Nobuhiko Kasai

Fatty acid composition and Shwartzman activity of lipopolysaccharides from oral bacteria.

The composition and the nature of the linkage of fatty acids and the Shwartzman activity of lipopolysaccharide (LPS) preparations derived from oral gram‐negative bacteria including Bacteroides gingivalis, Bacteroides loesheii, Eikenella corrodens, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans were examined. 3‐Hydroxylated and nonhydroxy fatty acids of various chain lengths were found in all of the LPS preparations. All nonhydroxy fatty acids were found to be ester‐bound, and part of the 3‐hydroxy fatty acids in the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans were shown to be involved in ester linkage. It was also suggested that the hydroxy group of the ester‐bound 3‐hydroxy fatty acid of the LPS of F. nucleatum and A, actinomycetemcomitans is at least partly substituted by another fatty acid, but in the LPS of B. gingivalis and E. corrodens it is not. The main amide‐linked fatty acid of the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans was 3‐hydroxyheptadecanoic, 3‐hydroxydodecanoic, 3‐hydroxyhexadecanoic, and 3‐hydroxytetradecanoic acid, respectively. The results of the Shwartzman assay showed that the E. corrodens LPS was the most active among the preparations tested, and that the Shwartzman toxicity of Bacteroides LPS is extremely low.

Pseudomonasdiminuta LPS with a new endotoxic lipid A structure

Lipid A that contains mainly 2,3-diamino-2,3-dideoxy-D-glucose, phosphate and fatty acids in the molar ratio 2:1:5-6 was found in Pseudomonas diminuta lipopolysaccharide. The lipid A was considered to have a diamino-sugar disaccharide structure that carries a nonglycosidic phosphomonoester group and amide-bound acyloxyacyl and 3-hydroxy fatty acyl groups. The lipopolysaccharide exhibited endotoxic activities including lethal toxicity, pyrogenicity, local Shwartzman activity, body weight-decreasing toxicity and Limulus activity. The free lipid A was also endotoxic.

Endotoxic Glycolipid as a Potent Depressor of the Hepatic Drug-Metabolizing Enzyme Systems in Mice*

Inhibitory effects of the endotoxic glycolipid from Salmonella minnesota R595 on hepatic drug‐metabolizing enzyme activities in mice were investigated, and the depressor activity of the glycolipid in the enzyme systems was confirmed. Among degradation products of lipopolysaccharides tested, lipid A preparations derived from the mild acetic acid hydrolysates of lipopolysaccharides were the most active, but the lipid A fractions prepared from the hydrolysates with 1 N‐HCl were almost inactive. A degraded polysaccharide fraction from E. coli lipopolysaccharide was inactive. The activities of the glycolipid and the lipid A preparation were markedly reduced by treatment with alkaline‐hydroxylamine, mild alkali or hydrazine. The data showed that the lipid A moiety of the glycolipid may be responsible for the inhibitory activity on the hepatic drug‐metabolizing enzyme systems.

An Endotoxin‐Induced Serum Factor that Depresses Hepatic δ‐Aminolevulinic Acid Synthetase Activity and Cytochrome P‐450 Levels in Mice

In the previous study we reported that lipid A moiety plays an important role in the activity of bacterial lipopolysaccharide (LPS) to depress hepatic microsomal drug-metabolizing enzyme activity in mice (5). Recent studies on the mechanism of its action showed that the decrease in the hepatic drug-metabolizing enzyme activity, as well as cytochrome P-450 levels seen on endotoxin administration, is closely related to metabolism of the heme moiety required for synthesis of cytochrome P-450 in the liver (2,3,8, 14). On the other hand, since various humoral factors, such as glucocorticoid antagonizing factor (GAF) (10), colony stimulating factor (CSF) (12), tumor necrosis factor (TNF) (4), and serum amyloid A mediator (SAA) (13), which mediate the host response to endotoxin have recently been found, it is also interesting to determine whether or not the effect of endotoxin on the hepatic heme metabolism and cytochrome P-450 is indirectly mediated by a humoral factor. In this study, therefore, we examined the effect of an endotoxin-induced mouse serum factor on hepatic t5-aminolevulinic acid synthetase (ALA synthetase) which is a rate-limiting enzyme for heme synthesis and cytochrome P-450 levels in mice. The endotoxin used in this study was a glycolipid preparation isolated from Salmonella minnesota R595 by the method of Galanos et al (6), and abbreviated as R595 GL or GL. Free lipid A preparation derived from the 1% acetic acid hydrolysate of R595 GL was used in a complex form with bovine serum albumin (Seikagaku-Kogyo Co., Tokyo) according to Galanos et al (7). Male, 5-weekold ddy mice (Shizuoka ]ikkendobutsu Co., Shizuoka) were used in this study and maintained on a laboratory diet (Oriental Yeast Co., Tokyo), and water which were given ad libitum throughout the experiment. Mice in each group were injected intraperitoneally with R595 GL or free lipid A suspended in saline and starved for about 24 hr prior to sacrifice. Endotoxin-induced serum factor, described as post GL serum or post lipid A serum, was prepared from mouse blood which was collected by cardiac puncture at various intervals after endotoxin administration. The serum was pooled from 5 mice per group and stored at -20 C. Normal mouse serum was prepared from control mice injected with the vehicle only. Eighteen hr after serum injection, the ALA synthetase activity was assayed

