lipid-a and Fever

E5531 is a specific lipid A antagonist and is expected to be a drug for the treatment of septic shock. The LAL (limulus amebocyte lysate) activity and pyrogenicity of E5531 were determined. The LAL activity of E5531 is large and confirmed that E5531 had a high affinity to the lipopolysaccharide receptor. The pyrogenic activity of E5531 is weak and the pyrogenic profile of E5531 is different from that of USP (United States Pharmacopoeia) reference standard endotoxin (ETX). E5531 suppressed the pyrogenicity of ETX in rabbits, indicating that E5531 is expected to be useful for the treatment of fever induced by ETX.

Topics: Analgesics, Non-Narcotic; Animals; Dose-Response Relationship, Drug; Endotoxins; Fever; Lipid A; Rabbits

The synthesis of a series of novel analogues of lipid A, the active principle of lipopolysaccharide, is reported. In these compounds, the 1-O-phosphono and (R)-3-hydroxytetradecanoyl moieties of native Salmonella minnesota R595 lipid A have been replaced with hydrogen and the length of the normal fatty acyl residues has been systematically varied. Normal fatty acid chain length in the 3-O-desacyl monophosphoryl lipid A (MLA) series is shown to be a critical determinant of iNOS gene expression in activated mouse macrophages and the induction of proinflammatory cytokines in human peripheral monocytes. Examination of pyrogenicity in rabbits and lethal toxicity in D-galactosamine-treated mice shows that toxic effects in the MLA series can be ameliorated by modifying fatty acid chain length. When used as an adjuvant for tetanus toxoid vaccines, certain MLA derivatives enhance the production of tetanus toxoid-specific antibodies in mice.

Topics: Adjuvants, Immunologic; Animals; Cytokines; Female; Fever; Humans; Immunoglobulin G; In Vitro Techniques; Lethal Dose 50; Lipid A; Macrophages; Mice; Monocytes; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rabbits; Salmonella; Structure-Activity Relationship; Tetanus Toxoid; Vaccination

In a previous study, a chemically synthesized disaccharide precursor of lipid A (406; identical to lipid IVA) was shown to have dramatically reduced lethality, B-cell mitogenicity, and tumor necrosis factor induction in macrophages when its hydroxyl groups were replaced with either succinyl or acetyl residues (K. Tanamoto, FEBS Lett. 351:325-329, 1994). Succinylated 406 was found to lose Limulus amoebocyte lysate gelation activity completely as a result of the modification (about 10(5)-fold), too, as expected. However, acetyl 406, surprisingly, exhibited activity comparable to that of the original 406. Both succinylated and acetylated 406 lost pyrogenicity completely. These results indicate that one of the typical endotoxic activities was dissociated from the others and that the ability to induce Limulus amoebocyte lysate gelation is not always representative of endotoxin activity.

Topics: Acetylation; Animals; Fever; Limulus Test; Lipid A; Pyrogens; Rabbits; Structure-Activity Relationship; Succinates

Topics: Animals; Carbohydrate Conformation; Carbohydrate Sequence; Endotoxins; Fever; In Vitro Techniques; Lethal Dose 50; Leukotriene C4; Lipid A; Lipopolysaccharides; Macrophages; Mice; Molecular Sequence Data; Mutation; Pyrogens; Rabbits; Rhodobacter capsulatus; Salmonella; Structure-Activity Relationship; Tumor Necrosis Factor-alpha; X-Ray Diffraction

