Endotoxin Inhibition Research Articles

Endotoxin inhibitor prevents sepsis-induced alterations in intestinal ion transport

The intestine is a target of septic insult. The aims of this study were to characterize sepsis-induced alterations in intestinal ion transport and to determine the role endotoxin plays in mediating these changes. Rats underwent cecal manipulation alone (control), cecal ligation and puncture (CLP), or CLP plus intraperitoneal injection of 0.2 mg of a recently synthesized endotoxin inhibitor. At 24 hours, distal ileum was harvested, and transport parameters were determined. Cecal ligation and puncture produced a significant increase in short-circuit current (Isc) that was attributable to the induction of chloride secretion. There were no alterations in transepithelial resistance or fluxes of mannitol and sodium. The sepsis-induced increase in Isc was prevented by administration of the endotoxin inhibitor. In this model of sepsis, the primary alteration in ileal ion transport is an induction of electrogenic chloride secretion. Endotoxin inhibition may represent a strategy for prophylaxis against the intestinal effects of sepsis.

Endotoxin inhibitor prevents sepsis-induced alterations in intestinal ion transport

BACKGROUND: The intestine is a target of septic insult. The aims of this study were to characterize sepsis-induced alterations in intestinal ion transport and to determine the role endotoxin plays in mediating these changes. METHODS: Rats underwent cecal manipulation alone (control), cecal ligation and puncture (CLP), or CLP plus intraperitoneal injection of 0.2 mg of a recently synthesized endotoxin inhibitor. At 24 hours, distal ileum was harvested, and transport parameters were determined. RESULTS: Cecal ligation and puncture produced a significant increase in short-circuit current (I,,) that was attributable to the induction of chloride secretion. There were no alterations in transepithelial resistance or fluxes of mannitol and sodium. The sepsis-induced increase in /,,was prevented by administration of the endotoxin inhibitor. CONCLUSIONS: In this model of sepsis, the primary alteration in ileal ion transport is an induction of electrogenic chloride secretion. Endotoxin inhibition may represent a strategy for prophylaxis against the intestinal effects of sepsis. Am J Surg. 1 g96;172:341-344.

Endotoxin inhibition of distension-stimulated gastric acid secretion in rat: mediation by NO in the central nervous system.

1. The involvement of nitric oxide in the acute inhibitory effects of low doses of endotoxin, following intracerebroventricular (i.c.v.) or intravenous (i.v.) administration, on gastric acid secretion stimulated by distension or i.v. infusion of pentagastrin has been investigated in the continuously perfused stomach of the anaesthetized rat. 2. The i.c.v. administration of E. coli endotoxin (800 ng kg-1) abolished the acid secretory response induced by gastric distension (20 cm water intragastric pressure) within 30 min of administration. 3. By contrast, submaximal rates of acid secretion induced by i.v. infusion of pentagastrin (8 micrograms kg-1 h-1) were not inhibited by i.c.v. administration of endotoxin (800 ng kg-1). 4. Prior i.c.v. administration of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 800 micrograms kg-1) restored the acid secretory responses to distension in rats treated with endotoxin (i.c.v.). 5. Likewise, i.v. administration of endotoxin (5 micrograms kg-1) abolished the acid secretory response induced by gastric distension within 30 min of administration. Prior i.c.v. injection of L-NAME (800 micrograms kg-1) or its i.v. administration (10 mg kg-1) restored acid secretory responses in rats receiving i.v. endotoxin. 6. The reversal by L-NAME (i.v.) of the acid inhibitory effects of endotoxin (i.v.) was prevented by L-arginine (12 mg kg-1, i.c.v. or 100 mg kg-1, i.v.), but not by its enantiomer D-arginine. 7. The present results imply the existence of an acute response to endotoxin involving NO synthesis in the brain. NO may act as a neuromodulator or neurotransmitter in a nervous reflex leading to the inhibition of acid secretion stimulated by gastric distension.

