Biomedical subjects
Publications and source records attributed to J A Rudbach.
Development in chicken embryos of increased resistance to lethal effects of endo-toxin could not be quantitatively accounted for by increased endotoxin-inactivating capacity of their sera.
Antigenic competition between bovine gamma globulin (BGG) and endotoxin from a smooth strain (S-ET) and a rough (R-ET) heptoseless mutant strain of Salmonella minnesota was studied in mice. Both endotoxins acted as adjuvants for enhancing the antibody response to BGG. However, other work showed that the R-ET had minimal antigenicity, and it was used as a control for the competition studies. Antigenic competition between BGG and endotoxin as expressed by a suppression of the antibody response to BGG could not be demonstrated when varying adjuvant doses of S-ET or R-ET were injected simultaneously with a small constant dose of BGG into normal mice. However, mice presensitized with S-ET several weeks before immunization with the S-ET and BGG combination produced anti-BGG levels which were four to eightfold lower than in normal mice. Nearly complete suppression of the anti-BGG response could be obtained in presensitized mice by reducing the BGG dose 10-fold or by increasing the adjuvant dose of endotoxin. Mice pretreated with R-ET and challenged with BGG plus S-ET or R-ET showed no depression of the anti-BGG response. These and other experiments confirmed the immunological basis of the competitive effect.
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Treatment with alkali is one of several methods for removing fatty acids from bacterial endotoxins and, in the process, detoxifying the material. Saponification of fatty acid esters is the major detectable chemical change produced by alkali; however, kinetic studies of mild alkaline hydrolysis of endotoxin failed to correlate rates of detoxification with rates of loss of ketodeoxyoctonates, heptose, O-acetyl groups, or fatty acid esters. The alterations occurring during the critical stages of hydrolysis apparently changed the essential chemical conformation of endotoxic particles before cleavage of a significant amount of material took place. The rates of both saponification and detoxification were markedly increased by carrying out the reaction in media of ethyl alcohol or dimethylsulfoxide instead of water.
Protoplasm separated from disrupted cells of gram-negative bacteria was extracted with hot phenol-water or was precipitated with ethyl alcohol after digestion with Pronase. These methods recovered about 10 times more endotoxin than was detectable in the untreated protoplasm. Inactivation of endotoxin by protoplasm also occurred in vitro when the endotoxin was first dissociated into subunits before reaction with protoplasm. Despite this increased yield from another source, the major proportion of endotoxin was still found in the cell walls.
Endotoxin dissociated into subunits by sodium deoxycholate treatment exhibited diminished capacity to kill chick embryos, protect mice against the lethal effects of infection with Salmonella typhi, evoke hemorrhagic necrosis in skin inoculated with epinephrine, prepare for and provoke the dermal Shwartzman reaction, and induce pyrogenic tolerance. Surfactant-treated material which had been allowed to reaggregate displayed activity equivalent to that of untreated material. These findings were consistent with the working hypothesis that a macromolecular complex of critical size is required in order for endotoxin to elicit its characteristic effects in the host.
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When endotoxins extracted from enteric bacteria were mixed in the presence of sodium deoxycholate, and the bile salt was subsequently removed by dialysis or by extraction with ethanol, a new type of endotoxin was formed. The latter material was as biologically active as the original endotoxins and possessed a combination of antigenic determinants that were previously unique to each of the individual endotoxins in the mixture. This hybrid formation between endotoxins was detected by immunodiffusion and radioautography and by quantitative precipitation procedures.
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Anacker, R. L. (Rocky Mountain Laboratory, Hamilton, Mont.), W. D. Bickel, W. T. Haskins, K. C. Milner, E. Ribi, and J. A. Rudbach. Frequency of occurrence of native hapten among enterobacterial species. J. Bacteriol. 91:1427-1433. 1966.-Smooth cultures of representative Enterobacteriaceae were screened for the presence of native hapten, a substance previously extracted with trichloroacetic acid from the protoplasmic fraction of one strain each of Escherichia coli O111:B4 and O113. Trichloroacetic acid extracts of protoplasmic fractions of the cells were analyzed for chemical composition, for constituent sugars by paper chromatography, for immunochemical relationship to endotoxin purified by gel filtration, for sedimentation behavior, and for pyrogenicity in rabbits and lethal toxicity in chick embryos. Extracts from two of three additional strains of E. coli O113, all five additional strains of E. coli O111:B4, and one strain each of E. coli O26:B6 and O55:B5 were similar to previously described native hapten in chemical composition, sedimentation properties (S(20,w), 3.7 to 5.2), biological potency (usually less than 0.1% that of corresponding endotoxin), and immunochemical relationship to endotoxin. Extracts of one strain each of E. coli O127:B8, Serratia marcescens, Citrobacter freundii, Salmonella enteritidis, and of two lipopolysaccharide-deficient mutants of S. enteritidis differed from typical native hapten. The biosynthetic relationship of native hapten to endotoxin has not yet been revealed.
Rudbach, Jon A. (The University of Michigan, Ann Arbor), and Arthur G. Johnson. Alteration of endotoxin activity after complexing with plasma proteins. J. Bacteriol. 92:892-898. 1966.-A substantial decrease in the ability of endotoxin to be precipitated by homologous antiserum and to cause fever occurred after incubation with human plasma or human plasma, Cohn fraction IV-1. The endotoxin, thus altered, also displayed decreased lethality for rabbits. These alterations in endotoxin activity could be restored when the endotoxin-plasma protein mixture was treated with a proteolytic enzyme, and the endotoxin was precipitated with ethyl alcohol. Inactivation of the antigenic and toxigenic properties of the endotoxin molecule by plasma is discussed as resulting from complexing with plasma proteins rather than from enzymatic degradation.
Ribi, E. (Rocky Mountain Laboratory, Hamilton, Mont.), R. L. Anacker, R. Brown, W. T. Haskins, B. Malmgren, K. C. Milner, and J. A. Rudbach. Reaction of endotoxin and surfactants. I. Physical and biological properties of endotoxin treated with sodium desoxycholate. J. Bacteriol. 92:1493-1509. 1966.-Endotoxins from three species of gram-negative bacteria were shown to be dissociated by the bile salt sodium deoxycholate (NaD) into nontoxic subunits with molecular weights of about 20,000. When the bile salt was removed by dialysis, the subunits reaggregated in an orderly manner to form a relatively uniform population of biologically active endotoxin particles with average molecular weights of 500,000 to 1,000,000. If a small amount of human plasma was added to the dissociated endotoxin before removal of the NaD, reassociation apparently did not occur and the preparation remained nonpyrogenic. However, the plasma protein could subsequently be removed from the endotoxin subunits, and reaggregation to the toxic form would then occur. The studies on the physical nature of endotoxin performed with biophysical solution techniques were supplemented and confirmed by direct examination of the endotoxin polymers by electron microscopy. The results of these studies were consonant with the theory that the biologically active endotoxic elements are composed of micellar aggregates of linear lipopolysaccharide subunits.