Alois Nowotny

RELATION OF STRUCTURE TO FUNCTION IN BACTERIAL O-ANTIGENS. V. NATURE OF ACTIVE SITES IN ENDOTOXIC LIPOPOLYSACCHARIDES OF SERRATIA MARCESCENS*

An understanding of the mechanism of action of endotoxic preparations must ultimately be related to their chemical and physical structures. In order to find those structural components or properties of bacterial lipopolysaccharides which are responsible for the biological reactions, acidic hydrolyses have been most often applied in the past (Boivin et al., 1933; Brinkley et al., 1945; Freeman, 1943; Morgan and Partridge, 1940; Bendich and Chargaff, 1946; Westphal and Liideritz, 1954). While Westphal and coworkers believe the biological effects to be elicited by the so-called “Lipid A” portion of the molecule (19601, Ribi and associates attempted to disprove this possibility. Their hypothesis, elaborated in several papers, states tha t a certain molecular size is required to exhibit endotoxic reactions (Haskins et al., 1961; Ribi et al . , 1962; Milner et al., 1963; Ribi et al., 1964). A critical evaluation of these claims and other experimental results has been published recently (Nowotny, 1965). Previous papers from this laboratory indicated tha t long-chain carboxylic acids esterified to hydroxyl groups of carbohydrates form the lipopolysaccharides and the presence of these 0-acyl groups may be involved in the characteristic biological effects of these preparations (Nowotny, 1963, 1964 and 1965). A continuation of the above studies was carried out by studying the kinetics of alkaline hydrolysis and its effects on endotoxic lipopolysaccharides. The aims of these kinetic investigations were two-fold: (1) To determine the smallest “building block” which still retained full potency. This could be visualized by considering a depolymerization of the large aggregates into smaller active fragments. (2) To obtain further information concerning the chemical nature of those groups which might be responsible for biological activity.

RELATION OF STRUCTURE TO FUNCTION IN BACTERIAL O‐ANTIGENS. IV. FRACTIONATION OF THE COMPONENTS

Satisfactory proofs of homogeneity are essential to studies of biosynthesis, chemical structure, or to the investigation of relationships between chemical structure and biological function. The use of a pure and homogeneous compound is required for the study of the mode of action through which endotoxins elicit a wide variety of biological reactions. Contaminants present in an incompletely purified preparation obviously may influence biological effects. Conventional proofs of homogeneity, such as the appearance of a single sharp band in free-boundary electrophoresis or in sedimentation in the ultracentrifuge, were often inconclusive if better resolving procedures, such as immunodiffusion or immunoelectrophoresis, were subsequently applied. However, using these latter methods, those components present in an antigen preparation which are not antigenic, or which will not form precipitates with the corresponding antibodies, will not be detected. I t was also observed in the authors’ laboratory tha t antigen-antibody systems which showed only one precipitin band after a few days of incubation gave two or more precipitin bands if they were incubated in the cold room for several weeks. Most of the procedures which have been described for the extraction of endotoxic 0-antigens from Gram-negative bacterial cells have also been used for the isolation of several different bacterial cell constituents. The two most frequently used extraction procedures are the cold trichloroacetic acid (TCA) procedure of Boivin, Mesrobeanu and Mesrobeanu (1933) and the phenol procedure of Palmer and Gerlough (1940), modified by Westphal, Luderitz and Biester (1952). After the application of any of these procedures, everything which is soluble in dilute TCA or in the water phase after phenol treatment is in the crude extract. Although a few further steps have been elaborated for the purification of endotoxins which unquestionably remove large amounts of impurities, i t must be assumed tha t complete homogeneity was not achieved. I t has been observed in the authors’ laboratory that the supernate obtained after the precipitation of endotoxin with rabbit 0-antiserum in slight antibody excess contained polysaccharides which were not precipitated with antiserum. A phenol-water extract of this supernate yielded carbohydrate-

Relation of structure to function in bacterial O-antigens—VII. Endotoxicity of ‘lipid A’