Lipid A is the active principle of lipopolysaccharide (LPS). Synthetic lipid A analogues with monosaccharide backbones, GLA-60, GLA-69 and GLA-58, which exhibit potent, weak and scarce agonistic activities of LPS, respectively, induced tolerance against LPS lethality in galactosamine-(GalN)-sensitized mice while none of them were pyrogenic in rabbits. The tolerance-inducing mechanisms were investigated focusing on the regulation of tumor-necrosis-factor-alpha(TNF-alpha)-mediated lethal pathway of LPS. Induction of serum TNF-alpha in LPS-challenged mice was suppressed by prior administration of these analogues as well as LPS. Prior treatment of murine macrophages with the substances suppressed LPS-stimulated TNF-alpha production in the culture supernatant and TNF-alpha mRNA expression in the cells as well. Lethal toxicity of TNF-alpha in GalN-sensitized mice was effectively suppressed by prior treatment with LPS, GLA-60 and GLA-69 but not by GLA-58. This protective effect was suggested to be mediated by endogenous TNF-alpha, which was induced by prior treatment with the effective substances, because either neutralization of endogenously induced TNF-alpha activity with an antibody or deletion of its induction by using LPS-resistant C3H/HeJ mice reduced the protective effect, and a detectable amount of TNF-alpha was produced by stimulating macrophages with the effective substances but not with GLA-58. These results indicated that multiple regulation steps (one is prior to and the others are following TNF-alpha production) are participating in the tolerance induction by LPS and some lipid A analogues and that GLA-58 is a characteristic compound which induces the tolerance by only blocking the step prior to TNF-alpha production.

Topics: Animals; Drug Tolerance; Fever; Galactosamine; Lipid A; Lipopolysaccharides; Macrophages, Peritoneal; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Molecular Structure; Rabbits; RNA, Messenger; Salmonella; Tumor Necrosis Factor-alpha

A pentaacyl precursor of lipid A biosynthesis, termed precursor Ib, and a structural isomer have been chemically synthesized. These compounds were, in comparison to synthetic Escherichia-coli type lipid A or lipopolysaccharide, analyzed for their activity in typical endotoxin test systems. It was found that both precursor Ib and the isomer exhibited similar or only slightly lower pyrogenic, lethal and Shwartzman-phenomenon-inducing activity than lipid A. All preparations were comparable in their B-lymphocyte mitogenicity, macrophage-activating capacity and immunoreactivity towards lipid A antisera. The proton nuclear magnetic resonance spectra of the 1-dephospho derivative of synthetic and bacterial precursor Ib were indistinguishable proving that the previously proposed structure for precursor Ib is correct.

Topics: Animals; Antigens; Endotoxins; Escherichia coli; Fever; Glycolipids; Hemolysis; Lethal Dose 50; Lipid A; Macrophage Activation; Magnetic Resonance Spectroscopy; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mitosis; Rabbits; Spleen

To investigate the structure-activity relationships, various biological activities, including pyrogenicity, lethal toxicity, elicitation of Shwartzman reaction, mitogenicity and tumor necrosis factor (TNF)-inducing activity, were compared among natural and synthetic lipid A's differing in fatty acid composition. In all these tests, natural lipid A's from Escherichia coli and Salmonella minnesota and synthetic LA-15-PP, which carries 3-hydroxy- and 3-acyloxy-tetradecanoyl groups at the 2, 3 and 2', 3' positions, respectively, showed the strongest activities among the tested lipid A's. In contrast, LA-16-PP, in which the amide-bound 3-hydroxytetradecanoic acid at position 2 of LA-15-PP is replaced by 3-hexadecanoyloxytetradecanoic acid, exhibited lower activity than LA-15-PP and natural lipid A's. Although LA-16-PP has been assumed to have a typical Salmonella lipid A structure (and, in fact, it has a structure corresponding to one of the components of Salmonella lipid A), the activity of this synthetic compound was not comparable to that of natural Salmonella lipid A. LA-17-PP, in which tetradecanoic acid is the sole fatty acid component, exhibited relatively strong mitogenicity and TNF-inducing activity, but very low pyrogenicity. The activities of LA-18-PP, which has ester-bound tetradecanoic acid and amide-bound 3-hydroxytetradecanoic acid, were lower than those of LA-17-PP. The results indicate that the differences in fatty acid composition of lipid A's have important influences on the biological activities studied.