Structural Heterogeneity Regarding Local Shwartzman Activity of Lipid A

The relation of chemical structure to local Shwartzman activity of lipid A preparations purified by thin‐layer chromatography from five bacterial strains was examined. Two lipid A fractions from E. coli F515—Ec‐A2 and Ec‐A3—exhibited strong activity, similar to that of previous synthetic E. coli‐type lipid A (compound 506 or LA‐15‐PP). The Ec‐A3 fraction contained a component that appeared to be structurally identical to compound 506, and the main component of Ec‐A2 fraction was structurally similar to compound 506 except that it carried a 3‐hydroxytetradecanoyl group at the C‐3′ position of the backbone in place of a 3‐tetradecanoyl‐oxytetradecanoyl group. Free lipid A (12 C) and purified lipid A fractions, Ec‐A2 (12 C) and Ec‐A3 (12 C), respectively, obtained from bacteria grown at 12 C, exhibited activity comparable to Ec‐A2 or Ec‐A3. In these preparations, a large part of the 3‐dodecanoyloxytetradecanoyl group might be replaced by 3‐hexadecenoyloxytetradecanoyl group. Salmonella minnesota R595 free lipid A also contained at least two active lipid A components as seen in E. coli lipid A, but the third component corresponding to the synthetic Salmonella‐type lipid A (compound 516 or LA‐16‐PP) exhibited low activity. A lipid A fraction, Cv‐A4 from Chromobacterium violaceum IFO 12614, which was proposed to have two acyloxyacyl groups at the C‐2 and C‐2′ positions with other acyl groups, exhibited weaker activity than the free lipid A or LPS. The purified lipid A fractions from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 contained an unusual backbone with 2,3‐diamino‐2,3‐dideoxy‐d‐glucose disaccharide phosphomonoester, and these lipid A (Pd‐A3 and Pv‐A3) exhibited strong activity comparable to the E. coli lipid A. Thus, the present results show that the local Shwartzman reaction can be expressed by partly different lipid A structures in both hydrophilic backbone and fatty acyl residues; when they have the same backbone the potency varies markedly depending on the structure of the acyl residues.

Tumor Necrosis Factor‐inducing Activities of Lipid A Preparations from Pseudomonas diminuta and Pseudomonas vesicularis

Tumor necrosis factor (TNP)‐inducing activities of lipid A preparations from P. diminuta and P. vesicularis, which contain mainly 2 mol of 2,3‐diamino‐2,3‐dideoxy‐D‐glucose and 1 mol of nonglycosidic phosphate as the backbone component and have partly different fatty acid compositions, were examined. TNF was induced by injecting various lipid A fractions into mice that had previously been sensitized with Mycobacterium bovis BCG vaccine. A major component of lipid A of both strains, referred to as A3 fraction, exhibited stronger TNF‐inducing activity than A2 fraction having incomplete acyl residues. The removal of ester‐linked fatty acyl groups by mild hydrazinolysis of the P. diminuta lipid A results in a marked decrease of the activity. These results suggest that the structure of the hydrophobic part, including the amide‐linked acyloxyacyl group(s), of the lipid A molecule play an important role in inducing TNF in the sera of mice.

Analysis of antigenic reactivity of synthetic monosaccharide lipid A analogues with monoclonal antibodies

In vitro antigenic reactivity of chemically synthesized monosaccharide analogues of nonreducing sugar moiety of lipid A with 4 monoclonal antibodies against Salmonella minnesota R595 lipid A was studied by the inhibition test of enzyme-linked immunosorbent assay (ELISA). In the assays with 2 monoclonal antibodies (mAb 5G and mAb36G), which have been suggested to recognize the Salmonella type lipid A structure, no antigenic reactivity of the tested analogues was observed. However, several monosaccharide analogues exhibited antigenic reactivities with other monoclonal antibodies (mAb161M and mAb19M), especially some structural differences of hydrophobic and/or hydrophilic parts among monosaccharide analogues were recognized by mAb161M.

