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PubMed2003

Structure and biological activities of beta-glucans from yeast and mycelial forms of Candida albicans.

We have achieved the extraction of cell wall beta-glucan from the mycelial form of Candida albicans (C. albicans) IFO 0579 (M-CSBG) by using acetic acid, sodium hypochlorite (NaClO), and dimethylsulfoxide (DMSO) treatments. The yield of M-CSBG was significantly lower (7.5% from dried mycelial cells) than that of the yeast form from C. albicans IFO 1385 (Y-CSBG, 25.9% from dried yeast cells). The properties of M-CSBG were similar to those of Y-CSBG in terms of nuclear magnetic resonance (NMR) spectra and limulus reactivity. Molecular weight (Mw) of M-CSBG was slightly higher than that of Y-CSBG. Both Y-CSBG and M-CSBG induced the production of comparable amounts of macrophage inflammatory protein-2 (MIP-2), a chemotactic factor, from mouse peritoneal exudate cells (PEC) in vitro. These findings suggest that the structure and properties of CSBG from yeast and mycelial cells are similar to each other.

Animals↗
PubMed1988

Endotoxinaemia in sickle cell disease.

Fifty-nine children with sickle cell anaemia (HbSS) or associated haemoglobinopathies were studied prospectively using a chromogenic Limulus amoebocyte lysate assay to detect circulating endotoxin. The 41 children with HbSS (mean age 8 years 9 months) had more serious disease than the 18 with HbSC disease (n = 14) or HbS-beta-thalassaemia (n = 4) (mean age 7 years 2 months), with a greater degree of splenomegaly, lower haemoglobin, and higher white cell counts, platelet counts and bilirubin values (P less than 0.05 for all). Twenty-nine children with HbSS had evidence of poor reticuloendothelial function, with red cell pitting of greater than or equal to 2%. Three of these 29 had low levels of endotoxin in plasma (0.12-0.24 endotoxin units (EU)/ml); two were clinically well, one had a painful crisis. Eight of 18 children with other sickle haemoglobinopathies had greater than or equal to 2% pitted red cells; none was endotoxinaemic. Therefore, in 37 patients with reticuloendothelial dysfunction, three were endotoxinaemic; all had sickle cell anaemia. Although not statistically significant, this suggests that endotoxinaemia may occur predominantly in patients with reticuloendothelial dysfunction, and is compatible with the hypothesis that systemic endotoxinaemia can derive from the intestine especially when reticuloendothelial function is depressed.

Adolescent↗
PubMed1994

Detection of lipopolysaccharide in the picogram range of tissue culture media by a kinetic chromogenic Limulus amebocyte lysate assay.

A kinetic chromogenic Limulus amebocyte lysate assay for detection of lipopolysaccharide in the picogram range is described. The kinetic Limulus assay was used to detect lipopolysaccharide in a tissue culture media containing interfering low-molecular-weight substances. The interfering substances were removed by ultrafiltration. The recovery of lipopolysaccharide from the ultrafiltrated tissue culture media was 61.6 +/- 21.4%. The kinetic Limulus assay was compared with the traditional end-point chromogenic Limulus assay. The results in the two assays showed a good correlation. The detection limit was 1 pg/ml in the chromogenic assay and 0.25 pg/ml in the kinetic assay.

Amino Acid Sequence↗
PubMed1988

Demonstration of soluble Plasmodium falciparum antigens reactive with Limulus amoebocyte lysate and polymyxin B.

To investigate whether soluble Plasmodium falciparum antigens possess endotoxin-like properties, the interaction of Limulus amoebocyte lysate (LAL) with defined soluble antigens from Plasmodium falciparum was studied by various crossed immunoelectrophoretic methods and immunoblotting. The soluble P. falciparum antigens were purified by affinity chromatography using human IgG from malaria-immune adults as ligand. Of eight possible antigens, at least three were affected by LAL, as indicated by disappearance of these antigens in the precipitation pattern, after the reaction with LAL. One of the LAL-reactive antigens is a heat-stable glycoprotein with the presence of both hydrophilic and hydrophobic regions in its structure. This antigen shows a strong reaction with polymyxin B, and antibodies against it have been shown to be inhibitory to the growth of P. falciparum in culture. It is concluded that LAL reacts with several soluble antigens from P. falciparum and it is suggested that these antigens participate in the induction of protective immunity to malaria, and consequently that one or more of the soluble antigens are candidates for a malaria vaccine.

