Recombinant Lipopolysaccharide Research Articles

Endogenous molecules released by haemocytes receiving Sargassum oligocystum extract lead to downstream activation and synergize innate immunity in white shrimp Litopenaeus vannamei

White shrimp Litopenaeus vannamei haemocytes receiving immunostimulating Sargassum oligocystum extract (SE) caused necrosis in haemocyte cells, which released endogenous EM-SE molecules. This study examined the immune response of white shrimp L. vannamei receiving SE and EM-SE in vitro and in vivo. Shrimp haemocytes receiving SE exhibited degranulation, changes in cell size and cell viability, necrosis and a release of EM-SE. Shrimp haemocytes receiving SE, EM-SE, and the SE + EM-SE mixture (SE + EM-SE) increased their phenoloxidase (PO) activity which was significantly higher in shrimp haemocytes receiving the SE + EM-SE mixture. Furthermore, shrimp haemocytes receiving EM-SE showed degranulation and changes in cell size and cell viability. Shrimp receiving SE, EM-SE, and SE + EM-SE all increased their immune parameters, phagocytic activity, clearance efficiency and resistance to Vibrio alginolyticus, being significantly higher in shrimp receiving SE + EM-SE. Meanwhile, the recombinant lipopolysaccharide- and β-1,3-glucan binding protein of L. vannamei (rLvLGBP) was bound to SE, EM-SE, and SE + EM-SE. We conclude that in shrimp haemocytes receiving a non-self molecule, SE in dying cells released EM-SE which led to downstream activation and synergization of the immune response. This study demonstrated that the innate immunity of shrimp was elicited and enhanced by a mixture of endogenous molecules and exogenous substances (or immunostimulants).

Bovine recombinant lipopolysaccharide binding protein (BRLBP) regulated apoptosis and inflammation response in lipopolysaccharide-challenged bovine mammary epithelial cells (BMEC)

Lipopolysaccharide-binding protein (LBP) is an acute-phase protein involved in host response to Gram-negative and Gram-positive pathogens. It has been reported to exert diverse biological activities, such as anti-inflammatory effects. However, what effects it has on bovine mastitis has not been investigated. The aim of this study was to verify the anti-inflammatory properties of LBP on the inflammatory response of primary bovine mammary epithelial cells (BMEC) induced by lipopolysaccharide (LPS), and to determine the underlying mechanism. Bovine mammary epithelial cells were treated with various concentrations of LPS (1, 10, 20, and 100μg/mL) for 3, 6, 12, and 24h. The results showed that LPS significantly inhibited cell viability in a dose-dependent manner. When cells were treated with LPS (10μg/mL) for 12h, the permeability of the cell membrane increased significantly. This promoted apoptosis. Various concentrations (10 and 20μg/mL) of bovine recombinant lipopolysaccharide binding protein (BRLBP) could weaken the inflammation injury of BMEC induced by LPS without cytotoxicity. Toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB), IL-1β, and tumor necrosis factor α (TNF-α) from BMEC were decreased. TLR4 and NF-κB P65 protein levels were down-regulated, and nuclear transcription factor κB activity was also weakened. All these results indicated that the protective effects of high concentrations of BRLBP on LPS-induced inflammation injury in BMEC were at least partially achieved by the decreased production of pro-inflammatory cytokines. BRLBP was found to directly inhibit LPS/TLR4-mediated NF-κB activation. One possible anti-inflammatory mechanism can be attributed to the negative role of BRLBP in suppressing TLR4/NF-κB activation mediated by LPS. These findings suggested that BRLBP may be a useful agent to treat LPS-induced mastitis.

