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PubMed1979

Isolation, characterization, and biological properties of an endotoxin-like material from the gram-positive organism Listeria monocytogenes.

The bacterial component responsible for the induction of transient cold agglutinin syndrome in rabbits after intravenous injection of heat-killed Listeria monocytogenes type 4B has been purified and biologically and chemically characterized. A purified immunoglobulin M cold agglutinin was prepared from high-titer sera resulting from the immunization of rabbits with heat-killed L. monocytogenes type 4B and was subsequently used to monitor the purification of the bacterial component responsible for its induction. The bacterial component was isolated from a hot phenol-water extract of lyophilized L. monocytogenes type 4B by multiple molecular sieve chromatography. Upon chemical analysis the purified material was found to be strikingly similar in chemical composition to gram-negative lipopolysaccharide endotoxins. The material contained 15% total fatty acid (of which 50% was beta-hydroxymyristic acid), 40 to 45% neutral sugar (glucose, galactose, and rhamnose), 11.5% amino sugar, 12% uronic acid, 2.5% 2-keto-3-deoxyoctonic acid, 2% heptose, 0.87% phosphorus, and 1.6% amino acid, thereby accounting for 85 to 90% of the weight of the component. Electron micrographs of the purified material were similar to those of lipopolysaccharide preparations from gram-negative organisms. The purified material exist in aqueous solutions as large aggregates, but can be dissociated into a single smaller subunit (3.1S) by dialysis against sodium dodecyl sulfate buffer. The listerial component was toxic and pyrogenic to rabbits, producing symptoms typical of gram-negative endotoxins. Activity in the limulus lysate gelation assay and in the carbocyanine dye assay provides a further link of this material with classical gram-negative endotoxins.

Agglutinins↗
PubMed1980

Preparation of a nontoxic and immunogenic polysaccharide fraction from a Haemophilus influenzae phenol-water extract.

A phenol-water extract from Haemophilus influenzae type a was hydrolyzed to decrease the toxicity without affecting the antigenicity of the preparation. We used partial hydrolysis for 15 h with ion exchangers in the presence of chloroform. The lipid fraction was collected into the organic solvent. The preparation obtained from the aqueous solution was designated the polysaccharide fraction. Rhamnose, glucose, galactose, mannose, and glucosamine were the major components of the polysaccharide fraction, and their molar ratios were determined by gas-liquid chromatography; 2.5% myristic acid was also found in the polysaccharide fraction. The mild hydrolysis of the polysaccharide fraction for 15 h caused a marked reduction in toxicity (50% lethal dose, 183 +/- 9 microgram/kg) and pyrogenicity. The generalized Sanarelli reaction was negative. The local Shwartzman phenomenon was not observed if chloroform and Dowex were exchanged three times during hydrolysis. Most of the antigenic components remained active after the hydrolytic process. The polysaccharide fraction could also induce the formation of circulating antibodies in rabbits and also increase the phagocytic process against H. influenzae from month 2 to 6.

Animals↗
PubMed1980

Lipophilic derivative of muramyl dipeptide is more active than muramyl dipeptide in priming macrophages to release superoxide anion.

Mouse peritoneal macrophages, when treated with a lipophilic derivative of muramyl dipeptide either in vitro or in vivo by intraperitoneal injection, showed a more than fivefold increase in their ability to generate superoxide anion after stimulation of the macrophages with phorbol myristate acetate. This response was more than twice that observed with the parent molecule, muramyl dipeptide (MDP). Unlike MDP, which has a systemic effect, the lipophilic derivative, [B30]-MDP, did not alter the response of peritoneal macrophages when given subcutaneously in the flank, suggesting that [B30]-MDP remains localized at the site of injection. The enhanced effect of [B30]-MDP over MDP appeared to be due to the inherent lipophilicity of the molecule, and was probably not due to either stimulation of T lymphocytes or activation of the alternative pathway of complement.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1981

Rabbit macrophages secrete two biochemically and immunologically distinct endogenous pyrogens.

