Rough Lipopolysaccharide Research Articles

Review: Bacterial endotoxin and the human monoclonal antibody HA-IA: specificity, potential mechanisms of action, and limits to its effectiveness

Bacterial endotoxins are lipopolysaccharides present in the outer membrane of all Gram-negative bacteria (GNB). Endotoxins consist of a lipid moiety, lipid A, that is covalently linked to highly variable, serotype O-specific polysaccharide lateral chains. In contrast, the endotoxin core, which includes lipid A, is better conserved and can be recognized by antibodies showing cross-reactivity among various GNB. Such polyclonal and monoclonal antibodies have been developed in an attempt to neutralize the biological and dele. terious effects of endotoxin, thus preventing lipid A from binding to macrophages. In fact, almost all the biological activities of endotoxin are elicited by lipid A, and there is substantial evidence to the effect that the monocyte-macrophage is the principal mediator of endotoxicity. Antiserum against LPS isolated from rough mutants of GNB (expressing virtually only the central core-lipid A), has been shown to counteract the lethal effects of endotoxin in animals and humans. However, such serum or plasma contains antibodies of different specificities and isotypes which represent different effector functions, insofar as LPS is a very complex and highly heterogenous macromolecule. Because of the difficulties encountered in investigating the nature and specificity of the protection afforded by these antisera, and their limited capacity of production for therapeutic use, specific anti-lipid A monoclonal antibodies have been produced in their stead. A variety of mouse and human monoclonal antibodies against LPS have been generated and selected for their ability to cross-react with many GNB species. The most recent clinical trials involving the treatment of septic patients with human HA-IA (Centoxin) or with murine (E5) anti-lipid A monoclonal antibody showed no difference in survival rates, as compared to treatment with a placebo. However, statistical significance was demonstrated in subsets of patients suffering from documented Gram-negative septicemia or Gram-negative sepsis without refractory shock. The usefulness of anti-lipid A antibodies will undoubtedly remain controversial, since they appear to benefit only a minority of all patients treated, and also because no consensus exists regarding their specificity and modes of action. The aim of this review is to describe results which demonstrate the requirements for, difficulties in and limits to, elucidating the ability of certain antibodies to recognize structural elements present in the lipid A domain of LPS. A clear demonstration of antibody cross-reactivity was obtained only when rough LPS bacteria were used, and was markedly enhanced when smooth bacteria had been pretreated with cell wall active antibiotics. Further, new data have recently demonstrated the specific involvement of HA-IA in the immunocytoadherence assay in the presence of human complement and human red blood cells. Such phenomena may form part of the potential role for natural or monoclonal human IgM anti-lipid A antibodies, which will be to remove IgM-lipid A immune complexes through transhepatic clearance via C3b binding to the CR1 present on circulating human erythrocytes. Insofar as immunocytoadherence is a multiparameter phenomenon, various limiting factors probably interfere with its mechanism of clearance. These factors may be absent in various subsets of septic patients under treatment, thus explaining therapeutic failures with HA-IA in humans or preclinical animal studies. Several clinical settings involving defects to CR1 expression, C3b production, LPS recognition and hepatic clearance dysfunction are described. Long term, however, it will impossible to specify the patient subsets suitable for monoclonal therapy without first defining their characteristics. HA-IA may be able to inhibit one of the earliest stages in activation of the cytokine cascade by sequestrating and eliminating biologically active lipid A. The major problem today in terms of using anti-lipid A antibodies is an efficiently early detection of specific pathway defects which detract from or nullify the HA-IA therapeutic effect.

