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PubMed2005

Complementary function of gamma delta T-lymphocytes and dendritic cells in the response to isopentenyl-pyrophosphate and lipopolysaccharide antigens.

Dendritic cells and gamma delta T-lymphocytes play a crucial role in the early response to microbial infections. Since both dendritic cells and gamma delta T-lymphocytes may be activated by specific microbial products, we analyzed their interplay in the presence of such respective ligands: lipopolysaccharide and isopentenyl-pyrophosphate. Activated gamma delta T-cells increased the maturational state of dendritic cells induced by lipopolysaccharide, increasing the expression of co-stimulatory and MHC class I and II molecules. IL-12 production by dendritic cells was strongly amplified in the presence of activated gamma delta T-cells and the Th1 polarization of naïve CD4(+) T-lymphocytes was significantly increased. On the other hand, dendritic cells enhanced gamma delta T-cell functions induced by isopentenyl-pyrophosphate and promote their IL-2 independent proliferation through CD86 contact. Altogether, dendritic cells and gamma delta T-cells exert a complementary function promoting an optimal immune response to non peptidic microbial antigens.

Antigens, CD↗

PubMed1983

Clinical significance of serum antibody responses to exotoxin A and type-specific lipopolysaccharides in patients with Pseudomonas aeruginosa infections.

Serum antibodies to Pseudomonas aeruginosa exotoxin A and immunotype-specific lipopolysaccharides were evaluated as diagnostic and prognostic markers in patients with Pseudomonas disease. Hemagglutination titers to exotoxin A were 1:1,024 or higher and/or showed a fourfold acute-to-convalescent increase in 17 of 25 (68 percent) patients infected with Pseudomonas compared with only one of seven (15 percent) colonized (p = 0.01) and two of 24 (8 percent) culture-negative patients (p less than 0.001). By comparison, hemagglutination titers to the lipopolysaccharide of patients' Pseudomonas isolates were 1:1,024 or higher or showed a fourfold increase in only four of 17 (24 percent) infected patients and in none of six (0 percent) colonized patients (p = 0.96). Serial antibody titers to exotoxin A provided serologic confirmation of invasive disease, distinguished infection from colonization, and, in the case of decreasing titers, indicated successful therapy. It is concluded that serum antibodies to exotoxin A are useful serologic markers for the clinical assessment of Pseudomonas infections in man.

ADP Ribose Transferases↗

PubMed1992

Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-alpha.

Quantification of intracellular and extracellular levels and production rates of reactive oxygen species is crucial to understanding their contribution to tissue pathophysiology. We measured basal rates of oxidant production and the activity of xanthine oxidase, proposed to be a key source of O2- and H2O2, in endothelial cells. Then we examined the influence of tumor necrosis factor-alpha and lipopolysaccharide on endothelial cell oxidant metabolism, in response to the proposal that these inflammatory mediators initiate vascular injury in part by stimulating endothelial xanthine oxidase-mediated production of O2- and H2O2. We determined a basal intracellular H2O2 concentration of 32.8 +/- 10.7 pM in cultured bovine aortic endothelial cells by kinetic analysis of aminotriazole-mediated inactivation of endogenous catalase. Catalase activity was 5.72 +/- 1.61 U/mg cell protein and glutathione peroxidase activity was much lower, 8.13 +/- 3.79 mU/mg protein. Only 0.48 +/- 0.18% of total glucose metabolism occurred via the pentose phosphate pathway. The rate of extracellular H2O2 release was 75 +/- 12 pmol.min-1.mg cell protein-1. Intracellular xanthine dehydrogenase/oxidase activity determined by pterin oxidation was 2.32 +/- 0.75 microU/mg with 47.1 +/- 11.7% in the oxidase form. Intracellular purine levels of 1.19 +/- 1.04 nmol hypoxanthine/mg protein, 0.13 +/- 0.17 nmol xanthine/mg protein, and undetectable uric acid were consistent with a low activity of xanthine dehydrogenase/oxidase. Exposure of endothelial cells to 1000 U/ml tumor necrosis factor (TNF) or 1 microgram/ml lipopolysaccharide (LPS) for 1-12 h did not alter basal endothelial cell oxidant production or xanthine dehydrogenase/oxidase activity. These results do not support a casual role for H2O2 in the direct endothelial toxicity of TNF and LPS.

Amitrole↗

PubMed1978

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide.

