C. Galanos

Chemical structure of E. Coli Lipid A: Linkage site of acyl groups in the disaccharide backbone

Abstract The structure of the lipid A component of E. coli lippopolysaccharide was determined by means of chemical and 2D-NMR methods unequivocally to be a glucosamine β(1′-6)-disaccharide 1,4′-diphosphate acylated at the two hydroxyl (positions C-3 and −3′) and the two amino groups.

Chemical structure of escherichia coli lipid A

The chemical structure of E. coli lipid A was elucidated to be 2 by determination of the nature of the individual acyl groups bound to the two hydroxyl groups in positions 3,3′ and the two amino groups of the D-glucosamine disaccharide phosphate backbone.

ELISA for antibodies to lipid A, lipopolysaccharides and other hydrophobic antigens

SummaryA 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.ZusammenfassungEine 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).

The Story of Bacterial Endotoxin

SUMMARY After the introduction of the term bacterial endotoxin early this century by Richard Pfeiffer, about 80 years elapsed until this biologically omnipotent and highly active principle could be chemically clearly defined. Endotoxin was found to reside in the lipopolysaccharide (LPS) of the bacterial cell envelope; it was then pinpointed as the lipid component of LPS, called lipid A . This lipid has an unusual structure - with certain variations on a characteristic backbone - which does not seem to exist anywhere else in nature. After the elucidation of the structure of some lipid As ( E. coli , Salmonella and others) it became more recently possible to identify the pathway of lipid A biosynthesis and to fully synthesize lipid A of E . coli . The synthetic product is chemically, physico-chemically and biologically identical with its natural lipid A prototype. With these achievements the old question as to the structure(s) responsible for endotoxicity has been solved - and a new chapter of research on endotoxin is open. The following review is an extended version of a lecture given by O.W. on May 7, 1985 at the 3rd International Conference on Immunopharmacology in Florence, Italy.

Identification of endotoxin-positive cells in the rat lung during shock

Following an intravenous administration into rats of a shock-inducing dose of endotoxin (2 mg) the lipopolysaccharide (LPS) was demonstrated immunohistochemically (light and electron microscopy) and determined quantitatively (radio-labelled LPS) in the lung tissue and in isolated alveolar macrophages. At different times after LPS injection morphological investigations of the pulmonary tissue and alveolar macrophages were carried out. One hour after endotoxin treatment 3% of the alveolar macrophages were already LPS-positive. The maximum extent of the immunoperoxidase reaction for endotoxin (100% cells involved) was observed on day 3, the vast majority (98%) of the alveolar macrophages being LPS-positive still on day 14. 0.9% of the injected radio-labelled LPS preparation was found to be associated with lung tissue on day 3. By this time 0.173 µg LPS/106 alveolar macrophages was detected. During the time of ultrastructural investigation endotoxin appeared in the lung only within cells. By their high capacity for storing endotoxin and their numerical superiority the mononuclear phagocytes are the leading LPS-positive cells in the lung, although granulocytes, endothelial cells, and alveolar epithelial cells were sometimes also involved. The accumulation of a high percentage of activated macrophages in the lung seen in the late stage of shock could represent at least one of the main factors leading to damage of pulmonary tissue. The correlation between appearance of LPS-positive macrophages and histological signs of lung tissue injury in the present investigation is striking.

The role of macrophages in the uptake of endotoxin by the mouse liver.

SummaryThe purpose of this investigation was to analyse the macrophage subpopulations involved in the uptake of endotoxin in the liver.The results show that in normal B10.D2 mice the liver macrophages constitute a heterogeneous population of cells which, depending on their state of differentiation, are distinguished by their differential distribution in the liver acinus and by their ability to phagocytose latex.Following the intravenous administration of endotoxin (lipopolysaccharide=LPS) from Salmonella abortus equi, endotoxin-carrying non-parenchymal cells of the liver (NPLC) were investigated immunohistochemically (in situ) and immunocytochemically (after isolation) between 1 h and 14 days after the injection. The endotoxin content of the blood and of isolated NPLC was also determined, using radioactively labeled LPS.Following LPS injection, the total number of macrophages in the liver increased, reaching a maximum after 3 days. There was a striking increase in the ratio of mature to immature macrophages. After day 3, the number of macrophages decreased again, returning to the pre-injection values by day 14.1 h after the administration of LPS, 41% of the isolated NPLC were already endotoxin-positive, a percentage which remained constant until the 3rd day. Thereafter, the number of LPS-bearing cells increased to a maximum of about 52% on the 5th day. This increase mostly involved macrophages which had taken up endotoxin. Concurrent with these changes there was a threefold increase in radioactively-labeled LPS from the 7th h to the 5th day after injection. Since the increase in the number of endotoxin-carrying macrophages in the liver occurred after endotoxin had been eliminated from the circulation, the increase must have resulted from endotoxin-carrying cells migrating into the liver from other organs.

Haemorrhagic tumour necrosis following endotoxin administration. I. Communication: morphological investigation on endotoxin-induced necrosis of the methylcholanthrene (Meth A) tumour in the mouse.