Cell Attachment Activity of Cementum Proteins and Mechanism of Endotoxin Inhibition

Cementum occupies a unique anatomical location where soft connective tissues of the periodontium are attached to root surfaces. Cell attachment properties of proteins present in cementum were studied. Human and bovine cementum were extracted with 0.5 mol/L CH3COOH followed by 4 mol/L guanidine, and proteins were separated by ion-exchange chromatography and SDS-polyacrylamide gel electrophoresis. Cells were labeled with radioactive amino acids and added to tissue-culture plastic plates incubated with cementum proteins, and attachment was measured. Results showed that cementum proteins promoted the attachment of smooth muscle cells, endothelial cells, and fibroblasts, but not epithelial cells. Fibroblasts attached more efficiently than other cell types, and they manifested spreading with re-organization of actin filaments. No attachment occurred to plates incubated with endotoxin from A. actinomycetemcomitans. Fewer fibroblasts attached to plates treated with cementum proteins in the presence of endotoxin, but cells pre-treated with endotoxin attached normally. Attachment was not inhibited when plates were incubated first with attachment proteins and then with endotoxin; however, it was decreased when endotoxin or bovine serum albumin preceded cementum proteins. Cementum proteins with Mr 68,000, 61,000, 55,000, and 36,000 (p68, p61, p55, and p36, respectively) manifested attachment activity, while protein(s) with Mr 23,000-24,000 did not. Western blots revealed that guanidine extracts contained three bands cross-reacting with anti-bovine sialoprotein-II antibody, but the p61, p55, and p36 were negative. We conclude that cementum contains bovine sialoprotein-II and at least four other fibroblast attachment proteins, and that they do not support epithelial cell attachment.(ABSTRACT TRUNCATED AT 250 WORDS)

Endotoxin-induced arachidonic acid metabolism requires de novo protein synthesis and protein kinase C activation

The mechanisms whereby bacterial endotoxins stimulate arachidonic acid metabolism in macrophages are uncertain. Both protein kinase C activation and de novo protein synthesis occur in macrophages in response to endotoxin. In this study we evaluated the time course and role of protein kinase C and de novo protein synthesis in endotoxin stimulated arachidonic acid metabolism in resident rat peritoneal macrophages. Thromboxane (TX) B2 was measured as the representative arachidonic acid metabolite synthesized in response to Salmonella enteritidis endotoxin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate (PMA). The effect of inhibition of protein kinase C by 1-(5-isoquinolinsulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine on endotoxin- and A23187-induced TXB2 synthesis was examined. The potential roles of transcriptional and translational events in endotoxin- and A23187-stimulated TXB2 synthesis were determined by utilizing the transcriptional inhibitors camptothecin (10 microM) or actinomycin D (0.08 microM), and the translational inhibitor cycloheximide (0.1 microM). Whereas, A23187 stimulated maximal TXB2 synthesis within 15 min, endotoxin showed a more prolonged time course with a 12-fold increase in TXB2 synthesis above basal levels after 3 h (P less than 0.05). PMA induced an approx. 8-fold increase above basal TXB2 levels that was blocked by inhibition of transcription with actinomycin D. H-7 (10 microM to 50 microM) inhibited endotoxin- and A23187-stimulated eicosanoid synthesis. Staurosporine (0.2 microM) produced a selective 66% inhibition of endotoxin, but not A23187-stimulated TXB2 synthesis. Endotoxin-induced TXB2 production was significantly (P less than 0.05) inhibited by staurosporine, camptothecin, actinomycin D or cycloheximide at intervals from 30 min prior to, through 60 min after endotoxin stimulation. These studies suggest a role for protein kinase C activation and de novo protein synthesis in endotoxin signal transduction events leading to increased macrophage arachidonic acid metabolism. These intracellular events are essential in sustaining the prolonged inflammatory response to endotoxin.

Mechanism of Endotoxin Inhibition of Human Gingival Fibroblast Attachment to Type I Collagen

Bacterial endotoxin inhibits the attachment of human gingival fibroblasts to collagen. The present study attempted to elucidate the possible mechanism of this inhibition. Two mechanisms were considered: direct toxicity to the cells and steric interference. Collagen substrates were prepared by rat type I collagen being air-dried in the wells of 24 multi-well plates. Experimental collagen substrates were treated with 50 micrograms of endotoxin/well, while untreated collagen substrates served as controls. Two mL of cell suspension (10(4) cells/mL) was added to each well, and these were incubated at 37 degrees C for two h. The average cell number/mm2 attached to experimental and control substrates was determined. Cell attachment to endotoxin-treated collagen was inhibited by 78%, compared with that to untreated collagen. The washing of the endotoxin-treated collagen for two h did not affect the inhibition of cell attachment, whereas after 24 h of washing, cell attachment was inhibited by 54%, compared with that to untreated collagen. Pre-incubation of the cells in endotoxin for two h did not affect their attachment to collagen. The addition of fetal calf serum (15%) to the experimental system completely reversed the inhibition of fibroblast attachment to endotoxin-treated collagen. These findings suggest that endotoxin interferes with fibroblast attachment to collagen through a steric phenomenon, possibly by blocking the binding sites on the collagen molecule recognized by the membrane receptor for collagen.

