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PubMed1995

Detection of Haemophilus ducreyi lipooligosaccharide by means of an immunolimulus assay.

A murine monoclonal antibody (MAb) directed against a surface-exposed epitope of the lipooligosaccharide (LOS) of Haemophilus ducreyi strain 35000 was shown to be reactive with all 37 strains of this pathogen tested in a colony blot-radioimmunoassay. The LOS epitope bound by this MAb appeared to be stably expressed by H. ducreyi growing in vitro. The use of this MAb in the immunolimulus system revealed that it could detect purified H. ducreyi LOS at a level of 25 pg/ml. Similarly, this immunolimulus system could detect as few as 1000 colony forming units of in vitro-grown H. ducreyi cells per ml of buffer. When this MAb was utilized in the immunolimulus system together with lesion material from rabbits infected with two different H. ducreyi strains, a positive reaction was obtained with every sample tested, even when no viable organisms were present in the lesion material. In contrast, this MAb yielded consistently negative results when used in the immunolimulus system with lesion material from animals infected with Staphylococcus aureus.

Animals↗
PubMed1994

Lipopolysaccharide-lipophorin complex formation in insect hemolymph: a common pathway of lipopolysaccharide detoxification both in insects and in mammals.

The formation of the lipophorin-lipopolysaccharide (LPS) complex in Bombyx mori hemolymph and its role in LPS detoxification were explored. LPS, an antibacterial protein inducer in insects, was injected into B. mori larvae. Analytical density gradient ultracentrifugation revealed that after injection the LPS peak shifts to a zone of lower density with time. The shifted peak was identified as the lipophorin-LPS complex. This complex formation was also achieved in an in vitro mixture of cell-free hemolymph and LPS at 25 degrees C but not at 1 degree C. The lipophorin-LPS complex had a significantly lower capacity to elicit the mRNA of cecropin B, an antibacterial protein. The biological activity of reextracted LPS from the complex was slightly reduced in the Limulus test and no structural modification was observed in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggested that the formation of lipophorin-LPS strikingly reduces the cecropin inducibility of LPS without any structural change in LPS. Similar serum lipoprotein-LPS complex formation and reduction of biological activities of LPS were also observed in mammals. We, therefore, suggest that the formation of the serum lipoprotein-LPS complex is a common pathway to inactivate LPS both in insects and in mammals.

Animals↗
PubMed1975

Possible pathogenic role of endotoxin in Reye's syndrome.

Evidence of circulating endotoxin was sought in children with Reye's syndrome, on the thesis that severe hepatic failure is likely to result in loss of capacity to detoxify intestinal endotoxins entering the circulation. A modification of the Limulus assay was used to demonstrate high levels of endotoxin-like activity (E.L.A.) in nine comatose patients with Reye's syndrome and in one of the two non-comatose patients. The symptom-free sibling of one patient had raised liver enzymes and a negative Limulus test. Plasma E.L.A. correlated significantly with degree of electroencephalographic disturbance early in the course of the illness. E.L.A. was also found in both of two cerebrospinal fluids evaluated. Preliminary in-vitro characterisation of this substance indicated that it resembled endotoxin derived from anaerobic intestinal bacteria. Intestinally derived endotoxin could be one factor in the pathogenesis of encephalopathy and other features of Reye's syndrome.

Bacteroides↗
PubMed1986

Biological activities of leptospiral lipopolysaccharide.

Lipopolysaccharide extracted with phenol-water from Leptospira interrogans serovar copenhageni strain Shibaura (L-LPS) showed various biological activities. In lethality for mice, L-LPS was active (LD 50, 3.4 mg/mouse) but about 12 times less potent than Escherichia coli LPS (E-LPS) per weight basis. L-LPS had pyrogenicity for rabbits, and the fever curves showed no evidence of the classical biphasic fever produced by E-LPS. In the bone marrow of mice, L-LPS caused hemorrhages and necrosis but less severe than those caused by E-LPS. Histopathologically, fresh hemorrhages were found in the intestine, spleen, lung and the other organs at 24 h after inoculation of L-LPS. Necrosis was also found in these organs and was particularly severe in mice inoculated with more than 2 mgL-LPS. Liver necrosis was found at 7th day after inoculation of L-LPS but not after inoculation of E-LPS. L-LPS had adjuvant activity just like E-LPS. L-LPS enhanced non-specific resistance to Salmonella infection and activated mouse peritoneal macrophages to kill these organisms. L-LPS was positive in limulus test just like E-LPS. These results demonstrated similarities of L-LPS and E-LPS. Some toxic effects of L-LPS were less than those of E-LPS, but some effects of L-LPS were more than those of E-LPS. L-LPS was antigenically active and the specificity was serogroup-associated. L-LPS was composed of carbohydrate (54%), lipid (12%), protein (5%). Arabinose, xylose and rhamnose were major sugars as detected by gas chromatography. 2-keto-deoxyoctanate (KDO) was not detectable.

