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Biomedical subjects

H Brade

Publications and source records attributed to H Brade.

At least 145 records · Page 8Linked to original sources
PubMed1990

Suppressive effect of lipid A partial structures on lipopolysaccharide or lipid A-induced release of interleukin 1 by human monocytes.

Experiments were designed to investigate the significance of lipid A partial structures, precursor Ia (compound 406), and lipid X (compound 401) to serve as antagonists of interleukin 1 (IL-1) release from human mononuclear cells and monocytes induced by lipopolysaccharide (LPS, endotoxin) of Salmonella abortus equi or synthetic Escherichia coli lipid A (compound 506). A definite inhibition mediated by lipid A partial structures on IL-1 release induced by LPS or lipid A was found in repeated experiments. The inhibitory effect was exerted not only on IL-1 release, but also on IL-1 peptide synthesis at the intracellular level. The results also show that lipid A partial structures have suppressive effects even when added 1-4 h after LPS or lipid A. We conclude from these results that lipid A partial structures (precursor Ia and lipid X) have potent immunomodulatory effects on LPS- and lipid A-induced IL-1 release and may become useful reagents to study the mechanism of interaction of LPS and lipid A with cells of the immune system.

Carbohydrate Sequence↗
PubMed1990

Characterization of murine monoclonal and murine, rabbit, and human polyclonal antibodies against chlamydial lipopolysaccharide.

Murine monoclonal and rabbit, murine, and human polyclonal antibodies against chlamydial lipopolysaccharide (LPS) were characterized by the passive hemolysis and passive hemolysis inhibition assays and by absorption experiments with LPSs of Chlamydia psittaci, Chlamydia trachomatis, and a recombinant strain of Salmonella minnesota Re (r595-207) expressing the chlamydia-specific LPS epitope, as well as natural and synthetic partial structures of chlamydial LPS. Eleven monoclonal antibodies of the immunoglobulin M and G classes were characterized as chlamydia-specific by their failure to react with Re-type LPS, binding to a similar epitope for which the trisaccharide alpha-3-deoxy-D-manno-2-octulosonic acid (KDO)-(2-8)-alpha-KDO-(2-4)-alpha-KDO was an absolute prerequisite. For optimal binding, parts of the lipid A moiety were also involved; however, phosphoryl and ester-linked acyl groups and the reducing glucosamine residue of lipid A were dispensable. A similar antibody specificity was detected in lapine and murine hyperimmune sera after immunization with chlamydia, in addition to those recognizing more complex (e.g., those requiring the presence of phosphoryl residues) and less complex epitopes. Among the latter were those cross-reacting with Re-type LPS, which could be removed by absorption. The titers of different antibody specificities, in particular the ratio of chlamydia-specific to cross-reactive antibodies, present in murine polyclonal antisera depended on the immunization protocol. The preferential formation of chlamydia-specific antibodies was observed after immunization with liposome-incorporated immunogens. Human sera from patients with suspected genital chlamydial infections were also found to contain chlamydia-specific and cross-reactive antibodies, the latter of which could be removed by absorption with Re-type LPS.

Antibodies, Bacterial↗
PubMed1989

Determination of the epitope specificity of monoclonal antibodies against the inner core region of bacterial lipopolysaccharides by use of 3-deoxy-D-manno-octulosonate-containing synthetic antigens.

Partial structures of enterobacterial lipopolysaccharides (LPS) of the Rechemotype, consisting of lipid A and 3-deoxy-D-manno-2-octulosonic acid (Kdo), as well as oligosaccharides and derivative of Kdo were synthesized and used to characterize the epitope specificity of monoclonal antibodies against Re-mutant LPS. High-molecular-weight antigens, obtained after copolymerization of the respective allyl glycosides with acrylamide, and the haptenic oligosaccharides were used in immunoprecipitation, immune hemolysis, and in inhibition assays. A monoclonal antibody (clone 20, igM) recognizing a terminal Kdop group was shown to require for its binding the alpha-anomeric configuration and OH-4 and OH-5 groups, whereas the C-7 - C-8 chain was of minor importance. Another monoclonal antibody (clone 25, IgG3), which recognizes a (2--4)-linked Kdo disaccharide, was shown to require for its binding the alpha-anomeric configuration of both residues. The isomer having a reducing beta-Kdo residue was significantly less active, and that with a terminal beta-Kdo group was completely inactive. The OH-5 group of the reducing residue was shown to be not important for the specificity of this antibody, since it could be replaced by a hydrogen atom without loss of serological reactivity. The alpha-(2--8)-linked Kdo disaccharide was strongly cross-reactive with its (2--4)-linked isomer. The antibody recognized also parts of the 2-amino-2-deoxy-D-glucose backbone of lipid A.