Animals↗
PubMed1989

Immunochemical and biological reactivity of human anti-lipopolysaccharide IgG obtained by screening of blood donors.

An anti-lipopolysaccharide (LPS) preparation for the intravenous treatment of septic endotoxic shock was prepared by purifying immunoglobulin G (IgG) from pooled serum from Danish blood donors. The sera were selected by the use of an enzyme-linked immunosorbent assay (ELISA) to screen blood donors for high concentrations of antibodies to a mixture of LPS from 11 different Gram-negative bacteria. ELISA was also used for indirect quantification of IgG antibodies to lipid A, and to rough LPS from Escherichia coli Ra and Salmonella minnesota R60 (Ra). The concentration of human antibodies to the LPS mixture correlated with the concentration of antibodies to the E. coli and S. minnesota rough LPS and to lipid A. The specificity of sera with high concentrations of anti-LPS IgG was investigated by immunoblotting. Sera from individual donors reacted with LPS from different bacteria and recognized different sites on the LPS molecules. The range of specificities to different LPS was increased by the pooling of selected sera. The IgG fraction from the high titre donor pool neutralized biological activities of LPS such as activation of the Limulus amoebocyte lysate reaction and induction of tumour necrosis factor secretion from human monocytes.

Absorption↗
PubMed1991

Hypotensive effects of stroma-free haemoglobin solutions attributable to adenine nucleotides.

Aqueous solutions of stroma-free human haemoglobin are being evaluated as potential oxygen-carrying resuscitation fluids. There are indications, however, that such solutions may produce toxic side-effects in vivo. Stroma-free haemoglobin solution produced a 50% fall in mean arterial pressure when infused into a small animal model despite containing very low levels of non-haem protein and phospholipid contaminants. This effect was not produced by haemoglobin solutions after extensive dialysis. Red cell-derived adenine nucleotides were found to be present in concentrations high enough to cause such a response (80-85 micrograms/ml). We have developed a chromatographic assay capable of predicting hypotension in our animal model and consider that the complete absence of adenine nucleotides must be confirmed in all studies concerning the possible toxic side-effects of stroma-free haemoglobin solutions.

Adenine Nucleotides↗
PubMed1990

Isolation and characterization of lipopolysaccharide from Centipeda periodontii ATCC 35019.

Lipopolysaccharide (LPS) was extracted from cells of Centipeda periodontii ATCC 35019 by a hot phenol-water method, purified by nuclease treatment and by repeated ultracentrifugations. The LPS was composed of 21.7% hexose, 9.1% hexosamine, 10.3% fatty acid, 0.5% protein, 2.5% phosphorus, 4.3% 2-keto-3-deoxyoctonate(KDO) and 2.3% heptose. The major fatty acids of the LPS were 3OH C13:0 and C13:0. C. periodontii LPS was able to gelatinize Limulus amebocyte and to produce the local Schwartzman reaction in rabbits. LPS from C. periodontii exhibited strong immunobiological activities, mitogenicity on splenocytes of C3H/HeN mice, and stimulation of interleukin 1 (IL-1) production in peritoneal macrophages of C3H/HeN mice.

Endotoxins↗
PubMed1997

Chemical and biological properties of lipopolysaccharide from Selenomonas sputigena ATCC 33150.

