lipid-a and Hemolysis

Conjugation with poly(ethylene glycol) ("PEGylation") is a widely used approach for improving the therapeutic propensities of peptide and protein drugs through prolonging bloodstream circulation, reducing toxicity and immunogenicity, and improving proteolytic stability. In the present study, we investigate how PEGylation affects the interaction of host defense peptides (HDPs) with bacterial lipopolysaccharide (LPS) as well as HDP suppression of LPS-induced cell activation. In particular, we investigate the effects of PEGylation site for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), a peptide displaying potent anti-inflammatory effects, primarily provided by its N-terminal part. PEGylation was performed either in the N-terminus, the C-terminus, or in both termini, keeping the total number of ethylene groups (

Topics: Cell Line; Gene Expression Regulation; Hemolysis; Humans; Lipid A; Lipopolysaccharides; Models, Molecular; NF-kappa B; Peptides; Polyethylene Glycols; Protein Binding; Protein Conformation; Transcription Factor AP-1

Gram-negative bacteria are becoming resistant to almost all currently available antibiotics. Systemically designed antimicrobial peptides (AMPs) are attractive agents to enhance the activities of antibiotics. We constructed a small Pro-scanning library using amphipathic model peptides. Measurements of minimum inhibitory concentration (MIC) against

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cell Membrane; Clarithromycin; Escherichia coli; Female; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Linezolid; Lipid A; Membrane Fluidity; Mice, Inbred ICR; Microbial Sensitivity Tests; Proline; Protein Binding; Protein Conformation, alpha-Helical; Rifampin

Liposomes containing cholesterol (Chol) have long been used as an important membrane system for modeling the complex interactions of Chol with adjacent phospholipids or other lipids in a membrane environment. In this study we utilize a probe composed of QS-21, a saponin molecule that recognizes liposomal Chol and causes hemolysis of erythrocytes. The interaction of QS-21 with liposomal Chol results in a stable formulation which, after injection into the tissues of an animal, lacks toxic effects of QS-21 on neighboring cells that contain Chol, such as erythrocytes. Here we have used liposomes containing different saturated phospholipid fatty acyl groups and Chol, with or without monophosphoryl lipid A (MPLA), as model membranes. QS-21 is then employed as a probe to study the interactions of liposomal lipids on the visibility of membrane Chol. We demonstrate that changes either in the mole fraction of Chol in liposomes, or with different chain lengths of phospholipid fatty acyl groups, can have a substantial impact on the detection of Chol by the QS-21. We further show that liposomal MPLA can partially inhibit detection of the liposomal Chol by QS-21. The Limulus amebocyte lysate assay is used for binding to and detection of MPLA. Previous work has demonstrated that sequestration of MPLA into the liposomal lipid bilayer can block detection by the Limulus assay, but the binding site on the MPLA to which the Limulus protein binds is unknown. Changes in liposomal Chol concentration and phospholipid fatty acyl chain length influenced the detection of the liposome-embedded MPLA.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Adjuvants, Immunologic; Animals; Cell Extracts; Cholesterol; Dimyristoylphosphatidylcholine; Dose-Response Relationship, Drug; Hemolysis; Horseshoe Crabs; Humans; Lipid A; Liposomes; Phosphatidylcholines; Phosphatidylglycerols; Saponins

Allium sativum is well known for its medicinal properties. The A. sativum lectin 50 (ASL50, 50 kDa) was isolated from aged A. sativum bulbs and purified by gel filtration chromatography on Sephacryl S-200 column. Agar well diffusion assay were used to evaluate the antimicrobial activity of ASL50 against Candida species and bacteria then minimal inhibitory concentration (MIC) was determined. The lipid A binding to ASL50 was determined by surface plasmon resonance (SPR) technology with varying concentrations. Electron microscopic studies were done to see the mode of action of ASL50 on microbes. It exerted antimicrobial activity against clinical Candida isolates with a MIC of 10-40 μg/ml and clinical Pseudomonas aeruginosa isolates with a MIC of 10-80 μg/ml. The electron microscopic study illustrates that it disrupts the cell membrane of the bacteria and cell wall of fungi. It exhibited antiproliferative activity on oral carcinoma KB cells with an IC50 of 36 μg/ml after treatment for 48 h and induces the apoptosis of cancer cells by inducing 2.5-fold higher caspase enzyme activity than untreated cells. However, it has no cytotoxic effects towards HEK 293 cells as well as human erythrocytes even at higher concentration of ASL50. Biological properties of ASL50 may have its therapeutic significance in aiding infection and cancer treatments.