Effects of Bacterial Endotoxin on Human Cross-Linked and Native Hemoglobins

Previous investigations have demonstrated that hemoglobin (Hb) is a binding protein for bacterial endotoxin (lipopolysaccharide, LPS) and that the structure and biological activity of LPS are altered in the presence of Hb. In the present study, the influence of LPS on the structure of native human HbA0 and covalently cross-linked Hb (alpha alpha Hb) was studied by analyzing the absorption and circular dichroic spectra of Hb in the wavelength region of 200-650 nm. Incubation of oxyHb with each of several LPSs resulted in a decrease in the intensity of the major Soret band at 414 nm with a shift in the maximum peak to 410 nm, decreases in the intensities of the major visible region peaks at 541 and 577 nm, and the appearance of increased absorbance in the visible region in the range of 630 nm. The resultant spectra are characteristic of methemoglobin formation. These spectral changes were time-dependent and LPS-concentration-dependent. Production of methemoglobin was prominent with chemically modified, partially deacetylated rough LPS, and was observed to a lesser extent both with native, complete rough and with native smooth LPSs. The influence of LPS on the absorption spectrum of methemoglobin also was directly tested. The conversion of methemoglobin to hemichrome in the presence of LPS was demonstrated and was shown to be reversible. Analysis of circular dichroic spectra of Hb demonstrated LPS-induced spectral changes in the visible and Soret regions consistent with the production of a substantial quantity of metHb, but did not demonstrate any alteration in the far-UV region (210-240 nm). Moreover, Hb oxygen affinity was only slightly altered after incubation with any of several LPSs. In conclusion, analyses of absorption and circular dichroic spectra reveal the potential of LPS to produce a facilitated oxidation of both alpha alpha-cross-linked human Hb and native human HbA0, without substantial changes in the secondary structure of the globin.

Thermotropic phase behavior of membranes made from Erwinia carotovora rough form lipopolysaccharide

The rough lipopolysaccharide (LPS) from Erwinia carotovora was found to form several μm diameter vesicles in aqueous solution. Spontaneous formation of spherical particles at ambient temperature was observed on addition of ethanol or a multivalent metal chloride to the solution. The LPS altered the gel-liquid crystalline phase transitions of the hydrocarbon chains in the temperature range 13–32°C. The vesicle formation indicated a marked increase of phase transition enthalpy ΔH up to three times greater than that of the LPS dispersed in pure water. The thermotropic phase behavior of the LPS was affected by metal ions, ionic strength and pH of the solution. Protonation of the LPS in acidic solution caused the transition temperature t m and ΔH to increase, whereas deprotonation in alkaline solution led to the opposite effect. Multivalent metal ions such as Mg 2+ and Ca 2+ raised the t m and increased the ΔH value. The LPS was organized to a greater degree in saline, acidic solution or divalent metal chloride solution than in pure water. The phase behavior varied according to the solvent conditions or metal chlorides that affect the phosphate and carboxyl groups, indicating that these charged moieties have an influence on the molecular organization within the membrane. Approximately two molar equivalents or more of Mg 2+ ion induced a prominent thermotropic peak at ≈ 13°C. This ionotropic phase property of the natural LPS is supposedly directed by cross-linkage through divalent metal ions and negatively charged moieties such as phosphate bound to the LPS molecule. The functions of the LPS membrane are thus susceptible to change according to the chemical and physical conditions of the environment.

Identification and Characterization of Immunodominant Antigens during the Course of Infection with Brucella Ovis

The seroresponse against Brucella ovis of 8 intrapreputially and 6 intravenously infected rams and 9 ewes infected through mating was analyzed by electrophoretic immunoblotting. Additionally, 87 sera from chronically infected rams that were shedding B. ovis in their semen, 226 sera from rams belonging to infected flocks, and 324 sera from false-positive complement fixation test (CFT) reactors were examined. In all infected animals, antibody reactivity was predominantly found against 5 B. ovis components of 8-12, 17, 19, 29, and 63 kD, of which the 29-kD antigen was most dominant in the seroresponse. Antibodies to the 29-kD component were present in 93-100% of the infected sheep in each infected group, whereas the frequency of antibodies to the 4 other components varied considerably among and within the different groups. No reactivity against the 29-kD antigen was found in the false-positive CFT reactors. By using monoclonal antibodies against known bacterial macromolecules, the immunodominant antigens were identified as rough lipopolysaccharide (8-12 kD), outer membrane proteins (17, 19, 29 kD), and a heat-shock protein (63 kD).

