Characteristics of the action of the biologically active factor from virulent Shigella flexneri strains in a study on mice and a cell culture]

The authors subjected to further study the biologically active factor revealed by them earlier in the virulent Sh. flexneri cultures by using the genetically bound triad of Sh. flexneri 5a-222 cultures and the corresponding couple of Sh. flexneri 2a-516. There was shown correlation of the strains virulence determined by the keratoconjunctival test, with the presence of genetically-determined production of the biologically active factor detectable in the culture filtrate, which produced toxic action of the continuous cell cultures in the virulen Sh. flexneri strains of different serovars (2a and 5a), and lethal action in intravenous injection to mice. Comparative study of toxicity of the preparations of the endotoxin, free endotoxin, and neurotoxin types showed the biologically active factor to resemble the neurotoxin, differing from it in the toxic action and thermolability. Filtrates of the virulent and genetically characterised avirulent strains differed in the protein and lipids content, this permitting to suggest participation of the protein and lipid complex in the toxic action of the biologically active factor.     

Surface Antigens of Smooth Brucellae

Surface antigens of smooth brucellae were extracted by ether-water, phenol-water, trichloroacetic acid, and saline and examined by immunoelectrophoresis and gel diffusion with antisera from infected and immunized rabbits. Ether-water extracts of Brucella melitensis contained a lipopolysaccharide protein component, which was specific for the surface of smooth brucellae and was correlated with the M agglutinogen of Wilson and Miles, a polysaccharide protein component devoid of lipid which was not restricted to the surface of smooth brucellae and was not correlated with the smooth agglutinogen (component 1), and several protein components which were associated with internal antigens of rough and smooth brucellae. Immunoelectrophoretic analysis of ether-water extracts of B. abortus revealed only two components, a lipopolysaccharide protein component, which was correlated with the A agglutinogen, and component 1. Component 1 from B. melitensis and B. abortus showed identity in gel diffusion tests, whereas component M from B. melitensis and component A from B. abortus showed partial identity with unabsorbed antisera and no cross-reactions with monospecific sera. Attempts to prepare monospecific sera directly by immunization of rabbits with cell walls or ether-water extracts were unsuccessful. Absorption of antisera with heavy fraction of ether-water extracts did not always result in monospecific sera. It was concluded (as has been described before) that the A and M antigens are present on a single antigenic complex, in different proportions depending upon the species and biotype, and that this component is a lipopolysaccharide protein complex of high molecular weight that diffuses poorly through agar gel. Components 1, A, and M were also demonstrated in trichloroacetic acid and phenol-water extracts. With all extracts, B. melitensis antigen showed greater diffusibility in agar than B. abortus antigens. After mild acid hydrolysis, B. abortus ether-water extract was able to diffuse more readily.     

Kinetics of the interaction of endotoxin with polymyxin B and its analogs: a surface plasmon resonance analysis

Lipopolysaccharide, the invariant structural component of Gram-negative bacteria, when present in minute amounts in the circulation in humans elicits 'endotoxic shock' syndrome, which is fatal in 60% of the cases. Polymyxin B (PMB), a cyclic cationic peptide, neutralizes the endotoxin, but also induces many harmful side effects. Many peptide-based drugs mimicking the activity of PMB have been synthesized in an attempt to reduce toxicity while still retaining the anti-endotoxic activity. The study attempts to use the recent technique of surface plasmon resonance (SPR), in determining the kinetics of association and dissociation involved in the interaction of endotoxin with a few selected peptides that have structural features resembling PMB. The results, in conjunction with the thermodynamic data derived using isothermal titration calorimetry (ITC), stress the vital role played by amphiphilicity of the peptides and hydrophobic forces in this biologically important interaction.     

