Tritiation of endotoxin.

Tritiated endotoxin was synthesized by three different methods: (1) sodium boro[3H]hydride reduction of native endotoxin; (2) sodium boro[3H]hydride reduction of endotoxin that had been oxidized previously with sodium metaperiodate; and (3) exposure of dry endotoxin to 3H2 gas. Sodium borohydride reduces aldehyde groups and sodium metaperiodate oxidizes vicinal glycol groups to aldehydes. Chromatographic analysis of the three tritiated endotoxins, using agarose, revealed that the biological activity associated with each labeled product appeared at the void volume, and in each case the biological activity coincided with a peak in radioactivity. The labeled product of the first method had a specific radioactivity of 0.18 mCi/g and a biological activity equal to that of native endotoxin. The labeled products of the second and third methods had specific activities of 2.1 mCi/g and 60.0 mCi/g, respectively, while their biological activities were one hundred-fold less than native endotoxin, as determined by the Limulus amebocyte lysate assay. These three labeled endotoxins are potentially ueled endotoxin.     

The biology of endotoxin

Endotoxin (lipopolysaccharide, LPS) is the major component of the outer leaflet of Gram-negative bacteria and has profound immunostimulatory and inflammatory capacity. The septic shock syndrome caused by endotoxin still has an unacceptably high mortality rate and, owing to increasing numbers of resistant strains, remains an ongoing threat throughout the world. However, the past years have provided new insights especially into the receptors of the innate immune system that are involved into the recognition of LPS and the initial signal transduction pathways that are engaged after the primary recognition on the cell surface. The knowledge about the molecular basis for the responses to endotoxin may eventually lead to the development of new drugs to fight the fatal effects of bacterial infections.     

The effect of endotoxin and endotoxin tolerance on inflammation induced by mycobacterial adjuvant

Peptidoglycan, the substance in mycobacteria thought to be responsible for inducing adjuvant arthritis, and endotoxin (lipopolysaccharide or LPS) share many inflammatory properties. Since repeated administration of LPS produces tolerance, i.e., resistance to the toxic and inflammatory effects of LPS, we tested whether LPS and/or LPS tolerance might influence inflammation due to mycobacterial adjuvant. Male Sprague-Dawley rats were injected with Escherichia coli LPS or saline intraperitoneally and then challenged with 100 micrograms killed Mycobacteria butyricum (adjuvant) in the footpad. A single dose of 100 micrograms LPS three or 24 hours before adjuvant markedly, but transiently, reduced the local footpad swelling that begins within hours of the adjuvant injection and histologically resembles a sterile abscess. Animals that received multiple doses of LPS and were therefore tolerant or animals that received LPS 72 hours before adjuvant demonstrated adjuvant-induced footpad swelling nearly equal to controls. The anti-inflammatory effect of LPS was transient since footpad swelling in all groups was nearly comparable six days after the adjuvant injection and LPS failed to inhibit consistently the arthritis that develops two or more weeks after adjuvant injection. These studies establish that LPS can markedly inhibit the prodrome of adjuvant arthritis (footpad swelling due to M. butyricum), that inhibition of this prodrome does not prevent the subsequent development of arthritis, and that LPS tolerance diminishes this anti-inflammatory effect of LPS.     

Molecular mechanisms of endotoxin activity

Endotoxin (lipopolysaccharide, LPS), a constitutent of the outer membrane of the cell wall of gramnegative bacteria, exerts a wide variety of biological effects in humans. This review focuses on the molecular mechanisms underlying these activities and discusses structure-function relationships of the endotoxin molecule, its interaction with humoral and cellular receptors involved in cell activation, and transmembrane and intra-cellular signal transduction pathways.     

Evaluation of the bacterial endotoxin test for quantification of endotoxin contamination of porcine vaccines.

We investigated the application of the bacterial endotoxin test for the quantification of the endotoxin contamination of various commercial porcine vaccines. In endotoxin-spiked samples, Freund's complete adjuvant and aluminum hydroxide gel adjuvant failed to interfere with the results of the endotoxin test, and both recovery ratios were within the permissible range mentioned in the Japanese Pharmacopoeia. At the various dilutions tested, none of the adjuvants in commercial porcine vaccines caused noteworthy interference in the test. In addition, none of the 39 samples of porcine vaccines approved in Japan induced an interfering effect in the endotoxin test. Our findings suggest that the bacterial endotoxin test using endotoxin-specific Limulus amoebocyte lysate (LAL) can detect endotoxin contamination in commercial porcine vaccines containing either oil or aluminum adjuvants.     

The action of endotoxin on complement

Действие эндотоксина на комплемент Я. Космка Я. шмерчъ Прибавление эндот оксина к сыворотке взрослых животных инактивирует комплемент. при иго прибавлении к сыворотке стерилъных поросят, вскормленных искусственно, без молозииа (в сыворотке которых имеется комплемент, но отсутствуют антитела), комплемент не инактииируется. это доказфвает, что энпотоксин не действует прямо на комплемент.     

Biological activity of phthalated endotoxin.

Glycolipid (GL) was extracted from a heptoseless mutant of Salmonella minnesota by a mixture of phenol, chloroform, and petroleum ether. The GL was subjected to treatment with either acetic anhydride or phthalic anhydride; a portion of the GL was untreated. Both of the chemically treated preparations as well as the parent GL were examined for biological activity in the following systems: mouse lethality assays, rabbit pyrogenicity assays, and rabbit skin assays. The results of these studies indicated that both treated preparations were less toxic in mice than the parent GL. Compared with saline-treated controls, rabbits pretreated with either of the modified preparations exhibited a reduced pyrogenic response to a subsequent challenge dose of the homologous material but no reduction when challenged with the parent GL. Pretreatment with the unaltered GL rendered rabbits tolerant to the homologous material and to some degree to the modified preparations. Rabbits immunized witn any of the three Gl preparations exhibited dermal toxicity responses comparable with those in untreated animals. Based on these findings, it was concluded that treating GL with either phthalic anhydride or acetic anhydride results in a product which is less toxic in mice and less pyrogenic in rabbits than the parent GL, but which also exhibits a loss of ability to render rabbits tolerant to challenge with untreated GL.