Maurice Landy

QUANTITATIVE STUDIES OF HUMAN LEUKOCYTIC AND FEBRILE RESPONSE TO SINGLE AND REPEATED DOSES OF PURIFIED BACTERIAL ENDOTOXIN

Bacterial endotoxins injected intravenously in small doses into animals and man produce, among other effects, a transient granulocytopenia followed by a marked granulocytosis (1-7). Several comprehensive reviews have recently appeared which deal with the wide spectrum of biologic effects of these substances, including their relationship to the pathogenesis of fever, the development of tolerance to repeated administration, and effects upon host resistance to bacterial infection (5, 8, 9). Recent studies utilizing radioisotopic cell-labeling techniques have increased considerably the understanding of granulocyte kinetics. Athens and his associates estimate the total blood granulocytes in man to be made up of nearly equal compartments of circulating cells and cells marginated and sequestrated within the capillary bed (10). The granulocytic elements in the marrow which are no longer capable of proliferation consist of a reserve of mature cells and cells in various stages of maturation which has been estimated to number 20 to 70 times the total blood granulocytes (7, 11, 12). Craddock, Perry and Lawrence consider this marrow granulocyte reserve to be the source of the granulocytes that enter the circulation in response to leukocytopheresis (11) and endotoxin (1). Athens and co-workers have added evidence that the source of the endotoxin granulocytosis is the bone marrow (10). No appreciable return of granulocytes to the circulation from extravascular spaces has been demonstrated. Craddock and others have suggested that the magnitude of the increase in circulating granulocytes after endotoxin administration may relate directly to the size of the marrow granulocyte reserve and

Estimation of Vi antibody employing erythrocytes treated with purified Vi antigen.

Summary 1. The preparation of a sensitive and specific Vi agglutination reagent by sensitization of human type O erythrocytes with Vi antigen isolated from E. colt has been described. 2. Vi antibody content of rabbit and human sera was determined by this method and the conventional bacterial agglutination test. The hemagglutination test provided a more sensitive measure of Vi antibody by a factor of 3 to 6 fold. Greater variability in Vi titers was encountered in testing the sera of typhoid carriers; however, both methods were in good agreement in the detection of Vi antibody in these sera. The advantages inherent in the Vi hemagglutination procedure are discussed.

Increased Synthesis of p-Aminobenzoic Acid associated with the Development of Sulfonamide Resistance in Staphylococcus aureus.

Sulfonamide-resistant strains of Staphylococcus aureus produce greater amounts of p-aminobenzoic acid than do their parent strains. This synthesis occurs both in the absence and in the presence of sulfonamides. The quantity of p-aminobenzoic acid synthesized by resistant strains appears sufficient to account for their resistance to sulfonamide drugs. On the basis of this evidence, it is suggested that the development of ability to synthesize p-aminobenzoic acid in excess of the normal metabolic requirements, as a result of continued exposure to sulfonamides, explains the phenomenon of sulfonamide fastness in Staphylococcus aureus.

OBSERVATIONS ON THE PYROGENIC RESPONSE AND ITS APPLICATION TO THE BIOASSAY OF ENDOTOXIN

Endotoxins prepared from gram-negative bacteria produce a wide variety of physiological reactions when administered to man and experimental animals. The intravenous injection of microgram quantities of these lipopolysaccharides into the appropriate host is followed by fever, leukopenia, thrombocytopenia, hyperglycemia, and irreversible shock. In addition, endotoxins are capable of producing the Shwartzman phenomenon , dermal necrosis, renal cortical necrosis, and hemorrhagic necrosis of certain tumors (1). The ubiquity of endotoxins has necessitated routine screening of all fluids and medications prepared for parenteral therapy, for which the pyrogenic response of the rabbit is commonly used. However, there have been few systematic studies of the dose-pyrogenic response relationships and little is known concerning the relative susceptibil-ities of rabbit and man to the pyrogenic and toxic properties of endotoxin (2-5). In this report the dose-febrile response curve for Serratia marcescens endotoxin is described. Data are also presented which show the relative susceptibilities of several species, including man, to the pyrogenic properties of endotoxin. An analysis of the data from an extensive series of experiments demonstrates that the febrile reaction of the rabbit provides a consistent and objective procedure for the bioassay of endotoxins. METHOD Endotoxin All experiments, except where otherwise stated, were done with a single endotoxin prepared by Perrault and Shear (6) from Serratia marcescens (lot P-45). Serratia endotoxin, prepared in this manner, has been characterized by Rathgeb and Sylven (7) as a phospholipid-poly-saccharide-protein complex in which the nitrogen content was 1.8 per cent and the carbohydrate moiety represented 76 per cent of the total dry weight. Animals Rabbits. All animals were New Zealand white rabbits of both sexes, weighing 2 to 3 kg, bred at the Animal Production Unit, National Institutes of Health. Prior to pyrogen tests the rabbits were trained in wooden stocks, with loosely fitting collars as the only restraining device. Before the injection was given, 1 hour was allowed for further acclimatization to the stocks. Only animals with baseline temperatures between 38.50 and 39.9° C were used for pyrogen assay. When individual rabbits were used more than once, a 2 week period was allowed between tests to avoid pyrogen tolerance. Cats. These were of mixed breeds and weighed 2.0 to 2.4 kg. They were kept in the laboratory for 1 week prior to use and were trained in the wooden stocks just as were the rabbits. They adapted rapidly to the restraining device and had very stable baseline temperatures. …

SEROLOGIC PROPERTIES OF BENTONITE PARTICLES COATED WITH MICROBIAL POLYSACCHARIDES.

