Lipid A, the endotoxic center of bacterial lipopolysaccharides: relation of chemical structure to biological activity.
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Animals↗
Biomedical subjects
L Brade
Publications and source records attributed to L Brade.
Biological activity of synthetic heptaacyl lipid A representing a component of Salmonella minnesota R595 lipid A.
A synthetic lipid A (preparation 516), containing seven acyl groups and representing one component of natural free lipid A of Salmonella minnesota R595, has been investigated for biological activity in a number of endotoxin test systems. It was found that the synthetic preparation was, in typical in vivo endotoxin tests (lethality, pyrogenicity, Shwartzman reactivity) as well as in its antigenicity and macrophage activation capacity, significantly less active than natural Salmonella lipid A. However, in other in vitro assay systems (B-cell mitogenicity, complement activation, Limulus amoebocyte lysate gelation) it expressed similar activity as Salmonella lipid A.
Animals↗
The effect of removal of D-fructose on the antigenicity of the lipopolysaccharide from a rough mutant of Vibrio cholerae Ogawa.
The lipopolysaccharide (LPS) of a rough mutant (95R) of Vibrio cholerae Ogawa has been investigated chemically and serologically. D-Fructose was released from LPS under conditions (10mM trifluoroacetic acid, 60 degrees, 1 h) that liberated no other sugar constituent of the LPS (2-amino-2-deoxy-D-glucose, D-glucose, L-glycero-D-manno-heptose). Upon periodate oxidation, D-fructose and D-glucose were oxidised quantitatively, whereas approximately 50% of heptose was periodate-resistant. The data indicate that D-fructose does not link the polysaccharide and lipid A portion as proposed earlier, and suggest that D-fructose is present as a branch. By passive hemolysis inhibition, it was shown that the release of D-fructose paralleled the exposure of an antigenic determinant cryptic in LPS.
Carbohydrates↗
Induction of human interleukin 1 by bacterial and synthetic lipid A.
Lipopolysaccharides (LPS) are well known inducers of interleukin 1 (IL 1). Here we show that synthetic heptaacyl Salmonella minnesota (compound 516) and synthetic E. coli type (compound 506) lipid A, as well as monodephospho part structures thereof, are able to induce IL 1 production in human mononuclear cells. The 4'-monodephospho part structure (compound 505) was found to be the most active IL 1 inducer as compared to compound 506 and the 1-monodephospho part structure (compound 504). Synthetic lipid A precursor Ia, lacking nonhydroxylated fatty acids (compound 406), and its 1- or 4'-monodephospho part structures (compounds 404 and 405) did not induce IL 1 production. IL 2 is not produced during stimulation with the synthetic compounds. IL 1 activity was partially heat labile and could be inhibited by a rabbit antiserum against human leukocytic pyrogen. These results show that the lipid A component of LPS is active in inducing IL 1 and that the presence of 3-acyloxyacyl residues is necessary for IL 1 induction.
Blood Physiological Phenomena↗
Immunogenicity and antigenicity of synthetic Escherichia coli lipid A.
The immunogenicity and antigenicity of synthetic Escherichia coli lipid A (compound 506) and its 1- and 4'-monophosphorylated derivatives (compounds 505 and 504, respectively) and nonphosphorylated derivative (compound 503) were compared with those of bis- and 4'-monophosphorylated natural free lipid A from E. coli. The synthetic compounds under study were either coated onto sheep erythrocytes (except for the water-insoluble preparation 503) or incorporated into liposomes and used for the immunization of rabbits. Both types of immunogens (the latter representing fully synthetic immunogens) resulted in high-titered polyclonal antisera which were characterized before or after absorption in a passive hemolysis assay as well as in a passive hemolysis inhibition assay with the synthetic compounds as test antigens. All antisera were found to react with their corresponding homologous antigens coated onto sheep erythrocytes, with titers of up to 2,048, and were comparable to those antisera obtained after immunization with natural lipid A exposed on the bacterial surface after acid hydrolysis. Antisera against bisphosphorylated compound 506 were highly specific for the homologous antigens, showing no interaction with compounds 504 and 505 in the passive hemolysis test. The same held true for the absorption experiments in which glutaraldehyde-fixed sheep erythrocytes were sensitized with the respective antigens. Antisera against monophosphorylated compounds 504 and 505 exhibited, besides their expected homologous reactivity, complete cross-reactivity with compound 506, but they did not cross-react with each other. Thus, anti-504 and anti-505 antibodies recognized distinct antigenic determinants, being related to the ester linked 4'-phosphate or the glycosidically linked 1-phosphate, respectively. Both antigenic determinants were also expressed by bisphosphorylated compound 506 used as an antigen; however, upon immunization, only antibodies against compound 506 were elicited.