Reaction of endotoxin and surfactants. I. Physical and biological properties of endotoxin treated with sodium deoxycholate

Ribi, E. (Rocky Mountain Laboratory, Hamilton, Mont.), R. L. Anacker, R. Brown, W. T. Haskins, B. Malmgren, K. C. Milner, and J. A. Rudbach. Reaction of endotoxin and surfactants. I. Physical and biological properties of endotoxin treated with sodium desoxycholate. J. Bacteriol. 92:1493-1509. 1966.-Endotoxins from three species of gram-negative bacteria were shown to be dissociated by the bile salt sodium deoxycholate (NaD) into nontoxic subunits with molecular weights of about 20,000. When the bile salt was removed by dialysis, the subunits reaggregated in an orderly manner to form a relatively uniform population of biologically active endotoxin particles with average molecular weights of 500,000 to 1,000,000. If a small amount of human plasma was added to the dissociated endotoxin before removal of the NaD, reassociation apparently did not occur and the preparation remained nonpyrogenic. However, the plasma protein could subsequently be removed from the endotoxin subunits, and reaggregation to the toxic form would then occur. The studies on the physical nature of endotoxin performed with biophysical solution techniques were supplemented and confirmed by direct examination of the endotoxin polymers by electron microscopy. The results of these studies were consonant with the theory that the biologically active endotoxic elements are composed of micellar aggregates of linear lipopolysaccharide subunits.     

Aggregates are the biologically active units of endotoxin

For the elucidation of the very early steps of immune cell activation by endotoxins (lipopolysaccharide, LPS) leading to the production and release of proinflammatory cytokines the question concerning the biologically active unit of endotoxins has to be addressed: are monomeric endotoxin molecules able to activate cells or is the active unit represented by larger endotoxin aggregates? This question has been answered controversially in the past. Inspired by the observation that natural isolates of lipid A, the lipid moiety of LPS harboring its endotoxic principle, from Escherichia coli express a higher endotoxic activity than the same amounts of the synthetic E. coli-like hexaacylated lipid A (compound 506), we looked closer at the chemical composition of natural isolates. We found in these isolates that the largest fraction was hexaacylated, but also significant amounts of penta- and tetraacylated molecules were present that, when administered to human mononuclear cells, may antagonize the induction of cytokines by biologically active hexaacylated endotoxins. We prepared separate aggregates of either compound 506 or 406 (tetraacylated precursor IVa), mixed at different molar ratios, and mixed aggregates containing both compounds in the same ratios. Surprisingly, the latter mixtures showed higher endotoxic activity than that of the pure compound 506 up to an admixture of 20% of compound 406. Similar results were obtained when using various phospholipids instead of compound 406. These observations can only be understood by assuming that the active unit of endotoxins is the aggregate. We further confirmed this result by preparing monomeric lipid A and LPS by a dialysis procedure and found that, at the same concentrations, only the aggregates were biologically active, whereas the monomers showed no activity.     

Role of the lymphatic system in the transport of biologically active substances in shock of various etiologies

The content of histamine, serotonin, adrenaline and noradrenaline in the thoracic and lymphatic duct lymph and blood as well as absolute quantity of lymph transported biogenic amines and mediators into the general circulation were studied on 68 dogs during anaphylactic and endotoxin shock (ASh and ESh, respectively). Both ASh and ESh were accompanied by considerable changes in the hemodynamics, lymph circulation, content of biologically active substances in lymph and blood and in their lymph transport to the blood stream. The most profound and early changes in the content of biologically active substances during ASh and ESh were found in the lymph, which shows an important role of the lymphatic system in their resorption and transport from organs and tissues into the general circulation.     

Rapid biological and chemical analyses of bacterial endotoxins separated by preparative thin-layer chromatography

Methods have been elaborated for rapid location of biologically active bacterial endotoxin components on preparative thin-layer chromatography (tlc) by Limulus lysate clotting assay. The separation of the components from silicic acid is not necessary. Further assays were established for the semiquantitative estimation of endotoxic activites (Shwartzman reactivity, chick embryo lethality, and non-specific tumor resistance enhancement) without complete removal of silicic acid.Quantitative chemical analytical procedures were also elaborated to determine the molar ratios of the biologically active components separated and detected in the above tlc system. These included phosphorus, hexosamine, heptose, 2-keto-3-deoxyoctonate, and long-chain carboxylic acid content measurements. Here again, the chemical determinations were carried out in the presence of silicic acid.     