The synthesis of non-histone chromatin proteins and nucleoplasmic proteins has been followed during lipopolysaccharide-induced division and differentiation of murine B lymphocytes. Synthesis was measured by pulse labelling with [3H]leucine, extraction of proteins was under conditions designed to prevent proteolysis and analysis of labelled proteins was by polyacrylamide gel electrophoresis. The average specific activity of non-histone chromatin proteins increased 3-fold, to a maximum, after culture for 24 h with lipopolysaccharide. Comparison of the relative synthesis of individual proteins (stimulation index) reveals three distinct responses: (1) those in the largest group show low stimulation indices, generally less than two; (2) a group of four proteins have indices between 4 and 5; (3) two proteins (molecular weights 21 000 and 22 000) both show an index of 5 at 24 h rising to between 7 and 8 by 48 h when the average specific activity is falling, coinciding with the period of rapid differentiation to high rate IgM secretion. Additionaly at this time, a newly labelled protein (Mr = 36 500) appears in the nucleoplasm followed by a second protein (Mr = 63 000) appearing between 48 and 72 h. The patterns of change are consistent with an overall increase in non-histone chromatin proteins synthesis, necessary for cell division, with superimposed specific changes in synthesis of non-histone chromatin proteins which could be related to regulation of cell differentiation.

Animals↗

PubMed1978

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide. II. Phosphorylation.

Stimulation of mouse lymphocytes with the B lymphocyte specific mitogen lipopolysaccharide results in an increased rate of phosphorylation of non-histone chromatin proteins. An initial small increase in phosphorylation occurs during the first 2 h and a much larger increase after 24 h of culture with mitogen. The phosphorylated nuclear and cytoplasmic proteins were analysed by polyacrylamide gel electrophoresis and the stimulation index of each prominent peak measured. It was inferred that selective stimulation of the phosphorylation of individual proteins had occurred from: (1) the range of stimulation indices for different proteins, and (2) the appearance, after 8 h stimulation of an apparently newly phosphorylated non-histone chromatin protein of molecular weight 115 000. The pool size of ATP was monitored and showed only small changes during the first 24 h of exposure to lipopolysaccharide. Phosphatase activity was found to be associated with lymphocyte chromatin and nucleoplasm and may help to regulate the level of phosphorylation of non-histone chromatin proteins in vivo. To preserve phosphorylated proteins during their isolation phosphatase activity was inhibited by Na2MoO4. The selective changes in phosphorylation of nuclear proteins precede, and continue during, the stimulation of immunoglobulin and DNA synthesis. Our results are thus consistent with the hypothesis that phosphorylation of non-histone chromatin proteins plays a role in the regulation of gene expression in B lymphocytes.

Animals↗

PubMed1985

Lipopolysaccharide and polyribonucleotide activation of macrophages: implications for a natural triggering signal in tumor cell killing.

There is evidence that activation of macrophages for tumor cell killing can involve either two signals (interferon/lipopolysaccharide, for example) or one signal (lipopolysaccharide or double-stranded RNA, for example). We investigated the apparent one-signal activation of bone marrow-derived macrophages for P815 mastocytoma killing by treatment with lipopolysaccharide (LPS) or by the synthetic double-stranded polyribonucleotide polyinosinic acid-polycytidylic acid (poly I:C). We found that "direct" activation of macrophages by either LPS or poly I:C was still a two-signal process. Based on antibody neutralizations, the first signal was probably mediated by LPS or poly I:C induced alpha/beta interferon in the macrophage cultures, and the second signal was that of a direct effect of the LPS or poly I:C on the cell. The fact that poly I:C can provide the triggering signal for macrophage activation suggests a possible role for double-stranded RNA structures in macrophage triggering. Such double-stranded RNA requirements could be met by single-stranded RNAs that possess significant double-strandedness in their structures.

Animals↗

PubMed1991

Inhibition of gastric mucosal laminin receptor by Helicobacter pylori lipopolysaccharide.

Laminin receptor was isolated from gastric epithelial cell membrane by the procedure involving membrane solubilization with octylglucoside followed by affinity chromatography on laminin-coupled Sepharose. The receptor protein, eluted from the matrix with cation-free EDTA buffer, yielded on SDS-PAGE a single 67kDa band. After radioiodination, the protein was incorporated into liposomes which displayed specific affinity toward the laminin-coated surface. The binding of liposomal receptor to the laminin-coated surface was inhibited by lipopolysaccharide from H.pylori. The inhibitory effect was proportional to the concentration of lipopolysaccharide up to 50 micrograms/ml at which point a 96% decrease in the receptor binding occurred. It is suggested that a similar process may account for the loss of mucosal integrity in the pathogenesis of H. pylori associated gastric disease.

Animals↗

PubMed1992

Polyclonal antibody against an inducible form of nitric oxide synthase purified from the liver of rats treated with Propionibacterium acnes and lipopolysaccharide.