Rabbit endogenous pyrogens occurred in two forms. One was an apparently single protein with a pI of 7.3; the other was a family of proteins with pI values of 4.5 to 5.0. We selected two of the latter, with pI values of 4.6 and 4.72, as representative of the group and compared them with the pI 7.3 pyrogen. Antisera raised in three goats completely neutralized the pyrogenic activity of the pI 7.3 pyrogen. Larger doses of these antisera did not block the pyrogenic activity of either of the pI 4.5 to 5.0 pyrogens. The pI 7.3 pyrogen contained a free --SH group which was essential to its biological activity. It was inactivated by 100 mM N-ethylmaleimide or 200 mM iodoacetamide, bound to Thiol-Sepharose columns, and could be eluted from them with mercaptoethanol. Neither of the pI 4.5 to 5.0 pyrogens was inactivated by N-ethylmaleimide or iodoacetamide, and neither bound to Thiol-Sepharose. Both endogenous pyrogens gave negative results in the Limulus lysate test for bacterial endotoxins. These results suggest that the pI 7.3 and pI 4.5 to 5.0 endogenous pyrogens are not closely related to each other and are consistent with the idea that they may not be related at all. Alternative hypotheses are discussed.

Animals↗
PubMed1982

Biological activity of a new synthetic muramyl peptide adjuvant devoid of pyrogenicity.

Immunostimulant activities of muramyl dipeptide (enhancement of specific immune responses and of nonspecific resistance to infection) were retained by its N-acetylmuramyl-L-alanyl-D-glutaminyl-n-butyl ester derivative, although very large amounts administered intravenously, or even by the very sensitive intracerebroventricular route, did not elicit fever in the rabbit. This analog also appeared to be devoid of other secondary effects which have been observed after administration of muramyl dipeptide.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1982

Pyocin-resistant lipopolysaccharide mutans of Neisseria gonorrhoeae: alterations in sensitivity to normal human serum and polymyxin B.

Pyocins from Pseudomonas aeruginosa were used to select several lipopolysaccharide (LPS) mutants of Neisseria gonorrhoeae strain FA19. Three classes of LPS mutans were found in the initial group selected for study. The LPS of one class lacked galactose. That of a second group lacked the typical heptose found in the parental LPS, was reduced in glucose, galactose, and N-acetylglucosamine content, appeared to contain a new unidentified sugar component, and consisted of two species of LPS separable on sodium dodecyl sulfate-polyacrylamide gels. The LPS of a third strain lacked the heptose, glucose, galactose, and N-acetylglucosamine found in the oligosaccharide portion of parental FA19 LPS. The minimal inhibitory concentration for polymyxin B of the mutant strains was 3 to 4 times that of the parental strain. The strains lacking only galactose were as resistant as the parent to the bactericidal action of normal human serum, but cells of the other two classes were quickly killed by serum. Gonococcal LPS thus appears to be important in determining phenotypic properties of the cells.

Blood Bactericidal Activity↗
PubMed1983

Crossed immunoelectrophoretic analysis of Legionella pneumophila serogroup 1 antigens.

By crossed immunoelectrophoresis, 85 different antigens were demonstrated in sonicated preparations of Legionella pneumophila serogroup 1 (Lp1). The precipitin patterns of 82 anodic-migrating antigens were numbered and were designated the Lp1 reference system. Eleven antigens were stable to boiling, and seven of these were shown to be surface antigens. One heat-stable surface antigen (antigen no. 61) was highly reactive with limulus amoebocyte lysates and formed a precipitin resembling lipopolysaccharide. Serum from an isolation confirmed case of Lp1 infection and serogroup-specific rabbit antiserum reacted specifically with antigen no. 61, which was designated the serogroup-specific antigen. Normal human and rabbit sera commonly had antibodies to antigen no. 66 of the Lp1 reference system. This antigen is antigenically related to the "common antigen" of Pseudomonas aeruginosa.

Animals↗
PubMed1983

Preparation and physicochemical and immunological characterization of polysaccharide-outer membrane protein complexes of Neisseria meningitidis.