Free radical-mediated transgene inactivation of macrophages by endotoxin

Endotoxin, the lipopolysaccharide component of gram-negative bacteria, is a common contaminant of plasmid DNA preparations. The present study investigated the effect of endotoxin on gene transfection efficiency and the role of reactive oxygen species (ROS) in this process. Gene transfection studies were performed in various cell types with cytomegalovirus-luciferase as a reporter plasmid and cationic liposome as a transfecting agent. The presence of endotoxin in plasmid DNA preparations severely limited transgene expression in macrophages but had little or no effect in other cell types tested. This decreased transfection was dependent on ROS-mediated cellular toxicity induced by endotoxin. Neutralizing the endotoxin by the addition of polymyxin B effectively increased transfection efficiency and reduced toxicity. Electron spin resonance studies confirmed the formation of ROS in endotoxin-treated cells and their inhibition by free radical scavengers. The ROS scavenger N-t-butyl-alpha-phenylnitrone, the H(2)O(2) scavenger catalase, and the.OH scavenger sodium formate effectively inhibited endotoxin-induced effects, whereas the O(2)(-) scavenger superoxide dismutase had lesser effects. These results indicate that multiple oxidative species are involved in the transfection inactivation process and that.OH formed by H(2)O(2)-dependent, metal-catalyzed Fenton reaction play a major role in this process.     

Serological analysis of eleven strains ofRhizobium japonicum

The present communication reports a serological analysis of eleven strains ofRhizobium japonicum. The slow-diffusing thermostable antigens were found to be suitable for the basic differentiation of the somatic serogroups inRhizobium japonicum. One to three precipitation bands of the slow-diffusing thermostable antigens, one to two bands of the fast-diffusing thermostable antigens and one to three bands of the thermolabile antigens were detectable in the whole cell cultures ofR. japonicum by means of the immunodiffusion technique. Two basic somatic serogroups were differentiated on the basis of the slow-diffusing thermostable antigens. The thermolabile antigens were identical in most of the strains.The author is greatly indebted to Mrs. M. Kabelovà for technical assistance.This investigation forms part of a contribution prepared by the Czechoslovak National Committee for the International Biological Programme (Section PP: Production Processes).     

Neutralization of endotoxin toxicity in chick embryos by antibiotics

Rifkind, David (University of Colorado Medical Center, Denver), and John D. Palmer. Neutralization of endotoxin toxicity in chick embryos by antibiotics. J. Bacteriol. 92:815-819. 1966.-Three cationic cyclic polypeptide antibiotics, polymyxin B sulfate colistin sulfate, and tyrocidine hydrochloride, were shown to neutralize endotoxin lethality in chick embryos. The neutralizing potency of these antibiotics was approximately equivalent, 0.06 to 0.11 mumole of antibiotic per mug of endotoxin. Methane sulfonation of colistin resulted in a 13-fold decrease in endotoxin-neutralizing potency. Other cationic cyclic polypeptide antibiotics were inactive, as well all other classes of antibiotics tested, including the neutral cyclic polypeptides. Several nonantibiotic polycationic proteins and polymers tested were also inactive. It is suggested that certain cationic cyclic polypeptide antibiotics neutralize by combining directly with the toxic moiety of the endotoxin molecule. Possibly this combination involves the cationic groups of the antibiotics and the polyphosphate groups of the phospholipid component of endotoxin.     