Diagnosis and follow-up of genital chlamydial infection by direct methods and by detection of serum IgG, IgA and secretory IgA

Purpose: To determine the prevalence of Chlamydia trachomatis infection in a high-risk population by direct and indirect methods and to evaluate the diagnosis of secretory immunoglobulin A (sIgA). Patients and Methods: Urethral or endocervical specimens from 78 patients (48 females and 30 males) were examined by cell culture, direct fluorescence assay, PCR Cobas Amplicor (Roche Molecular Diagnostics), and sIgA was detected by the recombinant lipopolysaccharide (LPS)-enzyme-linked immunoassay (rELISA). Serum from each patient was also obtained and analysed for the presence of IgG and IgA antibody by in-house microimmunofluorescence (MIF) and by the rELISA method (Medac, Hamburg, Germany). Results: The overall C. trachomatis prevalence determined by direct methods was 28%. The detection of sIgA antibodies was significantly higher in the group of patients with a positive direct detection (50%) than in the group of negative direct detection (10.7%). The Chlamydia-specific IgA antibodies were detected by the rELISA in 40.9 and 53.6% of group I (positive direct detection) and group II patients (negative direct detection), respectively. The species-specific IgA antibodies were detected by the MIF method in 18.2 and 16.1% of group I and II patients, respectively. Chlamydia genus-specific IgG antibodies were detected by the rELISA in 86.4 and 83.9% of group I and group II patients and, C. trachomatis specific IgG were present in 81.8 and 73.2% of group I and group II patients, respectively, as assessed by the MIF test. Conclusion: Combining the positive direct methods and/or positive sIgA antibody results from cervical or urethral specimens had an indication of current C. trachomatis infection.

Characterization of serum antibody response to chlamydiae in patients with sexually acquired reactive arthritis.

Sera from patients with sexually acquired reactive arthritis (SARA) with antibodies reacting with C. trachomatis and C. pneumoniae (group 1; n = 20) and also with C. psittaci (group 2; n = 19) were analyzed for antibody specificity. Sera from group 2 reacted significantly more often with C. trachomatis serotype E, H and K and had higher antibody titers to serotype E, as tested by microimmunofluorescence tests. Cross-reactivities occurring in microimmunofluorescence tests were related to the presence of antichlamydial lipopolysaccharide antibodies, adsorption of which by recombinant lipopolysaccharide removed microimmunofluorescence reactivity with C. psittaci antigen. In group 2, significantly more sera had antibodies to C. pneumoniae, remaining after lipopolysaccharide adsorption, as proved by adsorption with viable C. trachomatis and C. pneumoniae organisms. None of the sera had antibodies to Yersinia enterocolitica, Shigella flexneri, Sh. sonnei and Salmonella spp. It was observed that the frequency and titer of cross-reacting antibodies to chlamydial serotypes and species were related to the time period between the diagnosis of genital chlamydial infection and of SARA. Cross-reactivities were also related to the presence of lipopolysaccharide, but not heat shock protein 60- or neutralizing antibodies to chlamydiae. Antibody reactivity induced by antichlamydial lipopolysaccharide antibodies can be removed by lipopolysaccharide adsorption.

ChlamydiaAntibody Response in Healthy Volunteers Immunized with Nonchlamydial Antigens: A Randomized, Double‐Blind, Placebo‐Controlled Study

Serological analysis is often used for the diagnosis of chlamydial infections. However, an increase in Chlamydia antibodies has been reported in patients with parvovirus and Mycoplasma infections. Whether this antibody response is the result of dual infection or nonchlamydial antigen stimulation is unknown. In a randomized study, 48 healthy volunteers either were immunized against yellow fever, polio, diphtheria, and tetanus (the group receiving intervention with nonchlamydial antigen) or received saline injections (the placebo group). The change in antibody levels was compared between the 2 groups. The Chlamydia recombinant lipopolysaccharide enzyme-linked immunosorbent assay (Medac) showed an increase in the antibody titer in the intervention group, compared with that in the control group (for immunoglobulin M, P=.004; for immunoglobulin A, P=.038; and for immunoglobulin G, P=.056), but no differences between study groups was found when the C. pneumoniae enzyme immunoassay (EIA; ThermoLabsystems), the C. pneumoniae EIA (Medac), and the microimmunofluorescence test (MRL) were used. An increase in antibodies to Chlamydia organisms can be measured after exposure to nonchlamydial antigens, depending on the test used.