A crude complex containing group C polysaccharide, outer membrane proteins, and lipopolysaccharide (LPS) was isolated from the cell-free culture liquid of Neisseria meningitidis serogroup C, serotype 2a. Group C polysaccharide and LPS were removed from this complex, resulting in an outer membrane complex and a purified complex, respectively. Analysis by electron microscopy showed the outer membrane origin of the crude complex and the outer membrane complex, whereas such a structure was absent in the purified complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of the three complexes were identical. Pyrolysis-mass spectrometry data correlated well with those obtained by the biochemical assays and suggested a low LPS content in the purified complex and a low polysaccharide content in the outer membrane complex. The purified complex was shown to be nonpyrogenic and could be prepared with the same yield as that of purified polysaccharide. The immunogenic activities of the complexes were studied in mice. The antibodies were measured by the enzyme-linked immunosorbent assay; and the bactericidal antibody assay. All complexes induced immunoglobulin G antibodies to group C polysaccharide as well as to the serotype antigen, although the removal of polysaccharide and LPS resulted in a reduction of the immunogenic activities of outer membrane complex and purified complex, respectively. A second dose of all complexes produced a clear booster effect of both antibody responses. The antibodies were bactericidal.

Animals↗
PubMed1983

Induction of endotoxin tolerance with nonpyrogenic O-antigenic oligosaccharide-protein conjugates.

We prepared a dodecasaccharide, specific for the O-antigenic polysaccharide chain of Salmonella typhimurium (O-antigens 4 and 12), by the partial hydrolysis of the O-polysaccharide chain, utilizing bacteriophage 28B endo-alpha-L-rhamnosidase. The dodecasaccharide was shown by chemical and spectroscopical analyses to be totally devoid of lipid A and core oligosaccharide. By coupling this dodecasaccharide to human serum albumin, a glycoconjugate (DODECA-4809-ITC-HSA) was prepared and found to be (i) nonpyrogenic, (ii) unable to gelate a Limulus amoebocyte lysate, and (iii) unable to induce early-phase pyrogenic tolerance to endotoxin. Rabbits immunized either intravenously (with the glycoconjugate suspended in saline) or intrapopliteally (with the glycoconjugate suspended in Freund complete adjuvant) developed a significant although modest pyrogenic tolerance against challenge with the O-antigenic homologous S. typhimurium lipopolysaccharide (P less than 0.025 and P less than 0.01 for immunized and control rabbits, respectively). The evoked tolerance was O-antigen specific since no pyrogenic tolerance against challenge with lipopolysaccharide from S. thompson (possessing identical lipid A and core oligosaccharide structures but differing in the O-antigen polysaccharide chain) could be seen (P greater than 0.1). These results demonstrate that a nonpyrogenic O-antigenic polysaccharide hapten, when coupled to an immunogenic carrier protein, evokes immune responses which mediate significant, although modest, late-phase tolerance and is capable of partly reducing the pyrogenic activity of the O-antigenic homologous lipopolysaccharide.

Animals↗
PubMed1984

A Cytophaga species endotoxin as a putative agent of occupation-related lung disease.

A previous study suggested that a biologically active bacterial endotoxin was a putative agent of lung disease in a textile-producing facility. The endotoxin was isolated from the biomass growing in a chilled-water spray air humidification system. The bacterial flora of the air humidification system were isolated and taxonomically identified to the genus level. By using indirect immunofluorescence assays, a serologically reactive Cytophaga species was identified. A serologically reactive, biologically active (Limulus assay) endotoxin was purified from phenol extracts of the Cytophaga species. The endotoxin contained sugars, hexosamines, and lipids identical to those found in the humidifier biomass endotoxin. All subjects with biopsy-proven and suspected lung disease had antibodies directed toward the purified Cytophaga endotoxin. The data suggest that the Cytophaga endotoxin is the putative agent of lung disease in the textile facility.

Bacterial Infections↗
PubMed1984

Immunochemical characterization of high-molecular-weight polysaccharide from Fisher immunotype 3 Pseudomonas aeruginosa.

