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  • 1
    ISSN: 1439-0973
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Zusammenfassung Eine einfache, empfindliche und schnelle ELISA-Methode zur Bestimmung und Charakterisierung von Anti-Lipoid-A-Antikörpern wurde ausgearbeitet. Zum Beschichten der ELISA-Platten wird eine Lösung des Antigens in Chloroform/Äthanol (1:9 [v/v]) in die Löcher eingetragen und das Lösungsmittel dann mit einem Strom warmer Luft verdampft. Damit wird ein hoher Beschichtungsgrad erreicht, wodurch die Bestimmung sehr empfindlich wird. Gleichzeitig wird die unspezifische Adsorptionskapazität der Platten für Immunglobuline erniedrigt, so daß sich eine Blockierung unspezifischer Bindungsstellen erübrigt, wie sie üblicherweise mit BSA oder Gelatine durchgeführt wird. Zum Auffinden von Lipoid-A-Antikörpern sind 0,2 µg des entsprechenden Antigens pro Plattenloch bei Immunseren, und 1–2 µg bei Nicht-Immunseren und monoklonalen Antikörpern optimal. Die hier beschriebene Beschichtungsmethode kann generell bei Antigenen angewandt werden, die in organischen Lösungen direkt lösbar sind, wie zum Beispiel bei Re-LPS und Gangliosiden. Des weiteren ist diese Methode für Antigene geeignet, die zwar primär in Chloroform/Äthanol unlöslich sind, die aber nach Lösen in einem geeigneten wäßrigen Medium in die Chloroform/Äthanol-Mischung überführt werden können. Dies wird an mehreren Beispielen gezeigt (LPS der Klassen Ra-Rd und S-formen, sowie Lipoteichonsäure).
    Notes: Summary A simple, sensitive and rapid ELISA method for the quantification and characterization of antibodies to lipid A has been developed, which can also be applied to other hydrophobic antigens. For coating, antigens were applied to the wells of ELISA plates as solutions in a mixture of chloroform and ethanol 1:9 (v/v), and the solvent evaporated in a stream of warm air. Under these conditions a high coating efficiency was achieved, which made the assay highly sensitive. The use of the above organic solvent considerably reduced the nonspecific adsorption of immunoglobulins to the solid phase, making the usual blocking of unspecific binding sites with BSA or gelatine unnecessary. For screening of lipid A antibodies in the sera of immunized animals, coating with 0.2 µg of the corresponding antigen per well was found to be suitable. For optimal measurement of antibodies in pre-immune sera, sera of healthy human donors, and of monoclonal antibodies, higher amounts of antigen (1–2 µg/well) had to be used. The coating method described here proved excellent also for other antigens directly soluble in organic solvents, such as Re-lipopolysaccharide (LPS) or gangliosides. In addition, the method was successfully applied to less hydrophobic antigens, such as LPS of the classes Ra to Rd and S forms, and lipoteichoic acid. These could be brought into solution in chloroform/ethanol by diluting their aqueous solutions with a large volume of the organic mixture.
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  • 2
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] An invading pathogen must be held in check by the innate immune system until a specific immune response can be mounted. In the case of Gram-negative bacteria, the principal stimulator of the innate immune system is lipopolysaccharide (LPS), a component of the bacterial outer membrane. In vitro, ...
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  • 3
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] The IL-6 gene was disrupted in the second exon (first coding exon) by insertion of a neor cassette (Fig. \a). FI mice (C57Bl/6x 129 Sv) heterozygous for the mutation (IL-6+7) were interbred to obtain mice homozygous for the mutation. Loss of the wild-type alleles and IL-6 messenger RNA was ...
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  • 4
    ISSN: 1432-1831
    Keywords: Key words Cutaneous leishmaniasis ; Lipopolysaccharide responder ; Lipopolysaccharide nonresponder ; Cytokine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The course of cutaneous leishmaniasis was examined in mice from two genetically closely related strains, C57BL/10ScCr (Cr) and C57BL/10ScSn (Sn). Sn mice are able to heal Leishmania major infections, while Cr mice are unable to heal. The cutaneous lesions of the Cr mice progressed continuously and the increase in lesion size was paralleled by an unrestricted growth of the parasites in vivo. Cr mice, in contrast to their Sn counterparts, are highly resistant to all effects of lipopolysaccharide (LPS). The nonhealing L. major infection in Cr mice is in sharp contrast to the course of infection in another endotoxin-nonresponder mouse strain, C3H/HeJ, which heal infections with L. major. Cr mice exhibit, in addition to the defective LPS responsiveness, an impaired interferon-γ (IFN-γ) response after infection with a variety of microorganisms. The insufficient activation of parasitized macrophages to kill intracellular L. major could be due to the inability of splenocytes from infected Cr mice to secrete IFN-γ upon restimulation with L. major. IFN-γ is essential for the efficient activation of parasitized macrophages to kill intracellular L. major by producing nitric oxide (NO). Although bone marrow-derived Cr macrophages do not produce NO in response to LPS, both Sn and Cr macrophages release NO upon stimulation with IFN-γ and tumor necrosis factor, indicating that they are responsive to activation by these cytokines.
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  • 5
    ISSN: 1432-072X
    Keywords: Key wordsRhodospirillum salinarum ; Lipopolysaccharide ; Mixed lipid A ; 2 ; 3-Diamino-2 ; 3-dideoxy-d-glucose ; 4-O-Methyl- ; galacturonic acid ; Halophilic bacteria ; Lethal toxicity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The structural elucidation of lipid A of the cell wall lipopolysaccharide (LPS) of Rhodospirillum salinarum 40 by chemical methods and laser desorption mass spectrometry revealed the presence of a mixed lipid A composed of three different 1,4′bisphosphorylated β(1→6)-linked backbone hexosaminyl-hexosamine disaccharides, i.e. those composed of GlcN→GlcN, 2,3-diamino-2,3-dideoxy-d-Glc-(DAG)→DAG, and DAG→GlcN. Lipid A of R. salinarum contained preferentially 3-OH-18 : 0 and 3-OH-14 : 0 as amide-linked and cisΔ11–18 : 1 and c19 : 0 as ester-linked fatty acids. The mass spectra of the liberated acyl-oxyacyl residues proved the concomitant presence of 3-O-(cisΔ11–18 : 1)–18 : 0 and 3-O-(c19 : 0)-14 : 0 as the predominating diesters in this mixed lipid A. The glycosidically linked and the ester-linked phosphate groups of the backbone disaccharide were neither substituted by ethanolamine, phosphorylethanolamine, nor by 4-amino-4-deoxy-l-arabinose, in contrast to most of the enterobacterial lipid As. In the core oligosaccharide fraction, a HexA (1→4)HexA(1→5)Kdo-trisaccharide was identified by methylation analysis. The terminal HexA (hexuronic acid) is possibly 4-OMe-GalA, a component described here as an LPS constituent for the first time. LPS of R. salinarum showed a lethality in C57BL/10 ScSN (LPS-responder)-mice) of an order of 10–1–10–2 of that reported for Salmonella abortus equi LPS, and it was also capable of inducing TNFα and IL6 in macrophages of C57BL/10ScSN mice.
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