LPS-induced platelet response and rapid shock in mice: contribution of O-antigen region of LPS and involvement of the lectin pathway of the complement system

AbstractIntravenous injection of a lipopolysaccharide (LPS) into mice induces a rapid accumulation of platelets in the lung and liver. When degradation of the accumulated platelets occurs, anaphylactoid shock follows rapidly, the severity of the shock paralleling the quantity of platelets accumulated in the lung. Here we examined the contributions made by LPS structure and the complement system to the platelet response to LPS. BALB/c mice were injected with an LPS fromEscherichia coli O8, O9, O111, or K-12, or from recombinant mutants of K-12. The O-regions of the O8 and O9 LPSs consist of a mannose homopolysaccharide (MHP), while that of O111 consists of a heteropolysaccharide (not including mannose), and K-12 LPS lacks an O-region. O111 LPS was devoid of the ability to induce the platelet response or shock, while the ability of K-12 LPS was weak. The 2 recombinant LPSs—each having an O-region (from O8 or O9) linked to K-12 LPS—exhibited activities similar to or stronger than those of their original LPSs. Mannose-binding lectin (MBL) complexed with MBL-associated serine proteases (MASPs) bound strongly to LPSs containing MHP and caused C4 activation. Moreover, the abilities of these LPSs to activate the complement system corresponded well with their abilities to induce the platelet response and rapid shock. These results suggest that the structure of the O-antigen region is important for the platelet response to LPS, and that activation of the lectin pathway of the complement system is involved in this response.

Evaluation of a commercial test for antibodies to the chlamydial lipopolysaccharide (MedacTM ) for serodiagnosis of acute infections by Chlamydia pneumoniae (TWAR) and Chlamydia psittaci

A commercial test (rELISA) based on a recombinant chlamydial lipopolysaccharide (LPS) antigen has been evaluated for the diagnosis of acute infections caused by Chlamydia pneumoniae (TWAR) and Chlamydia psittaci. This test and a microimmunofluorescence test (MIF) were compared in 160 patients with community-acquired pneumonia. Seventeen of nineteen cases with significant titre changes detected by rELISA were confirmed by MIF. The two remaining cases not confirmed by MIF were considered false-positive reactions. One case positive by MIF only was judged not to be a true-positive reaction. All three cases occurred in patients with Mycoplasma pneumoniae infection and may be the result of a mitogenic effect. High antibody titres have been used to indicate acute C. pneumoniae infection. We found high MIF or rELISA titres to be equally common in patients and controls; no association between the two tests was detected. An unexpected cross-reactivity between the rELISA antigen and parvovirus was observed, which might have diagnostic implications. Both MIF and rELISA detected acute C. pneumoniae and C. psittaci infection, and there was good agreement between the tests. Single serum diagnosis was generally not feasible with either MIF or rELISA.

Plasma Lipoproteins Promote the Release of Bacterial Lipopolysaccharide from the Monocyte Cell Surface

When bacterial lipopolysaccharide (LPS) enters the bloodstream, it is thought to have two general fates. If LPS binds to circulating leukocytes, it triggers innate host defense mechanisms and often elicits toxic reactions. If instead LPS binds to plasma lipoproteins, its bioactivity is largely neutralized. This study shows that lipoproteins can also take up LPS that has first bound to leukocytes. When monocytes were loaded with [(3)H]LPS and then incubated in plasma, they released over 70% of the cell-associated [(3)H]LPS into lipoproteins (predominantly high density lipoprotein), whereas in serum-free medium the [(3)H]LPS remained tightly associated with the cells. The transfer reaction could be reproduced in the presence of pure native lipoproteins or reconstituted high density lipoprotein. Plasma immunodepletion experiments and experiments using recombinant LPS transfer proteins revealed that soluble CD14 significantly enhances LPS release from the cells, high concentrations of LPS-binding protein have a modest effect, and phospholipid transfer protein is unable to facilitate LPS release. Essentially all of the LPS on the monocyte cell surface can be released. Lipoprotein-mediated LPS release was accompanied by a reduction in several cellular responses to the LPS, suggesting that the movement of LPS from leukocytes into lipoproteins may attenuate host responses to LPS in vivo.

Microglial activation by components of gram-positive and -negative bacteria: distinct and common routes to the induction of ion channels and cytokines.

