Limulus Lysate Gelation Research Articles

Low endotoxic activity of lipopolysaccharides isolated from Bradyrhizobium, Mesorhizobium, and Azospirillum strains

The endotoxic activities of lipopolysaccharides (LPS) isolated from different strains of rhizobia and rhizobacteria (Bradyrhizobium, Mesorhizobium, and Azospirillum) were compared to those of Salmonella enterica sv. Typhimurium LPS. The biological activity of all the examined preparations, measured as Limulus lysate gelation, production of tumor necrosis factor (TNF), interleukin-1β (IL-1β), and interleukin-6 (IL-6), and nitrogen oxide (NO) induction in human myelomonocytic cells (line THP-1), was considerably lower than that of the reference enterobacterial endotoxin. Among the rhizobial lipopolysaccharides, the activities of Mesorhizobium huakuii and Azospirillum lipoferum LPSs were higher than those of the LPS preparations from five strains of Bradyrhizobium. The weak endotoxic activity of the examined preparations was correlated with differences in lipid A structure compared to Salmonella.

Detection of Pyrogens in Intravenous IgG Preparations

Five different intravenous IgG (i.v. IgG) preparations were assessed for their capacity to modify the pyrogenic response to bacterial lipopolysaccharide (LPS) of rabbits under the conditions of a pharmacopoeal test. Four of the five preparations were found to mitigate the reaction rendering the result “non-pyrogenic” with an LPS dose proved pyrogenic when administered in saline or in albumin. Bacterial LPS was found readily detectable by a simple Limulus amoebocyte lysate (LAL) gelation test. Four of six brands of i.v. IgG were found reactive in the test under conditions adjusted to detect the FDA limit. The reaction obtained upon addition of standard LPS to the negative preparations supported the validity of the assay. The LAL reactivity of two of the reactive preparations was inhibited by laminarin, a compound known to inhibit Limulus lysate gelation by β-D-glucan, but not by Polymyxin B. Specific detection of bacterial endotoxins in i.v. IgG solutions requires inhibition of the β-D-glucan pathway of the Limulus lysate coagulation. Using an appropriate inhibitor, the LAL gelation test is suitable to detect a potential endotoxin contamination in i.v. IgG which might have not been unravelled by the in vivo test for pyrogens. Copyright 2002 Published by Elsevier Science Ltd on behalf of The International Association for Biologicals

MALP-2, a Mycoplasma lipopeptide with classical endotoxic properties: end of an era of LPS monopoly?

Although some activities of LPS are shared by other bacterial components, for half a century LPS has been regarded as unique in displaying many pathophysiological activities. Here we report on a synthetic lipopeptide, MALP-2 from Mycoplasma fermentans, which expresses potent endotoxin-like activity and whose lethal toxicity is comparable to that of LPS. With the exception of the Limulus lysate gelation test, in which MALP-2 was approximately 1000-fold less active than LPS, the synthetic lipopeptide induced all activities tested for, and in most cases to an extent comparable to that of LPS. Unlike LPS, the biological activities of MALP-2 were expressed both in LPS-responder and in LPS-non-responder mice (BALB/c/l, C57BL10/ScCr), indicating that MALP-2 signaling, unlike that of LPS, is not transduced via the Toll-like receptor (Tlr) 4 protein.MALP-2 expressed no toxicity in normal or sensitized Tlr2 knockout (Tlr2(-/-)) mice indicating that its toxic activity is induced via Tlr2 signaling. The phenomenology of the lethal shock induced by MALP-2 in normal or sensitized mice, i.e. the kinetics of its development and symptoms of illness exhibited by the treated animals, was very reminiscent of the lethal shock induced by LPS.

Low endotoxic potential of Legionella pneumophila lipopolysaccharide due to failure of interaction with the monocyte lipopolysaccharide receptor CD14.

