Lipopolysaccharide Antigens Research Articles

Localization of the lipopolysaccharide recognition complex in the human healthy and inflamed premature and adult gut

Microbiota in the intestinal lumen provide an abundant source of potentially detrimental antigens, including lipopolysaccharide (LPS), a potent immunostimulatory product of Gram-negative bacteria recognized by the host via TLR-4 and MD-2. An aberrant immune response to LPS or other bacterial antigens has been linked to inflammatory bowel disease (IBD) and necrotizing enterocolitis (NEC). We investigated which cells express MD-2 in the normal and inflamed ileum from neonates and adults by immunohistochemistry. Moreover, MD-2 and TLR4 mRNA expression in normal adult ileum was studied by reverse-transcription polymerase chain reaction (RT-PCR) on cells isolated by laser capture microdissection. Premature infants did not show MD-2 expression either in epithelial cells or in the lamina propria. Similarly, MD-2 was absent in epithelial cells and lamina propria inflammatory cells in preterm infants with NEC. MD-2 protein in the healthy term neonatal and adult ileum was predominantly expressed by Paneth cells and some resident inflammatory cells in the lamina propria. MD-2 and TLR-4 mRNA expression was restricted to crypt cells. Also in IBD, Paneth cells were still the sole MD-2-expressing epithelial cells, whereas inflammatory cells (mainly plasma cells) were responsible for the vast majority of the MD-2 expression. The absence of MD-2 in the immature neonatal gut suggests impaired LPS sensing, which could predispose neonates to NEC upon microbial colonization of the immature intestine. The apparent expression of MD-2 by Paneth cells supports the critical concept that these cells respond to luminal bacterial products in order to maintain homeostasis with the intestinal microbiota in vivo.

The role of CDR H3 in antibody recognition of a synthetic analog of a lipopolysaccharide antigen

In order to explore the structural basis for adaptability in near germline monoclonal antibodies (mAb), we have examined the specificity of the promiscuous mAb S67-27 to both naturally derived carbohydrate antigens and a variety of synthetic nonnatural antigens based on the bacterial lipopolysaccharide component 3-deoxy-alpha-D-manno-oct-2-ulosonic acid (Kdo). One such analog, a 7-O-methyl (7-O-Me) Kdo disaccharide, was found to bind to the antibody with at least 30-fold higher affinity than any other antigen tested. The structure of S67-27 in complex with this analog and three other naturally occurring Kdo antigens revealed that the enhanced affinity of the mAb for the synthetic analog was accomplished by the strategic positioning of CDR H3 away from a conserved Kdo binding pocket that allowed the formation of new antibody-antigen contacts. Furthermore, the comparison of this structure with the structures of related mAbs revealed how the position and structure of CDR H3 influence the specificity or promiscuity of near-germline carbohydrate-recognizing antibodies by altering the architecture of the combining site.

Genome‐wide screens: novel mechanisms in colicin import and cytotoxicity

Only two new genes (fkpA and lepB) have been identified to be required for colicin cytotoxicity in the last 25 years. Genome-wide screening using the 'Keio collection' to test sensitivity to colicins (col) A, B, D, E1, E2, E3, E7 and N from groups A and B, allowed identification of novel genes affecting cytotoxicity and provided new information on mechanisms of action. The requirement of lipopolysaccharide for colN cytotoxicity resides specifically in the lipopolysaccharide inner-core and first glucose. ColA cytotoxicity is dependent on gmhB and rffT genes, which function in the biosynthesis of lipopolysaccharide and enterobacterial common antigen. Of the tol genes that function in the cytoplasmic membrane translocon, colE1 requires tolA and tolR but not tolQ for activity. Peptidoglycan-associated lipoprotein, which interacts with the Tol network, is not required for cytotoxicity of group A colicins. Except for TolQRA, no cytoplasmic membrane protein is essential for cytotoxicity of group A colicins, implying that TolQRA provides the sole pathway for their insertion into/through the cytoplasmic membrane. The periplasmic protease that cleaves between the receptor and catalytic domains of colE7 was not identified, implying either that the responsible gene is essential for cell viability, or that more than one gene product has the necessary proteolysis function.

A Changing Gastric Environment Leads to Adaptation of Lipopolysaccharide Variants in Helicobacter pylori Populations during Colonization

The human gastric pathogen Helicobacter pylori colonizes the stomachs of half of the human population, and causes development of peptic ulcer disease and gastric adenocarcinoma. H. pylori-associated chronic atrophic gastritis (ChAG) with loss of the acid-producing parietal cells, is correlated with an increased risk for development of gastric adenocarinoma. The majority of H. pylori isolates produce lipopolysaccharides (LPS) decorated with human-related Lewis epitopes, which have been shown to phase-vary in response to different environmental conditions. We have characterized the adaptations of H. pylori LPS and Lewis antigen expression to varying gastric conditions; in H. pylori isolates from mice with low or high gastric pH, respectively; in 482 clinical isolates from healthy individuals and from individuals with ChAG obtained at two time points with a four-year interval between endoscopies; and finally in isolates grown at different pH in vitro. Here we show that the gastric environment can contribute to a switch in Lewis phenotype in the two experimental mouse models. The clinical isolates from different human individuals showed that intra-individual isolates varied in Lewis antigen expression although the LPS diversity was relatively stable within each individual over time. Moreover, the isolates demonstrated considerable diversity in the levels of glycosylation and in the sizes of fucosylated O-antigen chains both within and between individuals. Thus our data suggest that different LPS variants exist in the colonizing H. pylori population, which can adapt to changes in the gastric environment and provide a means to regulate the inflammatory response of the host during disease progression.