A high-molecular-weight polysaccharide (PS) was isolated from the culture supernatant of a Fisher immunotype 3 (IT-3) strain of Pseudomonas aeruginosa. Consistent with previously reported findings for IT-1 and IT-2 PS, the preparation of IT-3 PS was found to be an immunogenic, nontoxic form of the O polysaccharide side chain on the lipopolysaccharide (LPS). The IT-3 PS was mainly carbohydrate in composition. It was serologically and chemically identical to LPS O side chain, but distinct from that structure in molecular size and immunogenicity. The IT-3 PS was nontoxic in mice and guinea pigs, nonpyrogenic in rabbits, and greater than 1,000-fold less reactive than IT-3 LPS in gelation of the Limulus amoebocyte lysate. Preliminary analyses by gas-liquid chromatography and 13C nuclear magnetic resonance have established the structural identity of IT-3 high-molecular-weight PS and the IT-3 O side chain. IT-3 PS was immunogenic in rabbits and mice. After active immunization, mice were protected against P. aeruginosa IT-3 intraperitoneal infection and burn wound sepsis. IT-3 PS also elicited protection against challenge with an IT-5 strain of P. aeruginosa, indicating that low-level contamination of the IT-3 PS with IT-3 LPS was not responsible for the immunogenic activity. These findings demonstrate the feasibility of preparing nontoxic immunogenic IT-3 PS capable of eliciting serotype-specific protective antibodies, employing methods similar to those previously described for the isolation of PS from other P. aeruginosa immunotypes.

Animals↗
PubMed1985

Biochemical and immunobiological properties of lipopolysaccharide (LPS) from Bacteroides gingivalis and comparison with LPS from Escherichia coli.

Lipopolysaccharides (LPSs) were isolated from Bacteroides gingivalis and Escherichia coli by the phenol-water and butanol-water procedures. The phenol-water-extracted LPS from B. gingivalis 381 was composed of 46% carbohydrate, 23% hexosamine, 18% fatty acid, and 5% protein. The major component sugars of this preparation were glucose, glucosamine, rhamnose, galactose, galactosamine, and mannose, and their molecular ratio was 1:0.9:0.7:0.6:0.6:0.4, respectively. Neither heptose nor 2-keto-3-deoxyoctonate was detected. The butanol-water-extracted LPS from this strain was composed of 76% glucose, 7% fatty acid, and 13% protein, and it was associated with a number of polypeptides (13 to 56 kilodaltons). The main fatty acid of both LPS preparations was palmitic acid. It was found that biological activities of LPS from B. gingivalis were comparable to those of LPS from E. coli in terms of activation of the clotting enzyme of Limulus amebocyte lysate, mitogenicity, polyclonal B cell activation, and stimulation of interleukin 1 production in BALB/c mice. Furthermore, LPS-nonresponsive C3H/HeJ spleen cells were found to yield good mitogenic responses to both phenol-water-extracted LPS and butanol-water-extracted LPS from B. gingivalis or butanol-water-extracted LPS from E. coli. On the other hand, spleen cells from LPS-responsive C3H/HeN mice responded well to all these LPS preparations.

Amino Acids↗
PubMed1985

Immunopharmacological activities of a synthetic counterpart of a biosynthetic lipid A precursor molecule and of its analogs.