Legionella pneumophila, a gram-negative bacterium causing Legionnaires' disease and Pontiac fever, was shown to be highly reactive in in vitro gelation of Limulus lysate but not able to induce fever and the local Shwartzman reaction in rabbits and mice. We analyzed the capacity of purified L. pneumophila lipopolysaccharide (LPS-Lp) to induce activation of the human monocytic cell line Mono Mac 6, as revealed by secretion of proinflammatory cytokines and desensitization to subsequent LPS stimulation. We showed that despite normal reactivity of LPS-Lp in the Limulus amoebocyte lysate assay, induction of cytokine secretion in Mono Mac 6 cells and desensitization to an endotoxin challenge required LPS-Lp concentrations 1,000 times higher than for LPS of Salmonella enterica serovar Minnesota. Therefore, we examined the interaction of LPS-Lp with the LPS receptor CD14. We demonstrated that LPS-Lp did not bind to membrane-bound CD14 expressed on transfected CHO cells, nor did it react with soluble CD14. Our results suggest that the low endotoxic potential of LPS-Lp is due to a failure of interaction with the LPS receptor CD14.

Immunosuppressive structural entity exists in endotoxin

Both lipopolysaccharides and lipid A moiety prepared fromVibrio el-tor exhibited almost equal endotoxic activities in eliciting the local Shwartzman reaction, in Limulus-lysate gelation, in chick-embryo lethality and pyrogenicity. The structural part responsible for these activities of lipopolysaccharides is the lipid A moiety. The high anti-complementary activities present in lipopolysaccharides and lipid A moiety was destroyed by alkaline digestion of the samples. Lipid A antiserum can protect against some of the toxic activities of the endotoxin in animals.

Purification and characterization of lipooligosaccharides from four strains of "Haemophilus somnus".

Lipooligosaccharides (LOSs) from four strains of "Haemophilus somnus" were purified and their electrophoretic profile, composition, endotoxic activity, and antigenic properties were analyzed. The LOSs were most efficiently purified by enzyme digestion, hot aqueous phenol extraction, and ultracentrifugation. Each LOS could be separated into two to six distinct bands with apparent Mrs of 3280 to 4960, following electrophoresis in polyacrylamide gels. Each LOS contained dodecanoic, tetradecanoic, and 3-hydroxytetradecanoic fatty acids; a high proportion of hexose, 3-deoxy-D-manno-octulosonic acid, and phosphate; and a small amount of heptose; glucosamine was present in both the oligosaccharide and the lipid A. Each "H. somnus" LOS demonstrated endotoxic activity, as determined by gelation of Limulus ameobocyte lysate, the dermal Schwartzman reaction, and mouse lethality. Antiserum to purified "H. somnus" LOS cross-reacted with all strains of "H. somnus" tested by enzyme-linked immunosorbent assay (ELISA), but not to any Enterobacteriaceae, Pseudomonas, or Pasteurella species tested. "H. somnus" LOS was a poor immunogen, but inhibition, dot blot, and sandwich ELISA data indicated that antibodies made to LOS were predominantly, though not exclusively, to lipid A. Monoclonal antibodies directed to "H. somnus" LOS confirmed that lipid A and non-lipid A determinants were present.

Chemical and biological properties of a phenol-water extract from leptospira interrogans. Evidence for the absence of lipopolysaccharide

Leptospira interrogans, serovar copenhageni was extracted by the phenol-water method and the resulting preparation examined for chemical composition and endotoxic activity. Chemical analysis revealed that a number of sugars were present, however, the amount of lipid content was very low. Further, the preparation was devoid of characteristic endotoxic properties, like lethal toxicity, pyrogenicity and the property to induce the local Shwartzman reaction. The extract, however, was active in the limulus lysate gelation test and in the induction of monocyte activation. It is concluded that the leptospira preparation is devoid of endotoxin properties, both from the chemical and from the biological point of view.

Neutralization and lipoprotein binding of lipopolysaccharides in tolerant rabbit serum.

We examined the ability of sera drawn from rabbits made tolerant to lipopolysaccharide (LPS) from Escherichia coli O18 to inhibit heterologous LPS-induced gelation of Limulus lysate. Compared with a pool of pretolerant sera, a pool of tolerant sera from these rabbits neutralized 5.5-fold more LPS from Salmonella typhimurium, 4.9-fold more LPS from Pseudomonas aeruginosa, and 10.0-fold more LPS from Klebsiella pneumoniae. Because previous studies have found that LPS bound to lipoprotein is less toxic than unbound LPS, we also studied the binding of LPS lipoprotein fractions in the serum pools by using fractionation with a cesium chloride-equilibrium density gradient. Radiolabeled LPS from E. coli O113 bound much more rapidly to lipoprotein fractions in tolerant serum than in pretolerant serum (after 11 min of incubation, 66.1% vs. 16.5% binding, respectively).

