabolism in inbred mice.

Effects of endotoxin on carbohydrate metabolism were studied in A/HeJ (endotoxin-sensitive) and C3H/HeJ (endotoxin-resistant) inbred mice. A/HeJ mice developed hypoglycemia within two hours after endotoxin injection, yet liver glycogen content did not differ from controls. Similarly treated C3H/HeJ mice did not develop significant hypoglycemia. Administration of glucagon to endotoxin-treated A/HeJ mice failed to elevate their blood glucose concentrations, while endotoxin-treated mice of the same strain did respond to dibutyryl cyclic AMP with a significant elevation of blood glucose. C3H/HeJ mice on the other hand responded to glucagon and dibutyryl cyclic AMP with elevated blood glucose. Endotoxin-treated C3H/HeJ but not A/HeJ mice were able to carry out gluconeogenesis induced by prednisolone, while both inbred strains showed active glycogenesis after administration of an exogenous glucose load. Administration of glucagon resulted in diminished liver glycogen concentrations in A/HeJ endotoxin-treated mice suggesting no impairment of glycogenolysis. The inability of endotoxin-treated A/HeJ mice to respond to glucagon could be due to impairment of gluconeogenesis. Although endotoxin interfered with the capacity of both inbred strains to respond to glucagon administration with elevation of liver cyclic AMP, the effect was significantly more severe in A/HeJ mice. The susceptibility of A/HeJ mice to the lethal effect of endotoxin may be related to the apparent sensitivity of carbohydrate metabolic pathways to disturbance by endotoxin.

Animals↗
PubMed1986

Endotoxin and lung injury.

Two things are certain: endotoxin has dramatic effects on the structure and function of the lungs in intact animals and also on isolated lung cells, and both the in vivo and in vitro effects of endotoxin are complex. In whole animals, endotoxin causes obvious and subtle effects on functions of both airways and the pulmonary circulation. These effects include diffuse lung inflammation and injury of pulmonary vascular endothelium. Endotoxin can also directly injure endothelial cells in vitro. In vivo, lung injury caused by endotoxin is at least partly dependent on the presence of granulocytes, and some evidence also suggests that both lymphocytes and macrophages may participate in the response either directly or by directing cell traffic. At least in the sheep preparation, platelets do not seem to play a major role in the lungs' response to endotoxemia. Although endotoxin can activate complement and activated complement infused into whole animals affects the lungs, it seems unlikely that complement activation alone is sufficient to explain the severe and prolonged lung injury caused by endotoxin. Cyclooxygenase metabolites of arachidonic acid appear to mediate both changes in lung mechanics and pulmonary vasoconstriction after endotoxemia. Lipoxygenase products may play a role in these responses as well as the inflammatory response and increases in vascular permeability, although evidence for these latter speculations is not firm. Lung cell injury caused by endotoxin probably is mediated at least in part by generation of free radicals. Inflammatory cells, especially neutrophils, are one source of these toxic oxygen species, but intracellular generation of free radicals within lung cells per se may also be stimulated by endotoxin and account for some of the lung injury. Likewise, inflammatory-cell-derived proteinases may mediate endotoxin-induced injury of lung cells and, as with chronic lung diseases, balance between proteinases and antiproteinases could be important. The fact that free radicals can inactivate antiproteinases, and antiproteinases can act as free radical scavengers, may suggest a complex relationship among the several possible mediators of toxicity. Cyclic nucleotide metabolism is affected in whole animals and isolated lung cells by endotoxin and these classic second messengers could be involved in the pathogenetic sequence, but exactly how is unclear. Chronic effects of endotoxin on the lungs may provide a pathogenetic link between acute lung injury and chronic changes in lung structure and function.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

The kinetics of the production of granulocyte-monocyte colony stimulating activity (GM-CSA) by isolated human monocytes: response to bacterial endotoxin.