Chemical and biological studies were performed on lipopolysaccharide isolated from Selenomonas sputigena ATCC 33150T, a possible causative agent of periodontal diseases. The sugar components of the lipopolysaccharide of S. sputigena were mannose, galactose, glucose, L-glycero-D-mannoheptose (heptose), 2-keto-3-deoxy-octonic acid, glucosamine and galactosamine in a molar ratio of 0.3:1.0:1.0:1.0:0.2:3.0:3.2 (mol/mol heptose). Sephadex G-50 chromatography of the polysaccharide portion of the lipopolysaccharide obtained by partial hydrolysis yielded three fractions: the O-polysaccharide chain attached to the core oligosaccharide, the core oligosaccharide and monosaccharides. Compositional analysis of these fractions revealed that lipopolysaccharide of S. sputigena carries a short O-polysaccharide chain consisting of galactose and glucosamine and that the core oligosaccharide consisted of glucose, heptose, glucosamine and 2-keto-3-deoxyoctonic acid. It is of particular interest that galactosamine was detected as a component sugar of the lipid A moiety in addition to glucosamine, which is a usual component sugar of the lipid A of most gram-negative bacteria. Thus, the lipid A of S. sputigena might have a unique backbone that differs from that of the lipid A of other gram-negative bacteria. Lipid A of S. sputigena consisted mainly of fatty acids such as undecanoic, tridecanoic, tridecenoic, 3-hydroxytridecanoic and 3-hydroxytetradecanoic acid in a molar ratio of 0.4:1.0:0.3:4.0:0.5 (mol/mol tridecanoic acid). Lipopolysaccharide and lipid A from S. sputigena both exhibited biological activity in activating the clotting enzyme of Limulus amebocytes, the Schwartzman reaction, mitogenicity for murine lymphocytes and in inducing interleukin-1 alpha and interleukin-6 production in murine macrophages to the same extent as those observed for lipopolysaccharide of the Salmonella serovar typhimurium used as a positive control. The results suggested that the lipopolysaccharide of S. sputigena is a virulent factor in human periodontal diseases.

Animals↗
PubMed1986

Lipopolysaccharide-sensitive serine-protease zymogen (factor C) found in Limulus hemocytes. Isolation and characterization.

Bacterial endotoxin (lipopolysaccharide, LPS) induces coagulation of horseshoe crab hemolymph. Our previous studies had demonstrated that a hemolymph factor, designated factor B, was associated with the LPS-mediated activation of the Limulus clotting system [Ohki et al. (1980) FEBS Lett. 120, 318-321]. On further purification of factor B we found that an additional component, designated factor C, was required to generate factor B activity in the presence of LPS in order to activate the proclotting enzyme. To elucidate the role of factor C in the LPS-mediated reaction, factor C was isolated and characterized from the hemocyte lysate under sterile conditions. The preparation exhibited a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of 2-mercaptoethanol, while two protein bands on SDS-PAGE were observed after reduction. Thus, factor C had a Mr of 123 000 consisting of a heavy chain of Mr = 80 000 and a light chain of Mr = 43 000. Factor C was converted to an activated form in the presence of LPS with a Mr = 123 000, designated factor C. Upon activation, cleavage of the light chain occurred resulting in the accumulation of two new fragments of Mr = 34000 and 8500 on reduced SDS-PAGE. A diisopropylfluorophosphate-sensitive active site was localized in the light chain (Mr = 34000) of factor C. The reconstitution experiments, using factor C, factor B, proclotting enzyme and LPS, demonstrated that all of these proteins are essential for the endotoxin-mediated coagulation system. On the basis of these results we propose that a cascade pathway of LPS-induced activation of the Limulus clotting system consists of three sequential activations of hemolymph serine protease zymogens.

Animals↗
PubMed2004

Biophysical characterization of the interaction of Limulus polyphemus endotoxin neutralizing protein with lipopolysaccharide.

Endotoxin-neutralizing protein (ENP) of the horseshoe crab is one of the most potent neutralizers of endotoxins [bacterial lipopolysaccharide (LPS)]. Here, we report on the interaction of LPS with recombinant ENP using a variety of physical and biological techniques. In biological assays (Limulus amebocyte lysate and tumour necrosis factor-alpha induction in human mononuclear cells), ENP causes a strong reduction of the immunostimulatory ability of LPS in a dose-dependent manner. Concomitantly, the accessible negative surface charges of LPS and lipid A (zeta potential) are neutralized and even converted into positive values. The gel to liquid crystalline phase transitions of LPS and lipid A shift to higher temperatures indicative of a rigidification of the acyl chains, however, the only slight enhancement of the transition enthalpy indicates that the hydrophobic moiety is not strongly disturbed. The aggregate structure of lipid A is converted from a cubic into a multilamellar phase upon ENP binding, whereas the secondary structure of ENP does not change due to the interaction with LPS. ENP contains a hydrophobic binding site to which the dye 1-anilino-8-sulfonic acid binds at a K(d) of 19 micro m, which is displaced by LPS. Because lipopolysaccharide-binding protein (LBP) is not able to bind to LPS when ENP and LPS are preincubated, tight binding of ENP to LPS can be deduced with a K(d) in the low nonomolar range. Importantly, ENP is able to incorporate by itself into target phospholipid liposomes, and is also able to mediate the intercalation of LPS into the liposomes thus acting as a transport protein in a manner similar to LBP. Thus, LPS-ENP complexes might enter target membranes of immunocompetent cells, but are not able to activate due to the ability of ENP to change LPS aggregates from an active into an inactive form.