Animals↗
PubMed1989

Synthesis of trisaccharides containing 3-deoxy-D-manno-2-octulosonic acid residues related to the KDO-region of enterobacterial lipopolysaccharides.

Glycosylation of methyl (allyl 7,8-O-carbonyl-3-deoxy-alpha-D-manno-2-octulo-pyranosid)o nate with an alpha-(2----4) linked per-O-acetylated KDO-disaccharide bromide derivative under Helferich conditions afforded a 2:1 mixture of the alpha- and beta-linked trisaccharide derivatives in 50% yield. Removal of the protecting groups gave sodium O-[sodium (3-deoxy-alpha-D-manno-2-octulopyranosyl)onate]-(2----4)-O-[ sodium (3-deoxy-alpha- and -beta-D-manno-2-octulopyranosyl)onate]-(2----4)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate. Radical copolymerization of the allyl glycosides afforded artificial antigens, suitable for defining antibody specificities directed against the KDO-region of enterobacterial lipopolysaccharides.

Carbohydrate Sequence↗
PubMed1989

IL-1 induction-capacity of defined lipopolysaccharide partial structures.

Natural and synthetic lipid A as well as natural and synthetic oligosaccharide partial structures of LPS were examined in dose-response experiments to define the minimal structure necessary for IL-1 induction and release in cultures of human mononuclear cells. Wild type LPS (S. abortus equi) and rough mutant LPS was active in minimal-doses of 1 to 100 pg/ml, whereas synthetic heptaacyl and hexaacyl lipid A (Salmonella minnesota and Escherichia coli lipid A, respectively) induced IL-1 in minimal-doses of 100 to 1,000 pg/ml and 10 to 1,000 pg/ml, respectively. Nanogram amounts (0.1 to 10 ng/ml) of synthetic monodephospho partial structures of E. coli lipid A were necessary for IL-1 induction. Synthetic pentaacyl partial structures induced IL-1 very weakly. Synthetic tetraacyl and bisacyl partial structures lacking non-hydroxylated fatty acids were not active. Compared to LPS million-fold higher doses of natural and synthetic 3-deoxy-D-manno-octulosonic acid containing core oligosaccharides were necessary for IL-1 induction. Dose-response investigations with LPS and natural or synthetic partial structures established the following hierarchy in IL-1 induction-capacity: LPS greater than lipid A much greater than lipid A partial structures greater than core oligosaccharides greater than oligoacyl lipid A. Lipid A was shown here to be the portion of LPS mainly responsible for induction of IL-1 activity. The high potency of lipid A in inducing IL-1 release and the failure of the precursor Ia of lipid A to induce IL-1 production and release was also observed measuring intracellular IL-1 activity after freeze-thawing the cells. Levels of IL-1 beta mRNA in extracts of mononuclear cells correlated with biologic activity. In co-incubation experiments, precursor Ia of lipid A produced dose-dependent inhibition of production and release of IL-1 activity induced by lipid A or LPS, but not by Staphylococcus epidermidis or PHA. Incubation of cells with precursor Ia for 1h, followed by a medium change and further incubation of stimulus without precursor Ia of lipid A also resulted in inhibition. We conclude that lipid A is the main portion of LPS responsible for induction of IL-1, and that specific activation- and/or binding-mechanisms are involved in stimulation of cells with LPS and/or lipid A.

Acinetobacter↗
PubMed1989

Induction of tumor necrosis factor-alpha release by lipopolysaccharide and defined lipopolysaccharide partial structures.

