Detoxification of Bacterial Endotoxin with Retention of Ability to Stimulate Non-Specific Resistance to Infection

Interest in the bacterial endotoxins arises, not only from their specific antigenicity and from the phenomenon of tolerance, but, equally, from the remarkable ability of these pyrogenic and highly toxic complexes to stimulate, at microgram doses, substantial refractoriness to a variety of ordinarily lethal insults.Endotoxin treatment has been shown to protect against the consequences of infection with Gram-negative, Gram-positive, or acid-fast bacteria (1-4), and to promote resistance to the lethality of hemorrhagic or traumatic shock (5-7) or wholebody x-irradiation (8, 9). Despite their wide spectrum of activities (10, 11), no unequivocal evidence for experimental dissociation of the in ~ivo non-specific properties of endotoxins by chemical modification of the molecule has been reported.The presence of lipid, carbohydrate, and, usually, protein moieties within the endotoxin molecule has encouraged hydrolytic approaches designed to recover one or another of these components. Hydrolysis with acid or base leads to destruction of all in ~ivo activities, but, where active material has been recovered following hydrolysis, the whole range of properties, toxic and protective, has been present. This is perhaps best illustrated by the current difference of opinion on the role of the lipid component (12, 13). To avoid hydrolytic or oxidative alterations, studies on the chemical modification of endotoxin were undertaken using reactions resulting in substitution at functional groups known to be present in the molecule. This and the following paper (14) describe the acylation of endotoxin, with recovery of a fraction exhibiting in the gross reduced pyrogenicity and lethality but retaining the ability to stimulate so-called non-specific host resistance to infection. A preliminary report of our findings has been published (15). While our manuscripts were in preparation, Noll and Braude (16) reported the detoxification of endotoxin by reductive cleavage of ester bonds yielding a preparation of high immunogenic potency, which, unlike our preparation, confers tolerance to the parent endotoxin. Materials and MethodsChemicaL--Several endotoxin preparations have been used as starting materials for a variety of substitution reactions. This and the following report (14) describe results obtained with wcetylafion of a Boivin-type endotoxin derived from Salmonella typhosa 0-901 (Difco 929 on

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confidence: 71%

Section: Methodsmentioning

confidence: 99%

Dissociation of the Biological Properties of Bacterial Endotoxin by Chemical Modification of the Molecule

Freedman

Sultzer

1962

“…The way in which lipopolysaccharide particles initiate properdin activation is not known, although the importance of primary structure for the biologic activity of lipopolysaccharide has been illuminated by studies in which chemical modifications of lipopolysaccharide resulted in loss or diminution in endotoxic potency. Diverse treatments, such as acetylation (30), alkaline hydrolysis (43), and deacylation (31), have been shown to diminish mouse lethality and complementconverting ability of lipopolysaccharide without destroying antigenicity. Such treatment also appears to compromise the capacity of lipopolysaccharide to activate the properdin system.…”

Section: N G E S T I O N Of Paraffin Oil Emulsified W I T H E Colimentioning

confidence: 99%

“…(c) The lipopolysaecharide was acetylated (30) or deacylated with pyridine-formic acid or BF~-methanol (31).…”

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confidence: 99%

Serum-Dependent Phagocytosis of Paraffin Oil Emulsified With Bacterial Lipopolysaccharide