Synthetic lipid A analogs (compounds 404 through 406) were examined for their immunopharmacological activities. These compounds had two amide-bound and two ester-bound (R)-3-hydroxytetradecanoyl groups at the C-2 and C-2' and the C-3 and C-3' positions, respectively, of beta (1-3)glucosamine disaccharide. In all of the in vitro assays, these synthetic compounds exhibited high activities comparable to those of a reference lipid A prepared from Escherichia coli O8:K27 Re-mutant strain F515. The compounds activated the clotting enzyme cascade of the horseshoe crab, activated the human complement via the classical pathway, caused polyclonal B-cell activation, stimulated the phagocytosis of sheep erythrocytes by murine peritoneal macrophages, and enhanced the migration of human polymorphonuclear leukocytes. They also increased the thymidine uptake of splenocytes of BALB/c nu/nu and C3H/HeN mice but not those of C3H/HeJ (a nonresponder to lipopolysaccharide). A dephosphorylated derivative, compound 403, was barely active in all of the above assays except for the enhancement of polymorphonuclear leukocyte migration. However, compounds 404 through 406 were feeble in pyrogenicity and could not prepare the local Shwartzman reaction, although they were very lethal to galactosamine-loaded mice. Therefore, synthetic lipid A analogs described here were fully immunopharmacologically active in in vitro assays, but all of them were far less active than natural E. coli F515 lipid A regarding the biological activities characteristic of endotoxic lipopolysaccharides and lipid A's. The high lethal toxicity of compound 406 (1,4'-bisphosphate) to the galactosamine-loaded mice may not reflect its real toxicity to normal mice. In all activities examined, compound 406 was quite comparable to a biosynthetic lipid A precursor, a natural counterpart of compound 406. The immunopharmacological activities of these newly synthesized lipid A analogs, especially compound 406, were much stronger than those of compounds that had been synthesized earlier by using the originally proposed model of the lipid A structure. The findings described in this report justify the acylation pattern of a disaccharide backbone of lipid A, revised on the basis of recent analytical studies. The low in vivo endotoxic activities of the present lipid A analogs are most probably due to the fact that the kinds of acyl groups were different from those of the complete lipid A from E. coli, although there were no differences in the acylation positions on the disaccharide backbone.

Adjuvants, Immunologic↗
PubMed1985

Lipopolysaccharide variation in Coxiella burnetti: intrastrain heterogeneity in structure and antigenicity.

We isolated lipopolysaccharides (LPSs) from phase variants of Coxiella burnetii Nine Mile and compared the isolated LPS and C. burnetii cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The LPSs were found to be the predominant component which varied structurally and antigenically between virulent phase I and avirulent phase II. A comparison of techniques historically used to extract the phase I antigenic component revealed that the aqueous phase of phenol-water, trichloroacetic acid, and dimethyl sulfoxide extractions of phase I C. burnettii cells all contained phase I LPS, although the efficiency and specificity of extraction varied. Our studies provide additional evidence that phase variation in C. burnetii is analogous to the smooth-to-rough LPS variation of gram-negative enteric bacteria, with phase I LPS being equivalent to smooth LPS and phase II being equivalent to rough LPS. In addition, we identified a variant with a third LPS chemotype with appears to have a structural complexity intermediate to phase I and II LPSs. All three C. burnetii LPS contain a 2-keto-3-deoxyoctulosonic acid-like substance, heptose, and gel Limulus amoebocyte lysates in subnanogram amounts. The C. burnetii LPSs were nontoxic to chicken embryos at doses of over 80 micrograms per embryo, in contrast to Salmonella typhimurium smooth- and rough-type LPSs, which were toxic in nanogram amounts.

Bacterial Proteins↗
PubMed1985

Activation and inhibition of Limulus amebocyte lysate coagulation by chemically defined substructures of lipid A.

Recent work with lipid mutants of Escherichia coli and Salmonella typhimurium has helped to elucidate the correct structure of lipid A and has suggested a biosynthetic pathway. Precursor molecules include diacylglucosamine 1-phosphates and tetraacyl disaccharide bis-phosphates. The activities of several of these compounds and of their derivatives were measured by Limulus amebocyte lysate (LAL) assay. We report that (i) both mono- and disaccharide precursors of lipid A activate LAL, (ii) two acyl chains on the monosaccharide subunit of lipid A are necessary for activation of LAL, and (iii) the monosaccharide, 2-monoacylglucosamine 1-phosphate can competitively inhibit LAL activation by diacyl monosaccharide lipid A precursors. However, 2-monoacylglucosamine 1-phosphate did not inhibit endotoxin activation of LAL. One unanticipated finding was that the activities of the monosaccharides were reduced upon storage even though their covalent structures were unchanged. Perhaps this is due to alterations in physical state. Thus, these lipid A precursors and derivatives offer some insight into the structural features required for activation of the LAL assay and may in the future provide derivatives which are competitive inhibitors of endotoxin.