Neutralization of Shwartzman‐Inducing Activity by Antibodies Recognizing the Re Core or Lipid A Structures of Lipopolysaccharide from Salmonella minnesota R595 and Pseudomonas vesicularis JCM1477

Antibodies recognizing the Re core or lipid A structures of lipopolysaceharide (LPS) derived from Salmonella minnesota R595 and Pseudomonas vesicularis JCM1477 were tested for the ability to neutralize the preparatory activity of endotoxin using the local Shwartzman reaction. Shwartzman‐inducing activity of R595 LPS (Reform) was strongly suppressed when the LPS was incubated with the rabbit anti‐R595 antiserum or the purified IgG antibody which recognizes core region of the LPS. The antiserum also suppressed the preparatory activity of LPS from S. typhimurium SL1102 (Re) and Escherichia coli F515 (Re), but not that of either S. typhimurium LT‐2 (S) LPS or R595 lipid A. Moreover, it was found that the murine monoclonal antibody (MAb), SmRe100G (IgG2a) which recognizes the core region of R595 LPS, significantly suppressed the preparatory activity of R595 LPS. Both conventional antibodies specific to R.595 lipid A, which contains a 1, 4‐bisphosphorylated β‐D‐glucosaminyl‐α‐D‐glucosamine disaccharide structure, and JCM 1477 lipid A, which contains a monophosphorylated 3‐amino‐D‐glucosamine disaccharide structure, neutralized the preparatory activity of homologous and a closely related lipid A, but not that of LPS. In addition, it was observed that MAb Sm5G (IgG2b) specific to enterobacterial lipid A preparations (especially R595 lipid A) neutralized the preparatory activity of R595 lipid A, although the effect was somewhat weak as compared with that of rabbit antiserum. These results suggest that anti‐Re LPS antibody binding to the core of Re LPS is involved in suppressing the endotoxic activity of Re LPS, and that the direct binding of anti‐lipid A antibody to some specific epitopes of lipid A is important in neutralizing the endotoxic activity.

Hepatic Drug-Metabolizing Enzyme System and Endotoxin Tolerance: Structural Requirement of LPS in Induction of an Early Tolerance

The alteration of hepatic drug‐metabolizing enzyme activities in mice given Salmonella endotoxin by single or multiple intraperitoneal injections was investigated. An essentially the same biphasic, early and late phase, endotoxin tolerance was observed in the animals receiving a single injection of endotoxin or repetitive daily injections. The results of reciprocal cross tolerance tests using lipopolysaccharide and free lipid A preparations derived from Salmonella minnesota, Salmonella typhimurium, E. coli, Pseudomonas aeruginosa, and Chromobacterium violaceum suggested that lipid A moiety plays an important role in the induction of early endotoxin tolerance to endotoxin response.

Immunochemistry of lipid A

We previously suggested (1, 2, 8–10) that lipid A epitopes are composed of the backbone and acyl groups of the lipid A molecule, and that lipid A has specific and common or cross-reactive epitopes, in which the specificities are derived from the chemical and conformational structures of the backbone and/or acyl groups. In these studies, the in vitro antigenic reactivity of a number of chemically synthesized lipid A analogs with free lipid A preparations from many strains including E. coli, Salmonella minnesota, Klebsiella pneumoniae, Chromobacterium violaceum, Plesiamonas shigelloides and Pseudomonas diminuta was analyzed by enzyme-linked immunosorbent assay (ELISA) and ELISA inhibition test with monoclonal and conventional antibodies against the free lipid A from S. minnesota R595. During these studies, we found that the development of monoclonal antibodies against lipid A having different backbone and/or hydrophobic structures, and the evaluation of antibody-specificity by various assay systems was important to confirm our hypothesis concerninglipid A epitopes.In the presentstudy, therefore, we examined the in vitro antigenic reactivity of synthetic lipid A analogs and bacterial lipid A with monoclonal and conventional antibodies against the lipid A of E. coli F515, E. coli J5 and P. diminuta JCM 2788, as well as S. minnesota R595 by the ELISA, ELISA inhibition test, and the immunodot assay.