Animals↗
PubMed1997

Immunobiological activities of chemically defined lipid A from Helicobacter pylori LPS in comparison with Porphyromonas gingivalis lipid A and Escherichia coli-type synthetic lipid A (compound 506).

Helicobacter pylori lipid A, characterised by a glucosamine beta (1-6) disaccharide 1-(2-aminoethyl)phosphate acylated by (R)-3-hydroxyoctadecanoic acid and (R)-3-(octadecanoyloxy)octadecanoic acid at the 2- and 2'-positions, respectively, exhibited no or very low endotoxic activities, i.e. lethal toxicity in galactosamine-loaded mice, pyrogenicity for rabbits and the activity of the Limulus test compared with Escherichia coli-type synthetic lipid A (compound 506), which possesses beta-(1-6)-linked glucosamine disaccharide 1,4'-bisphosphate, with two acyloxyacyl groups at the 2'- and 3'-positions and two 3-hydroxytetradecanoyl groups at the 2- and 3-positions. The endotoxic properties of H. pylori lipid A were also a little weaker than those of the low endotoxic lipid A of P. gingivalis, which has 1-phospho beta-(1-6)-linked glucosamine disaccharide with 3-hydroxy-15-methylhexadecanoyl and 3-hexadecanoyloxy-15-methylhexadecanoyl groups at the 2- and 2'-positions, respectively. Further, the mitogenic activity of H. pylori lipid A in murine splenic mononuclear cells was also less than those of P. gingivalis lipid A and compound 506. However, H. pylori lipid A induced comparable production of interleukin-6 (IL-6) by human peripheral blood mononuclear cells (PBMC) compared with P. gingivalis lipid A and compound 506. H. pylori lipid A also increased human natural killer cell activity, and strongly agglutinated rabbit erythrocytes. However, the lipid As of H. pylori and P. gingivalis showed lower activities in inducing tumour necrosis factor alpha (TNF-alpha) production by human PBMC and IL-8 production by human gingival fibroblasts than that of compound 506. The structural feature of H. pylori lipid A may be associated with low endotoxic properties and potent immunobiological activities.

Animals↗
PubMed1985

Endotoxin in middle ear effusions: in cases with persistent effusion after acute otitis media.

Endotoxin in persistent middle ear effusions (MEEs) from children with acute otitis media (AOM) whose acute inflammation had cleared was assayed by Limulus test, parallel with the detection of bacteria. Seventy-five percents of MEEs were endotoxin positive, and the concentration of endotoxin in MEE was not related to the interval between the onset of AOM and the date of sample collection. However, the cases with MEE that had high concentration of endotoxin tended to have persistent MEE for more than one month after sample collection. Therefore, the presence of endotoxin may be one of the causal factors of a delayed recovery of AOM and of the development of otitis media with effusion.

Acute Disease↗
PubMed1999

Correlation between the serum level of endotoxin and periventricular leukomalacia in preterm infants.

The objective of our study was to determine the relation between the serum level of endotoxin at birth and the development of periventricular leukomalacia (PVL) in preterm infants. We studied 68 preterm infants whose gestational ages ranged between 27 and 33 weeks, and birthweights between 1000 and 2000 g. The serum endotoxin level was measured in the blood immediately after birth by means of a conventional chromogenic Limulus test. Serum endotoxin was taken as present when it was >5 pg/ml (indicated by ENDO(+)). Premature rupture of membranes (PROM), the mode of delivery, EEG findings and the development of PVL were investigated. Six infants were diagnosed as having cystic PVL on ultrasonography. Endotoxin was detected in 28 infants. PROM was observed in 18 ENDO(+) infants and 16 ENDO(-) infants (P = 0.085). Caesarean section was performed in nine ENDO(+) infants and 14 ENDO(-) infants (P = 0.29). PVL was observed in two ENDO(+) infants and four ENDO(-) infants (P > 0.99). EEG abnormalities were recognized in five ENDO(+) infants and six ENDO(-) infants (P > 0.99). Between ENDO(+) infants and ENDO(-) infants, no significant difference was observed in any of the four parameters. These results may indicate that endotoxin itself does not directly cause PVL.