Summary A flocculation test utilizing polysaccharide-coated bentonite particles is described. The conditions for coating the particles and performance of the test are reported. The method offers advantages such as simplicity, reproducibility, stability and specificity. A number of polysaccharide antigens, which fail to coat erythrocytes, readily adsorb onto bentonite, permitting serologic tests with these antigens.

INACTIVATION OF ENDOTOXIN BY A HUMORAL COMPONENT. VII. ENZYMATIC DEGRADATION OF ENDOTOXIN BY BLOOD PLASMA

The pathogenesis of the febrile response that follows the intravenous injection of bacterial endotoxin has been the subject of extensive investigations. In the course of these studies it became evident that blood serum contains factors which, when interacted in vitro with endotoxin, may either enhance or inactivate the fever-producing effects. LeQuire demonstrated that a brief period of interaction between serum and endotoxin results in augmentation of pyrogenicity (3). Others have confirmed this observation and have, in addition, defined some of the conditions necessary for demonstrating the pyrogen-enhancing effect of serum (4-8). In seeking to explain the termination of endotoxin fever, Hegemann postulated that this was effected by components of serum capable of detoxifying endotoxin. He demonstrated the presence of such agents in serum by incubating endotoxin and serum for prolonged periods at physiological temperature (9-12). However, these experiments did not provide evidence that this effect of serum was operative in the termination of endotoxin fever. The observations of LeQuire and of Hegemann were confirmed by Cluff and Bennett (8), who clearly distinguished the two effects of serum by demonstrating that pyrogen augmentation was not dependent upon the time or temperature of incuba-

Studies on the O Antigen of Salmonella typhosa IV. Endotoxic Properties of the Purified Antigen

Summary A study was made of the endotoxic properties of a protein-free lipopolysac-charide isolated from the 0-901 strain of S. typhosa. The product was lethal for rabbits in a dose range of 20-50 μg while 300-500 μg were required to kill mice. The minimum pyrogenic dose in rabbits was 0.0002 μg, characterizing it as a pyrogen of extremely high potency. In a quantitative study of the local Shwartzman reaction, 1 μg or less, was effective as a preparatory or provocative dose. Its activity in eliciting bilateral renal cortical necrosis, the lesion characteristic of the generalized Shwartzman reaction, was in the range of 5-20 μg. Thus, very small quantities of a lipopolysaccharide, freed of protein, are capable of producing the various biological alterations generally attributed to endotoxins. Addendum. Since this report was submitted for publication preliminary tests of the effects of the typhosa lipopolysaccharide in man were undertaken(17). These indicate that when injected i.v. the minimum pyrogenic dose for a 70 kg subject lies in the range of .01-.04 μg.

Effect of Bacterial Endotoxin in Germ-free Mice

Summary Germ-free and conventionally reared mice of similar genetic stock maintained on the same sterilized diet were compared as to their reaction to bacterial endotoxin. No significant difference was found in the following: elevation in levels of antibody to Gram-negative bacteria, stimulation of glycolytic activity of spleen cells and peritoneal macrophages, susceptibility to infection with Salmonella typhosa, production by endotoxin of increased resistance to infection, and LD50 of endotoxin.

Bacterial Synthesis of p-Aminobenzoic Acid.

Summary Representative cultures of many bacterial genera were grown in culture media of known composition free of p-aminobenzoic acid. Culture filtrates and hydrolized whole cultures were assayed for p-aminobenzoic acid content employing the Acetobacter suboxydans microbiological method. All organisms successfully cultured in the p-aminobenzoic acid free media elaborated p-aminobenzoic acid in readily measurable amounts, indicating that p-aminobenzoic acid is truly an “essential metabolite” synthesized by many bacteria.

Effect of Purines on Sensitivity of Acetobacter suboxydans Assay for p-Aminobenzoic Acid

It has been demonstrated that Acetobacter suboxydans requires p-aminobenzoic acid as a growth essential. 1 The p-aminobenzoic acid requirement of this organism forms the basis of a microbiological method for its determination. 2 We have observed that when the casein hydrolysate employed in the basal medium for the above assay method is treated with Darco G-60 charcoal (10% of weight of casein) at pH 2.5 for one half hour, growth response to lower concentrations of p-aminobenzoic acid is variable and generally subnormal. Of a variety of compounds examined for ability to increase growth at lower concentrations of p-aminobenzoic acid the purine bases∗ adenine, guanine, and xanthine were found to be effective. Not only was the quantity of growth increased, but the sensitivity of the Acetobacter response to p-aminobenzoic acid was increased considerably. While in the absence of purines the test organism responds to 0.01 μ of p-aminobenzoic acid, the presence of purines brings about readily measurable growth with 0.001 μ per culture. The purines are without effect in the absence of p-aminobenzoic acid or when p-aminobenzoic acid is supplied in amounts above 0.03 μ per culture, indicating that they are not essential for growth but act instead as growth accessories.† The results of a typical experiment are given in Table I. Adenine possesses the greatest activity and 150 /xg per culture can generally replace the combination of the 3 purines. Results with the 3 bases have, however, been more uniform and in the absence of detailed information on the purine requirements of this organism it seems desirable to employ all 3 purines. Hypo-xanthine was not available for test. The pyrimidine base uracil exerted no effect on growth under these test conditions. Parallel assays on bacterial extracts with and without purines in the basal medium yielded values in agreement within ±15%.