Biologically Active Endotoxins from Salmonella Mutants Deficient in O- and R-Polysaccharides and Heptose

Well-characterized Salmonella mutants formerly used in biosynthetic studies of lipopolysaccharides were used to study the toxic portion of the complex endotoxin. Endotoxins prepared from wild types and their mutants were tested for their biological activities, including pyrogenicity, lethality, and immunogenicity. There was little difference either in the endotoxin yields or in the toxicities between endotoxins from the wild-type and O-antigen deficient mutants. Endotoxin containing mostly lipid A and keto-deoxyoctonate (KDO) prepared from the mutant deficient in both O- and R-antigens and the backbone sugar, heptose, was biologically active. Possibly because of the difference in solubility in water, the yield of endotoxin from the heptoseless mutant was about 10% of the wild type. There was complete reciprocal cross-immunity between all endotoxins tested. These observations suggest that the common toxic moiety is not present in the O- and R-polysaccharides or the backbone sugar heptose, but rather is associated with the lipid portion of the molecule which includes mostly lipid A and KDO.     

Chemical composition of <Emphasis Type="Italic">Desulfovibrio desulfuricans</Emphasis> lipid A

Lipopolysaccharides also called endotoxins are an integral component of the outer membrane of Gram-negative bacteria. When released from the bacterial surface, they interact with a host immune system, triggering excessive inflammatory response. Lipid A is the biologically most active part of endotoxin, and its activity is modulated by the quantity, quality and arrangement of its fatty acids. Desulfovibrio desulfuricans is sulfate-reducing, Gram-negative bacterium that is supposed to be opportunistic pathogens of humans and animals. In the present study, chemical composition of lipid A from various strains of D. desulfuricans was analyzed by gas chromatography/mass spectrometry. It was found that the fatty acid component of the lipid A contains dodecanoic, tetradecanoic, 3-hydroxytetradecanoic and hexadecanoic acids, and its carbohydrate core is composed of glucosamine. The analysis of 3-acyloxyacyl residue of the lipid A revealed the presence of amide-bound 3-(dodecanoyloxy)tetradecanoic and 3-(hexadecanoyloxy)tetradecanoic acids and ester-bound 3-(tetradecanoyloxy)tetradecanoic acid. It was concluded that both fatty acid and 3-acyloxyacyl residue profiles of the lipid A from the studied bacteria were similar to those of E. coli and S.enterica.     

Tissue distribution and clearance of soluble murine TNF receptors in mice

In this study, clearance of sTNFR was investigated. The data show that bilateral nephrectomy results in an increase of the levels of both sTNFR after which a new steady state situation develops, suggesting that other organs, apart from the kidneys, are involved in clearing of sTNFR. Bilateral nephrectomy also leads to an increase in circulating TNF. This TNF was detected by ELISA and appeared to be not biologically active. To investigate whether the endotoxin induced increase in sTNFR is dependent of renal function, endotoxin was injected in nephrectomized mice. The data show that nephrectomy followed by endotoxin injection resulted in a further increase of the levels of both sTNFR. However, the endotoxin induced increase in nephrectomized mice was similar to the situation in normal mice after LPS indicating that the endtoxin induced increase is kidney independent in these mice. To investigate the relative participation of various organs in sTNFR clearance, ¹²⁵I labelled sTNFR-P75 was injected. The data reveal that the majority of the sTNFR is removed from the circulation by the kidneys although indications for involvement of the liver and the lungs were also obtained. Calculation of the parametric clearance revealed that nephrectomy resulted in a 50% reduction of sTNFR-P75 clearance. Furthermore, the data presented strongly suggest that sTNFR release seems to be a continuous process, which is in balance with clearance of the sTNFR by the kidney, although other organs such as the liver and the lungs are involved.     

Partial purification of α-glycerotoxin,a presynaptic neurotoxin from the venom glands of the polychaete annelid glycera convoluta

The venom secreted from glands appended to the jaws of Glycera convoluta, a Polychaete Annelid, increases the spontaneous quantal release of transmitter from nerve terminals. The component that is biologically active on vertebrate cholinergic nerve terminals has recently been shown to be a high molecular weight protein. In the present work, the crude extract from the venom apparatus was shown to be toxic for mammals and crustaceans. It was fractionated by gel filtrations and ion exchange chromatographies. The biologically active component at frog neuromuscular junctions, α-glycerotoxin, was purified more than 1,000-fold. It is distinct from the components that are toxic for crustaceans. Purified α-glycerotoxin is a globular protein of 300,000 ± 20,000 mol wt. It has a Stokes radius of 65 Å and a sedimentation coefficient of 11 S. By its molecular properties, α-glycerotoxin appears distinct from other neurotoxins such as α-latrotoxin, which also trigger transmitter release.     