Topics: Amino Acid Sequence; Anti-Infective Agents; Antineoplastic Agents; Apoptosis; Cell Proliferation; Garlic; HEK293 Cells; Hemolysis; Humans; KB Cells; Lipid A; Molecular Weight; Plant Lectins; Plant Stems; Sequence Analysis

We previously described a cell surface anionic polysaccharide (APS) in Porphyromonas gingivalis that is required for cell integrity and serum resistance. APS is a phosphorylated branched mannan that shares a common epitope with posttranslational additions to some of the Arg-gingipains. This study aimed to determine the mechanism of anchoring of APS to the surface of P. gingivalis. APS was purified on concanavalin A affinity columns to minimize the loss of the anchoring system that occurred during chemical extraction. (1)H nuclear magnetic resonance spectroscopy of the lectin-purified APS confirmed the previous structure but also revealed additional signals that suggested the presence of a lipid A. This was confirmed by fatty acid analysis of the APS and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the lipid A released by treatment with sodium acetate buffer (pH 4.5). Hence, P. gingivalis synthesizes two distinct lipopolysaccharide (LPS) macromolecules containing different glycan repeating units: O-LPS (with O-antigen tetrasaccharide repeating units) and A-LPS (with APS repeating units). Nonphosphorylated penta-acylated and nonphosphorylated tetra-acylated species were detected in lipid A from P. gingivalis total LPS and in lipid A from A-LPS. These lipid A species were unique to lipid A derived from A-LPS. Biological assays demonstrated a reduced proinflammatory activity of A-LPS compared to that of total LPS. Inactivation of a putative O-antigen ligase (waaL) at PG1051, which is required for the final step of LPS biosynthesis, abolished the linkage of both the O antigen and APS to the lipid A core of O-LPS and A-LPS, respectively, suggesting that WaaL in P. gingivalis has dual specificity for both O-antigen and APS repeating units.

Topics: Carbohydrates; Carbon-Oxygen Ligases; Cell Line; Chromatography, Affinity; Gas Chromatography-Mass Spectrometry; Gene Deletion; Hemolysis; Humans; Lipid A; Lipids; Lipopolysaccharides; Magnetic Resonance Spectroscopy; Microscopy, Electron, Transmission; O Antigens; Porphyromonas gingivalis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Lipopolysaccharides (LPS), otherwise termed "endotoxins", are outer membrane constituents of Gram-negative bacteria. Lipopolysaccharides play a key role in the pathogenesis of "septic shock", a major cause of mortality in the critically ill patient. Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time. We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS and, using animal models of sepsis, have shown that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy. In this paper, the interactions of a series of acylated homologated spermine compounds with LPS have been characterized. The optimal acyl chain length for effective sequestration of LPS was identified to be C(16) for the monoacyl compounds. The most promising of these compounds, 4e, binds LPS with an ED(50) of 1.37 muM. Nitric oxide production in murine J774A.1 cells, as well as TNF-alpha in human blood, is inhibited in a dose-dependent manner by 4e at concentrations orders of magnitude lower than toxic doses. Administration of 4e to d-galactosamine-sensitized mice challenged with supralethal doses of LPS provided significant protection against lethality. Potent antiendotoxic activity, low toxicity, and ease of synthesis render this class of compounds candidate endotoxin-sequestering agents of potential significant therapeutic value.

Topics: Amides; Animals; Cell Line; Cell Survival; Escherichia coli; Female; Hemolysis; Humans; In Vitro Techniques; Lipid A; Lipopolysaccharides; Mice; Nitric Oxide; Sepsis; Spermine; Structure-Activity Relationship; Surface Properties; Tumor Necrosis Factor-alpha