Apgar Score↗
PubMed1999

Use of quadrupole GC-MS and ion trap GC-MS-MS for determining 3-hydroxy fatty acids in settled house dust: relation to endotoxin activity.

Gas chromatography-mass spectrometry (GC-MS) using a quadrupole instrument and GC-tandem MS (GC-MS-MS) using an ion trap instrument were applied to determine 3-hydroxy fatty acids (3-OH FAs) with 10-18 carbon chain lengths, specific components of the endotoxin (lipopolysaccharide, LPS) of Gram-negative bacteria, in 30 house dust samples. The two methods provided similar detection sensitivity for methyl ester/trimethylsilyl derivatives of the 3-OH FAs and allowed these acids to be distinguished from co-eluting 2-OH FA derivatives. The correlation coefficients between endotoxin activity (Limulus test) and the combined amounts of 3-OH C10, 3-OH C12, and 3-OH C14 were 0.60 and 0.61 when using GC-MS and GC-MS-MS, respectively. The superior selectivity of GC-MS-MS was illustrated in analyses of sub-milligram amounts of dust, where the chromatograms achieved by GC-MS were difficult to interpret due to a high background and several closely eluting compounds. GC-MS-MS is therefore preferable to GC-MS for determining 3-OH FAs in minute (sub-milligram) amounts of dust.

Air Pollution, Indoor↗
PubMed2002

Biophysical characterization of the interaction of high-density lipoprotein (HDL) with endotoxins.

The interaction of bacterial endotoxins [lipopolysaccharide (LPS) and the 'endotoxic principle' lipid A], with high-density lipoprotein (HDL) from serum was investigated with a variety of physical techniques and biological assays. HDL exhibited an increase in the gel to liquid crystalline phase transition temperature Tc and a rigidification of the acyl chains of the endotoxins as measured by Fourier-transform infrared spectroscopy and differential scanning calorimetry. The functional groups of the endotoxins interacting with HDL are the phosphates and the diglucosamine backbone. The finding of phosphates as target groups is in accordance to measurements of the electrophoretic mobility showing that the zeta potential decreases from -50 to -60 mV to -20 mV at binding saturation. The importance of the sugar backbone as further target structure is in accordance with the remaining negative potential and competition experiments with polymyxin B (PMB) and phase transition data of the system PMB/dephosphorylated LPS. Furthermore, endotoxin binding to HDL influences the secondary structure of the latter manifesting in a change from a mixed alpha-helical/beta-sheet structure to a predominantly alpha-helical structure. The aggregate structure of the lipid A moiety of the endotoxins as determined by small-angle X-ray scattering shows a change of a unilamellar/inverted cubic into a multilamellar structure in the presence of HDL. Fluorescence resonance energy transfer data indicate an intercalation of pure HDL, and of [LPS]-[HDL] complexes into phospholipid liposomes. Furthermore, HDL may enhance the lipopolysaccharide-binding protein-induced intercalation of LPS into phospholipid liposomes. Parallel to these observations, the LPS-induced cytokine production of human mononuclear cells and the reactivity in the Limulus test are strongly reduced by the addition of HDL. These data allow to develop a model of the [endotoxin]/[HDL] interaction.

Calorimetry, Differential Scanning↗
PubMed1995

Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm.

The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin.

Acrosome↗
PubMed1975

Comparative evaluation of the Limulus assay and the direct Gram stain for detection of significant bacteriuria.

A double-blind study comparing the Limulus in-vitro endotoxin assay with the direct Gram stain of uncentrifuged urine for detection of significant bacteriuria was performed. One-thousand seventy-seven urine specimens were examined by the two methods and the results compared with results of quantitative urine cultures. Two hundred three samples produced growth of greater than 10-5 organisms per ml. urine. The Limulus assay detected 86.2% of these specimens, and 98.8% of urines that contained greater than 10-5 Gram-negative bacilli per ml. The Gram stain procedure detected only 69.5% of urines containing greater than 10-5 organisms per ml. and 74.5% of specimens with greater than 10-5 Gram-negative bacteria per ml. urine. The Limulus assay demonstrated both greater sensitivity and greater specificity than the Gram stain procedure. Moreover, the Limulus test is much less susceptible to errors of interpretation than methods involving microscopy.