Animals↗
PubMed2006

Determination of the inflammatory potential of bioaerosols from a duck-fattening unit by using a limulus amebocyte lysate assay and human whole blood cytokine response.

Inhalation of bioaerosols from animal houses can induce acute inflammatory reactions in the respiratory tract. Determination of the concentration of airborne endotoxins is widely used to characterize this risk. In this study, the activity of bioaerosol samples from a duck-fattening unit to induce interleukin-1beta (IL-1beta) in human blood and to react with Limulus Amebocyte Lysate (LAL) was investigated. The activity detected in the whole blood assay correlated well with the endotoxic activity found in the LAL assay (Spearmen's rho = 0.902). However in all samples, the inflammation-inducing potential was overestimated by the LAL assay. It is assumed that this overestimation could be, in part, a result of an overestimation of the inflammatory potential of endotoxins originating from Pseudomonadaceae by the LAL assay. Pseudomonadaceae were regularly isolated from the air of the duck-fattening unit. The results presented here indicate that the whole blood assay can be used besides the LAL assay as an additional method to characterize the inflammation-inducing potential of bioaerosols.

Air Microbiology↗
PubMed1984

Role of dialyzer contaminants in the allergic epiphenomena of hemodialysis.

Cuprophan hollow-fiber dialyzers contain contaminants including 1,2,3-propanetriol, carbohydrates, Limulus amebocyte lysate-reactive material, and particulates. In a clinical study, the role of these substances in the allergic-type response seen in some hemodialysis patients was examined. Patients were dialyzed three times per week for 6-week intervals with each of four dialyzer preparations designed to vary the burden of contaminants presented to the patient. Predialysis eosinophil counts and serum immunoglobulin (Ig) E levels were obtained weekly. White cell and platelet counts and plasma C3a and C5a levels were measured during dialysis for each dialyzer preparation. Dialyzer preparation had no effect on predialysis eosinophil counts or IgE levels. All patients demonstrated transient leukopenia and complement activation during dialysis, the magnitudes of which were unaffected by the type of dialyzer preparation. At the levels found in the dialyzers studied, it was questioned whether water-soluble extractables or particulates play any role in the allergic epiphenomena of hemodialysis.

Cellulose↗
PubMed2005

Endotoxin level measurement in hemodialysis biofilm using "the whole blood assay".

Biofilms have been found on the inner surface of silicone tubing inside dialysis machines. Endotoxin releasing from those biofilms increases the bioincompatibility of dialysis liquids and leads to long-term inflammatory complications among dialysis patients. Endotoxin measurement is recommended for the control of dialysis liquids. This article describes the use of a new method, the Whole Blood Assay (WBA), for endotoxin quantification in dialysis biofilms. Biofilms were suspended in sterile water by scraping the tubing samples. Diluted blood samples from healthy donors were stimulated overnight with the contaminated suspension. Stimulated mononuclear cells released IL-1beta in response to endotoxins. IL-1beta level was then measured using an ultrasensitive ELISA method. We demonstrated a semilogarithmic model in which the optical densities measured after the ELISA assay increases linearly with the levels of endotoxin. This model allowed the determination of the amount of endotoxins in biofilm samples with a detection limit of 0.032 EU/mL. Most of the time, the amounts of endotoxin measured by the WBA were higher than those measured by the Limulus Amoebocyte Lysate (LAL) assay. This study suggested the presence of "endotoxin-like" compounds different from the lipopolysaccharides that are not detected by the LAL assay. We concluded that the LAL is necessary but insufficient to have a representative quantification of endotoxins that could be hazardous to patient health.

Biofilms↗
PubMed1995

Cytokine-inducing glycolipids in the lipoteichoic acid fraction from Enterococcus hirae ATCC 9790.