Endotoxin inhibition of drinking behaviour in the rat

Intravenous (i.v. 320 and 640 μg/kg) and intracerebroventricular (i.c.v.; 1 μg/rat) injection of Escherichia coli lipopolysaccharide (LPS) powerfully inhibited drinking induced by 24 h water deprivation. Pretreatment with acetylsalicylic acid (ASA) into the preoptic area (POA) completely abolished the effect induced by i.v. LPS, but did not modify that elicited by i.c.v. LPS. Intraperitoneal ASA injections significantly reduced the antidipsogenic effect of i.c.v. LPS. Electrolytic ablation of the subfornical organ (SFO) did not modify the effect induced by either i.v. or i.c.v. LPS. Present findings indicate that: (1) the antidipsogenic effect of i.v. LPS is mediated by prostaglandin synthesis into the POA, (2) the SFO is not involved in this effect, and (3) prostaglandins in other brain areas, besides POA, modulate the effect of i.c.v. LPS. It is suggested that at least two different brain sites, inside the blood-brain barrier, might be involved in the antidipsogenic effect of LPS.

Endotoxin inhibition of glucocorticoid enzyme induction and in vivo3H-dexamethasone labelling of rat liver nuclei

1. 1. The effect of endotoxin on glucocorticoid (GC) induction of liver TO and TAT was investigated. 2. 2. It was found that endotoxin inhibited not only TO GC induction, but also that of TAT, though to a lesser extent (17.41%). 3. 3. Endotoxin did not influence the binding capacity of liver cytosol for 3H-dexamethasone at the second hour after the toxin administration. 4. 4. In in vivo experiments endotoxin inhibited with 57.2% the binding of 3H-dexamethasone to hepatic nuclei. 5. 5. It is suggested that the lower extent of endotoxin inhibition of GC induction of TAT may be due to the counteracting action of some inductor(s) for TAT only.

Barium and Calcium Block Bacillus Thuringiensis Subspecies Kurstaki δ-Endotoxin Inhibition of Potassium Current Across Isolated Midgut of Larval Manduca Sexta

Ba2+ and Ca2+ prevent and reverse the Btk delta-endotoxin inhibition of the short-circuit current in isolated lepidopteran midgut. These findings support the K+ pump-leak steady-state model for midgut K+ homeostasis and the K+ channel mechanism of Bt toxin action. They provide a new tool with which to study the interactions between Bt toxin and midgut cell membranes.

Tumor necrosis factor inhibits a polymorphonuclear leukocyte-dependent airway edema in guinea pigs.

Intravenously administered endotoxin inhibits the polymorphonuclear leukocyte (PMN)-dependent airway edema produced in guinea pigs exposed to toluene diisocyanate (TDI). Tumor necrosis factor (TNF) is produced in vivo by peripheral blood monocytes and tissue macrophages stimulated with endotoxin and has been shown to activate PMN's and vascular endothelial cells. To determine whether the inhibition of airway edema is mediated by TNF, guinea pigs were treated with intravenous saline or 75,000 U/kg recombinant human TNF 1.5 h before exposure to air or 3 ppm TDI for 1 h. Animals were then injected intravenously with 50 mg/kg Evans blue dye as a marker of protein extravasation. Saline-treated animals exposed to TDI had a significant increase in tracheal Evans blue dye extravasation (85 +/- 6.5 micrograms dye/g trachea, mean +/- SE) compared with saline-treated animals exposed to air (31.3 +/- 2.5, P less than 0.001). The tracheal extravasation of Evans blue dye was significantly inhibited (P less than 0.05) in TDI-exposed animals treated with TNF (64.7 +/- 7.5). Neither heat-inactivated TNF (104.9 +/- 9.5) nor TNF neutralized with a monoclonal antibody against TNF (99.7 +/- 17.9) inhibited TDI-induced airway edema. In addition, treatment with 15,000 U/kg (99.9 +/- 21.3) or 150,000 U/kg (103.2 +/- 17.6) interleukin 1, a monokine also produced in response to endotoxin, did not prevent airway edema. These results suggest that TNF released in response to endotoxin mediates endotoxin's inhibition of a PMN-dependent airway edema.