Arthropods↗
PubMed1994

Suppression by methylprednisolone of augmented plasma endotoxin-like activity and interleukin-6 during cardiopulmonary bypass.

It has been reported that plasma endotoxin, measured by Limulus amoebocyte lysate assays, markedly increased during extracorporeal circulation (ECC). Therefore, this study was undertaken to see if pretreatment with methylprednisolone 30 mg kg-1 modifies the endotoxaemia and associated increases in plasma cytokines in 17 patients undergoing cardiac surgery requiring ECC. We found that methylprednisolone suppressed significantly the ECC-induced increases in plasma endotoxin, measured by a conventional Limulus amoebocyte lysate assay (Toxicolor), and interleukin-6. Plasma concentrations of endotoxin, measured by a highly specific chromogenic Limulus test (Endospecy test), tumour necrosis factor-alpha and interleukin-1 beta did not increase significantly in either the control or methylprednisolone groups.

Adult↗
PubMedDate not supplied

Lipopolysaccharide-induced elaboration of interleukin 1 by human monocytes: use for detection of lipopolysaccharide in serum and the influence of serum-lipopolysaccharide interactions.

An assay of interleukin 1 (IL-1) production by human monocytes was used to detect minute amounts of bacterial lipopolysaccharide (LPS) in sera or serum components designed for use in experiments or for injection into humans. Elaboration of IL-1 is a more sensitive criterion for the presence of LPS than is the rabbit pyrogenicity assay, and contamination of a given solution with LPS can be semiquantitatively determined by comparison with a standard. Interaction of (exogenously added) LPS with serum interferes with the recognition of LPS by monocytes but in a manner different from that encountered in the limulus test. Recognition of LPS by monocytes was not affected by the presence of naturally occurring antibodies to LPS. With use of IL-1 elaboration as an indicator, the presence of functionally active LPS in the sera of some patients with sepsis could be demonstrated.

Adult↗
PubMed1993

Induction of inflammatory cytokines by a soluble moiety prepared from an enzyme lysate of Actinomyces viscosus cell walls.

A carbohydrate-rich and immunobiologically active component, M1Av, was prepared from an M-1 endo-N-acetylmuramidase digest of cell walls of Actinomyces viscosus ATCC 19246 by CM Sephadex C-25 and Sephadex G-100 chromatography. M1Av stimulated thioglycolate-induced peritoneal macrophages from C3H/HeN and C3H/HeJ mice to release cell-free tumour necrosis factor (TNF) and interleukin (IL)-6, and a cell-associated thymocyte activating factor, probably IL-1. An intravenous (i.v.) injection of M1Av induced increased levels of TNF and IL-6 in the serum of C3H/HeN mice that had been primed with 100 micrograms of muramyldipeptide (MDP) i.v. However, M1Av did not induce TNF release in C3H/HeJ mice similarly primed with MDP. Simultaneous administration of M1Av (100 micrograms, i.v.) and galactosamine (18 mg, intraperitoneally) killed C3H/HeN, but not C3H/HeJ mice. M1Av was shown to be practically free of endotoxin by the Limulus test. These findings indicate that the solubilised A. viscosus cell-wall carbohydrate moiety induced inflammatory cytokines both in vitro and in vivo.

Actinomyces viscosus↗
PubMed1979

A B lymphocyte mitogen extracted from a fungus Peziza vesiculosa.

A water-soluble mitogen was extracted with hot-water from the fruiting bodies of a fungus, Peziza vesiculosa, collected in the wild. The active substance, named vesiculogen, was able to stimulate selectively murine B cells because mitogenic activity was observed in the spleen cell cultures of congenitally athymic nude mice, but not in the thymus cell cultures. The possibility that the mitogenicity of vesiculogen was due to lipopolysaccharide was denied completely by the following evidence: 1) lipopolysaccharide in vesiculogen was undetectable(less than 0.001% in the Limulus test), 2) vesiculogen was able to stimulate strongly DNA synthesis of spleen cells from C3H/HeJ mice, and 3) the mitogenic activity of vesiculogen was not inhibited by polymyxin B. Vesiculogen increased antigen-nonspecifically the number of direct plaque forming cells to sheep erythrocytes, horse erythrocytes, and trinitrophenylated-horse erythrocytes. This result shows that vesiculogen acts as a polyclonal B cell activator on murine spleen cells.