Gram-positive Streptococcus pneumoniae is the major pathogen causing lethal meningitis in adults. We used pneumococcal cell walls (PCW) to investigate microglial consequences of a bacterial challenge and to determine the role of serum in the activation process. PCW caused the characteristic induction of an outwardly rectifying K+ channel (IK+(OR)), together with a concomitant suppression of the constitutively expressed inward rectifier K+ current, and evoked the release of tumor necrosis factor-alpha (TNF alpha), interleukin-6 (IL-6), IL-12, KC, macrophage inflammatory protein (MIP) 1alpha and MIP-2. Serum presence strongly facilitated the PCW effects, similarly as observed for lipopolysaccharide (LPS) from gram-negative Escherichia coli. The inflammatory cytokine, interferon-gamma (IFNgamma) induced the same electrophysiological changes, but independent of serum. Recombinant LPS binding protein (LBP) could partially replace serum activity in LPS stimulations. In contrast, neither LBP nor an antibody-mediated blockade of the LPS receptor, CD14 had significant influences on PCW-inducible changes. Cell surface interactions and cofactor involvement in microglial activation by gram-positive bacteria are thus distinct from the mechanisms employed by LPS. Moreover, tyrphostin AG126, a protein kinase inhibitor that prevents activation of the mitogen-activated protein kinase, p42MAPK (ERK2), potently blocked the PCW-stimulated cytokine release while having only a limited effect on LPS-inducible cytokines. In contrast, AG126 did not influence IK+(OR) inductions. This indicates that PCW recruits more than 1 intracellular signaling pathway to trigger the various responses and that different bacterial agents signal through both common and individual routes during microglial activation.

Detection of Chlamydia trachomatis antibodies by 2 novel tests: rELISA and peptide EIA.

The performance of 2 newly developed enzyme immunoassays (EIAs) intended for the routine serological diagnosis of chlamydial infections was evaluated. rELISA is based on a recombinant lipopolysaccharide antigen which detects chlamydia genus-specific antibodies, and Chlamydia trachomatis EIA is based on a peptide derived from major outer membrane protein and is therefore species-specific. Both tests distinguished patients with tubal factor infertility (TFI) or pelvic inflammatory disease (PID) from the controls. The prevalence of IgA antibodies was higher for the PID patients than for the TFI patients; the finding indicates a more active state of infections for the PID patients. Furthermore, C. trachomatis EIA detected more IgG antibodies in the TFI patients than in patients with non-tubal factor infertility. In conclusion, rELISA detected chlamydial antibodies in general, and C. trachomatis EIA detected species-specific antibodies. These EIA tests may be useful in the serodiagnosis of chlamydial infection.

Evaluation of PCR, culture, and serology for diagnosis of Chlamydia pneumoniae respiratory infections.

We prospectively studied 156 patients with a diagnosis of community-acquired pneumonia requiring admission. Several respiratory specimens were obtained for the detection of Chlamydia pneumoniae by cell culture and PCR. Three serum samples were obtained from each patient. Serological diagnosis of a C. pneumoniae infection was determined by the microimmunofluorescence (MIF) test, the complement fixation (CF) test, and recombinant lipopolysaccharide (LPS) enzyme-linked immunosorbent assay (ELISA; referred to as the rDNA LPS ELISA). Twenty-three patients (15%) had serological results compatible with acute C. pneumoniae infection; nine (39%) of these subjects were C. pneumoniae PCR positive. Twenty-two patients (14%) had positive PCR results without serological evidence of an acute C. pneumoniae infection. An attempt was made to calculate the sensitivities and specificities of the MIF test, rDNA LPS ELISA, and PCR for the diagnosis of chlamydial community-acquired pneumonia. Several "gold standards" were defined. Generally, the sensitivities of the rDNA LPS ELISA and MIF were comparable, while the sensitivity of the CF test was shown to be very low. Independent of the gold standard used, the best PCR results were obtained with nasopharyngeal specimens. However, the predictive value of a positive C. pneumoniae PCR result for patients with community-acquired pneumonia remains unknown and may be low. Although a widely accepted gold standard is still lacking, the rDNA LPS ELISA may currently be the preferred tool for diagnosing acute respiratory Chlamydia infections in routine clinical practice. However, the MIF test remains the method of choice for determining the prevalence of C. pneumoniae infections in a given community.