A murine model for studying endotoxemia and the efficacy of anti-LPS agents in an immunocompromised host

In most murine models of endotoxemia, an exogenous agent is injected to increase the sensitivity of the mouse to endotoxin (lipopolysaccharide, LPS). Here, a clinically encountered event, a bum, was found to reproducibly decrease the amount of LPS required to kill half of the mice (LD50). In this more clinically relevant model, the anti-LPS agents, monophosphoryl lipid A and polymyxin B sulfate, each increased the LD50 of burned mice challenged with LPS from Klebsiella pneumoniae, while the LPS-directed monoclonal antibody E5 did not. However, E5 did protect burned mice challenged with smooth or rough LPS from Salmonella typhimurium and S. minnesota, respectively. Hence, in vivo protection was dependent upon both the anti-LPS agent and the chemical composition of the LPS used for intoxication. The differences in protection observed in this intoxication model may explain some protection discrepancies reported when these anti-LPS agents have been tested for protection against Gram negative sepsis.

Anti-bacteroides lipopolysaccharide IgG levels in healthy adults and sepsis patients

Members of the genus Bacteroides greatly outnumber enterobacteria in the human colon and therefore represent a vast potential pool of biologically active LPS. An enzyme-linked immunosorbent assay was developed to estimate the distribution of IgG levels to LPS from B. fragilis, B. vulgatus, B. thetaiotaomicron and to a mixture of rough LPS from three enterobacteria and Pseudomonas aeruginosa in sera from 641 adult blood donors. By inhibition ELISA some cross-reactivity was demonstrated between the different anti-bacteroides LPS IgG, but with very little between the anti-bacteroides LPS IgG and the anti-enterobacterial/ Pseudomonas LPS IgG. Serum IgG was measured daily over 5–9-day periods in 12 sepsis patients (6 survivors, 6 non-survivors) and in a healthy individual. In all patients IgG levels fluctuated to a greater extent than levels in a healthy subject. Variations all followed similar overall trends and indicated that exposure to bacteroides LPS had occurred. In 5 out of 6 survivors, IgG levels were rising at the end of the period, while 4 of the 6 non-survivors showed falls, with an exception showing increasing levels to B. fragilis LPS. In 5 out of 6 non-survivors, IgG levels against B. fragilis LPS were substantially higher than those against the other LPSs. In this small sample some trends in antibody kinetics have been recognised which suggest bacteroides LPS may be significant in sepsis, and indicate that this study should be extended.

An easy microtiter assay for quantitation of cytokine induction by lipopolysaccharide (LPS) and activity of LPS-binding serum components.

Lipopolysaccharide (LPS, endotoxin) is a major inducer of cytokines, such as interleukin 1 (IL1), IL6, IL8 and tumor necrosis factor (TNF). A convenient microtiter assay was developed to measure such activity. LPS coated onto a plastic surface was used to stimulate purified human mononuclear cells (MNC) in microtiter plates. Following stimulation the supernatants were assayed for presence of TNF by ELISA. Purified rough and smooth LPS from Pseudomonas aeruginosa gave a dose-dependent TNF release over a range of 0.1-1.0 microgram LPS/well. The assay was subsequently used to investigate the biological activity of anti-LPS antibodies and other LPS-specific serum components in sera from patients with cystic fibrosis (CF). As a group, sera from 10 CF patients chronically infected with P. aeruginosa did not affect the LPS-induced TNF release, while sera from normal controls inhibited this biological activity. When individual CF patients with or without chronic lung infection are considered, the antibodies appear to either enhance or inhibit the LPS-stimulated TNF release (range: 73-120%), while all antibodies from healthy controls inhibit the activity of LPS (range: 76-97%). Only a weak correlation (rho = 0.491, p = 0.037, n = 19) was found between the antibody titer in ELISA and the biological activity of sera. This new assay is suggested for convenient measurement of interference with cytokine induction from human MNC by patient or therapeutic anti-LPS antibodies and other LPS-specific serum components.

Human hemoglobin increases the biological activity of bacterial lipopolysaccharides in activation of Limulus amebocyte lysate and stimulation of tissue factor production by endothelial cells in vitro