Endotoxin induced necrosis of the Meth A mouse tumour has been investigated using macroscopic, histological and ultrastructural examination methods. On the 8th day after tumour cell transplantation, the animals received a relatively non-toxic dose of the Salmonella abortus equi endotoxin intravenously. The natural history of the tumour necrosis took the following course: 1. The earliest morphological changes could be seen with the electron microscope 90 min after administration of the endotoxin, and were seen as an interstitial oedema with separation of the tumour cells. 2. Haemmorrhagic necrosis of the tumour was complete 4 hours after injection, and could be easily recognized with the naked eye. 3. Rejection of the necrotic malignant tumour was complete two weeks after LPS administration. Only minor residual scarring of the belly-wall remained. The earliest morphological changes could be seen with the electron microscope 90 min after administration of the endotoxin, and were seen as an interstitial oedema with separation of the tumour cells. Haemmorrhagic necrosis of the tumour was complete 4 hours after injection, and could be easily recognized with the naked eye. Rejection of the necrotic malignant tumour was complete two weeks after LPS administration. Only minor residual scarring of the belly-wall remained. Haemorrhagic tumour necrosis due to endotoxin can be compared with the localized Shwartzman reaction and probably involves tumour necrotizing factor (TNF). For complete destruction of a tumour by haemorrhagic necrosis the size of the tumour is critical. Certain regression after endotoxin administration depends upon additional T-cell-mediated immunity (provided the tumour is immunogenic). In contrast to the haemorrhagic necrosis, BCG-induced tumour regression is accompanied by granulomatous inflammation, which may be responsible for destruction of the tumour.

Identification and percentage frequency of isolated non-parenchymal liver cells (NPLC) in the mouse

SummaryThe aim of the present study was to identify non-parenchymal liver cells (NPLC) in B10.D2 mice and to determine their percentage frequency. The isolation of NPLC was carried out using the collagenase/pronase technique. Using functional techniques (latex phagocytosis, immunocytochemical detection of surface-bound and intracytoplasmic antigens) and morphological methods (light and electron microscopy), the following cell types were identified, and their percentage frequency in the NPLC determined: endothelial cells (50%), macrophages (23%), desmin-positive cells (14%), immunocompetent cells (10%, including T-, B-cells, pit and large vacuolated cells-both immunopositive to the asialo-GM 1 antigen) and unidentified cells (3%). These results show that, apart from the more familiar varieties of NPLC, two groups of cells exist in the liver which have not yet been fully identified and in which the immunocompetent cells predominate numerically.

Chemical and biological properties of a phenol-water extract from Leptospira interrogans. Evidence for the absence of lipopolysaccharide.

SummaryLeptospira interrogans, serovar copenhageni was extracted by the phenol-water method and the resulting preparation examined for chemical composition and endotoxic activity. Chemical analysis revealed that a number of sugars were present, however, the amount of lipid content was very low. Further, the preparation was devoid of characteristic endotoxic properties, like lethal toxicity, pyrogenicity and the property to induce the local Shwartzman reaction. The extract, however, was active in the limulus lysate gelation test and in the induction of monocyte activation. It is concluded that the leptospira preparation is devoid of endotoxin properties, both from the chemical and from the biological point of view.ZusammenfassungLeptospira interrogans serovarcopenhageni-Bakterien wurden mit dem Phenol-Wasser-Verfahren extrahiert. Das so isolierte Material wurde auf seine chemische Zusammensetzung und mögliche endotoxische Aktivität untersucht. Es enthielt verschiedene Zucker, der Anteil an Lipid-Bestandteilen war jedoch sehr gering. Die für Endotoxine charakteristischen Eigenschaften wie Pyrogenität, letale Toxizität und Induktion des Shwartzman-Phänomens waren in dem Extrakt nicht nachweisbar. Das Präparat zeigte jedoch eine starke Limulus-Lysat gelierende Aktivität, die mit der von klassischen Lipopolysacchariden ausSalmonella vergleichbar war. Der Extrakt war auch in der Lage, eine mäßige Aktivierung von Monozyten zu induzieren. Aus diesen Ergebnissen schließen wir, daß das isolierte Material keine endotoxischen Eigenschaften, weder in chemischer noch biologischer Hinsicht, besitzt.

Investigations into the origin of mouse liver sinusoidal cells

The possibility that liver sinusoidal cells are derived from the bone-marrow was investigated in chimeric mice. H2k-positive bone-marrow cells from F1 (B10.BR × B10.D2) hybrid mice were transplanted into irradiated H2k-negative parental mice (B10.D2), and the liver examined immunohistochemically for the presence of H2k-positive cells, with the help of an anti-H2k monoclonal antibody. With the passage of time (from the fifth week onwards), increasing numbers of transplanted bone-marrow cells enter the liver sinusoids, undergo alteration in their shape, and remain there, probably replacing sinusoidal lining cells. DNA-synthesising cells in the sinusoids were observed, suggesting, in addition, local cell proliferation. The replacement of sinusoidal cells from bone-marrow was greatly accelerated after liver damage had been induced by sublethal doses of endotoxin (LPS), and proliferation was also enhanced after treatment with LPS. These results strongly suggest that the bone-marrow participates in the replacement of liver sinusoidal cells.