Characterization of serum antibody response to chlamydiae in patients with sexually acquired reactive arthritis

Sera from patients with sexually acquired reactive arthritis (SARA) with antibodies reacting with C. trachomatis and C. pneumoniae (group 1; n=20) and also with C. psittaci (group 2; n=19) were analyzed for antibody specificity. Sera from group 2 reacted significantly more often with C. trachomatis serotype E, H and K and had higher antibody titers to serotypes E, as tested by microimmunofluorescence tests. Cross-reactivities occurring in microimmunofluorescence tests were related to the presence of antichlamydial lipopolysaccharide antibodies, adsorption of which by recombinant lipopolysaccharide removed microimmunofluorescence reactivity with C. psittaci antigen. In group 2, significantly more sera had antibodies to C. pneumoniae, remaining after lipopolysaccharide adsorption, as proved by adsorption with viable C. trachomatis and C. pneumoniae organisms. None of the sera had antibodies to Yersinia enterocolitica, Shigella flexneri, Sh. sonnei and Salmonella spp. It was observed that the frequency and titer of cross-reacting antibodies to chlamydial serotypes and species were related to the time period between the diagnosis of genital chlamydial infection and of SARA. Cross-reactivities were also related to the presence of lipopolysaccharide, but not heat shock protein 60- or neutralizing antibodies to chlamydiae. Antibody reactivity induced by antichlamydial lipopolysaccharide antibodies can be removed by lipopolysaccharide adsorption.

Construction and characterization of genetically-marked bivalent anti- Shigella dysenteriae1 and anti- Shigella flexneriY live vaccine candidates

Bivalent vaccine candidates were developed against Shigella dysenteriae1 and Shigella flexneri, which are among the most frequent causative agents of shigellosis in developing countries. The rfpand rfbgene clusters, which code for S. dysenteriaeserotype 1 O-antigen biosynthesis, were inserted into an arsenite resistance minitransposon and randomly integrated into the attenuated S. flexneri aroDserotype Y strain SFL124. Nine recombinant clones that efficiently expressed both homologous and heterologous O-antigens were obtained. Southern blot analysis showed that in one clone the S. dysenteriae1 genes had integrated into the chromosome, whereas in all the others they had integrated into the virulence plasmid. All recombinant clones exhibited normal growth characteristics, were able to invade and survive within eukaryotic cells to the same extent as the parental strain, and expressed efficiently the recombinant lipopolysaccharide within invaded cells. Immunization of mice with two of the recombinant clones resulted in the production of antibodies specific for both homologous and heterologous O-antigens. The recombinant clones constitute promising vaccine candidates which can readily be distinguished from endemic shigellae by their non-antibiotic resistance marker.

Cloning and characterization of the galE locus of Pasteurella haemolytica A1

The enzyme UDP-galactose 4-epimerase (GalE) is involved in one of the major steps of galactose metabolism in bacteria. In many cases, GalE is required for the biosynthesis of extracellular polysaccharide materials such as lipopolysaccharide (LPS) and capsule. Mutants defective in galE have been shown to exhibit reduced virulence. Here we describe the cloning and characterization of the galE gene from the bovine pathogen Pasteurella haemolytica A1. This was achieved by the complementation of a Salmonella typhimurium galE mutant with a P. haemolytica A1 plasmid bank. Analysis of six clones recovered on minimal media with galactose as the carbon source showed that they all contained the same recombinant plasmid with a 5-kbp DNA insert. The galE-complementing activity was localized to a 2.2-kbp DNA region by subcloning. Biochemical, immunological, and phage sensitivity analyses of the recombinant LPS in S. typhimurium showed that it is essentially identical to that of the wild type. In vivo expression studies showed that a 37-kDa protein is expressed from the complementing plasmids, and the presence of GalE activity was confirmed by an assay for epimerase activity. Nucleotide sequence analysis of the cloned DNA identified the galE gene. Comparison of the deduced amino acid sequence analysis of P. haemolytica A1 GalE with published data showed high-level homology, 81.6%, with the GalE of Haemophilus influenzae type b. However, the sequences flanking galE do not show similarity with any other gal gene, suggesting that P. haemolytica A1 galE is not linked to the other genes of the gal operon, as is the case for Neisseria meningitidis, Neisseria gonorrhoeae, and H. influenzae. The separation of galE from the classical gal operon genes was confirmed by Southern blot hybridization studies, and a physical map showing the relative positions of galE, galT, and galK was constructed.