The analysis of lipopolysaccharide (endotoxin) in meningococcal polysaccharide vaccines by silver staining following SDS-polyacrylamide gel electrophoresis

A simple physical-chemical method for estimating the lipopolysaccharide (LPS, i.e. endotoxin) content of meningococcal polysaccharide vaccines is described. The low molecular weight LPS was first separated from the high molecular weight polysaccharide by SDS-polyacrylamide gel electrophoresis. The LPS was then selectively visualized by a highly sensitive silver stain which can detect 5 ng of meningococcal LPS. The method can detect concentrations of LPS in meningococcal polysaccharides as low as 0.01%, and was used to measure semiquantitatively the LPS in bulk powders of meningococcal polysaccharides and in vaccines in final containers. The LPS in the bulk powders and in the vaccines was also assayed by the gelation of Limulus polyphemus ameobocyte lysate and the results of the two methods were in agreement.

Bacterial endotoxins in umbilical cord blood of neonates.

We tested umbilical cord blood from 255 infants for evidence of bacterial endotoxins. Using a Limulus lysate gelation technique, endotoxin-like activity (ELA) was detected in 13 (9.1%) of 142 term infants and in 23 (20.3%) of 113 preterm infants (p less than 0.05), in concentrations ranging from 30 to 3,000 pg Escherichia coli equivalent activity per milliliter of plasma. No factors predisposing to endotoxemia could be detected in term pregnancies but among ELA+ premature infants, placental cultures more frequently revealed gram-negative species (p less than 0.05). Cord blood endotoxemia in the concentrations observed was without obvious consequence in mothers and term infants. Our data confirm the existence of natal endotoxemia, a potential mechanism of perinatal injury.

Lipopolysaccharides of the cyanobacterium Microcystis aeruginosa

Lipopolysaccharides (LPS) of two isolates of Microcystis aeruginosa were extracted with phenol/water and purified. Cesium chloride gradient ultracentrifugation of these preparations yielded only one fraction. The LPS contained significant amounts of 3-deoxy-D-manno-octulosonic acid, glucose, 3-deoxy sugars, glucosamine, fatty acids, fatty acid esters, hexoses, and phosphate. Heptose, a characteristic sugar component of the polysaccharide moiety of LPS of most gram-negative bacteria was absent. Lipopolysaccharides and lipid A hydrolysate of LPS preparations were active in mouse lethality and Limulus lysate gelation. The lipid A moiety was slightly less active in toxicity and Limulus lysate gelation assays than the intact LPS. The LPS and lipid A moiety of the two isolates of M. aeruginosa were less active in toxicity in mice and Limulus test than LPS of Salmonella abortus equi.

Polymyxin B nonapeptide complexes with lipopolysaccharide

We have recently shown that a polymyxin derivative, polymyxin B nonapeptide (PMBN), which lacks the fatty acid part, the bactericidal activity and the toxicity of polymyxin [1,2] nevertheless causes disorganization of the enterobacterial outer membrane (OM). Thus, PMBN makes the OM of smooth encapsulated Escherichia coil and smooth Salmonella permeable to hydrophobic antibiotics such as actinomycin D, novobiocin, fusidic acid, erythromycin and others, and sensitizes the bacteria to all those agents. Furthermore, PMBN sensitizes the PMBN to the attack of the complement cascade (M. Vaara and T. Vaara, manuscripts submitted for publication). Electron microscopic studies showed that fingerlike projections appear on the OM of bacteria treated with PMBN (M. Vaara and T. Vaara, submitted). These projections appear similar to those caused by polymyxin [3]. Polymyxin interacts with acidic macromolecules, including the lipopolysaccharide (LPS) present in the outer leaflet of the OM [4-8]. We have previously shown that the LPS of polymyxin-resistant pmrA mutants of Salmonella typhimurium binds less polymyxin and is less acidic (due to the nearly stoicheiometric occurrence of 4-amino-4-deoxy-arabinose in the lipid A part of the LPS) than the LPS of the polymyxin sensitive parent strains [8]. In the present communication we show that PMBN does not sensitize the pmrA strain (in contrast to the parent strain) to hydrophobic antibiotic~ Furthermore, we show that PMBN, like polymyxin, inhibits biological activities of isolated LPS (gelation of Limulus-lysate, consumption of complement). These results suggest that PMBN causes a disorganization of the OM by binding to the acidic groups of LPS, as does polymyxin.