When mononuclear phagocytes are stimulated by bacterial endotoxins, they produce Granulocyte-Monocyte Colony-Stimulating Activity (GM-CSA). In order to study the kinetics of the production of GM-CSA by human monocytes, we prepared suspensions of these cells and studied their response to Salmonella typhi endotoxin. We found that when human monocytes were exposed to this preparation of endotoxin, they synthesized GM-CSA de novo and subsequently secreted it into the extracellular environment; however, within hours, the cells became highly refractory to further stimulation by endotoxin. The resistance to endotoxin which these cells rapidly acquired in vitro could not be accounted for by cell attrition, the accumulation of toxic metabolites in the cultures, negative feedback inhibition by newly synthesized GM-CSA, depolarization of the plasma membrane of the cells, or by degradation of endotoxin. When we studied the binding of tritium-labeled endotoxin to viable monocytes, we found that after monocytes were initially exposed to endotoxin, their subsequent ability to bind lipopolysaccharide molecules onto the surface of the plasma membrane was reduced. When we subjected concentrated supernates from endotoxin-stimulated monocyte cultures to gel filtration and isoelectric focusing, we noted that GM-CSA derived from human monocytes had an apparent molecular weight of approximately 42,000 daltons. Our results indicate that resistance to bacterial endotoxins acquired by mononuclear phagocytes may play a role in the immunologic phenomenon of immediate endotoxin tolerance observed in vivo and may in part be due to down-regulation of endotoxin binding to the outer surface of the cell.

Cells, Cultured↗
PubMed1993

[Endotoxin-inactivating activity in normal and D-galactosamine induced liver failure rats].

Hepatic failure model rats were prepared by administration of D-Galactosamine (Gal) to clarify the endotoxemia under the hepatic failure. Endotoxin was injected from the femoral vein and the endotoxin level in blood was measured periodically in normal and Gal induced liver failure rats. Further endotoxin was added to plasma, incubated and periodically the endotoxin level in plasma was measured. After the injection of endotoxin, liver biopsy was made and by immunohistochemical staining with Factor C, the localization of endotoxin in the liver was studied. The endotoxin level in blood before the injection was within the normal range in both groups. In the in vivo test, the endotoxin level of the Gal group was significantly higher and its disappearance rate was also significantly lower than the control after the injection of endotoxin. In the in vitro test, the endotoxin level of the Gal group was significantly higher. After one hour, endotoxin was stained on Kupffer cells and hepatocytes in the control group only. In the Gal group, no intrinsic endotoxemia was noted, but it was presumed that endotoxemia is easily caused due to the lowered inactivation of endotoxin in plasma and in the liver.

Animals↗
PubMed1997

Impaired activation of nuclear factor-kappaB in endotoxin-tolerant rats is associated with down-regulation of chemokine gene expression and inhibition of neutrophilic lung inflammation.

We postulated that repeated injections of endotoxin could induce a state of endotoxin tolerance in rats that is mediated at least partially through a nuclear factor (NF)-kappaB-dependent mechanism. We treated rats with four doses of endotoxin (0.06 or 0.6 mg/kg/day) and evaluated the ability of these treatments to modulate activation of the transcription factor NF-kappaB induced by treatment with high dose endotoxin (6 mg/kg). In lung tissue, NF-kappaB activation in response to i.p. injection of high dose endotoxin was blocked by treatment with four daily doses of endotoxin at 0.6 mg/kg/day. Endotoxin tolerance in rats was associated with diminished endotoxin-induced mRNA expression of the NF-kappaB-dependent rat chemokine, cytokine-induced neutrophil chemoattractant. Treatment of rats with four daily doses of 0.6 mg/kg/day of endotoxin almost completely abolished the neutrophilic alveolitis that occurs in rats following i.p. injection of high dose endotoxin. These data indicate that in vivo tolerance to endotoxin challenge is associated with and possibly mediated by inhibition of NF-kappaB activation. Potentially, endotoxin tolerance could be exploited to limit inflammation in disease states whose pathobiology is related to excessive or unregulated inflammation.

Animals↗
PubMed1995

Activation of complement by human hemoglobin and by mixtures of hemoglobin and bacterial endotoxin.