Inhibition and reversal of endotoxin-, aggregated IgG- and paf-induced hypotension in the rat by SRI 63-072, a paf receptor antagonist

Platelet activating factor (paf) given intravenously produces systemic hypotension in the rat. Similar effects can be induced using endotoxin or heat-aggregated IgG challenges, which are thought to involve endogenous paf release. Extending this concept, we have examined the ability of the paf antagonist SRI 63-072 to inhibit or reverse systemic hypotension induced with paf, heat-aggregated IgG or endotoxin 0111-B4 in rats. At 100 ng kg−1 paf, there occurred a 38.6 ± 5.1% decrease in carotid mean arterial pressure (MAP) followed by a 3.2 ± 0.7 min recovery period (RP) to return to normal pressure values. The ED50 of SRI 63-072 was 0.16 mg kg−1 i.v. (MAP) and 0.25 mg kg−1 (RP) when given 1–5 min before the paf challenge. Endotoxin (15 mg kg−1 i.v.) produced a hypotensive response (54 ± 8% decreased in MAP) and a corresponding 80% decrease in mesenteric artery blood flow. When given 2–8 min after endotoxin, 1.0 mg kg−1 i.v. SRI 63-072 totally restored blood pressure and artery blood flow. SRI 63-072 similarly reversed heat-aggregated IgG (10 mg kg−1) induced reduction of MAP, with an ED50 of 0.05 mg kg−1 i.v. The observations that SRI 63-072 can inhibit or reverse systemic vascular effects produced from paf and other provocators of endogenous paf release strongly implicates paf as a common final mediator of hypotension and shock. As SRI 63-072 is a competitive receptor antagonist, the hypotensive effects of these provocators appear to be mediated by vascular receptors for paf.

Zymosan activation of plasma reduces hypoxic pulmonary vasoconstriction

Small doses of endotoxin (15 μg/kg IV) inhibit the pulmonary vascular pressor response to alveolar hypoxia in the anesthetized dog. One of the actions of endotoxin is to initiate the alternate pathway of complement activation. Incubation of human plasma with zymosan (ZAP) will activate this pathway. We wished to see if ZAP would mimic the effect of endotoxin. Prior to ZAP, hypoxia (F i O 2 12%) in 5 anesthetized dogs increased pulmonary vascular resistance (PVR: mm Hg/L/min) from 3.7±0.8 to 7.1±1.5. After 50 ml ZAP IV the PVR change with hypoxia was only from 3.6±0.6 to 3.9±0.8.Plasma heated to destroy complement prior to ZAP incubation in one experiment did not reduce the pressor response. In a further 5 dogs pretreated with meclofenamate (2 mg/kg IV) the PVR increased from 3.7±0.4 to 7.5±0.4 with hypoxia prior to IV ZAP and from 4.4±0.5 to 6.5±0.6 after ZAP. The effect of ZAP indicates that endotoxin may work through the activation of complement. The protection of the hypoxic pressor response by meclofenamate suggests that the ZAP inhibition (like endotoxin inhibition) may involve dilator prostaglandin-like substances.

Polymyxin B suppresses the endotoxin inhibition of concanavalin a-mediated erythrocyte agglutination.

The lectin agglutinability of human erythrocytes has been utilized to examine interactions of gram-negative endotoxin with mammalian cell plasma membranes. Erythrocytes treated in buffer with Escherichia coli 0127:B8 lipopolysaccharide (LPS) or Salmonella minnesota Re595 glycolipid for 1 h became resistant to agglutination by the lectin concanavalin A (ConA) in buffer free of LPS or glycolipid. Polymyxin B, a cationic cyclic lipopeptide which specifically binds to the lipid A toxophore, was tested for possible effects on the LPS and glycolipid inhibition of Con A erythrocyte agglutination. The presence of polymyxin B during the initial 1-h treatment with LPS or glycolipid blocked the ability of the endotoxins to render erythrocytes refractory to agglutination by ConA. Inhibition by polymyxin B was stoichiometric, and in repeated experiments, LPS was completely suppressed in the hemagglutination assay at a polymyxin B:LPS weight ratio of 1:4.1 (increasing polymyxin concentration, constant LPS concentration) and 1:5.1 (constant polymyxin concentration, increasing LPS concentration). These stoichiometry values are similar to values obtained for inhibition by polymyxin B of LPS lymphoid cell activation. It was concluded, therefore, that endotoxin inhibition of ConA erythrocyte agglutination reflects interactions of erythrocyte membranes with the lipid A region of endotoxin. In addition, the stoichiometry of polymyxin B inhibition suggests a similar extent of lipid A-dependent LPS interaction with erythrocytes and lymphoid cells.

Endotoxin inhibition of macrophage-mediated bone resorption.