We have investigated the release of tumor necrosis factor-alpha (TNF-alpha) by human mononuclear cells (MNC) and isolated human monocytes/macrophages stimulated with S- and R-form lipopolysaccharide (LPS), natural lipid A, and natural and synthetic partial structures thereof. We found that LPS of Salmonella minnesota (S. min.) Rb2, which represents a partial structure of wildtype LPS of Salmonella abortus equi (S.a.e.) lacking the O-chain and parts of the outer core region, was the most active inducer of all substances tested, even more active than the wildtype LPS. Lipid A also induced the production of TNF-alpha by monocytes/macrophages but was less active than wildtype LPS. The natural Escherichia coli (E. coli) type hexaacyl lipid A (compound 506) was more active than the natural S. min. type heptaacyl lipid A (compound 516). The 1- and 4'-monodephospho partial structures (compounds 505 and 504) of E. coli lipid A were less active and represented the smallest structures tested that were able to induce TNF-alpha release by monocytes/macrophages. Synthetic tetraacyl lipid A precursor Ia of E. coli lipid A, lacking non-hydroxylated fatty acids (compound 406), and the monosaccharide precursor lipid X did not induce the release of TNF-alpha in MNC or isolated monocytes/macrophages. This might indicate that the ability of a lipid A structure to induce the release of TNF-alpha is closely connected with the conditions to be at least hexaacylated and/or to contain hydroxylated fatty acids. These results demonstrate a structure-dependent hierarchy of LPS and natural or synthetic partial structures in their capacity of inducing TNF-alpha release by monocytes/macrophages.

Humans↗
PubMed1989

Epitope specificities of murine monoclonal and rabbit polyclonal antibodies against enterobacterial lipopolysaccharides of the Re chemotype.

Murine monoclonal and rabbit polyclonal antibodies raised against the lipopolysaccharides (LPS) of Re mutants of Salmonella minnesota, Proteus mirabilis, and Escherichia coli were serologically characterized. Using natural Re LPS and natural and synthetic partial structures thereof, representing the 3-deoxy-D-manno-2-octulosonic acid (KDO) or lipid A region or both, the epitope specificities of four monoclonal antibodies were defined. Clones 20 (immunoglobulin M [IgM]) and 25 (IgG3) recognize a terminal alpha-pyranosidically linked KDO monosaccharide residue and the alpha-2,4-linked KDO disaccharide, respectively, as the immunodominant group. Therefore, these two antibodies are core antibodies which do not require the presence of lipid A constituents for binding. The minimal structure enabling binding of clone 17 (IgG2b) is a pseudotetrasaccharide of the sequence alpha-KDO-(2----4)-alpha-KDO-(2----6)-beta-glucosamine-(1----6)- glucosaminitol with two amide-linked 3-hydroxytetradecanoic acid residues. The smallest structure with which clone 22 (IgG3) reacted was de-O-acylated Re LPS. Therefore, clones 17 and 22 are LPS antibodies requiring both the lipid A and the KDO region for binding. Phosphoryl residues of the lipid A moiety in Re LPS are dispensable for the reaction with clone 17, whereas they are necessary for that with clone 22. These four different antibody types were also detected in polyclonal rabbit antisera and could be distinguished from each other by absorption experiments. It was found that type 20 and 25 antibodies either were not present or were present only in small amounts and that the majority of the antibodies were of types 17 and 22. From these data, we conclude that the immunodominant structures of Re LPS comprise both the KDO and lipid A domains.

Animals↗
PubMed1989

Interleukin 1 and tumor necrosis factor: studies on the induction by lipopolysaccharide partial structures.

Minimal concentration ranges of lipopolysaccharides, natural and synthetic lipid As, and partial structures were established for the induction of release of IL-1 and TNF from human MNC. LPS was more potent than lipid A. Partial structures of lipid A lacking nonhydroxylated fatty acids were inactive, but in combination with LPS or lipid A exhibited time-dependent synergistic or antagonistic effects.

