The development of a simple technique for labelling E. coli endotoxin with radioactive chromium has presented the opportunity for tracer experiments (1). Such experiments have been conducted in different species of animals, using various doses of endotoxin tagged with either hexavalent chromium as Na2Cr510, or trivalent chromium, as Cr1Cl,. One of the objectives was to observe the physiologic changes which unfold as the endotoxin is removed from the blood and deposited in various organs. This paper presents the first of a series of studies describing these changes; it deals with the injection into rabbits of lethal doses of endotoxin tagged with hexavalent radiochromium. METHODSEndotoxin, prepared from bacteria grown in synthetic medium, was labelled by direct incubation with Na.CrmO4 by the method described in the preceding paper (1). When injected into rabbits, there were 223.4 counts per second per mg. and the LD. for mice before and after tagging was 0.2 mg.Three groups of adult rabbits weighing 2.5 to 3.0 kg. were injected intravenously with Cr' as follows:Group I-Endotoxin labelled with NaCrO4. Group II-NaCr'O4 in one vein followed by the injection of unlabelled endotoxin in another vein.Group III-Na2Cr'O4 only (no endotoxin). The dose of endotoxin and chromium is given for each group in Tables IA, IB, Before the injection of endotoxin, leukocyte counts were made on blood from the ear vein, and rectal temperatures and blood pressures were measured repeatedly until the rabbits were stabilized. The blood pressures were measured by placing a human blood pressure cuff around the abdomen, inflating it in the usual manner, and palpating the femoral arterial pulse until it was obliterated. This method for measuring blood pressure in rabbits has been checked by simultaneous determinations with the ear-chamber apparatus of Grant and Rothschild (2), and the results were identical when the chamber and cuff were connected to the same manometer. Although the abdominal-cuff method may not be as precise as direct intra-arterial measurement, it provides a simple and satisfactory non-surgical technique for demonstrating changes in pressure, and does not require anesthesia which might complicate the studies on endotoxin.When the animals were about to be sacrificed, determinations of blood pressure, temperature, and leukocyte count were repeated, and 30 to 50 ml. of blood was drawn from the heart. The blood was citrated with one-fourth its volume of ACD (1.32 gm. sodium citrate, 0.48 gm. citric acid, and 1.47 gm. dextrose per 100 ml.) solution and separated into its three components; plasma, red cells, and buffy coat. The plasma was easily removed after centrifugation at 5,000 r.p.m. in the refrigerated ultracentrifuge for 20 minutes. Buffy coat was next removed in the form of a continuous membrane with a Pasteur pipette. The buffy coat and accompanying red cells were suspended in saline in a small test tube (75 X 9 mm.) and centrifuged at approximately 2,000 r.p.m.for 10 minutes. After several washings with saline, the buf...
The endotoxins in different species of gram negative bacteria exhibit remarkably similar biologic effects which have been observed in both experimental animals and'man (1-5). These effects may be seen after the injection of living bacteria," killed bacteria, or endotoxin itself; they consist of fever, shock, leukocytosis preceded by leukopenia, hypoglycemia, diarrhea, and prostration.. Although there are sometimes hemorrhages in the intestine and other viscera, the findings postmortem are not enough to explain the cause of death or to indicate where the endotoxin acts.As a means of locating the site of action of endotoxin, as well as its fate in tivo, a series of experiments have been carried out using endotoxin labelled with radioactive chromium. The endotoxin of Escherichia coli was selected for this study because it can be conveniently prepared in large quantities without the hazards associated with more highly pathogenic bacteria. Radioactive chromium proved to be satisfactory by virtue of its firm attachment to endotoxin, its relative safety in handling, and its half-life of 26.5 days. This report deals with the technique of labelling of E. coli endotoxin and with the properties of labelled toxin; those which follow describe its distribution in vivo under various experimental conditions. EXPERIMENTAL METHODS I. Preparation of E. coli EndotoxinThe method of preparation of E. coli endotoxin was based on Boivmi and Mesrobeanu's (6) principle that endotoxin can