Drug Stability↗
PubMed1991

Endotoxin shedding by enterobacteria: free and cell-bound endotoxin differ in Limulus activity.

The endotoxin activities of gram-negative bacteria and their lipopolysaccharides (LPS) have been quantitated by a chromogenic Limulus amocbocyte lysate (CLAL) assay. When bacterial cell exposing various cell surface structures were compared, the highest Limulus activities were found in R strains of Escherichia coli and Salmonella typhimurium mutants. E. coli with K antigens did not differ from K-negative strains. By measuring beta-hydroxymyristic acid (3-OH tetradecanoic acid, beta-OHC14:0), it was possible to compare the CLAL activities of LPS bound to bacterial cells, LPS shed into the culture medium, and purified LPS. After 16 h of growth, the cell-free culture supernatants of three E. coli O1K1 strains and S. typhimurium showed CLAL activities 14.3 to 20.3 times higher than did the corresponding bacterial cell suspensions in relation to their beta-OHC14:0 contents. Four other E. coli strains (O serotypes O14, O24, and O75) and the S. typhimurium 395 R mutants MR5 and MR6 showed CLAL values 2.8 to 7.9 times higher in their culture supernatants. LPS of E. coli O1K1 and S. typhimurium had lower CLAL activities than the culture supernatants (1/10 and 1/4, respectively). Although the beta-OHC14:0 concentrations of the culture supernatants were approximately half those of the corresponding bacterial cells, all had CLAL values that were 2 to 21 times higher. The bacterial cell suspension, culture supernatant, and purified LPS of S. typhimurium MS were compared by CLAL assay and a quantitative enzyme-linked immunosorbent assay based on monoclonal antibodies to the O5 antigen. Endotoxin shed into the culture medium was the most CLAL-active form of LPS, while purified LPS was the most antigen-active form. The results emphasize the importance of appropriate standards when quantifying endotoxin in various states. In conclusion, E. coli and S. typhimurium bacteria shed significant amounts of endotoxin into the surrounding medium during growth. This form of LPS is more CLAL active than the cell-bound or purified LPS.

Animals↗
PubMed1991

Enzymatically deacylated Neisseria lipopolysaccharide (LPS) inhibits murine splenocyte mitogenesis induced by LPS.

Acyloxyacyl hydrolase is a leukocyte enzyme that selectively removes the secondary acyl chains from the lipid A moiety of gram-negative bacterial lipopolysaccharides (LPS). As predicted by the reported contribution of secondary acyl chains to the bioactivities of lipid A analogs, enzymatic deacylation of Salmonella typhimurium Rc LPS substantially reduces its potency in the dermal Shwartzman reaction and in several in vitro assays that measure responses of human endothelial cells and neutrophils, whereas the potency of this LPS for inducing murine splenocyte mitogenesis is affected much less. In the experiments described here, we studied the impact of acyloxyacyl hydrolysis on the bioactivities of several LPS that differ from Salmonella LPS in carbohydrate and lipid A structures. Deacylated LPS from Escherichia coli, Haemophilus influenzae, Neisseria meningitidis, and S. typhimurium were similarly reduced in potency in the Limulus lysate test (30- to 60-fold reduction in potency relative to the corresponding mock-treated LPS), and the ability of all of these deacylated LPS to stimulate neutrophil adherence to human endothelial cells was reduced by a factor of 100 or more. For LPS from E. coli, H. influenzae, and Pseudomonas aeruginosa, the impact of deacylation on spleen cell mitogenesis was also similar to that observed for S. typhimurium LPS: deacylation reduced potency by less than 15-fold. Unexpectedly, the potency of Neisseria LPS in the murine splenocyte mitogenicity test was reduced over 100-fold by deacylation, and deacylated Neisseria LPS could block the mitogenic activity of Neisseria and Salmonella LPS. These studies indicate that the contribution of secondary acyl chains to the bioactivities of a given LPS cannot be predicted with confidence from the reported structure-activity relationships of lipid A or from the behavior of other deacylated LPS.

Acetylation↗