Previous studies have demonstrated that hemoglobin (Hb) and bacterial endotoxin (lipopolysaccharide, LPS) form stable complexes and result in disaggregation of macromolecular LPS. To examine the effect of complex formation on LPS biological activity, we investigated the ability of Hb to alter LPS-induced activation of the coagulation cascade of Limulus amebocyte lysate (LAL) and expression of tissue factor from human endothelial cells. Both native HbAo and derivatized (covalently cross-linked) hemoglobin resulted in prominent enhancement of LAL activation and endothelial cell tissue factor production by Proteus mirabilis LPS. No substantial differences were observed between the enhancement effect of Hb on P. mirabilis smooth and rough LPS, indicating a dominant role for the lipid A component of LPS. Rough (Re) Salmonella min nesota 595 LPS also demonstrated both enhanced activation of LAL and stimulation of endothelial cell tissue factor in the presence of Hb. In contrast, neither lipid A nor singly dephosphorylated or partially deacylated Re LPS manifested significant enhancement of LAL activation by Hb, and partially deacylated Re LPS showed no enhancement of endothelial cell tissue factor by Hb. These results suggest that the Kdo moieties, as well as the phosphate residues and fatty acyl moieties of lipid A, may be involved in the interaction of Hb with LPS. Comparison of Hb with other endotoxin binding proteins for ability to cause enhancement of LPS biological activity demonstrated more prominent enhancement with lipopolysaccharide binding protein (LBP) than that observed with Hb, lesser enhancement with albumin, and no enhancement effect with IgG or transferrin.

Monoclonal antibodies that distinguish inner core, outer core, and lipid A regions of Pseudomonas aeruginosa lipopolysaccharide.

In order to examine the immunochemistry of the core-lipid A region of Pseudomonas aeruginosa lipopolysaccharide (LPS), monoclonal antibodies (MAbs) specific for this region were produced in mice. Immunogen was prepared by coating a rough mutant of P. aeruginosa with column-purified core oligosaccharide fractions in order to enhance the immune response to the LPS core-lipid A region. Fourteen hybridoma clones were isolated, characterized, and further divided into three groups on the basis of their reactivities to rough LPS antigens in both enzyme-linked immunosorbent assays and Western immunoblots. In addition, another MAb, 18-19, designated group 1, was included in this study for defining core-lipid A epitopes. MAb 18-19 recognizes the LPS core-plus-one O-repeat unit of the serologically cross-reactive P. aeruginosa O2, O5, and O16. Group 2 MAbs are specific for the LPS outer core region and reacted with P. aeruginosa O2, O5, O7, O8, O10, O16, O18, O19, and O20, suggesting that these serotypes share a common outer core type. Group 3 MAbs recognize the inner core region and reacted with all 20 P. aeruginosa serotypes as well as with other Pseudomonas species, revealing the conserved nature of this region. Group 4 MAbs are specific for lipid A and reacted with all gram-negative organisms tested. Immunoassays using these MAbs and well-defined rough mutants, in addition to the recently determined P. aeruginosa core structures, have allowed us to precisely define immunodominant epitopes within the LPS core region.

The identification of cryptic rhamnose biosynthesis genes in Neisseria gonorrhoeae and their relationship to lipopolysaccharide biosynthesis

Neisseria gonorrhoeae synthesizes a rough lipopolysaccharide that does not contain any of the repetitive units characteristic of the smooth lipopolysaccharide of members of the family Enterobacteriaceae. Three gonococcal homologs of Salmonella serovar typhimurium genes involved in the synthesis of the rhamnose component of the repetitive subunits have been isolated. Gonococcal homologs for rfbB, rfbA, and rfbD were found downstream of the galE gene in a region of the chromosome which shows overall homology with the meningococcal capsule gene complex region D. Sequence alignment demonstrated that the gonococcal gene products have 69, 65, and 54% amino acid identity with the Salmonella proteins RfbB, RfbA, and RfbD. The gonococcal RfbB and RfbA amino acid sequences share even more identical residues (73 and 65%, respectively) with the amino acid sequences derived from Escherichia coli genes o355 and o292, respectively. These genes are clustered with the genes involved in the biosynthesis of enterobacterial common antigen, and o355 is listed in the GenBank and Swiss Protein data banks as rffE (encoding UDP-GlcNAc-2-epimerase). However, complementation studies demonstrated that o355 does not encode the enzyme UDP-GlcNAc-2-epimerase. Gonococcal strains constructed with null mutations in the rfbBAD genes were unchanged in lipopolysaccharide phenotype and in the synthesis of gonococcal carbohydrate-containing C antigen. We were unable to detect any changes in gonococcal phenotype with respect to lipopolysaccharide sialylation, monoclonal-antibody binding, serum sensitivity, or interaction with eukaryotic cells in vitro. We conclude that the absence of a homolog for rfbC precludes the existence of a functional dTDP-rhamnose biosynthesis pathway in the gonococcal strains examined and that these genes are only maintained in N. gonorrhoeae either because of the presence of the galE gene or because of another as yet unrecognized function.