Lipid A is responsible for the endotoxic activities of gram-negative bacteria. Binding of lipid A (50 micrograms/ml) to RBC was studied using a passive hemolysis test. RBC from adults, cord and venous RBC from full-term infants and RBC from preterm infants were studied. Lipid A sensitized RBC were hemolysed with anti-lipid A and guinea pig complement. Hemolysis was expressed as hemoglobin concentration (absorbance at 546 nm) in the supernatant after centrifugation. 50 micrograms/ml lipid A did not increase spontaneous hemolysis (< 3%) after 60 min of incubation in any of the four groups. The passive hemolysis test did not result in additional hemolysis (5%) of umbilical cord RBC. RBC of preterm infants showed a significant increase in hemolysis (24%) after 60 min of incubation. In RBC of full-term neonates, increased hemolysis (14 and 46%) was detected after 30 and 60 min of incubation. Adult RBC hemolysed stronger (26 and 64%) after 30 and 60 min than neonatal RBC. We conclude that lipid A binds less to neonatal RBC compared to adults.

Topics: Adult; Endotoxins; Erythrocyte Membrane; Hemolysis; Humans; Infant; Infant, Newborn; Infant, Premature; Lipid A

Sixty-two rabbit anti-lipid A serum samples were compared with respect to the immunogens used (synthetic lipid A and partial structures, natural lipid A, or acid-treated bacteria). Immunoglobulin (Ig) type-specific differences in rabbit response between liposomal membrane-embedded (LME) and other lipid A immunogens were found: LME lipid A elicited predominantly IgM antibodies. Previous findings of equally good immune responses to synthetic lipid A and acid-treated bacteria (L. Brade, E.T. Rietschel, S. Kusumoto, T. Shiba, and H. Brade, Infect. Immun. 51:110-114, 1986, and L. Brade, E.T. Rietschel, S. Kusumoto, T. Shiba, and H. Brade, Prog. Clin. Biol. Res. 231:75-97, 1987) turned out to be restricted to complement-fixing antibodies; IgG titers of sera against free lipid A (whether synthetic or natural) were significantly lower than those raised with bacteria. The results indicated an increase in IgG content of sera from LME lipid A over other free lipid A immunogens to acid-treated bacteria. These data underline the importance of the physicochemical environment for the immunogenicity of lipid A. As a second objective, the presence of various lipid A antibody specificities was tested with synthetic lipid A antigens. Antibodies to monophosphoryl lipid A were detected only in sera raised with monophosphoryl immunogens. Reactivity with monosaccharide partial structures of lipid A was found both in sera against monophosphoryl lipid A and in 60% of sera against bisphosphoryl lipid A. In the former, monosaccharide reactivity was of a magnitude similar to that of reactivity with lipid A; in sera against bisphosphoryl lipid A, it was lower. No reactivity or only marginal reactivity was found with phosphate-free lipid A, thus emphasizing the role of phosphate substitution for the lipid A epitopes recognized.

Topics: Animals; Antibodies, Bacterial; Epitopes; Hemolysis; Immune Sera; Immunoenzyme Techniques; Immunoglobulin G; Immunoglobulin M; Lipid A; Mercaptoethanol; Rabbits

Histatins are histidine-rich polypeptides secreted in human saliva. They were found to inhibit lipopolysaccharide (LPS)-mediated gelation of Limulus amoebocyte lysate, and to reverse the anti-complement action of LPS or lipid A. Histatins also gave precipitate bands in agarose gels with various LPS. The results indicate that histatins neutralized the activity of LPS by binding to the lipid A moiety of LPS.

Topics: Escherichia coli; Hemolysis; Humans; Limulus Test; Lipid A; Lipopolysaccharides; Proteins; Pseudomonas aeruginosa; Saliva; Salivary Proteins and Peptides; Salmonella

A series of monoclonal antibodies directed against lipid A was characterized by using synthetic lipid A analogs and partial structures. These compounds vary in phosphate substitution, acylation pattern (type, number, and distribution of fatty acids), and, in the case of monosaccharides, in their backbone glycosyl residue. The monoclonal antibodies tested could be subdivided into five groups according to their reactivity patterns. One group reacted exclusively with 1,4'-bisphosphoryl lipid A, and a second also reacted with 4'-monophosphoryl lipid A. Two further groups recognized either 4-phosphoryl or 1-phosphoryl monosaccharide partial structures of lipid A. The fifth group reacted with 4-phosphoryl monosaccharide structures and with phosphate-free compounds. Antibodies reactive with monosaccharide structures also recognized their epitopes in corresponding phosphorylated disaccharide compounds. Both groups of monosaccharide and monophosphoryl lipid A-recognizing antibodies have access to their epitopes in bisphosphoryl compounds as well. Because of this unidirectional reactivity with more complex structures, the various specificities cannot be distinguished by using bisphosphoryl lipid A (e.g., Escherichia coli lipid A) as a test antigen. The epitopes recognized by the various monoclonal antibodies all reside in the hydrophilic backbone of lipid A, and there was no indication that fatty acids were part of the epitopes recognized. Nevertheless, the reactivities of compounds in the different test systems are strongly influenced by their acylation patterns; i.e., acyl groups may modulate the exposure of lipid A epitopes.