Evidence for pulmonary release of 5-hydroxyeicosatetraenoic acid (5-HETE) during endotoxemia in unanesthetized sheep

Leukocyte trapping in the pulmonary circulation may be an important component of the lung vascular injury response to endotoxin, but mediators of the pulmonary leukostasis and increased lung vascular permeability are unknown. The leukocyte 5-lipoxygenation pathway of arachidonic acid metabolism yields highly biologically active products including leukotrienes C₄ and D₄ (formerly slow reacting substance of anaphylaxis) and the potent chemotaxin, leukotriene B₄. A major product of 5-lipoxygenation is 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), for which a sensitive, stable isotope dilution assay employing combined gas chromatography-mass spectrometry is available. This assay was used to test the hypothesis that 5-lipoxygenation products might participate in pulmonary vascular responses to endotoxin. We measured 5-HETE concentrations in lung lymph at three intervals during endotoxemia in unanesthetized sheep. Concentrations of 5-HETE in lung lymph exceeded those in aortic blood plasma. Lymph 5-HETE concentrations increased from 1.7±0.3 (mean ± SEM, N = 7) ng/ml during baseline to peak values of 6.1±1.8 ng/ml (p < 0.05) during the hours after endotoxemia and preceeding the steady state increased lung vascular permeability response. During the increased permeability steady state from 240 to 270 minutes after endotoxin, lymph 5-HETE concentrations (1.4±0.3 ng/ml) and lymph 5-HETE flow (i.e., 5-HETE concentration x lung lynph flow rate) returned to baseline values. Although these observations are consistent with the hypothesis that 5-lipoxygenation products participate in the pulmonary vascular injury response to endotoxin, lymph 5-HETE concentrations did not correlate with any of the other experimental measurements. It may be only coincidence that the increase in lymph 5-HETE concentrations appeared contemporaneous with the onset of lung vascular injury.     

Potentiation of lethal endotoxin shock by streptococcal pyrogenic exotoxin in rabbits: Possible relevance of hyperreactivity of macrophages to endotoxin

Abstract Streptococcal pyrogenic exotoxin (SPE) potentiates lethal shock induced by endotoxin. We have previously reported that macrophages derived from SPE-treated rabbits showed hyperreactivity to endotoxin, and that the effect of SPE on macrophages was mediated by a lymphokine(s). Here we show that culture supernatants of SPE-stimulated lymphocytes, when administered into rabbits three hours before or together with endotoxin, potentiate a variety of endotoxin-induced pathophysiological changes and even lethal shock. These results suggest that SPE-induced lymphokine(s) mediates the potentiating effect of SPE on the lethal endotoxin shock through enhancing endotoxin reactivity of macrophages which play the central role in mediating endotoxin toxicity.     

Endotoxin activity of Moraxella osloensis against the grey garden slug,Deroceras reticulatum