Abstract The ‘lipid A’ precipitate obtained from endotoxic lipopolysaccharide (LPS) by acidic hydrolysis is highly heterogenous, it consists of more than 30 components as shown by chromatography. This preparation represents approximately 1 10 of the original endotoxic activity of the parent endotoxin. The crude ‘lipid A’ was separated into three major fractions: (1) ethyl acetate-soluble (ETAC); (2) chloroform-soluble (CHLO); and (3) insoluble residue (RESI). Investigating the biological activity of these and comparing them to the activity of the parent endotoxin as well as to the crude, nonfractionated ‘lipid A’ preparation, it was found that almost all the biological activity residing in the crude ‘lipid A’ preparation can be found in the RESI. Chemical analysis as well as biological studies of this fraction strongly suggested that it contains incompletely degraded endotoxin derivatives. The insolubility of RESI in dilute acids or in water is due to partial degradation of the hydrophilic polysaccharide moiety of the endotoxic lipopolysaccharide molecule. While this degredation rendered this endotoxin derivative less soluble in water, it was apparently not strong enough to completely abolish its biological activity. Further separation of the fraction RESI on preparative thin-layer chromatography provided a chromatographically homogeneous component which is apparently equally or more active than the parent endotoxin. Biological activities and molecular ratios of this subtance reveal some similarities between endotoxic glycolipids and this material. This substance represents a relatively acid resistance active fragment of the parent endotoxin. This study also compared various preparations, all called ‘lipid A’, and established chemical as well as biological differences among them. The more limited use of the term ‘lipid A’ and more accurate description of the various lipid-rich endotoxin derivatives is therefore recommended.

RELATION OF STRUCTURE TO FUNCTION IN BACTERIAL O-ANTIGENS. VI. NEUTRALIZATION OF ENDOTOXIC O-ANTIGENS BY HOMOLOGOUS O-ANTIBODY*

Protection against the toxic manifestations of endot,oxins with 0antibodies cannot be achieved in a manner similar t o that in which corresponding antibodies can protect against Gram-positive toxins or certain animal poisons. The first experiments described in this field were published by Boivin and Mesrobeanu in 1938. Antibodies were produced in rabbits by Shigella endotoxin injections. This immune rabbit serum was subcutaneously injected into mice which were challenged with Shigella endotoxin intraperitoneally. Very little protection was observed. Similar results were published by Weil et al . (1939). Morgan (1941) combined Salmonella typhosa endotoxin in uitro with the corresponding antiserum in the zone of antibody excess. The antigen-antibody complex obtained was tested in pyrogenicity and in the Shwartzman skin test. The extremely large doses injected in these experiments resulted in a pyrogenic response and a weak Shwartzman skin reactivity. His conclusion was that combination of the immunogenic sites of endotoxins with the corresponding 0-antiserum did not eliminate the toxic effects. Recently Kim and Watson (1965a and 1965b) showed that endotoxin tolerance can be passively transferred by injection of isolated 19s immunoglobulins of a tolerant rabbit serum, but they claim that 0-antigenic sites of the lipopolysaccharides are not involved in toxic effects. Stetson and coworkers (1961, 1964) likened the toxic manifestations of endotoxins to hypersensitivity. In their working hypothesis, endotoxins do not have separate toxophore groups but their toxic effects are elicited by the 0antigenic structures. I n a previous paper, the authors reported tha t endotoxic 0-antigens of Serratia marcescens precipitated with homologous 0-antibodies did not show toxic effects in pyrogenicity, Shwartzman reactivity, and mouse lethality assays (Nowotny, Radvany, Neale, 1965). Continuation of this work is aimed a t investigating whether toxic and separate immunogenic sites exist in the endotoxic 0-antigen preparations.

Neutralization of toxic bacterial O-antigens with O-antibodies while maintaining their stimulus on non-specific resistance

Abstract Reaction of endotoxic O-antigens with rabbit O-antisera results in a neutralization of toxic effects, determined in mice lethality, pyrogenicity, and Shwartzman skin reactivity assays, while the enhancement of non-specific resistance of these preparations remains unchanged. These results show that enhancement of non-specific resistance requires neither antigenic stimulus nor toxic action on the host organism. Complete saturation of all antibody receptors of an endotoxin molecule is not necessary in order to neutralize its biological activities. While 2 3 or more of the antigenic sites are still available for antibodies, the above endotoxic reactions are already eliminated. It could be shown that not all phases of the biological reactions were inhibited by antibodies to the same extent. The three-hour peak of the pyrogenicity curve was considerably reduced with relatively low amounts of antibodies, while the first hour peak was practically unchanged.

Fate and Effect of Endotoxin Derivatives in Tumor-Bearing Mice

Summary 1. A toxic endotoxin, its nontoxic chemical derivative called endotoxoid, and endotoxic glycolipids were tested in tumor-hemorrhage and lethality assays in tumor-bearing mice. All three materials were active in the tumor-hemorrhage assay. This indicates that neither toxicity nor the presence of O-antigenic polysaccharides is necessary to induce tumor hemorrhage. 2. No evidence of a direct action of endotoxin on tumors or of altered reticuloendothelial function could be obtained by the fluorescein antibody technique, which was used to detect the presence of endotoxin preparations in the different tissues. 3. Basic amines such as putrescine, spermine, and cadaverine, found in incompletely purified endotoxin preparations, and vasoactive agents such as histamine and serotonin could not duplicate the hemorrhagic activity of the endotoxic materials. 4. The tumor-bearing state increases the susceptibility of mice to the lethal action of all three materials. 5. Various inbred mouse strains, with or without tumor, showed a significantly different degree of endotoxin sensitivity.