Characterization of the lipopolysaccharide from Vibrio cholerae 395 (Ogawa).

The chemical structure and biological properties of the lipopolysaccharide (LPS) from Vibrio cholerae 395 (Ogawa), isolated by the phenol-water procedure, were studied. Upon acid hydrolysis, the LPS was split into its polysaccharide and lipid A moieties. The polysaccharide contained both neutral (glucose, heptose, fructose) and amino (glucosamine, quinovosamine) sugars. The LPS contained the acid-labile amino sugar, 4-amino-arabinose, which was absent in the Inaba serotype of V. cholerae. The LPS differed from the LPSs of Enterobacteriaceae by the absence of 2-keto-3-deoxyoctonate and the presence of fructose. Analysis of the methylated polysaccharide by gas-liquid chromatography and mass spectrometry showed that it had a branched structure with glucose and heptose residues primarily appearing at the nonreducing-end groups. Interactions with lectins, concanavalin A. and wheat germ agglutinin suggested that terminal glucose residues were alpha linked, whereas terminal glucosamine residues were connected by alpha-1,3 linkages. The major fatty acids of the LPS were C14:0, C16:0, C12h:0, and C14h:0 compounds, of which only the C14h:0 were amide linked, the remainder being ester linked to the backbone. Biological studies showed that the LPS possessed endotoxic properties such as lethality, pyrogenicity, limulus lysate gelation, and ability to induce non-specific resistance to infection. Thus, the LPS from V. cholerae 395 (Ogawa) possessed both common and distinct features as compared with the LPSs from the Enterobacteriaceae.

Gelatin of Limulus amoebocyte lysate by simple polysaccharides.

The Limulus lysate gelatin activity of several simple polysaccharides including yeast mannans and bacterial dextrans was investigated. The mannans from Saccharomyces cerevisiae wild and mutant strains possessing dense branches showed positive gelatin activity at concentrations of 1 microgram/ml or more regardless of differences in their chemical structure. However, two synthetic mannans possessing linear structures with alpha 1 leads to 2 and alpha 1 leads to 6 linkages also gave positive reactions at concentrations of 10 microgram/ml or more and 500 microgram/ml or more, respectively. The dextran from Leuconostoc mesenteroides IAM 1046 consisting of a dense branching moiety displayed reactivity at concentrations of 100 microgram/ml or more, while the dextrans devoid of such branches were negative in this reaction. The optimal concentration for Limulus lysate gelatin could not be determined for any of the polysaccharides and lipopolysaccharides (LPS) tested in this study. The gelation activity of the polysaccharides was stable to treatment with 100 mM NaOH at 30 C for 72 hr. The minimum concentration for the gelation activity of LPS treated with 100 mM NaOH under the same conditions was reduced from 10(-6) approximately(-9) microgram/ml to 1-10 microgram/ml. The above findings demonstrate that the major part of Limulus lysate gelation activity of LPS depends on the alkali-degradable lipid A moiety, and that such simple polysaccharides are also able to participate in this activity even though the extent of participation is very low.

Application of Limulus Lysate Gelation Reaction to Environmental Pollution Analyses : Cross-Gelation Reaction with Several Simple Polysaccharides (Regular Presentations) (Proceedings of the 8th Symposium on Environmental Pollutants and Toxicology)

Application of Limulus Lysate Gelation Reaction to Environmental Pollution Analyses : Cross-Gelation Reaction with Several Simple Polysaccharides (Regular Presentations) (Proceedings of the 8th Symposium on Environmental Pollutants and Toxicology)

Endotoxin-induced hyperinsulinemia and hyperglucagonemia after experimental liver injury.