Stossel

Alper

Rosen

1973

The ability of fresh serum to opsonize microorganisms was recognized nearly 70 yr ago (1). Subsequent studies identified synergistic effects of both antibody and complement on opsonizatiou (2) and demonstrated that particles to which C3 was affixed by the sequential participation of antibody, C1, C4, and C2 became opsonized (3-6). In 1968, a patient was found with marked susceptibility to infection who spontaneously inactivated C3 in vivo. His serum had normal functional levels of antibody, CI, C4, and C2, but failed to opsonize bacteria even when fortified with purified C3 (7). Further studies of his serum revealed a defect in his properdin pathway (8, 9). This patient, and the good health of humans deficient in C2 (10) and of guinea pigs deficient in C4 (~ 1), have emphasized the functional significance of this alternate pathway of C3 activation, first recognized by Pillemer and his associates (12). The properdin system has been shown to participate in bacteriolysis, protozoal inactivation, virus neutralization, and lysis of erythrocytes in paroxysmal nocturnal hemoglobinuria (13-16). Unlike the classical complement pathway, the properdin system can be activated by polysaccharides in the absence of antibody (17).This report describes quantitative studies of the phagocytosis of paraffin oil droplets coated with bacterial lipopolysaccharide. Opsonization of these particles occurs in the absence of C4 and C2, but is dependent upon C3 and the properdin pathway. Measurement of the opsonization of these particles thus constitutes a quantitative functional assay for this system. Materials and MethodsPreparation of Phagocytes.--Suspensions containing over 95% polymorphonuclear leukocytes were obtained from guinea pig peritoneal exudates. The exudates were elicited with sodium caseinate, and the cells were collected and washed as previously described (18). Human peripheral blood from normal laboratory personnel or patients with infections and leukocytosis was collected with acid-citrate dextrose, National Institutes of Health formula A; the erythrocytes were removed by dextran sedimentation and lysis with ammonium chloride; and leukocytes were washed as described previously (19). The celts were suspended in KrebsRinger phosphate medium, pH 7.4 (medium), at a concentration of 2-5 mg of guinea pig

“…One such problem of primary interest is whether the lethal and pyrogenic effects of these materials are necessarily related to their property of increasing resistance of the host to infection. With the development of a relatively detoxified endotoxin, this subject became amenable to direct investigation (1,2).…”

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confidence: 99%

The Stimulation of Non-Specific Host Resistance to Infection by Chemically Modified Endotoxin

Sultzer

Freedman

1962

In recent years the use of endotoxins to alter the natural equilibrium between host and parasite has engendered m a n y intriguing questions. One such problem of primary interest is whether the lethal and pyrogenic effects of these materials are necessarily related to their property of increasing resistance of the host to infection. With the development of a relatively detoxified endotoxin, this subject became amenable to direct investigation (1, 2).The experiments reported in this paper deal with several properties of the detoxified derivative in stimulating host defense mechanisms as directly compared to the original endotoxin. The essentially equivalent capacity to acutely increase resistance to various bacterial infections has been demonstrated, as well as the differences produced b y varying the methods of treatment and infection. Studies dealing with the passive transfer of protection and the effects on the level of circulating leucocytes in the mouse are also discussed. Materials and MethodsAnimals.--Male mice of the ICR strain weighing 16 to 18 gm (Bellewood Farm, Englishtown, New Jersey) were housed 10 to a metal cage, fed Purina lab chow and water ad libitum. All animals were used routinely within 1 week after receipt.Bacterial Cultures.--Three representative Gram-negative bacteria were employed, including strains of Escherichia coli and Salmonella typhimurium previously described (3) and a strain of Pseudomonas aeruginosa isolated in this laboratory. A strain of Staphylococcus aureus (Smith) known to be virulent for mice was kindly provided by Dr. Ian M. Smith. Each organism was grown in brain heart infusion broth (BHI) (Difco Laboratories, Inc., Detroit) for 18 hours at 35°C prior to each experiment. A standard inoculum was determined for E. coli, Ps. aeruginosa, and S. aureus which produced 70 per cent to 80 per cent mortality within 3 days in those animals injected intraperitoneally. In those mice infected with S. typhimurium, half the deaths would occur within I day, or, with a smaller inoculum, by 3 days. In certain experiments, an E. coli bacteremia was produced by intravenous injection of 0.25 ml of an 18 hour culture into one of the tail veins, a dose which produced 90 per cent to 100 per cent deaths within 3 days. For the Gram-negative cultures, appropriate dilutions were made in sterile BHI broth. The staphylococcal culture was centrifuged, the cells washed once, and resuspended to their original volume in sterile saline. Viable numbers of organisms were determined by triplicate plate counts on BHI agar. All inocula were kept chilled in an ice bath during the challenge procedure and 0.25 ml of the appropriate dilutions was used for intraperitoneal injections.