Identification of an ATP‐binding cassette transport system required for translocation of lipopolysaccharide O‐antigen side‐chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1

The rfbKpO1 gene cluster of Klebsiella pneumoniae O1 directs synthesis of the D-galactan I component of the lipopolysaccharide O-antigen. The first two genes in the rfbKpO1 cluster encode RfbAKpO1 and RfbBKpO1, with predicted sizes of 29.5 or 30.0 kDa and 27.4 kDa, respectively. RfbBKpO1 contains a consensus ATP-binding domain and shares homology with several proteins which function as ATP-binding components of cell surface polysaccharide transporters. RfbAKpO1 is predicted to be an integral membrane protein with five putative membrane-spanning domains and its transmembrane topology was confirmed by TnphoA mutagenesis. The hydropathy plot of RfbAKpO1 resembles KpsM, the transcytoplasmic membrane component of the capsular polysaccharide transporter from Escherichia coli K-1 and K-5. These relationships suggest that RfbAKpO1 and RfbBKpO1 belong to a family of two-component ABC (ATP-binding cassette) transporters. E. coli K-12 containing a plasmid carrying an rfbKpO1 gene cluster deleted in rfbAKpO1 and rfbBKpO1 expresses rough lipopolysaccharide molecules on its surface and accumulates cytoplasmic O-antigen. When RfbAKpO1 and RfbBKpO1 are supplied in trans by a compatible plasmid, O-polysaccharide transport is restored and smooth D-galactan I-substituted lipopolysaccharide is produced. RfbAKpO1 and RfbBKpO1 are, therefore, proposed to constitute a system required for transport of D-galactan I across the cytoplasmic membrane, where RfbAKpO1 represents the membrane-spanning translocator and RfbBKpO1 couples the energy of ATP hydrolysis ot the transport process.

Binding of surfactant protein A to the lipid A moiety of bacterial lipopolysaccharides.

Surfactant protein A (SP-A) enhances the phagocytosis of opsonized and non-opsonized bacteria by alveolar macrophages, but it is not known with which component of the bacterial surface it associates. We investigated the interaction of SP-A with lipopolysaccharides (LPS), which are important biologically active constituents of the outer membranes of Gram-negative bacteria. Flow cytometry was used to study the binding of fluorescein isothiocyanate-labelled SP-A either to LPS of various chain lengths coupled to magnetic beads or to Gram-negative bacteria. The binding of SP-A to LPS-coated beads was saturable, both time- and concentration-dependent, and required both Ca2+ and Na+. SP-A bound to the lipid A moiety of LPS and to LPS from either the Re-mutant of Salmonella minnesota or the J5-mutant of Escherichia coli. In contrast, it did not bind to O111 LPS of E. coli, suggesting that SP-A binds only to rough LPS. The binding of SP-A to LPS was not affected by mannan and heparin or by deglycosylation of the SP-A, indicating that the carbohydrate-binding domain and the carbohydrate moiety of SP-A are not involved in its interaction with LPS. We also observed saturable and concentration-dependent binding of SP-A to the live J5 mutant of whole E. coli, but not to its O111 mutant. In addition, Re LPS aggregated in the presence of SP-A, Ca2+ and Na+. We conclude that SP-A associates with LPS via the lipid A moiety of rough LPS and may be involved in the anti-bacterial defences of the lung.