Topics: Acylation; Animals; Antibodies, Monoclonal; Epitopes; Hemolysis; Immunoenzyme Techniques; Lipid A; Mice; Mice, Inbred BALB C; Phosphates; Rabbits; Structure-Activity Relationship

A detailed characterization of binding specificity and cross-reactivity of three antilipid A murine mAb was performed. Binding characteristics of these three mAb were investigated against Ag (ReLPS, lipid A, derivatives of lipid A) in solid phase (ELISA) and in fluid phase (C consumption, inhibition studies), and upon incorporation in membranes (E: passive hemolysis assay, and liposomes: inhibition studies). Cross-reactivity with heterologous Ag was investigated in ELISA (LPS, Gram-negative bacteria) and immunoblot experiments (LPS). The binding specificity of mAb 26-5 (IgG2b), raised against synthetic lipid A, was located in the hydrophilic region of biphospholipid A and was also exposed after membrane incorporation of lipid A or after preincubation of lipid A with polymyxin B (PMX). mAb 26-20 (IgM), also raised against synthetic lipid A, showed binding specificity for the hydrophobic region of lipid A: no binding to membrane-associated lipid A could be demonstrated, and binding in ELISA could be blocked very efficiently by PMX. The reaction pattern of mAb 8-2 (IgM), raised against the heat-killed Re mutant of Salmonella typhimurium, was in part similar to that of mAb 26-20. However, inhibition of binding with PMX was less efficient and a high specificity for ReLPS, also after membrane incorporation of this Ag, was demonstrated. In contrast to mAb 26-5 and 26-20, mAb 8-2 showed extensive cross-reactivity with heterologous LPS preparations and heat-killed as well as live Gram-negative bacteria. It is concluded that each of the three mAb binds to a different antigenic epitope in lipid A and that exposure of those epitopes for antibody binding is restricted in a differential manner, depending on mode of Ag presentation. The here defined reaction patterns provide a basis for the interpretation of potential inhibitory effects on in vitro and in vivo biologic (and toxic) activities of endotoxins and Gram-negative bacteria.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Binding Sites, Antibody; Binding, Competitive; Complement System Proteins; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Gram-Negative Bacteria; Hemolysis; Hot Temperature; Immunoblotting; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred BALB C

Twenty-nine murine monoclonal antibodies (MAbs) were prepared against antigenic determinants in the core and lipid A regions of Escherichia coli and Salmonella minnesota lipopolysaccharide (LPS). At least eight distinct MAb specificities were identified. Epitopes recognized by MAbs bearing these specificities were localized in the hexose, heptose, and 2-keto-3-deoxy-D-manno-octulosonic acid regions of the core oligosaccharide and on lipid A. Two groups of MAbs exhibited multispecificity for similar but distinct core- and lipid A-related epitopes. Some core-reactive MAbs cross-reacted with corresponding E. coli and Salmonella rough mutant chemotypes; others were specific for E. coli J5 LPS. Lipid A-specific MAbs reacted with free lipid A from diverse sources. Few MAbs reacted with smooth LPS. Antibody cross-reactivity was restricted by inter- and intraspecies differences in covalent core structure and by epitope concealment by overlying O-side chain and core sugars. The putative cross-reactive and antiendotoxic properties of MAbs specific for the core-lipid A complex may be limited by the inability of such MAbs to recognize determinants on "native" LPS.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Bacteria; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Hemolysis; Immunoblotting; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Species Specificity

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Cross Reactions; Endotoxins; Enzyme-Linked Immunosorbent Assay; Epitopes; Hemolysis; Immunochemistry; Lipid A; Lipopolysaccharides; Mice

Topics: Animals; Antigen-Antibody Complex; Epitopes; Escherichia coli; Hemolysis; Immune Sera; Lipid A; Salmonella; Sheep; Species Specificity