Augmented immunological activities of recombinant lipopolysaccharide possessing the mannose homopolymer as the O-specific polysaccharide.

Recombinant lipopolysaccharide possessing the mannose homopolymer as the O-specific polysaccharide was manufactured genetically by transforming Escherichia coli K-12 with various rfb genes capable of synthesizing the mannose homopolymer. Recombinant lipopolysaccharide exhibited levels of anticomplement activity, adjuvant activity, and regional lymph node-enlarging activity much higher than those exhibited by the original rough-type lipopolysaccharide from E. coli K-12 or lipopolysaccharide possessing the heteropolysaccharide from E. coli O111. Immunological activities of recombinant lipopolysaccharide were as strong as those of wild-type lipopolysaccharide possessing the mannose homopolymer. Characteristic activities of wild-type lipolysaccharide possessing the mannose homopolymer were exhibited by recombinant lipopolysaccharide. The abilities of lipopolysaccharide to activate B cells polyclonally and to produce cytokines did not seem to be related to the presence of the mannose homopolymer. Therefore, it was suggested that the mannose homopolymer in the O-specific polysaccharide might exclusively enhance anticomplement activity, adjuvant activity, and regional lymph node-enlarging activity among various lipid A activities.

Cloning and expression of rfb genes from Vibrio anguillarum serotype O2 in Escherichia coli: evidence for cross-reactive epitopes

Vibrio ordalii and Vibrio anguillarum O2 express lipopolysaccharide (LPS) O antigens containing both specific and cross-reactive epitopes. The localization of these epitopes on the O antigen is not known. We have cloned and expressed the rfb gene cluster for O-antigen synthesis from V. anguillarum O2 (rfbVaO2) in Escherichia coli. E. coli DH5 alpha containing the recombinant plasmid pAM86 expressed O antigens which reacted with polyclonal antisera to V. ordalii and to V. anguillarum O2 LPS and with monoclonal antibody (MAb) 7B4, which is specific for V. anguillarum O2 O antigens. The recombinant strains were also protected from bactericidal killing by normal fish serum. Surprisingly, the LPS expressed from the cloned rfbVaO2 genes also reacted with MAb A16, which is specific for V. ordalii O antigens. Western immunoblot analysis revealed that MAb 7B4 reacted with recombinant LPS bearing shorter O-antigen repeat units, while MAb A16 reacted with the longer O antigens. Similar results were obtained when pAM86 was transformed into E. coli CLM4, which has a deletion spanning the sbcB-rfb region, indicating that the changes in antigenic profiles of O antigens from the recombinant strains were not due to genes within the E. coli rfb cluster. These data suggest that the epitope recognized by the MAb A16 is expressed by V. anguillarum O2 strains but it is apparently not accessible to the antibody in the native O polysaccharide. Cloning of the rfbVaO2 gene cluster resulted in expression of a novel O antigen. The modification(s) which leads to the alterations in antigenic profile of these recombinant LPS remains to be determined.

PBC-like lesions is induced by immunization with recombinant PDC-E2 BCOADH-E2 hybrid molecule and lipopolysaccharide injection in neonatally thymectomized mice

PBC-like lesions is induced by immunization with recombinant PDC-E2 BCOADH-E2 hybrid molecule and lipopolysaccharide injection in neonatally thymectomized mice

Lipopolysaccharide (LPS)-binding protein is carried on lipoproteins and acts as a cofactor in the neutralization of LPS.