Immunogenicity of Toxic and Detoxified Endotoxin Preparations

Summary Several-times repeated injections of heat-killed cells, toxic endotoxins, or CH3OK detoxified derivative thereof (endo-toxid-2) result in a comparable antibody titer after 8-10 injections. The antibody titer produced by toxic preparations shows a rapid initial increase, while endotoxoid-2 produces a lower titer of circulating antibodies in the first 10 days. While toxic preparations produce both 2-Me resistant and sensitive antibodies from the beginning of the immunization, endotoxoid-2 produces almost exclusively 2-Me sensitive antibodies in the first 10 days. Simultaneous with the appearance of 2-Me resistant antibodies, the level of total antibodies rises rapidly. A single injection of toxic endotoxin produces a rapid antibody production which starts to decline after approximately 2 weeks. Booster injections bring this antibody level back to previous highs, but hardly any additional effect can be observed. A single injection of endotoxoid-2 gives a very slow output of antibodies, which seems to remain stable for 1 month. Booster injection of endotoxoid-2 results in a strong increase of antibody production.

Comparisons of Five Toxicity Parameters of Serratia marcescens Endotoxins

Summary Phenol-water purification of TCA extracted Serratia marcescens endotoxin results in an apparent enhancement of toxicity as measured by two methods for testing biological activity, whereas three other methods indicate only an insignificant increase in toxicity. Partial degradation of endotoxin by NaOH shows a rapid loss of chick embryo lethality both by the iv and CAM routes of inoculation. Mouse lethality persists longer, almost complete detoxification occurring between 1 and 6 hours of NaOH treatment. The FI40 figures in rabbits tend to show an increase in pyrogenicity after 5 min of treatment and a progressive decrease between 30 min and 24 hours, although a low level of pyrogenicity is retained at the latter time. Shwartzman reactivity increases up to 1 hour of NaOH treatment, followed by a decrease to approximately the initial level after 24 hours of treatment and thus would appear to be a less reliable indicator of detoxification when compared to the other testing procedures. It is thus seen that one method of testing endotoxin may indicate an active preparation whereas another parameter may indicate otherwise to a greater or lesser extent. As a result, it is important to recognize the inherent danger involved in using a single assay technique for describing the toxic potency of an endotoxin preparation. The observed discrepancies in biological assays also indicate that the mode of endotoxic action is obviously triggered and channeled by a number of different pathways in the various host systems.

Rapid biological and chemical analyses of bacterial endotoxins separated by preparative thin-layer chromatography

Abstract Methods have been elaborated for rapid location of biologically active bacterial endotoxin components on preparative thin-layer chromatography (tlc) by Limulus lysate clotting assay. The separation of the components from silicic acid is not necessary. Further assays were established for the semiquantitative estimation of endotoxic activites (Shwartzman reactivity, chick embryo lethality, and non-specific tumor resistance enhancement) without complete removal of silicic acid. Quantitative chemical analytical procedures were also elaborated to determine the molar ratios of the biologically active components separated and detected in the above tlc system. These included phosphorus, hexosamine, heptose, 2-keto-3-deoxyoctonate, and long-chain carboxylic acid content measurements. Here again, the chemical determinations were carried out in the presence of silicic acid.

DISCUSSION PAPER: DUAL EFFECTS OF TUMOR, ANTIGENS: INDUCTION OF TUMOR RESISTANCE, OR TUMOR GROWTH ENHANCEMENT*

TA3-HA nonspecific tumors growing in ascites form shed their membrane components into the ascites fluid. This product can induce tumor growth enhancement if given together with or after i.p. tumor inoculation in relatively large quantities, but if given i.m. in small quantities, several days before tumor challenge, the mice show an elevated resistance to the tumor. The strictly strain-specific TA3-St line does not release such membrane components into the ascites fluid. It has also been found that the TA3-HA cells have a larger membrane fluidity than the TA3-St cells, as measured by membrane microviscosity, using the Shinitzki procedure. Isolation and gross chemical analysis of the TA3-Ha ascites fluid component was carried out. Results, so far, indicate that the active site of this product is probably carbohydrate in nature. The possible mode of action of such component in tumor growth enhancement is most probably by overwhelming and neutralizing immunocytes capable of recognition and eventually cytotoxic action.