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

The immunogenicity and antigenicity of synthetic Escherichia coli lipid A (compound 506) and its 1- and 4'-monophosphorylated derivatives (compounds 505 and 504, respectively) and nonphosphorylated derivative (compound 503) were compared with those of bis- and 4'-monophosphorylated natural free lipid A from E. coli. The synthetic compounds under study were either coated onto sheep erythrocytes (except for the water-insoluble preparation 503) or incorporated into liposomes and used for the immunization of rabbits. Both types of immunogens (the latter representing fully synthetic immunogens) resulted in high-titered polyclonal antisera which were characterized before or after absorption in a passive hemolysis assay as well as in a passive hemolysis inhibition assay with the synthetic compounds as test antigens. All antisera were found to react with their corresponding homologous antigens coated onto sheep erythrocytes, with titers of up to 2,048, and were comparable to those antisera obtained after immunization with natural lipid A exposed on the bacterial surface after acid hydrolysis. Antisera against bisphosphorylated compound 506 were highly specific for the homologous antigens, showing no interaction with compounds 504 and 505 in the passive hemolysis test. The same held true for the absorption experiments in which glutaraldehyde-fixed sheep erythrocytes were sensitized with the respective antigens. Antisera against monophosphorylated compounds 504 and 505 exhibited, besides their expected homologous reactivity, complete cross-reactivity with compound 506, but they did not cross-react with each other. Thus, anti-504 and anti-505 antibodies recognized distinct antigenic determinants, being related to the ester linked 4'-phosphate or the glycosidically linked 1-phosphate, respectively. Both antigenic determinants were also expressed by bisphosphorylated compound 506 used as an antigen; however, upon immunization, only antibodies against compound 506 were elicited.

Topics: Epitopes; Escherichia coli; Hemolysis; Lipid A; Structure-Activity Relationship

Antisera were raised in rabbits with acid-treated Re mutant bacteria from Salmonella minnesota and Escherichia coli and tested in a passive hemolysis assay with di- and monophosphorylated free lipid A of E. coli (LipA-Ac and LipA-HCl, respectively) coated onto sheep erythrocytes. Depending on the acid used to prepare the immunogen (acetic versus hydrochloric acid), different antibody specificities were obtained. Antiserum prepared against HCl-treated bacteria was found to react with both antigens to the same extent (i) in the passive hemolysis test, (ii) in the passive hemolysis inhibition test, and (iii) in absorption experiments, suggesting that antibodies in this antiserum recognize an antigenic determinant equally present in LipA-Ac and LipA-HCl. Antiserum raised against acetic acid-treated bacteria reacted with the homologous antigen (LipA-Ac) in the passive hemolysis and passive hemolysis inhibition test as well as in absorption experiments. However, the antiserum failed to react with the heterologous antigen (LipA-HCl) in the hemolysis test and during absorption, whereas in inhibition studies interaction of this antiserum with both antigens was observed. The inhibiting capacity of LipA-Ac was lower compared with that of LipA-HCl, indicating that the antigenic determinant of LipA-Ac is partly expressed by LipA-HCl in solution, but not when fixed on the surface of sheep erythrocytes. The role of glycosidically linked phosphate in lipid A is discussed with respect to antigenicity.

Topics: Animals; Antibodies, Bacterial; Antibody Specificity; Epitopes; Escherichia coli; Hemolysis; Immunization; Lipid A; Rabbits

The effect of synthetic lipid A analogs on murine spleen cells was studied. The preparations represented D-glucosamine and D-glucosaminyl-beta 1,6-D-glucosamine disaccharide derivatives substituted in different combinations by ester- and amide-bound fatty acids and by phosphate groups. Significant mitogenic activity was demonstrated with a number of synthetic disaccharide preparations; however, their potency was lower than that of lipid A. The synthetic preparations were not mitogenic for spleen cells from C3H/HeJ mice. Furthermore, the mitogenicity of the synthetic preparations was abolished after binding with polymyxin B. The results indicate that for expression of mitogenicity, a phosphate group at position 1 of the reducing glucosamine and amide-bound acyloxyacyl residues are important factors. Some of the synthetic preparations containing the diglucosamine backbone and expressing relatively low mitogenicity suppressed B-cell mitogenicity of lipid A. Although these preparations were lytic for erythrocytes, they did not affect the viability of the splenic lymphocytes. Suppression was seen when the synthetic preparations were added simultaneously with or after the lipid A mitogen, but optimal suppression was expressed when the preparations were added to the system 3 h before lipid A. Washing of the cells before the addition of lipid A did not affect the results. The suppression was not due to the induction of suppressor cells by the synthetic preparations. The disaccharide preparations did not inhibit T-cell mitogenicity of concanavalin A. In contrast to the disaccharide preparations, the monosaccharide preparations suppressed mitogenicity of both lipid A and concanavalin A, probably because of their direct toxicity for lymphocytes.