Topics: Animals; Antibodies; Cross Reactions; Epitopes; Fatty Acids; Female; Fever; Glycoproteins; Limulus Test; Lipid A; Lymphocyte Activation; Male; Mice; Mice, Inbred Strains; Rabbits; Salmonella; Shwartzman Phenomenon; Structure-Activity Relationship; Tumor Necrosis Factor-alpha

A synthetic compound (506), beta (1-6) D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated at 2'-amino and 3'-hydroxyl groups with (R)-3-dodecanoyloxytetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl groups, respectively, and has (R)-3-hydroxytetradecanoyl groups at 2-amino and 3-hydroxyl groups, exhibited full endotoxic activities identical to or sometimes stronger than those of a reference lipid A from an Escherichia coli Re-mutant (strain F515). Endotoxic activities tested include pyrogenicity and leukopenia-inducing activity in rabbits, body weight-decreasing toxicity in normal mice, lethal toxicity in galactosamine-sensitized mice and chicken embryos, and the preparation and provocation of the local Shwartzman reaction in rabbits. Compound 406, a synthetic counterpart of a biosynthetic precursor of lipid A molecule, showed by contrast only weak activities in all of the above assay systems except for the lethality in galactosamine-loaded mice. This finding strongly suggests that the presence of acyloxyacyl groups at the C-2' and C-3' positions of the disaccharide backbone is one of the most important determinant structures of the lipid A molecule for exhibition of strong biological activities characteristic of lipopolysaccharide and its lipid A moiety. The activities of the corresponding 4'-monophosphate (compound 504) and 1-monophosphate (505) analogs were considerably less than those of the parent molecule 506 and the reference F515 lipid A. Regarding other biological activities, not only compound 506 but also compounds 504, 505, and 406 showed definite activities, sometimes comparable to those of F515 lipid A and other reference natural products. These are the activation of Tachypleus tridentatus amoebocyte clotting enzyme cascade and human complement via the classical pathway, mitogenic and polyclonal B-cell activation of murine splenocytes, stimulation of peritoneal macrophages in a guinea pig, enhancement of migration of human blood polymorphonuclear leukocytes, and induction of a serum factor that is cytostatic and cytocidal to L-929 cells in Mycobacterium bovis BCG-primed mice. Relative potencies of test synthetic compounds depended on the assay systems and varied from one system to another. Dephospho-compound 503 lacked most of the biological activities that were definitely observed with phosphorylated compounds, probably because of its insolubility. This study demonstrates the successful chemical synthesis of an E. coli-type

Topics: Adjuvants, Immunologic; Animals; Body Weight; Chemotaxis, Leukocyte; Complement Activation; Endotoxins; Escherichia coli; Fever; Glycoproteins; Leukopenia; Limulus Test; Lipid A; Lymphocyte Activation; Macrophage Activation; Mice; Mice, Inbred Strains; Shwartzman Phenomenon; Structure-Activity Relationship; Tumor Necrosis Factor-alpha

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 diff

Topics: Adjuvants, Immunologic; Animals; Complement Activation; Female; Fever; Galactosamine; Guinea Pigs; Limulus Test; Lipid A; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mitogens; Rabbits; Shwartzman Phenomenon; Structure-Activity Relationship

A lipopolysaccharide (LPS) was isolated from the Lyme disease spirochete by a modification of the hot phenol-water method. The material was composed of 45% carbohydrate, 8% protein, 44% lipid A, and 1% 3-deoxy-D-mannooctulosonic acid and accounted for approximately 1.5% of the cellular dry weight. The isolated LPS possessed several biologic activities characteristic of endotoxins. The LPS was pyrogenic for rabbits, mitogenic for human mononuclear cells and murine splenocytes, capable of clotting limulus lysate, and cytotoxic for murine macrophages. LPS extracted from Borrelia burgdorferi by the petroleum-ether:chloroform:liquid-phenol procedure was also characterized. The results show that the Lyme disease spirochete contains a hitherto unknown LPS that is biologically active in vitro, and the expression of such activities in vivo may play an important role in the pathogenesis of Lyme disease. Some of the clinical manifestations of other spirochetal disease may be explained by similar endotoxins in those organisms. To our knowledge this is the first report of an LPS extracted from a spirochete that is known to be a human pathogen.