Purified human hemoglobin is being developed as an alternative to transfusions of homologous erythrocytes. However, toxicity associated with infusion of hemoglobin has limited the development of this resuscitation fluid. Some observed toxicities, including activation of the complement cascade, have been associated with contamination of hemoglobin solutions by bacterial endotoxin. Recent studies have demonstrated complex formation between hemoglobin and endotoxin, and have documented a resultant increase in the ability of endotoxin to activate coagulation, stimulate tissue factor production by human peripheral blood mononuclear cells, and stimulate tissue factor activity and protein synthesis in cultured human endothelial cells. The process of hemoglobin enhancement of endotoxin toxicity suggests a possible mechanism by which the consequences of endotoxin contamination of hemoglobin solutions, including complement activation, could be magnified. Therefore, we studied the potential of hemoglobin to either fix complement directly, or modify the ability of endotoxin to fix complement. Human crosslinked and native hemoglobins, at concentrations between 0.2 mg/ml and 3 mg/ml, were shown to fix complement. Complement fixation by hemoglobin was identical in normal human serum or in factor B-depleted serum, suggesting that fixation occurred via the classical pathway of complement activation. Complement fixation then was examined with a battery of smooth and rough endotoxins tested in the absence and presence of hemoglobin. Addition of hemoglobin to a solution of a rough Salmonella endotoxin partial structure, from which a single fatty acid had been hydrolyzed from the lipid A portion of the macromolecule, resulted in decreased efficiency of complement fixation. However, addition of hemoglobin had little or no effect on the intrinsic complement fixing abilities of eight other smooth endotoxins, rough endotoxins, or endotoxin partial structures. Our results demonstrated the ability of hemoglobin to fix complement at hemoglobin concentrations which would be achieved during infusion for resuscitation, but failed to demonstrate a reproducible effect of hemoglobin on the activation of complement by endotoxin.

Bacterial Toxins↗
PubMed1985

Skeletal muscle perfusion and metabolism during canine endotoxin shock.

Conflicting data exist in literature about the effects of endotoxin on skeletal muscle perfusion and metabolism during canine endotoxin shock. In 12 dogs we therefore studied (six control and six endotoxin treated, 1.5 mg X kg-1) under etomidate (4 mg X kg-1 X h-1) anaesthesia muscle blood flow (radioactive microspheres) in fore limb, thorax, diaphragm and hind limb (five different muscles) and skin blood flow before (t = 0) and 90 and 120 min after endotoxin. We also measured blood flow in the femoral artery and vein (electromagnetic flow transducers) and the arteriovenous differences of oxygen, lactate, glucose and FFA over the femoral vascular bed (at t = 0, 30, 90 and 120 min). Endotoxin administration caused a fall of flow in the femoral artery and vein (by 65 and 63%, respectively at t = 15). After t = 60 flow in the femoral artery and vein increased slowly but the flows were still below the preshock values at t = 20 (by 33 and 50%, respectively). Skeletal muscle and skin flow did not decrease or even increased after endotoxin but decreased in the control group. Percentage of cardiac output distributed to brachial, intercostal and hind limb muscle and skin increased after endotoxin (by 163, 167, 111 and 120%, respectively at t = 20). The five muscles of the hind limb did not respond differently to endotoxin. In spite of diminished arterial inflow, skeletal muscle perfusion was thus maintained in the hind limb, probably due to closing of shunts and redistribution of blood away from bone. Oxygen extraction but also lactate release by the femoral bed had increased during endotoxin shock. After endotoxin femoral glucose extraction was only elevated at t = 30 when arterial glucose concentration had also increased. The femoral bed produced free fatty acids (FFA) but during endotoxin shock the arteriovenous concentration difference of FFA decreased. Our data suggest that skeletal muscle flow nor oxygen consumption and glucose metabolism is affected during 2 h of canine endotoxin shock. Lactate production, however, tended to increase.

Animals↗
PubMed1997

Myocardial and vascular adrenergic alterations in a rat model of endotoxin shock: reversal by an anti-tumor necrosis factor-alpha monoclonal antibody.