Topics: Animals; Cells, Cultured; Hemolysis; Immunosuppression Therapy; Lipid A; Lipopolysaccharides; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred Strains; Polymyxin B; Salmonella; Sheep; Structure-Activity Relationship

A new antigenic specificity, referred to here as common lipopolysaccharide (LPS) specificity, is described in the LPSs of gram-negative bacteria belonging to various families. The specificity is present in S- and R-form LPS but absent in Re mutants of different enterobacterial genera. By the use of purified LPS and monospecific antibodies obtained by immunoabsorption, the specificity is differentiated from the known core specificities of the genus Salmonella and the lipid A specificity by aid of the passive hemolysis and passive hemolysis inhibition test. In Salmonella minnesota R-form LPS, the specificity may be cryptic (R345, Rb2 mutant) or partly exposed in the intact molecule (R7, Rd1 mutant). The specificity is either demasked or completely exposed after mild acid hydrolysis for a short time, whereas it is destroyed after prolonged hydrolysis. Periodate oxidation, reduction, and hydrolysis under conditions that do not affect the ketosidic linkages of 2-keto-3-deoxyoctulosonic acid destroy the specificity in R4 (Rd2 mutant) LPS, but do not do so in R7 LPS. It is suggested that 2-keto-3-deoxyoctulosonic acid and a following neutral sugar are the compositional requirements for expressing the specificity.

Topics: Bacteroidaceae; Enterobacteriaceae; Epitopes; Gram-Negative Bacteria; Hemolysis; Lipid A; Lipopolysaccharides; Neisseriaceae; Pseudomonadaceae; Rhodospirillaceae; Vibrionaceae

Topics: Adolescent; Animals; Antibodies, Bacterial; Child; Child, Preschool; Hemolysis; Humans; Infant; Lipid A; Lipopolysaccharides; Mexico; Rabbits; Salmonella

Anti-lipid-A, anti-endotoxin, antibodies have been measured by a passive haemolysis test using antigen from an E. coli Re mutant. Titres in the normal population are low but do rise in situations in which there has been gram-negative sepsis. Absence of raised titres in other situations has profound implications.

Topics: Animals; Antibodies, Bacterial; Child, Preschool; Hemolysis; Humans; Lipid A; Lipopolysaccharides; Sepsis; Sheep

Lipid A isolated from lipopolysaccharide of Yersinia pseudotuberculosis was used for immunization of rabbits to afford antisera to lipid A with titers of 1:640 in the passive hemolysis test. Exhaustion of immune serume with sheep erythrocytes decreased antibody titers up to 1:160. Authentic samples of 2-(DL-3-hydroxytetradecanoyl)amino-2-deoxy-D-glucose 6-phosphate, 2-tetradecanoylamino-2-deoxy-D-glucose 6-phosphate and 2-acetamido-2-deoxy-D-glucose 6-phosphate have been synthesized in order to carry out a comparative study of inhibitory activity of these compounds and lipid A using a system of lipid A and antiserum to lipid A. As a result, the immunodominant moiety of the lipid A of Y. pseudotuberculosis proved to contain a D-glucosamine residue acylated with 3-hydroxytetradecanoic acid at the amino group. The nature of the fatty acid acylating the amino group of glucosamine does not play an important role in the structure of immunodominant moiety of lipid A.

Topics: Animals; Carbohydrates; Epitopes; Hemolysis; Lipid A; Lipopolysaccharides; Rabbits; Yersinia

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Erythrocytes; Female; Hemolysis; Interferon Inducers; Lipid A; Lipopolysaccharides; Mice; Pseudomonas aeruginosa; Sheep; Spleen