Parameters that influence the efficiency of processing antigenic epitopes expressed in Salmonella typhimurium

We investigated parameters that affect the efficiency with which antigenic epitopes from Salmonella typhimurium are processed for presentation to T lymphocytes. As a model system, the hen egg white lysozyme 52-61 [HEL(52-61)] epitope, which binds the murine major histocompatibility complex class II (MHC-II) molecule I-Ak, was expressed in soluble fusion proteins in S. typhimurium. Murine peritoneal macrophages mediated phagocytic processing of viable S. typhimurium expressing fusion proteins of the HEL epitope for presentation via I-Ak regardless of the bacterial compartment in which the epitope was contained (i.e., surface exposed, facing the periplasmic space, or in the cytoplasm). Minor differences in processing efficiency observed with different epitope compartmentalizations could be overcome by altering the relative expression level, indicating that epitope abundance is an important factor for efficient processing of epitopes from S. typhimurium. This processing pathway required phagocytosis of bacteria followed by passage through an acidic compartment, suggesting a pathway involving phagolysosomal degradation of the bacteria to liberate epitopes that bind MHC-II. HEL(52-61) was processed more efficiently from heat-killed S. typhimurium than from viable bacteria, and in addition, the HEL epitope was processed more efficiently from a rough lipopolysaccharide (LPS) strain than from its isogenic smooth LPS counterpart, most likely because of enhanced phagocytosis of the rough LPS strain. These data suggest that the efficiency of epitope processing from S. typhimurium for presentation via MHC-II is affected by bacterial viability, epitope abundance, and LPS phenotype, factors which may be important to consider in development of recombinant S. typhimurium vaccine strains.

Characterization of mutants of Caulobacter crescentus defective in surface attachment of the paracrystalline surface layer

Strains of Caulobacter crescentus express a paracrystalline surface layer (S-layer) consisting of the protein RsaA. Mutants of C. crescentus NA1000 and CB2, isolated for their ability to grow in the absence of calcium ions, uniformly no longer had the S-layer attached to the cell surface. However, RsaA was still produced, and when colonies grown on calcium-sufficient medium were examined, large two-dimensional arrays of S-layer were found intermixed with the cells. Such arrays were not found in calcium-deficient medium even when high levels of magnesium ions were provided. The arrays could be disrupted with divalent ion chelators and more readily with the calcium-selective ethylene glycol-bis (beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). Thus, the outer membrane surface was not needed as a template for self-assembly, but calcium likely was. The cell surface and S-layer gene of assembly-defective mutants of NA1000 were examined to determine the basis of the S-layer surface attachment defect. Mutants had no detectable alteration in the rough lipopolysaccharide (LPS) or a characterized capsular polysaccharide, but another polysaccharide molecule was greatly reduced or absent in all calcium-independent mutants. The molecule was shown to be a smooth LPS with a core sugar and fatty acid complement identical to those of the rough LPS and an O polysaccharide of homogeneous length, tentatively considered to be composed of 4,6-dideoxy-4-amino hexose, 3,6-dideoxy-3-amino hexose, and glycerol in equal proportions. This molecule (termed SLPS) was detectable by surface labeling with a specific antiserum only when the S-layer was not present. The rsaA genes from three calcium-independent mutants were cloned and expressed in an S-layer-negative, SLPS-positive strain. A normal S-layer was produced, ruling out defects in rsaA in these cases. It is proposed that SLPS is required for S-layer surface attachment, possibly via calcium bridging. The data support the possibility that calcium binding is required to prevent an otherwise lethal effect of SLPS. If true, mutations that eliminate the O polysaccharide of SLPS eliminate the lethal effects of calcium-deprived SLPS, at the expense of S-layer attachment.

Brucella melitensis cell envelope protein and lipopolysaccharide epitopes involved in humoral immune responses of naturally and experimentally infected sheep.

Cell envelope fraction (CEF) of Brucella melitensis B115 was used to investigate antibody responses of B. melitensis naturally and strain H38 experimentally infected sheep by immunoblotting, indirect enzyme-linked immunosorbent assay (ELISA) (I-ELISA), and competitive ELISA (C-ELISA) with monoclonal antibodies (MAbs). MAbs used were directed to outer membrane proteins with molecular masses of 10, 16.5, 19, 25 to 27, 31 to 34, 36 to 38, and 89 kDa; to the heat shock protein DnaK, to O-polysaccharide (O-PS) common (C) and M epitopes; and to rough lipopolysaccharide (R-LPS) epitopes. In immunoblotting, all infected sheep sera tested recognized a large number of protein bands, including the above-cited proteins and other proteins for which MAbs have not been defined. The antibody response pattern was different from one animal to another, even within the experimentally infected sheep which were infected under the same experimental conditions. A number of protein bands were recognized by the sheep sera prior to experimental infection and by other uninfected sheep sera. The antibody reactivity to these antigens and others might explain the nonspecific antibody reactivity of sera in I-ELISA with CEF. C-ELISA confirmed also the individual variability of the antibody responses of infected sheep to protein antigens. Antibody responses to O-PS C and M epitopes were detected in all experimentally infected sheep and in half of the naturally infected sheep, but these responses were also heterogeneous in relation to their intensities. Antibody responses to R-LPS epitopes detected by use of C-ELISA with the anti-R-LPS MAbs were low or negative in most of the infected animals. Despite antibody response heterogeneity for CEF antigens, immunoblot and C-ELISA results indicated that, among the CEF antigens, the O-PS epitopes (C and M epitopes) and epitopes of the major 25- to 27- and 31- to 34-kDa outer membrane proteins seem to be the most promising for detecting B. melitensis infection in sheep.