A polyclonal antibody was raised in the rabbit against an inducible form of nitric oxide (NO) synthase (EC 1.14.23) purified from the liver of rats with acute liver necrosis induced by i.v. administration of Propionibacterium acnes and lipopolysaccharide. The antibody immunoprecipitated NO synthase activities in the soluble extract of the liver from treated rats. Western blot analysis showed that the cytosols of the liver, lung and spleen from the treated rats but not from non-treated rats, and that of murine macrophages cultured in the presence of lipopolysaccharide and interferon-gamma, contained immunoreactive protein with a molecular weight of 125 kDa. The antibody, however, does not cross-react with a 150 kDa constitutive form of NO synthase present in the brain of rats, indicating that the inducible and constitutive enzymes are immunologically distinguishable.

Amino Acid Oxidoreductases↗

PubMed1992

Lipopolysaccharide induces prostaglandin H synthase-2 in alveolar macrophages.

Prostaglandin H synthase is a key enzyme in the formation of prostaglandins and thromboxane from arachidonic acid. The recent cloning of a second prostaglandin H synthase gene, prostaglandin H synthase-2, which is distinct from the classic prostaglandin H synthase-1 gene, may dramatically alter our concept of how cells regulate prostanoid formation. We have recently shown that the enhanced production of prostanoids by lipopolysaccharide-primed alveolar macrophages involves the induction of a novel prostaglandin H synthase (J. Biol. Chem., (1992), 267, 14547-14550). We report here that the novel PGH synthase induced by lipopolysaccharide in alveolar macrophages is prostaglandin H synthase-2.

Animals↗

PubMed1992

Down regulation of CD4 expression in cultured microglia by immunosuppressants and lipopolysaccharide.

Microglia, brain macrophages, are thought to be the primary target of HIV-1 infection in the brain, because they exclusively express the CD4 antigen which is effectively used for viral entry. The expression of CD4 mRNA in cultured microglia could be detected by the reverse-PCR method. Using this and immunohistochemical staining, we found that the immunosuppressants cyclosporin A and FK506 decreased CD4 expression in cultured murine microglia without causing any significant decrease in cell viability. FK506 was more potent than cyclosporin A. Lipopolysaccharide also decreased CD4 mRNA expression in microglia. The effects of immunosuppressants and lipopolysaccharide seemed to be specific for microglia since these chemicals did not alter the CD4 expression in lymphocytes or peritoneal macrophages. These agents, if modified to pass through the blood-brain barrier, may prevent viral spread of HIV-1 infection in the central nervous system and the AIDS-dementia complex.

Animals↗

PubMed1995

Effect of lipopolysaccharide on nitric oxide synthase activity in rat proximal tubules.

Renal proximal tubules isolated from the rat possess nitric oxide synthase (NOS) activity that is calcium/calmodulin dependent and stereoselectively inhibited by NG-monomethyl-arginine (NMMA). In the absence of added Ca2+ and calmodulin, activity was reduced 84 +/- 13% compared with the activity in the presence of 2 mM Ca2+ and 25 micrograms/mL calmodulin. Inhibition by EGTA (10 mM) was 95 +/- 5% and by calmidazolium (R24571, 250 microM) was 99 +/- 1%. Inhibition by L-NMMA (100 microM) was 78 +/- 13% and by D-NMMA (100 microM) was 7 +/- 7%. The majority of NOS activity was found in the soluble fraction. NOS activity in isolated proximal tubules was also examined 6 hr after a single i.v. injection of lipopolysaccharide. Activity was increased significantly (P < 0.05) in the soluble fraction by 2-fold [from 0.320 +/- 0.052 to 0.648 +/- 0.046 (nmol/mg protein/30 min)] and in the particulate fraction by 3-fold [from 0.081 +/- 0.030 to 0.256 +/- 0.034 (nmol/mg protein/30 min)]. All activities were inhibited by EGTA. These data demonstrate that proximal tubules express a calcium/calmodulin-dependent NOS activity that is increased in vivo by lipopolysaccharide.

Amino Acid Oxidoreductases↗

PubMed1983

The structure of an R-type oligosaccharide core obtained from some lipopolysaccharides of Neisseria meningitidis.

The main structure of a lipopolysaccharide R-type core oligosaccharide common to a number of different strains of Neisseria meningitidis has been elucidated. Methylation analysis, specific degradations, and nuclear magnetic resonance spectroscopic analysis of the dephosphorylated cores indicated that they all have the following structure. (formula see text) The determinants responsible for the L3, L7, and L9 meningococcal lipopolysaccharide serotypes are situated in this oligosaccharide.

Lipopolysaccharides↗

PubMed1983

Chemical and immunochemical studies on lipopolysaccharides from pyocin 103-sensitive and -resistant Neisseria gonorrhoeae.