Topics: Animals; Bacterial Proteins; Borrelia; Borrelia burgdorferi; Carbohydrates; Cell Survival; Fever; Humans; Limulus Test; Lipid A; Lipopolysaccharides; Lyme Disease; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred C3H; Rabbits; Sugar Acids

Chemically synthesized lipid A analogs were investigated for several endotoxic activities, including pyrogenicity, lethal toxicity, anticomplement activity, and the capacity to gelate Limulus amoebocyte lysate in comparison to natural lipid A. The synthetic preparations contained D-glucosamine or D-glucosamine-beta-1,6-D-glucosamine disaccharide substituted by ester- and amide-bound hydroxylated or non-hydroxylated fatty acids and by phosphate groups in different combinations. Some preparations which were insoluble in water were succinylated and thus rendered more soluble. Strong biphasic pyrogenic responses with a maximal increase in body temperature of 1 to 2 degrees C were obtained with 50 micrograms/kg doses of 3 disaccharide preparations of 15 tested. With two preparations (50 micrograms/kg) moderate pyrogenicity with monophasic fever curves and a maximal temperature increase of about 0.6 degrees C was obtained. Lethal toxicity tests were carried out in galactosamine-sensitized mice. Of 15 synthetic preparations, 4 exhibited lethal toxicity under these conditions. The effective doses of the lipid A analogs in both in vivo tests were, however, several hundred times higher than those of bacterial lipid A. For the activities in vivo, hydroxyacyl residues seemed to be important. Anticomplement activity was demonstrable in seven preparations, one of which expressed an activity comparable to that of lipid A. Preparations containing non-hydroxylated fatty acids seemed to be most active in this test. None of the synthetic preparations was found to exhibit gelation activity for Limulus amoebocyte lysate when tested in doses up to 0.4 micrograms, whereas bacterial free lipid A was active in doses of about 2 pg. None of the monosaccharide derivatives exhibited any of these activities.

Topics: Animals; Complement Fixation Tests; Complement Inactivator Proteins; Fever; Galactosamine; Guinea Pigs; Limulus Test; Lipid A; Male; Mice; Mice, Inbred C57BL; Pyrogens; Structure-Activity Relationship; Time Factors

Topics: Acinetobacter; Animals; Complement Inactivator Proteins; Fever; Lethal Dose 50; Limulus Test; Lipid A; Lipopolysaccharides; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Shwartzman Phenomenon

1. Previous evidence purporting to show that lipid A is the pyrogenic moiety of endotoxin is demonstrably inconclusive. 2. We have extracted lipid A from endotoxin of Salmonella typhosa and tested the pyrogenic action of the lipid A, the residual polysaccharide and the parent endotoxin, by intravenous injection in conscious rabbits. 3. Lipid A dissolved in an aqueous solution of rabbit serum albumin induced a significant pyrexia of short latency, while neither rabbit serum albumin alone, nor the polysaccharide from S. typhosa, affected body temperature. The physical presence in the injectate of the polysaccharide from S. typhosa did not enhance the pyrogenicity of the lipid. 4. Dose-response curves for lipid A and the parent endotoxin, over the dose range 10 ng-20 micrograms, showed that lipid A incorporated in endotoxin was much more pyrogenic than pure lipid A in solution. When separated from the polysaccharide component of endotoxin, lipid A lost more than 99.9% of its pyrogenic activity, at threshold doses.