Endotoxin-induced early gene expression in C3H/HeJ (Lpsd) macrophages.

C3H/HeJ (Lpsd) macrophages have been shown to respond to certain LPSs, especially from rough mutant bacteria. C3H/OuJ (Lpsn) macrophages are induced by wild-type LPS, rough LPS, or lipid A to express many genes, including TNF-alpha, TNFR-2, IL-1 beta, IP-10, D3, and D8. C3H/HeJ macrophages failed to induce any of these genes when cultured with wild-type LPS or synthetic lipid A, even when pretreated with IFN-gamma. However, rough mutant Salmonella minnesota Ra, Rc, and Rd LPS, and Escherichia coli D31 m3 Rd LPS induced Lpsd macrophages to express a subset of genes within the gene panel. Because bioactive preparations contained trace quantities of endotoxin protein(s), a deoxycholate-modified, phenol-water method was used to repurify rough LPS into an aqueous phase, and extract endotoxin proteins into a phenol phase. Repurified LPS failed to stimulate Lpsd macrophages; however, phenol fractions were approximately 10% as potent in Lpsd macrophages as crude rough LPS. Full potency was restored in C3H/HeJ macrophages when aqueous phase LPS and phenol-phase proteins were co-precipitated, suggesting that LPS and endotoxin proteins interact synergistically. Endotoxin proteins alone induced TNF-alpha, TNFR-2, and IL-1 beta, but not IP-10, D3, and D8 genes in both Lpsd and Lpsn macrophages. Tyrosine phosphorylation of three 41- to 47-kDa proteins was induced by endotoxin proteins, but not by LPS, in Lpsd macrophages. Thus, endotoxin proteins seem to activate a signaling pathway(s) that converges (distal to the Lps gene product) with a subset of LPS-signaling pathways.

An anti-idiotype antibody which mimics the inner-core region of lipopolysaccharide protects mice against a lethal challenge with endotoxin

Recently, we described the generation and characterization of an Armenian hamster Ab2 beta anti-idiotype monoclonal antibody (MAb4G2) specific for the binding site of a mouse monoclonal antibody, MAbY1-4A6, directed against the conserved 2-keto-3-deoxyoctulosonate (Kdo)-containing inner-core region of lipopolysaccharide (LPS) (S. K. Field, M. Pollack, and D. C. Morrison, Microb. Pathog. 15:103-120, 1993). In that study, mice and hamster immunized with MAb4G2 generated serum immunoglobulin G and M (IgG and IgM) antibodies which cross-react with Salmonella minnesota R595-chemotype rough mutant LPS (Re-LPS). In this report, we demonstrate that in C3Heb/FeJ mice, MAb4G2 elicits an immune response which is characterized by specific binding of antibody to Re-LPS, as assessed by enzyme-linked immunosorbent assay. The practical use of MAb4G2 as a potentially effective therapeutic agent against gram-negative bacterial sepsis is suggested by the demonstration that immunization of these mice with MAb4G2 results in significant protection of D-galactosamine-sensitized animals against an otherwise lethal dose of Re-LPS. Assessment of the temporal changes in Re-LPS-specific serum antibody titers from mice immunized with MAb4G2 or Re-LPS over a 40-day period indicates that immunization with Re-LPS elicits significantly higher titers of serum IgM antibodies compared with those in animals immunized with MAb4G2. Conversely, two immunizations with MAb4G2 result in an up to 10-fold increase in anti-Re-LPS-specific IgG serum antibody titers relative to those obtained in mice immunized with Re-LPS. Nineteen days after the secondary boost with MAb4G2, anti-Re-LPS-specific IgG serum antibody titers were significantly higher (three- to fourfold) compared with those in Re-LPS-treated animals. Initial immunization with the anti-idiotype antibody primes animals for enhanced secondary responses to Re-LPS, as assessed by the titers of anti-Re-LPS-specific IgG profiles. These data suggest the potential utility of MAb4G2 as a candidate vaccine against the lethal properties of gram-negative bacterial LPS.