Basal portal and systemic venous hyperinsulinemia and hyperglucagonemia were present in fasted rats at 24 h after experimental liver injury by partial (67%) hepatectomy, carbon tetrachloride gavage, and intravenous D-galactosamine administration. Both enhanced pancreatic hormone secretion and depressed hepatic hormone extraction were likely responsible for the insulin and glucagon oversupply. Endogenous gut-derived endotoxin is proposed as the causative factor for the exaggerated hormonal response because intravenous exogenous endotoxin elicited an identical elevation of insulin and glucagon. Systemic endotoxemia at 24 h after liver injury was indicated by marked (78-100%) lethality in lead-sensitized rats and positive Limulus lysate gelation tests of plasma samples. Furthermore, antiendotoxin treatments, including endotoxin tolerance, polymyxin B, and gut sterilization, significantly reduced both lead-sensitized lethality and hyperinsulinemic and hyperglucagonemic responses at 24 h in most liver-injury groups. Portal versus systemic venous administration of endotoxin at a low dose implied that normal endotoxin phagocytosis by the liver suppressed the pancreatic endocrine response. A physiological negative-feedback control system involving gut-derived systemic endotoxemia after liver damage with insulin and glucagon hypersecretion by the pancreas for stimulation of hepatic regeneration is hypothesized.

Binding of bacterial endotoxin (LPS) to encephalitogenic myelin basic protein and modulation of characteristic biologic activities of LPS.

Myelin basic protein, isolated from central nervous system tissue and an inducer of experimental allergic encephalomyelitis in animals, has been demonstrated to form a stable molecular complex with the lipid A region of gram-negative bacterial lipopolysaccharides (endotoxins). This binding of endotoxin with myelin basic protein results in generation of lower m.w. aggregates with decreased isopycnic density. A number of lipid A-induced characteristic properties of endotoxin, such as B lymphocyte proliferative response in C3H/St mice, complement activation of normal human serum, Limulus lysate gelation, and lethal effects in mice, are modified as a result of binding of myelin basic protein with lipopolysaccharides.

Isolation and partial characterization of a lipopolysaccharide from phase II Coxiella burnetii

A lipopolysaccharide (LPS) was isolated by hot phenol–water extraction from phase II Coxiella burnetii. The LPS was isolated from organisms that had a history of 95 serial yolk sac passages and others that were cloned. The chemical composition of the LPS was partially determined and compared with that of LPS previously isolated from phase I organisms. Most of the sugars present in phase I LPS were detected in phase II LPS; however, there were some quantitative and qualitative differences. The fatty acid profiles of phase I and II LPS were identical. The amount of LPS extracted from phase II organisms was about [Formula: see text] the amount extracted from phase I C. burnetii. Limulus lysate gelation was observed with subnanogram amounts of both phase I and II LPS. In complement fixation tests, both LPSs reacted only with antisera containing antibodies versus phase I antigen. Ouchterlony immunodiffusion tests demonstrated immunological identity of the two LPSs.

Studies on Limulus amoebocyte lysate. III. Purification of an endotoxin-binding protein from Limulus amoebocyte membranes.

A protein that has been isolated from Limulus polyphemus amoebocyte membranes binds endotoxin. The protein was purified by two independent methods, organic solvent extraction and affinity chromatography, both followed by gel filtration. Immunologic studies confirm that the protein is a component of amoebocyte membranes. Although without enzymatic activity, the binding protein enhances Limulus lysate gelation. As a membrane-associated endotoxin binding "protein," it may be involved in Limulus lysate coagulation, which is initiated by minute amounts of Gram-negative bacterial endotoxin. The protein has an apparent molecular weight of 80,000.

Purification of a Mannan from Candida albicans which Activates Serum Complement

Candida species activate complement by the alternative pathway, induce leukocyte migration and, when applied epicutaneously, cause epidermal microabscesses of neutrophils in man and experimental animals. Complement activation by C. albicans appears to be a property of the cell wall. To biochemically identify the complement-activating constituent(s) of C. albicans, an ethyleneglycol extract of growth phase blastospores was prepared. Acid hydrolysis and neutral sugar analysis revealed mannose (82%), fucose (7%), and glucose (11%). The soluble, mannose-rich cell wall polysaccharide of C. albicans activates serum complement via the alternative pathway, induces neutrophil chemotaxis and is antigenically reactive with antisera to C. albicans. This constituent exhibits in vitro endotoxin-like activity as measured by Limulus lysate gelation, but is nonpyrogenic in rabbits. The extracts produced precipitin lines in double immunodiffusion studies against serum from patients with invasive candidiasis and rabbit antisera to mycelial and blastospore preparations of C. albicans, but not against normal serum. Thus, pathogenic properties and reactive phenomenon of C. albicans are in part attributable to a cell wall polysaccharide, mannan.