OBJECTIVES: a) To investigate responsiveness to exogenous catecholamines in rat endotoxin shock by studying both myocardial and vascular functional parameters, and to determine the relationship of these parameters with other relevant biological parameters of the adrenergic pathway, such as myocardial beta-adrenergic receptors and cyclic adenosine monophosphate (cAMP); b) to investigate the role of tumor necrosis factor (TNF)-alpha via prophylactic anti-TNF-alpha monoclonal antibody administration. DESIGN: Experimental, comparative hospital. SETTING: Laboratory in a university hospital. SUBJECTS: Male Sprague-Dawley rats, weighing 280 to 340 g. INTERVENTIONS: Intravenous injection of Escherichia coli endotoxin (5 mg/100 g) in the first group; injection of the same dose of endotoxin preceded by 2 mg/100 g of anti-TNF-alpha monoclonal antibody in the second group; injection of saline in the third (control) group. MEASUREMENTS AND MAIN RESULTS: TNF-alpha concentration was measured before and during the first 3 hrs in all three groups. Myocardial and vascular functional parameters were obtained, respectively, from Langendorff perfused hearts and isolated aortic rings. Adrenergic biochemical parameters (catecholamines, density and affinity of beta-receptors, and isoproterenol-stimulated myocardial cAMP) were determined 3 hrs after injections in the three groups. After endotoxin injection, serum TNF-alpha concentrations peaked at 60 mins (2496 +/- 412 pg/mL) and returned slowly to control values at 3 hrs; serum TNF-alpha concentrations remained under the limit of detection in the other two groups. When compared with the control group, plasma concentrations of epinephrine and norepinephrine were significantly (p < .05) increased. Baseline values for differential left ventricular pressure and coronary flow were significantly (p < .001, p < .01, respectively) reduced in the endotoxin group; heart rate remained unchanged. In the endotoxin and control groups, isoproterenol induced a similar increase in differential left ventricular pressure and in heart rate. Anti-TNF-alpha antibody increased cardiac response by partially preventing the decrease by endotoxin in differential left intraventricular pressure. Maximal specific binding of 125iodocyanopindolol and myocardial cAMP accumulation were significantly (p < .01) reduced in the endotoxin group in comparison with the control group. Anti-TNF-alpha antibody prevented the endotoxin-induced decrease in cAMP synthesis (p < .05) but did not modify the density of receptors. Affinity of receptors was similar in the three groups. In aortic rings, endotoxin administration significantly (p < .01) shifted the dose-response curve to norepinephrine to the right, both in the presence and absence of endothelium. NG-monomethyl-L-arginine significantly increased the contractions to attain the control level: p < .001 in the presence of endothelium; p < .05 in the absence of endothelium. Anti-TNF-alpha antibody did not prevent endotoxin-induced vascular hyporeactivity to norepinephrine in either endothelium-intact or -denuded rings, but partially attenuated the decrease in maximal response. CONCLUSIONS: In ex vivo experiments, 3 hrs after endotoxin injection, vascular responsiveness was sharply decreased. This impaired response was improved in vitro by the inhibition of nitric oxide. The heart response to isoproterenol, nevertheless, was maintained, even though there was an obvious decrease in receptor density and an impaired myocardial accumulation of cAMP. Anti-TNF-alpha antibody partially prevented the alteration of both myocardial pressure response to isoproterenol and biochemical parameters, and was not efficacious in preventing vascular hyporeactivity to vasoconstrictor agents.

Animals↗
PubMed1992

Reduction of endotoxin-induced vascular permeability by monoclonal antibodies against lipopolysaccharide determinants.

Endotoxin, a bacterial lipopolysaccharide implicated in the pathogenesis of septic shock, markedly alters vascular permeability following intravenous injection in rabbits. We investigated the ability of murine monoclonal antibodies to confer protection against endotoxin-induced increases in a rabbit model of ocular vascular permeability. Four monoclonal antibodies of differing specificities as well as polymyxin B were compared for their effects on endotoxin from either Escherichia coli or Pseudomonas aeruginosa. Preincubation of endotoxin with antibodies directed against Pseudomonas O side chain or core glycolipid resulted in marked attenuation of vascular permeability due to Pseudomonas endotoxin, but not E. coli endotoxin. Antilipid A antibodies were not significantly effective in neutralizing either endotoxin with in vitro preincubation. Low avidity of the antilipid A antibody, low density of lipid A binding sites, or inaccessibility of the lipid A may have prevented more marked interactions. When administered intravenously prior to endotoxin challenge, none of the antibodies demonstrated the ability to provide specific protection to subsequent endotoxin in this model. They did provide partial nonspecific protection against endotoxins regardless of epitope specificity. When administered prophylactically, polymyxin B, an antibiotic that binds to lipid A, was highly effective in neutralizing the toxic effects of endotoxin. Since antibodies to lipid A reduce mortality in septic shock, the failure to demonstrate efficacy in this study may be due to the marked sensitivity of the rabbit eye to endotoxin. Alternatively, beneficial effects from antiendotoxin antibodies in septic shock may be unrelated to the inhibition of vascular permeability. Some protection from antiendotoxin antibodies may be due to enhancement of nonspecific mechanisms.

Animals↗