Animals↗
PubMed1989

Endotoxic properties of chemically synthesized lipid A analogs. Studies on six inflammatory reactions in vivo, and one reaction in vitro.

Biological activities of two groups of synthesized lipid A analogs, the counterpart of biosynthetic precursor, Lehmann's Ia type, 406, and E. coli lipid A type, 506, as well as their non-phosphorylated, and mono-phosphorylated analogs were investigated. The activities employed included four bone marrow cell reactions in mice, mice skin reaction, leukocytes migration in rabbits' cornea, and hemagglutination. Compound 406 and 506 elicited bone marrow reactions in mice and hemagglutination of mouse RBC, although 406 failed to elicit hemorrhage and necrosis also in mice skin. Compound 406 did not elicit corneal reaction in rabbits. The results suggest that for elicitation of this reaction and mice skin reaction, acyloxyacyl structure is required. Cytotoxicity and thromboplastin production of four bone marrow reactions had been reported by us to be endotoxic reactions, since these had not been elicited by peptidoglycan of Lactobacillus and Staphylococcus (1981) and 300 series synthesized analogs (1984) which did not have endotoxic structures. From these results, it seems that these two marrow reactions and hemagglutination require, as does the limulus test, the lipid A part structure as is present in 406.

Animals↗
PubMed1988

Endotoxin-stimulated human monocyte secretion of interleukin 1, tumour necrosis factor alpha, and prostaglandin E2 shows stable interindividual differences.

The secretions of interleukin 1 (IL-1), tumour necrosis factor alpha (TNF), and prostaglandin E2 (PGE2) of low-dose E. coli lipopolysaccharide (LPS)-stimulated human monocytes (M phi) were investigated in an endotoxin (ET)-free milieu (less than 1.6 pg LPS/ml). Human M phi cultures from nine healthy men were stimulated with 0, 12.5-500, and 250,000 pg LPS/ml as measured by a very sensitive Limulus test. The IL-1 activity was tested by the mouse costimulatory thymocyte (LAF) assay, which was thoroughly standardized and characterized (interassay variation 22-24%, intra-assay variation 3-7%). Spontaneous M phi secretions of IL-1, TNF, and PGE2 were negligible, but 12.5 pg LPS/ml significantly stimulated the secretions of these M phi products and the monokine responses to 500 and 250,000 pg LPS/ml were almost in the same range. It was demonstrated that the secretions of IL-1-TNF and TNF-PGE2 were strongly correlated. Pronounced interindividual differences in LPS responsiveness were demonstrated, and two low-responders, one of whom was HLA-DR1,2-positive, were identified. Three first-degree relatives of the DR1,2-positive low-responder had similar low responses. Furthermore, M phi cultures were prepared weekly for 4 weeks from four HLA-DR different men and the only DR2,2 homozygous individual had low monokine responses. In conclusion, stable interindividual differences in in vitro monokine and PGE2 secretions of LPS-stimulated M phi were demonstrated. It is suggested that HLA-DR2-positive individuals may be low responders.

Biological Products↗
PubMed2000

Immunobiological activities of a chemically synthesized lipid A of Porphyromonas gingivalis.

A synthetic lipid A of Porphyromonas gingivalis strain 381 (compound PG-381), which is similar to its natural lipid A, demonstrated no or very low endotoxic activities as compared to Escherichia coli-type synthetic lipid A (compound 506). On the other hand, compound PG-381 had stronger hemagglutinating activities on rabbit erythrocytes than compound 506. Compound PG-381 also induced mitogenic responses in spleen cells from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, as well as LPS-responsive C3H/HeN mice. The addition of polymyxin B resulted in the inhibition of mitogenic activities, however, compound 506 did not show these capacities. Additionally, compound PG-381 showed a lower level of activity in inducing cytokine production in peritoneal macrophages and gingival fibroblasts from C3H/HeN mice, but not C3H/HeJ mice, in comparison to compound 506. Thus, this study demonstrates that the chemical synthesis of lipid A, mimicking the natural lipid A portion of LPS from P. gingivalis, confirms its low endotoxic potency and immunobiological activity.

Animals↗