Lipoproteins isolated from normal human plasma can bind and neutralize bacterial lipopolysaccharide (LPS) and may represent an important mechanism in host defense against gram-negative septic shock. Recent studies have shown that experimentally elevating the levels of circulating high-density lipoproteins (HDL) provides protection against death in animal models of endotoxic shock. We sought to define the components of HDL that are required for neutralization of LPS. To accomplish this we have studied the functional neutralization of LPS by native and reconstituted HDL using a rapid assay that measures the CD14-dependent activation of leukocyte integrins on human neutrophils. We report here that reconstituted HDL particles (R-HDL), prepared from purified apolipoprotein A-I (apoA-I) combined with phospholipid and free cholesterol, are not sufficient to neutralize the biologic activity of LPS. However, addition of recombinant LPS binding protein (LBP), a protein known to transfer LPS to CD14 and enhance responses of cells to LPS, enabled prompt binding and neutralization of LPS by R-HDL. Thus, LBP appears capable of transferring LPS not only to CD14 but also to lipoprotein particles. In contrast with R-HDL, apoA-I containing lipoproteins (LpA-I) isolated from plasma by selected affinity immunosorption (SAIS) on an anti-apoA-I column, neutralized LPS without addition of exogenous LBP. Several lines of evidence demonstrated that LBP is a constituent of LpA-I in plasma. Passage of plasma over an anti-apoA-I column removed more than 99% of the LBP detectable by ELISA, whereas 31% of the LBP was recovered by elution of the column. Similarly, the ability of plasma to enable activation of neutrophils by LPS (LBP/Septin activity) was depleted and recovered by the same process. Furthermore, an immobilized anti-LBP monoclonal antibody coprecipitated apoA-I. The results described here suggest that in addition to its ability to transfer LPS to CD14, LBP may also transfer LPS to lipoproteins. Since LBP appears to be physically associated with lipoproteins in plasma, it is positioned to play an important role in the neutralization of LPS.

Cloning and surface expression of Pseudomonas aeruginosa O antigen in Escherichia coli.

As a step toward developing recombinant oral vaccines, we have explored the feasibility of expression of O polysaccharide antigens from Pseudomonas aeruginosa by Escherichia coli. We cloned in E. coli HB101 a 26.2-kilobase DNA fragment from P. aeruginosa strain PA103 that specifies the production of the O polysaccharide of Fisher immunotype 2 (IT-2) strains. The recombinant organism incorporated the P. aeruginosa IT-2 O polysaccharide onto the core of the E. coli lipopolysaccharide (LPS). Transfer of the recombinant plasmid to three LPS-rough strains of P. aeruginosa resulted in synthesis of IT-2 O antigen, and two of these transconjugant strains also synthesized a second O polysaccharide, presumably representing expression of a repressed, or an incomplete, set of genes for an endogenous O polysaccharide. Rabbits injected with the purified recombinant LPS made antibody specific for P. aeruginosa IT-2 O side chains, as did mice fed the recombinant E. coli strain. Expression of P. aeruginosa O antigens by enteric bacteria makes it possible to study these recombinant strains as oral vaccines to prevent P. aeruginosa infections.

Defective Tolerance to the Toxic and Metabolic Effects of Interleukin 1

The effect on food intake, body weight, and survival of mice given recombinant lipopolysaccharide (LPS), tumor necrosis factor/cachectin (TNF), or interleukin 1 (IL-1) (5 micrograms/mouse, ip, twice daily) was studied. All agents induced a rapid reduction of food intake and body weight after 1 day of treatment. Unlike TNF and LPS, IL-1 given as two daily administrations of 5 micrograms was lethal within 3 days. Mice treated with LPS or TNF rapidly developed tolerance to their anorectic effect, whereas tolerance to IL-1 required a longer time to develop and was not complete. We investigated the possible roles of changes in serum corticosterone and glucose in the effects of LPS, TNF and IL-1. A single injection of LPS, TNF, or IL-1 markedly increased serum corticosterone levels after 2 h. After only 2 days of chronic treatment, mice given LPS or TNF were refractory to induction of serum corticosterone by a subsequent injection of LPS or TNF, but mice given IL-1 for 2 days were still fully responsive to IL-1. IL-1, unlike TNF and LPS, induced a marked hypoglycemic response. Repeated administration of IL-1 sensitized to its hypoglycemic effect. This lack of adaptation to the increase of serum corticosterone and hypoglycemia was also observed when IL-1 was given at lower, nonlethal doses (0.25-1.0 microgram) and for a longer period (up to 8 days). The defective tolerance to the metabolic and toxic effects of IL-1 in this experimental model indicates that there are major differences between the in vivo biological responses to IL-1 and TNF.