Topics: Animals; Dose-Response Relationship, Drug; Endotoxins; Fever; Injections, Intravenous; Lipid A; Lipopolysaccharides; Pharmaceutical Vehicles; Pyrogens; Rabbits; Salmonella typhi

Endotoxin from fresly sedimented Bordetella pertussis cells, isolated by the phenol/water procedure when submitted to kinetically controlled, mild acidic hydrolysis released a polysaccharide (polysaccharide 1), a complex lipid (lipid X), and a glycolipid. When treated with somewhat stronger acid, the glycolipid yielded a second polysaccharide (polysaccharide 2) and another complex lipid (lipid A). The intact pertussis endotoxin had all the usual properties of endotoxins extracted from enteric bacteria. Lipid X and the intermediary glycolipid retained all the endotoxic properties of the unfractionated endotoxin. In lipid A, pyrogenicity was reduced to a very low level and toxicity and Shwartzman reactivity were absent; however, this fraction retained most of the endotoxin's antiviral activity, and its adjuvant power was considerably higher than that of the intact endotoxin. Lipid A elicited nonspecific resistance against challenge with certain bacteria, but not against others.

Topics: Adjuvants, Immunologic; Bacterial Toxins; Bordetella pertussis; Endotoxins; Fever; Lipid A; Lipopolysaccharides; Shwartzman Phenomenon; Viruses

The antiendotoxic activity of lipid A antiserum was studied in rabbits, using lipid A (lipopolysaccharide)-induced fever and skin necrosis as test systems. It was found that lipid A antiserum had no significant antipyretic effect when it was incubated with lipid A or injected intravenously before lipid A challenge. However, in animals that were pretreated (day 0) with a single dose of lipid A (lipopolysaccharide), a significant protective effect of passively transferred antiserum (day 1) to lipid A (lipopolysaccharide fever) (day 2) was observed. Also, the lipid A (lipopolysaccharide)-induced local shwartzman reaction could be prevented by lipid A antiserum. In the fever system, the degree of protection depended on the preparative and the challenge doses as well as on the amount of antiserum transferred. The fever protection mediated by lipid A antiserum seemed to be lipid A (lipopolysaccharide) specific with regard to both the preparative and the challenge injections. Lipid A specificity of the protective factor present in the antiserum was indicated by the fauggest that the factor might be identical with lipid A-specific immunoglobulin. The significance of the preparative injection is not understood at the present time. Iti is concluded, however, that in the fever protection system described, besides specific humoral factors, other factors, perhaps cellular, are involved.

Topics: Animals; Dose-Response Relationship, Immunologic; Female; Fever; Immunization, Passive; Lipid A; Lipopolysaccharides; Male; Rabbits; Shwartzman Phenomenon; Time Factors

The chemical properties and the general biological activities of lipopolysaccharide (LPS) and Boivin-type endotoxin obtained respectively by phenol-water and trichloroacetic acid extraction from Yersinia enterocolitica serotypes O3 and O9 were studied. The yield of LPS from the O9 strain was about 10% of the O3 strain possibly because of the lower solubility of O9-LPS in aqueous phase. However, the chemical composition of O9-LPS was similar to that of O3-LPS in the proportions of reducing sugar, glucosamine, heptose, KDO, and lipid A. In pyrogenicity and local Shwartzman reactivity in rabbits and lethality for mice, there was also no difference between O3 and O9-LPS. The anthrone-positive carbohydrate and lipid A contents of Boivin-type endotoxin from O3 were higher than those of the endotoxin from O9. The biological activities of Boivin-type endotoxin from O3 were also remarkably higher than those of the endotoxin from O9. It seems that endotoxin of Y. enterocolitica serotype O3 may play an important role in infection by this organism.

Topics: Adjuvants, Immunologic; Animals; Bacterial Proteins; Carbohydrates; Endotoxins; Fever; Glucosamine; Lipid A; Lipopolysaccharides; Mice; Polysaccharides, Bacterial; Pyrogens; Rabbits; Shwartzman Phenomenon; Yersinia