Glycolipids↗
PubMed1988

Synthesis of a trisaccharide of 3-deoxy-D-manno-2-octulopyranosylonic acid (KDO) residues related to the genus-specific lipopolysaccharide epitope of Chlamydia.

The disaccharides, O-(sodium 3-deoxy-alpha- and -beta-D-manno-2-octulopyranosylonate)-(2----8)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate, were prepared via glycosylation of methyl (allyl 4,5,7-tri-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosid)onat e with methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-D-manno-2-octulopyranosyl bromide)onate under Helferich and Koenigs-Knorr conditions, respectively. Based on g.l.c.-m.s. data of the alpha- and beta-(2----8)-linked disaccharide derivatives, obtained after carbonyl- and carboxyl-group reduction, followed by methylation, the alpha-anomeric configuration was assigned to the terminal KDO-residue in the KDO-region of Chlamydial lipopolysaccharide. The trisaccharide O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----8)-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----4)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate was obtained via block synthesis using an alpha-(2----8)-linked disaccharide bromide derivative as the glycosyl donor. Copolymerization of the allyl glycosides with acrylamide gave water-soluble macromolecular antigens, suitable for defining epitope specificities of monoclonal antibodies directed against Chlamydial LPS.

Acrylamide↗
PubMed1988

Chemical structure of the lipopolysaccharide of Haemophilus influenzae strain I-69 Rd-/b+. Description of a novel deep-rough chemotype.

The chemical structure of the lipopolysaccharide of a deep-rough mutant (strain I-69 Rd-/b+) of Haemophilus influenzae was investigated. The hydrophilic backbone of lipid A was shown to consist of a beta-(1',6)-linked D-glucosamine disaccharide with phosphate groups at C-1 of the reducing D-glucosamine and at C-4' of the non-reducing one. Four molecules of (R)-3-hydroxytetradecanoic acid were found directly linked to the lipid A backbone, two by amide and two by ester linkage (positions 2,2' and 3,3', respectively). Laser-desorption mass spectrometry showed that both 3-hydroxytetradecanoic acids linked to the non-reducing glucosamine carry tetradecanoic acid at their 3-hydroxyl group, so that altogether six molecules of fatty acid are present in lipid A. The lipopolysaccharide was the first described to contain only one sugar unit linked to lipid A. This, sugar in accordance with a previous report [Zamze et al. (1987) Biochem. J. 245, 583-587], was shown to be a dOclA phosphate. The phosphate group was found at position 4, but the analytical procedures employed (permethylation and methanolysis followed by gas-liquid chromatography/mass spectrometry) also revealed dOclA 5-phosphate. Since a cyclic 4,5-phosphate could be ruled out by 31P-NMR, we conclude that, in this lipopolysaccharide, a mixture of dOclA 4- and 5-phosphate is present. By methylation analysis of the dephosphorylated, deacylated and reduced lipopolysaccharide the attachment site of the dOclA was assigned to position C-6' of the non-reducing glucosamine of lipid A. The anomeric linkages present in the lipopolysaccharide were assessed by 1H-NMR and 13C-NMR of deacylated lipopolysaccharide. The saccharide backbone of this Haemophilus influenzae lipopolysaccharide possesses the following structure: (Formula; see text)

Gas Chromatography-Mass Spectrometry↗
PubMed1988

Structure of the K74 antigen from Escherichia coli O44:K74:H18, a capsular polysaccharide containing furanosidic beta-KDO residues.

The structure of the capsular K74 antigen of E. coli H702c (O44:K74:H18) was elucidated by determination of the composition, 1H- and 13C-n.m.r. and c.d. spectroscopy, periodate oxidation, and methylation analysis of the polysaccharide and of a trisaccharide obtained by mild acid hydrolysis. The K74 antigen has the repeating unit----3)-beta-D-Ribf-(1----2)-beta-D-Ribf-(1----6)-beta-++ +KDOf-(2----. Of the repeating units, approximately 65% are O-acetylated, most probably at C-2 of the 3-linked ribose.

Acetylation↗
PubMed1988

Isolation and chemical analysis of 7-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-heptose as a constituent of the lipopolysaccharides of the UDP-galactose epimerase-less mutant J-5 of Escherichia coli and Vibrio cholerae.