Moraxella osloensis is a gram-negative bacterium associated with Phasmarhabditis hermaphrodita, a slug-parasitic nematode that has prospects for biological control of mollusk pests, especially the grey garden slug, Deroceras reticulatum. This bacterium-feeding nematode acts as a vector that transports M. osloensis into the shell cavity of the slug, and the bacterium is the killing agent in the nematode-bacterium complex. We discovered that M. osloensis produces an endotoxin(s), which is tolerant to heat and protease treatments and kills the slug after injection into the shell cavity. Washed or broken cells treated with penicillin and streptomycin from 3-day M. osloensis cultures were more pathogenic than similar cells from 2-day M. osloensis cultures. However, heat and protease treatments and 2 days of storage at 22 degrees C increased the endotoxin activity of the young broken cells but not the endotoxin activity of the young washed cells treated with the antibiotics. This suggests that there may be a proteinaceous substance(s) that is structurally associated with the endotoxin(s) and masks its toxicity in the young bacterial cells. Moreover, 2 days of storage of the young washed bacterial cells at 22 degrees C enhanced their endotoxin activity if they were not treated with the antibiotics. Furthermore, purified lipopolysaccharide (LPS) from the 3-day M. osloensis cultures was toxic to slugs, with an estimated 50% lethal dose of 48 microg per slug, thus demonstrating that the LPS of M. osloensis is an endotoxin that is active against D. reticulatum. This appears to be the first report of a biological toxin that is active against mollusks.     

Variations in the carbohydrate regions of Bordetella pertussis lipopolysaccharides: electrophoretic, serological, and structural features.

Structural and immunological differences between the two components that are usually present in unequal quantities in Bordetella pertussis endotoxin preparations and are visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been studied by using strains 1414, A100, and 134, all in phase I. According to analyses by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer chromatography, the minor (8%) component of the endotoxin of strain 1414 (endotoxin 1414) appeared to be the predominating component of endotoxins A100 and 134. The masses of the carbohydrate chains isolated from endotoxin A100 and from the major component of endotoxin 1414 were 1,649 and 2,311 atomic mass units, respectively, as determined by 252Cf plasma desorption mass spectrometry. Comparison of the 1H nuclear magnetic resonance spectra of these chains established that four N-acetyl groups, an N-methyl group, and a 6-deoxy function, which characterize the nonreducing, distal trisaccharide of the glycose chain of strain 1414, were absent from that of strain A100. The antigenicity of endotoxin 1414, as measured by enzyme-linked immunosorbent assay, was higher than that of endotoxin A100, but fell below it when the glycose chain of endotoxin 1414 was deprived of seven sugars by treatment with nitrous acid. This observation suggests that at least three (distal, proximal, and intermediate) regions of the glycose chain of endotoxin 1414 carry antigenic determinants. One of these, located in the distal trisaccharide, is absent from both endotoxins A100 and 134.     

Endotoxins stimulate neutrophil adhesion followed by synthesis and release of platelet-activating factor in microparticles

Lipopolysaccharides and triacyl-cysteine-modified proteins of Gram-negative and positive organisms are potent endotoxins. Animal models show that the receptor for platelet-activating factor (PAF) is responsible for many of the deleterious effects of endotoxin, where regulated, localized PAF production localizes the inflammatory response. In contrast, biologically active analogs of PAF (PAF-like lipids) are generated by oxidative attack on phospholipids by chemical reactions that are unregulated and unlocalized. The identity and distribution of the PAF receptor ligand in endotoxemia is unknown. We found human polymorphonuclear leukocytes (PMNs) were a significant source of PAF receptor agonists after stimulation by either class of endotoxin. Production of PAF receptor agonists required that the PMN adhere to a surface, and adhesion (and therefore accumulation of PAF-like bioactivity) in response to endotoxic stimulation was delayed for several minutes. PAF-like oxidized phospholipids were found by mass spectroscopy, but biosynthetic PAF accounted for most of the phospholipid agonists arising from endotoxic stimulation. A significant portion of the PAF made by PMNs was secreted, in contrast to its near complete retention by other inflammatory cells. Endotoxic stimulation induced a respiratory burst with the production of superoxide and the formation and shedding of microparticles. Free and microparticle-bound PAF appeared in the media, and blocking microvesiculation with calpeptin blocked PAF release. The released material activated platelets, and platelets co-aggregated with endotoxin-stimulated PMNs. Adherent PMNs therefore behave differently than suspended cells and are a significant source of free PAF after endotoxin exposure. Leukocytes can couple endotoxic challenge to the widespread circulatory and inflammatory effects of endotoxin.