The mechanisms by which bacterial endotoxins (ETX) elicit bone loss in septic osteolytic lesions and in organ cultures of bone rudiments have never been clearly established. The possible mechanisms for ETX action include: (a) the stimulation of osteoclast proliferation; (b) the stimulation of synthesis of secondary agents known to elicit bone resorption, e.g., prostaglandins; (c) the stimulation of the resorptive activity of osteoclasts. In the absence of extant methods for isolating and culturing osteoclasts, we have explored the last possibility by evaluating the action of ETX in a bone resorption system consisting of a putative osteoclast precursor, the macrophage, cocultured with isotopically labeled devitalized bone. We have observed the following: We suggest the ETX elicits bone loss in vivo by stimulating osteoclast proliferation or prostaglandin synthesis, and not by directly evoking enhanced bone resorption by osteoclasts or other osteolytic cells.

Endotoxin inhibition of dexamethasone induction of tryptophan oxygenase in suspension culture of isolated rat parenchymal cells: Involvement of the hepatic nonparenchymal cell fraction

The effect of endotoxin on the induction of tryptophan oxygenase activity by dexamethasone has been studied in suspension cultures of isolated rat hepatic parenchymal cells incubated alone or mixed with hepatic nonparenchymal cells. Hepatic cellular fractions were isolated from untreated rats and from animals injected 2 hr prior to killing with 2 mg/kg of endotoxin. Endotoxin (400 μg/ml) added to suspensions of parenchymal cells from untreated animals did not decrease the induction of tryptophan oxygenase by dexamethasone. Endotoxin addition to cell suspension containing parenchymal and nonparenchymal cells from untreated rats caused a significant diminution in the induction of enzyme activity elicited by 0.1 μM, but not by 20 μM, dexamethasone. The nonparenchymal cell fraction from untreated animals had no effect on enzyme induction in the absence of added endotoxin. However, nonparenchymal cells isolated from rats pretreated in vivo with endotoxin diminished the induction of tryptophan oxygenase activity elicited by 0.1 and 20 μM dexamethasone even in the absence of added endotoxin. The inhibitory effect of nonparenchymal cells from endotoxin-pretreated rats was related to cell concentration and was accentuated by the in vitro addition of endotoxin. Parenchymal cells from the endotoxin-pretreated animals had an altered sensitivity to dexamethasone. The ec 50 concentration for dexamethasone induction of tryptophan oxygenase was 40 and 500 μm for parenchymal cells from untreated and endotoxin-pretreated animals respectively. Parenchymal cells from the pretreated animals were more sensitive to the inhibitory effect of the nonparenchymal cells from the pretreated animals. These results are interpreted as indicating that endotoxin does not directly antagonize the induction of tryptophan oxygenase. The results suggest that this inhibition of tryptophan oxygenase induction is a mediated event involving an interaction of endotoxin with cellular constituents of the hepatic nonparenchymal cell fraction.

In vitro inhibition of endotoxin induced platelet aggregation with hydrocortisone sodium succinate (Solu-Cortef).

Citrated canine platelet rich plasma stimulated with ADP, thrombin, collagen and E. coli endotoxin demonstrated platelet aggregation when measured with Payton aggregometer. Following incubation with hydrocortisone sodium succinate (Solu-Cortef) the same platelet rich plasma did not undergo platelet aggregation when stimulated by these agents. Solu-Cortef added to platelet rich plasma during ATP, thrombin, collagen and endotoxin stimulated platelet aggregation resulted in a reversal of the optical density suggesting deaggregation. These in vitro observations support the beneficial effects observed when steroids are administered in vivo to experimental or clinical shock.

Growth inhibition and pyocin receptor properties of endotoxin from Pseudomonas aeruginosa.

SummaryEndotoxin from Pseudomonas aeruginosa exhibited bacteriocinogenic activity against several auto-plaque positive strains and appeared to enhance auto-plaque production in the inhibited strains, whereas all auto-plaque negative strains were unaffected. However, chemically removed protein moiety of the endotoxin did not exhibit any bacteriocinogenic activity when tested against seventy strains of P. aeruginosa. In addition, endotoxin appeared to behave as specific receptor site for bacteriocin particles (pyocin) obtained from P. aeruginosa. Consequently, when uncontracted pyocin particles from strain C9 were incubated with endotoxin from the sensitive indicator strain W9, they adsorbed to the endotoxin and underwent contraction. However, pyocin from C9 did not adsorb or undergo contraction when incubated with endotoxin from C9.