Methyl 7-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-hepto+ ++ pyranoside (1) was released from the lipopolysaccharide of the UDP-galactose epimerase-less mutant J-5 of Escherichia coli by methanolysis and isolated by high-voltage paper electrophoresis. Its chemical structure was determined by chemical analysis, deamination with nitrous acid, g.1.c.-m.s., and 1H- and 13C-n.m.r. spectroscopy performed on its acetylated derivative. The disaccharide moiety of 1 was also detected in lipopolysaccharides of Vibrio cholerae.

Carbohydrate Epimerases↗
PubMed1988

Structural studies on the core and lipid A region of a 4-amino-L-arabinose-lacking Rc-type mutant of Proteus mirabilis.

The structure of the 4-amino-L-arabinose-lacking lipopolysaccharide of the Proteus mirabilis Rc-type mutant R4, derived from wild-type O28, was elucidated. The lipopolysaccharide core structure has previously been partially characterized. The linkage between heptose and deoxyoctulosonic acid(dOclA) is now reported, as well as the structure of the lipid A moiety of this mutant strain. Besides the tentative identification of an alpha-linked glucosamine disaccharide in the lipid A backbone accompanying the usual beta 1----6-linked glucosamine-disaccharide, the only significant structural variation to previous studies was the lack of substitution of the C-4' phosphate by 4-amino-L-arabinose. In addition, the substitution at C-8 of one dOclA unit by 4-amino-L-arabinose, previously reported for the R45 mutant of P. mirabilis 1959, is lacking in this R mutant. Also in addition to previous findings, the terminal unit of heptose was found to be substituted at C-7 with phosphorylethanolamine (PEtN) and not only with phosphate, although this substitution is not complete as demonstrated by the relevant signals in 31P-NMR. Additional studies with the wild-type strain P. mirabilis O28 revealed the presence of 4-amino-L-arabinose in both the core and the lipid A regions suggesting that the R4 mutant is defective in the biosynthesis of this amino sugar rather than in its transfer. Otherwise the lipid A regions of the mutant and the wild-type strain show no structural differences. The following formula is proposed for the lipopolysaccharide of 4-amino-L-arabinose-lacking mutant R4/O28 P. mirabilis: (Formula; see text)

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Prevention of lethal endotoxemia in actinomycin D-sensitized mice by incubation of Salmonella minnesota R595 lipopolysaccharide with monoclonal antibodies to R595.

Murine monoclonal antibodies reacting with lipopolysaccharide (LPS) of Salmonella minnesota strain R595 (Re chemotype) were prepared, and tested for their ability to protect actinomycin D-sensitized mice against lethal endotoxemia. Protection was found with some antibodies up to a 90-fold increase in LD50, whereas others exhibited no protection. The various protective antibodies did not all bind to the same epitope. The same applied for non-protective clones. Protective and non-protective clones could not be discriminated by ELISA. One protective monoclonal antibody (clone 20) was specific for ketodeoxyoctonate, a structural element common to various LPS. These findings show that the involvement of lipid A in the binding site of monoclonal antibodies is no prerequisite for protection.

Animals↗
PubMed1988

Mitogenic activities of synthetic Escherichia coli lipid A and a synthetic partial structure (tripalmitoyl pentapeptide) of E. coli lipoprotein.

Synthetic Escherichia coli lipid A and synthetic S-[2,3-bis-(palmitoyloxy)propyl]-N-palmitoylpentapeptide (tripalmitoyl pentapeptide [TPP]), representing the mitogenically active principles of bacterial lipopolysaccharide (LPS) and lipoprotein, respectively, were compared for their mitogenic activities on splenocytes of LPS responder (BALB/c) and LPS-low-responder (C3H/HeJ) mice. Whereas lipid A was active only in LPS-responder mice, TPP resulted in mitogenic activation of B lymphocytes from both LPS-responder and LPS-low-responder mice. When the mitogens were added simultaneously, mainly additive effects of both activators were observed. The data suggest that two different B-lymphocyte populations are responding to these two mitogens.

Animals↗