The chemical and immunochemical properties of lipopolysaccharides (LPS) isolated from pyocin 103-sensitive and -resistant Neisseria gonorrheae were investigated. Marked differences were found in immunochemical behavior of LPS from pyocin-sensitive gonococcal strain JW31 and its isogenic pyocin-resistant variant JW31R. JW31 LPS readily precipitated wheat-germ agglutinin, soybean lectin, and rabbit anti-Streptococcus faecalis or horse anti-type 14 pneumococcal antibody. In contrast, JW31R LPS precipitated only soybean lectin. The combining-site specificity of anti-S. faecalis cross-precipitated by JW31 LPS, or type 14 pneumococcal capsular polysaccharide, was examined by hapten inhibition, and lactose found to be the most potent inhibitor. Horse anti-pneumococcal type 14 antibodies, cross-precipitated by JW31 LPS and streptococcal lactose polymer, exhibited heterogeneity with respect to combining site specificity. Gel filtration of LPS-derived core oligosaccharide showed both strain JW31 and JW31 R to possess R-type lipopolysaccharide with cores having a Mr approximately 1800. JW31R LPS contains more galactose but less hexosamine than JW31 LPS. Both JW31 and JW31R core oligosaccharides possess D-glucosamine and D-galactosamine, probably N-acetylated, as the only nonreducing end-groups, and (1 leads to 4)-linked D-glucose residues. Chemical data support immunochemical findings which indicate that lactose units occur as a structural feature of JW31 gonococcal LPS.

Bacteriocins↗

PubMed1983

Somatic antigens of Shigella: the structure of the polysaccharide chain of Shigella boydii type 2 lipopolysaccharide.

The structure of the polysaccharide chain of Shigella boydii type 2 lipopolysaccharide was established using mainly 13C-n.m.r. spectroscopy, partial hydrolysis, Smith degradation, and methylation analysis. The repeating unit of the polysaccharide was concluded to be a branched hexasaccharide, as follows: (formula in text). Acetaldehyde was detected in the hyrolysate of the lipopolysaccharide, but no evidence was obtained to indicate that acetaldehyde is located in the polysaccharide moiety.

Carbohydrate Conformation↗

PubMed1983

Hydrolytic release, and identification by g.l.c.-m.s., of 3-deoxy-D-manno-2-octulosonic acid in the lipopolysaccharides isolated from bacteria of the Vibrionaceae.

The identification of the peracetylated methyl glycosides of 3-deoxy-D-manno-2-octulosonic acid (KDO) methyl esters was achieved by g.l.c.-m.s. These peracetylated methyl glycoside methyl esters were obtained from fully acetylated lipopolysaccharides and core oligosaccharides of representative strains of the Vibrionaceae family by the following sequence of mild reactions: acetolysis, methanolysis, and acetylation. KDO was shown to be present in all of the lipopolysaccharides (LPS), a result in direct contrast to the generally accepted view of the absence of this compound in LPS from this family of bacteria.

Gas Chromatography-Mass Spectrometry↗

PubMed1988

A core oligosaccharide component from the lipopolysaccharide of Rhizobium trifolii ANU843.

The major oligosaccharide from the core region of the lipopolysaccharide from R. trifolii ANU843 was isolated and its structure determined. It is a trisaccharide consisting of two galacturonic acid residues and one 3-deoxy-D-manno-2-octulosonic acid (KDO) residue. The two galacturonic acid residues are terminally linked alpha to the C-4 and C-7 atoms of KDO. This structure was determined through use of 1H- and 13C-n.m.r. spectroscopy, f.a.b.-m.s., and g.l.c.-m.s. techniques. This oligosaccharide had not previously been reported to be present in the lipopolysaccharides from Gram-negative bacteria.

Carbohydrate Conformation↗

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↗

PubMed1989

Elucidation of the structure of the Pasteurella haemolytica serotype T10 lipopolysaccharide O-antigen by n.m.r. spectroscopy.

The aqueous-phase lipopolysaccharide isolated from Pasteurella haemolytica serotype T10 cells by the phenol-water extraction method was found to be S-type lipopolysaccharide which possessed O-antigenic polysaccharide chains composed only of D-galactose residues. Structural analysis of the O-polysaccharide, using a combination of 1D and 2D 1H- and 13C-n.m.r. methods, led to the identification of the disaccharide repeating-unit as [----3)-alpha-D-Galp-(1----3)-beta-D-Galf-(1----]n. The serological cross-reactivity between P. haemolytica serotypes T4 and T10 can now be related to the structural similarity of the antigenic LPS O-polysaccharides.

Antigens, Bacterial↗