Epitopes↗
Chemical, biological, and immunochemical properties of the Chlamydia psittaci lipopolysaccharide.
The lipopolysaccharide (LPS) of Chlamydia psittaci was extracted from yolk sac-grown elementary bodies, purified, and characterized chemically, immunochemically, and biologically. The LPS contained D-galactosamine, D-glucosamine, phosphorus, long-chain fatty acids, and 3-deoxy-D-manno-2-octulosonic acid in the molar ratio of approximately 1:2:2:6:5. The antigenic properties of the isolated LPS were compared with those of the LPS from Chlamydia trachomatis and Salmonella minnesota Re by the passive hemolysis and passive hemolysis inhibition tests, absorption, hydrolysis kinetics, and Western blot analysis with rabbit polyclonal antisera against chlamydiae and with a mouse monoclonal antibody recognizing a genus-specific epitope of chlamydial LPS. Two antigenic determinants were identified, one of which was chlamydia specific and the other of which was cross-reactive with Re LPS. Both determinants were destroyed during acid hydrolysis, whereby a third antigen specificity was exposed which was indistinguishable from the lipid A antigenicity. In rabbit polyclonal antisera prepared against Formalin-killed elementary bodies or detergent-solubilized membranes, two antibody specificities were differentiated. One of these was chlamydia specific, and the other was cross-reactive with Re LPS. The LPS of C. psittaci was inactive within typical endotoxin parameters (lethal toxicity, pyrogenicity, local Shwartzman reactivity); it was, however, active in some in vitro assays, such as those testing for mouse B-cell mitogenicity and the induction of prostaglandin E2 in mouse peritoneal macrophages.
Abortion, Veterinary↗
Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities.
The recently chemically synthesized Escherichia coli lipid A and the natural free lipid A of E. coli were compared with respect to their endotoxic activities in the following test systems: lethal toxicity, pyrogenicity, local Shwartzman reactivity, Limulus amoebocyte lysate gelation capacity, tumour necrotizing activity, B cell mitogenicity, induction of prostaglandin synthesis in macrophages, and antigenic specificity. It was found that synthetic and natural free lipid A exhibit identical activities and are indistinguishable in all tests.
Animals↗
Antigenic properties of Chlamydia trachomatis lipopolysaccharide.
The antigenic properties of the lipopolysaccharide (LPS) of Chlamydia trachomatis L2 were investigated. By means of passive hemolysis, passive hemolysis inhibition, and absorption experiments, it was shown that antiserum raised against chlamydial elementary bodies contained at least two different antibody specificities which reacted with different antigenic determinants of chlamydial LPS. One of these antibodies cross-reacted with enterobacterial Re LPS, recognizing a structure which is shared by both LPSs, whereas the reactivity of the second antibody was restricted to chlamydial LPS. The former antibody could be absorbed with Salmonella minnesota Re LPS, whereas the latter was not affected by this absorption. Therefore, chlamydial LPS possesses two distinct antigenic determinants, one of which is C. trachomatis specific, the other of which is responsible for the cross-reactivity with enterobacterial Re-type LPS. Both antigenic determinants were destroyed during mild acid-catalyzed hydrolysis. It was further shown that free chlamydial lipid A exhibits antigenicity that cross-reacts with free enterobacterial lipid A. This antigenicity, however, as in enterobacterial LPS, is present in a cryptic form, i.e., it is unmasked only after acid hydrolysis of LPS.
Chlamydia trachomatis↗
Characterization of two different antibody specificities recognizing distinct antigenic determinants in free lipid A of Escherichia coli.
Antisera were raised in rabbits with acid-treated Re mutant bacteria from Salmonella minnesota and Escherichia coli and tested in a passive hemolysis assay with di- and monophosphorylated free lipid A of E. coli (LipA-Ac and LipA-HCl, respectively) coated onto sheep erythrocytes. Depending on the acid used to prepare the immunogen (acetic versus hydrochloric acid), different antibody specificities were obtained. Antiserum prepared against HCl-treated bacteria was found to react with both antigens to the same extent (i) in the passive hemolysis test, (ii) in the passive hemolysis inhibition test, and (iii) in absorption experiments, suggesting that antibodies in this antiserum recognize an antigenic determinant equally present in LipA-Ac and LipA-HCl. Antiserum raised against acetic acid-treated bacteria reacted with the homologous antigen (LipA-Ac) in the passive hemolysis and passive hemolysis inhibition test as well as in absorption experiments. However, the antiserum failed to react with the heterologous antigen (LipA-HCl) in the hemolysis test and during absorption, whereas in inhibition studies interaction of this antiserum with both antigens was observed. The inhibiting capacity of LipA-Ac was lower compared with that of LipA-HCl, indicating that the antigenic determinant of LipA-Ac is partly expressed by LipA-HCl in solution, but not when fixed on the surface of sheep erythrocytes. The role of glycosidically linked phosphate in lipid A is discussed with respect to antigenicity.