Five high molecular weight glycolipids capable of stimulating human peripheral whole-blood cell cultures to cause interleukin 6 (IL-6) and tumor necrosis factor (TNF)-alpha induction were isolated from one of the lipoteichoic acid fractions (LTA-2) extracted from Enterococcus hirae ATCC 9790 (Tsutsui et al., (1991) FEMS Microbiol. Immunol. 76, 211-218) by a combination of hydrophobic interaction and anion-exchange chromatographies. This purification procedure resulted in a remarkable increase in the cytokine-inducing activities on the weight basis of isolated glycolipids (a maximum of 36- and 17-fold increases of IL-6 and TNF-alpha induction, respectively). The total yield of these bioactive glycolipids amounted to 6 wt% of the parent LTA-2 fraction, while the recovery rate in terms of the cytokine-inducing activities was estimated to be sufficient. The chemical composition and the profile, using SDS-PAGE, revealed that all of the isolated bioactive components were high molecular weight glycolipids, which were distinct from each other and from the parent LTA-2 fraction. These findings suggest that the IL-6 and TNF-alpha-inducing activities previously noted in the parent LTA-2 fraction are not attributable to a chemical entity, the structure of which had been proposed elsewhere (Fischer, W. (1990) in Glycolipids, Phosphoglycolipids and Sulfoglycolipids (Kates, M. ed.) pp. 123-234, Plenum Press, New York), but to the other high molecular weight glycolipids described here.

Animals↗
PubMed1996

Kupffer cells play important roles in the metabolic degradation of a soluble anti-tumor (1-->3)-beta-D-glucan, SSG, in mice.

Metabolic degradation of a soluble highly branched (-->3)-beta-D-glucan, SSG, was examined in mice using a macrophage blocker, gadolinium chloride (GdCl3). Intraperitoneally administered SSG distributed in the liver was slowly degraded, and after 5 weeks about 30% of the SSG became anionic. In addition, it is suggested that the metabolites would contain fewer branching points as assessed by the reactivity to limulus factor G. On the other hand, in the spleen, the molecular weight and the degree of branching of SSG were not changed for at least 5 weeks. Blockade of Kupffer cells by GdCl3 did not significantly change the distribution ratio of SSG in the liver. However, the treatment significantly delayed the degradation of SSG. These results suggested that Kupffer cells play important roles, not in the distribution, but in the oxidative degradation of SSG in the liver. In addition, splenic macrophages did not significantly contribute to the metabolic degradation of SSG.

Animals↗
PubMed1996

Effects of murine lysozyme on lipopolysaccharide-induced biological activities.

We have demonstrated that egg-white lysozyme (EW-LZM) bound to lipopolysaccharide (LPS), reduced the lethal toxicity and the biological activity of LPS. In this study, the interaction of LPS with murine lysozyme (M-LZM) and the modulation of biological activities were investigated. M-LZM was prepared from the culture supernatant of the murine macrophage cell line RAW264.7 by ion-exchange and gel filtration chromatographies and dialysis. Two types of M-LZM, murine M lysozyme (MM-LZM) and murine P lysozyme (MP-LZM), were purified from the supernatant. The enzymatic activities of both MM-LZM and MP-LZM were inhibited by LPS and their effects were affected by the temperature and the ionic strength. TNF-alpha production from RAW264.7 by LPS was inhibited by mixing with MM-LZM and MP-LZM. MP-LZM inhibited TNF-alpha production stronger than MM-LZM. Considering these facts, we suggested that M-LZM, like EW-LZM, make a complex with LPS to reduce the toxicity of LPS together with inhibiting the enzymatic activity.

Amino Acid Sequence↗
PubMed1996

Biological characterization of Campylobacter fetus lipopolysaccharides.

Lipopolysaccharides (LPS) of three strains of Campylobacter fetus (subspp. fetus and venerealis, and serotypes A and B), a bacterium of veterinary importance but also a cause of various infections in humans, were assessed for their ability to induce mitogenicity, gelation of Limulus amebocyte lysate, lethal toxicity in mice, and pyrogenicity in rabbits. All C. fetus LPS exhibited activities lower than those of Salmonella typhimurium LPS. LPS of C. fetus subsp. fetus serotype A had the lowest activity in all assays. Since the majority of C. fetus subsp. fetus isolates from humans are serotype A, the lower biological activities of this LPS may aid the pathogenesis of such strains. The lower activities of C. fetus LPS compared with those of S. typhimurium LPS may reflect the presence of longer fatty acid chains in the lipid A of C. fetus LPS, whereas interstrain differences in C. fetus LPS bioactivities may be related to some property influenced by composition of the saccharide moiety.

Animals↗