Genes for TDP-rhamnose synthesis affect the pattern of lipopolysaccharide heterogeneity in Escherichia coli K-12.

The rough lipopolysaccharide (LPS) of commonly used strains of Escherichia coli K-12 has two distinctly different band patterns when analyzed by high-resolution polyacrylamide gel electrophoresis. The LPS of ancestral strains such as W1485F- consists primarily of a single broad gel band. In contrast, the LPS of strains derived from strain Y10 such as AB1133 or C600 gives three sharp gel bands. Complementation studies using DNA fragments from the rfb gene cluster of Shigella dysenteriae 1 indicated that the difference between the two gel patterns is due to a mutation in the gene encoding the TDP-rhamnose synthetase, the final enzyme involved in TDP-rhamnose biosynthesis. This mutation arose during the construction of strain Y10, and not in strain 679-680 as previously thought. The requirement for the rfaS gene for synthesis of the broad major band seen in W1485F- LPS and the shift in gel migration of a component of this band when an rfaQ mutation was introduced indicated that this broad band contained the unique form of rough E. coli LPS which has been termed lipooligosaccharide. This finding indicates that lipooligosaccharide is likely to contain rhamnose and suggests a model in which one of the functions of partial substituents such as rhamnose may be to direct core synthesis into different pathways to produce alternative forms of LPS.

Altered Colonizing Ability for the Ceca of Broiler Chicks by Lipopolysaccharide-Deficient Mutants of Salmonella typhimurium

Salmonella typhimurium strain 3333/O was used to assess the role of bacterial lipopolysaccharide (LPS) in intestinal colonization of broiler chicks by salmonellae. LPS-defective TnPhoA mutants of this strain were isolated. The sensitivities of the mutants to smooth and rough phages and LPS banding patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a defect in the polysaccharide side chain of the LPS molecule. Colonization was determined by orally administering 10(8) cells each of the wild-type and/or the mutant strains per chick and counting the colony-forming units (CFU) from the ceca 1 to 3 weeks after gavage. CFU of chicks given the LPS-deficient strains either were not detected or were significantly lower than the CFU from chicks given the wild-type strain. The incidence of the wild-type strain in spleens was higher than incidence of the mutant strains. In vitro binding studies with LPS-deficient mutants derived in this study and from S. typhimurium LT2 suggest that LPS side-chain components may shield the bacterial cell from entrapment in the chicken mucus. The LPS layer appears to enhance persistence of Salmonella in the avian intestinal tract.

The CD14 differentiation antigen mediates the development of endotoxin responsiveness during differentiation of mononuclear phagocytes.

The CD14 antigen was originally described as a differentiation antigen on mononuclear cells. The purpose of this study was to investigate the relationship between the appearance of surface CD14 and the acquisition of lipopolysaccharide (LPS) responsiveness during maturation of mononuclear phagocytes. Immature THP-1 cells responded poorly to LPS in the absence or presence of serum. Treatment with the maturational agent calcitriol caused a dose- and time-dependent increase in CD14 mRNA and surface CD14 and enhanced the responsiveness of THP-1 cells to smooth and rough form LPS, complexes of LPS and lipopolysaccharide-binding protein (LBP), and LPS in low concentrations of serum. Monoclonal antibodies to CD14 blocked the responses of THP-1 to LPS, LPS-LBP complexes and LPS in serum. Immunodepletion of LBP from serum also inhibited the effect of LPS in serum. The data show that maturation of the response of THP-1 cells to LPS and LPS-LBP complexes depends on the appearance of CD14 on the cell surface. Maturation of the response to LPS in serum depends in large part on the appearance of CD14 on the cell surface and the presence of LBP in serum.