Animals↗
A 28,000-dalton protein of normal mouse serum binds specifically to the inner core region of bacterial lipopolysaccharide.
Normal mouse serum was found to contain a protein, referred to here as factor, which binds to the inner core region of lipopolysaccharides (LPSs) of various bacterial families. Since factor-LPS interactions resulted in activation of guinea pig complement, factor activity could be assayed by a passive hemolysis test with sheep erythrocytes coated with LPS or lipid A from Acinetobacter calcoaceticus (which was found earlier to bind particularly well to factor). Factor was purified by G-50 and hydroxyapatite chromatography whereby the specific hemolytic activity was enriched 1,675-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed the presence of a 28,000-dalton protein as the main band. The identity of this band was determined by absorption experiments with LPS-coated sheep erythrocytes or latex beads, whereby the 28,000-dalton band disappeared after specific absorption and could be recovered from the absorbent. The binding specificity of factor was determined in a passive hemolysis inhibition assay with defined oligosaccharides representative for the inner core region of LPS. Thus, the di- and trisaccharides alpha-D-mannoheptopyranosyl-(1----5)-2-keto-3-deoxy-D-mannoocto nic acid and alpha-D-mannoheptopyranosyl-(1----3)-alpha-D-mannoheptopyranosy l-(1----5)-2- keto-3-deoxy-D-mannooctonic acid, respectively, were able to inhibit binding of factor to LPS. The results are in accordance with our earlier observation that the heptose-2-keto-3-deoxy-D-mannooctonic acid region represents a common antigen of bacterial LPS. Rabbit hyperimmune serum directed against this common antigen and purified factor was found to exhibit the same specificity for LPS. Factor activity was followed in mice in vivo after injection of LPS; it disappeared completely 15 min after the injection of LPS and reappeared within 1 h.
Animals↗
Newer aspects of the chemical structure and biological activity of bacterial endotoxins.
Gram-negative bacteria express at their surface various amphiphiles among which the lipopolysaccharides (endotoxins, O-antigens) have been studied most intensively. Lipopolysaccharides consist of a heteropolysaccharide portion (O-specific chain and core) which is responsible for the O- (and R-) antigenic properties and a covalently bound lipid component, termed lipid A, which contains the endotoxic principle of lipopolysaccharides. The detailed chemical structure of a large number of O-chains and the general architecture of the core oligosaccharide has been established. Recent analyses of the enterobacterial inner core region indicate the presence of a linear trisaccharide of alpha 2.4-linked 3-deoxy-D-manno-2-octulosonic acid (KDO) residues of which only the reducing group is believed to be located in the main core chain. This KDO residue which provides the link between the polysaccharide and the lipid A component appears to be involved in a recently detected ubiquitous immunodeterminant expressed by lipopolysaccharides of various origin. The chemical structure of enterobacterial lipid A's is now known in some detail. Lipid A of Salmonella, Escherichia coli and Proteus consists of a beta 1.6-linked D-glucosamine disaccharide which carries four (R)-3-hydroxytetradecanoyl groups in positions 2, 3, 2' and 3' and two phosphoryl residues in positions 1 and 4'. Up to three of the hydroxy fatty acids (positions 2, 2' and 3') are, at their 3-hydroxyl groups, acylated by non-hydroxylated acyl residues, and to phosphoryl groups non-acylated, nitrogen-containing residues such as 4-amino-4-deoxy-L-arabinopyranose and phosphorylethanolamine may be bound. The hydroxyl group in position 4 of the glucosamine disaccharide is free and that in position 6' serves as the attachment site for KDO (i.e. the polysaccharide component) in lipopolysaccharide. Based on this structure lipid A analogues have been chemically synthesized and analysed for endotoxic activity in vivo and in vitro. In two test systems (pyrogenicity, local Shwartzman reaction) the synthetic part structures exhibited weak or no endotoxic activity, as did a precursor of lipid A biosynthesis which is structurally identical to one of the analogues. In many other systems, however, including lethal toxicity, B-lymphocyte mitogenicity, macrophage activation, induction of cross tolerance, expression of lipid A antigenicity, the synthetic materials were of comparable activity as bacterial free lipid A. These findings support the structural proposals made for lipid A and they prove the previous hypothesis that the endotoxic principle is embedded in lipid A.(ABSTRACT TRUNCATED AT 400 WORDS)
Endotoxins↗
Endotoxic properties of chemically synthesized lipid A part structures. Comparison of synthetic lipid A precursor and synthetic analogues with biosynthetic lipid A precursor and free lipid A.