be extracted by trichloroacetic acid at cold temperatures. The bacteria were grown in a synthetic medium modified from that described by Gladstone (7) for growth of S. typhosa. For These salts were dissolved in 12 liters of distilled water and 700 ml. 1 N NaOH were added, the volume being made up to 15,000 ml. with distilled water. The reaction was adjusted to pH 7.6 by the addition of 1 N NaOH, 9125 ml. distilled water were added, and the medium was put in 4 liter quantities into 6 flasks of 6 liter size and autoclaved at 115°C. for 20 minutes at 15 lbs. pressure. To each flask the following sterile solutions were added: M/6 MgSOj-7H2O 40 ml., 50 per went glucose solution 20 ml., and M/NaHCOs 10 ml. The incubated at 370 C. for 48 hours and dialyzed four days against running tap water followed by 24 hours against distilled water to remove free Cr". The volume was concentrated to 50 ml. in vacuo and the suspension chilled to 0°C. The chilled suspension of bacteria was mixed with ice cold 0.25 M trichloroacetic acid, and the endotoxin extracted in the manner described. The yield was 39.5 mg.To test the stability of the bond between Cr" and endotoxin, the entire volume of tagged endotoxin solution was dialyzed against distilled water at 4°C. for 10 days.Under these circumstances less than 1 per cent of the radioactivity on the endotoxin appeared in the dialysate water during a period of 24 hours.C. Adding Na.Cr"04 and Cr"Cl, to the endotozin directly 1) Addition of NaCr"04 and Cr"Cl, to non-tagged endotoxin. E. coli endotoxin was prepared from bacteria ...
The repeated injection at frequent intervals of the same dose of a bacterial endotoxin is followed by gradual diminution in the severity of its toxic effects. The state of resistance that develops during the course of such injections has been designated tolerance and is considered to be independent of specific antibody formation, because tolerance for one endotoxin is effective against the toxic action of immunologically distinct endotoxins of other gram-negative bacilli (1-3). The importance of the reticuloendothelial system in the development of tolerance has been explored by Beeson (4,5), who demonstrated that reticuloendothelial "blockade" not only abolished immunity to the Shwartzman reaction, but also tolerance to the febrile response of sublethal doses of bacterial pyrogens.The heavy localization of radioactivity in the liver of animals injected intravenously with endotoxin labeled with Cr5' has provided evidence that the reticuloendothelial system removes the endotoxin from their circulation (6). In rabbits receiving massive lethal doses of endotoxin intravenously, it was found that the localization of endotoxin in the liver was not enough to prevent the persistent circulation of endotoxin in high concentrations in the plasma for several hours. Thus, 30 to 40 per cent of the total amount of injected radioactivity was present in the circulation for nearly three hours after the injection of 6.0 mg. of radioactive endotoxin in rabbits. Because it seemed likely that these large amounts of circulating endotoxin were responsible for the damag-
The generalized Shwartzman reaction (1) occurs in immature (2) rabbits following the second of two sublethal intravenous injections of gram-negative bacterial endotoxin, spaced 18 to 24 hours apart, and consists of widespread hemorrhage and necrosis. The reaction also occurs after a single injection of endotoxin in animals treated with intravenous thorotrast, trypan blue (3), colloidal iron, or colloidal carbon (4). Because these substances were thought to "blockade" the reticuloendothelial (R.E.) system, it was suggested that an analogous impairment of R.E. function may also be produced by the first or "preparing" injection of endotoxin, resulting in slower removal and detoxification of the second dose and directly or indirectly in the characteristic lesions in the kidney, heart, lungs, and liver.In the experiments to be reported, the distribution of radioactive Crl' labeled endotoxin in normal rabbits was compared with that in rabbits which had received a preparing injection of unlabeled endotoxin 24 hours earlier. The results indicate that, in the rabbit, the "prepared" state is characterized by impaired removal of a second injection of endotoxin by the liver and increased localization by the lung.METHODS AND PROCEDURE Animals. Young albino rabbits of a hybrid stock weighing between 0.77 and 1.3 kilo., used for all experi-
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.