Synthetic lipid A part structures corresponding structurally to a biosynthetic lipid A disaccharide precursor have been analyzed for endotoxic activity in several systems in vivo and in vitro. It was found that a synthetic beta-1,6-linked D-glucosamine disaccharide, which carries four molar equivalents of (R)-3-hydroxytetradecanoyl residues in positions 2, 3, 2' and 3' and phosphoryl groups in positions 1 and 4' (preparation 406), exhibited lethal toxicity, B lymphocyte mitogenicity, the capacity to engender prostaglandin formation in macrophages and to induce endotoxic tolerance, as well as serological lipid A antigenicity. On a weight basis, preparation 406 was of comparable activity to lipid A precursor and bacterial free lipid A. Preparation 406, like lipid A precursor, lacked, however, the ability to induce the local Shwartzman phenomenon and both preparations were of moderate pyrogenicity. Two further synthetic analogues which contained only one phosphoryl group (preparation 404 at C-4', preparation 405 at C-1) showed comparable or diminished activity depending on the test system employed, except in the capacity to inactivate complement where they exhibited, in contrast to preparation 406, significant activity. The results show that the endotoxic principle of lipopolysaccharides, as postulated previously is embedded in the lipid A component. Our results also suggest initial conclusions on the structural requirements for the expression of endotoxin activities.
Animals↗
Biochemical studies on structural and nonstructural proteins of the African green monkey B-lymphotropic papovavirus (LPV).
African green monkey (AGM) B-lymphotropic papovavirus (LPV) particles were separated from infected B-lymphoma BJA-B cells by neuraminidase and deoxycholate treatment and were further purified twice by CsCl density centrifugation. The SDS-PAGE analysis of virus particles banding at a density of 1.3510 g/ml and radioactively labeled by 125I revealed a major polypeptide of 40,000 and two minor polypeptides of 42,000 and 29,000. In addition, in infected BJA-B cells, all three viral structural polypeptides could be identified by immunoprecipitation. A nonstructural phosphopolypeptide of approximately 90,000 MW could be detected when sera against SDS-denatured SV40 T antigen or an AGM serum pool were used for immunoprecipitation. It is suggested that the 90K polypeptide of LPV represents an equivalent to T antigens of other papovaviruses.
Animals↗
Propagation of B-lymphotropic papovavirus (LPV) in human B-lymphoma cells and characterization of its DNA.
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B-Lymphocytes↗
B-lymphotropic papovavirus and possibility of infections in humans.
A novel member of the papovavirus group has been isolated from EBV-transformed African green monkey (AGM) B-lymphoblasts. The virus is characterized by its B-lymphotropic host range and has tentatively been named "lymphotropic papovavirus" (LPV). Seroepidemiological studies revealed that besides sera from African green monkeys a substantial proportion of human sera reacted with antigens of this virus. The specificity of this reaction observed in indirect immunofluorescence tests was underlined by immunoprecipitation (IP) and neutralization studies. By IP we demonstrated that AGM as well as human sera reacting in immunofluorescence with LPV antigens precipitated polypeptides of about 40 K. Those polypeptides were detected only in LPV-infected cultures. Sera of high reactivity in immunofluorescence tests also neutralized viral infectivity. These data suggest that an agent antigenetically closely related to AGM-LPV also infects part of the human population. The age distribution of the human antibody response revealed that a low percentage of sera reacted in age groups below 30 years. Thereafter, however, about 30% of all sera exhibited antibodies to LPV antigens. There was a slight increase in seroreactivity in sera from patients tested for infectious hepatitis when compared to non-selected human sera.
Age Factors↗
[The latex-IgM-test in virus diagnostic: determination of cytomegalovirus antibodies (author's transl)].
We examined the practical value of the Latex-IgM-test in diagnostic virology. Sera of patients suspected to have a cytomegalic infection were used for serological investigation. Cytomegalovirus produced in cell cultures was incubated with serum from patients. The resulting virus-antibody complex was removed by centrifugation and sensibilized Latex particles were added (anti IgG and anti IgM). The quantity and type of the bound antibody could be determined in this way. The sera were checked by parallel determination by another reliable method, the neutralization test. Immunoglobulins G and M separated and tested for antibodies. The usefulness of the Latex-IgM test for the routine diagnostics could be evaluated in the present study.
Antibodies, Viral↗
[Relative effectiveness of disinfectants against rotaviruses (author's transl)].
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Cell Survival↗
[Rotaviruses: origin of acute gastroenteriteis in newborns and infants].
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Adult↗