A proposed bailout for A-T patients?

A proposed bailout for A-T patients?

PDE4 inhibitors can induce pro‐inflammatory chemokine expression in human macrophages

Monocytes differentiate into macrophages upon exposure to GM‐CSF. Agonists that increase cAMP are known inhibitors of many inflammatory processes in macrophages activated by lipopolysaccharide and other antigens. We find however, that when cAMP is increased during the differentiation process there is a large increase in the mRNA and protein for several pro‐inflammatory chemokines including CXCL 1, 2, 5, and 7 as well as CCL2, and 13. These pro‐inflammatory effects on chemokine expression are mimicked by stimulation of the Epac pathway. One major phosphodiesterase (PDE) controlling cAMP in macrophages is PDE4. Several PDE4 inhibitors are currently in clinical trials for treatment of inflammatory lung diseases like chronic obstructive pulmonary disorder and asthma. We find that inhibition of PDE4 by rolipram potentiates expression of these pro‐inflammatory chemokines. We also find that under these conditions ATF3, a newly identified negative regulator of NF‐kB activation, is decreased by cAMP and that this also is potentiated by PDE4 inhibitors. These data suggest that co‐administration of chemokine receptor or Epac pathway antagonists should improve the efficacy of PDE4 inhibitor therapy.

Production and Characterization of a Cross-Reactive Monoclonal Antibody to Lipopolysaccharide

Lipopolysaccharide (LPS) is located on the cell wall of gram-negative bacteria and plays an important role in the pathogenesis of sepsis, which continues to be a leading cause of death in the intensive care units. There are many strains and serotypes of gram-negative bacteria and each individual has a unique kind of LPS. In addition, LPS belongs to thymus-independent (TI) antigen, making it difficult to produce high-affinity, cross-reactive monoclonal antibodies (MAb) against LPS. Here we report a novel method to produce cross-reactive murine MAbs against LPS by mixed immunization with whole cell bacteria, commercial LPS, and synthetic peptide, which simulates the structure of LPS. Using this approach, an MAb designated SMU-3A8 was generated, which can react with four commercial LPSs and seven gram-negative bacteria with high affinity suited for ELISA, dot-ELISA, Western blotting, immunofluorescence, and flow cytometry. Our results provide a new strategy for the generation of high-affinity, cross-reactive MAbs against LPS and other TI antigens.

Saccharomyces boulardii inhibits lipopolysaccharide-induced activation of human dendritic cells and T cell proliferation

Saccharomyces boulardii (Sb) is a probiotic yeast preparation that has demonstrated efficacy in inflammatory and infectious disorders of the gastrointestinal tract in controlled clinical trials. Although patients clearly benefit from treatment with Sb, little is known on how Sb unfolds its anti-inflammatory properties in humans. Dendritic cells (DC) balance tolerance and immunity and are involved critically in the control of T cell activation. Thus, they are believed to have a pivotal role in the initiation and perpetuation of chronic inflammatory disorders, not only in the gut. We therefore decided to investigate if Sb modulates DC function. Culture of primary (native, non-monocyte-derived) human myeloid CD1c+CD11c+CD123(-) DC (mDC) in the presence of Sb culture supernatant (active component molecular weight < 3 kDa, as evaluated by membrane partition chromatography) reduced significantly expression of the co-stimulatory molecules CD40 and CD80 (P < 0.01) and the DC mobilization marker CC-chemokine receptor CCR7 (CD197) (P < 0.001) induced by the prototypical microbial antigen lipopolysaccharide (LPS). Moreover, secretion of key proinflammatory cytokines such as tumour necrosis factor-alpha and interleukin (IL)-6 were notably reduced, while the secretion of anti-inflammatory IL-10 increased. Finally, Sb supernatant inhibited the proliferation of naive T cells in a mixed lymphocyte reaction with mDC. In summary, our data suggest that Sb may exhibit part of its anti-inflammatory potential through modulation of DC phenotype, function and migration by inhibition of their immune response to bacterial microbial surrogate antigens such as LPS.

LPS and Flagellin-Based Models for Serological Screening and Confirmation of Salmonella Infections in Chickens

Summary Avian salmonellosis due to Salmonella B and D serogroups has a great importance because of the public health and the economical losses in poultry industry. For accurate diagnosis of avian salmonellosis, a total of 126 chicken sera evaluated with a commercial Salmonella Enteritidis (SE) ELISA kit have been used to develop two indirect ELISA models using lipopolysaccharide (LPS) and purified flagellin (Flg) of SE as diagnostic antigens. After optimization, 154 and 263 sera were tested with indirect LPS- and Flg-ELISA, respectively. The performance of both indirect LPS- and Flg-ELISA was found high and similar for detecting anti-Salmonella antibody in comparison with a commercial LPS-ELISA kit. Nevertheless, a limited number of chicken sera were found discordant between Flg- and LPS-ELISAs. Western-blot analyses of the discordant sera demonstrated that they were found reactive with bacterial lysate of SE but only recognized LPS or Flg antigen. It was concluded from this study that the simultaneous use of both LPS- and Flg-ELISAs would allow differential diagnosis of avian specific salmonellosis from zoonotic Salmonella infections due to flagellated bacteria and Western blot models can be used as confirmatory serological test. .

Characterization of bacteriophages used in the Salmonella enterica serovar Enteritidis phage-typing scheme

The 16 Salmonella enterica serovar Enteritidis (S. Enteritidis) typing phages (SETPs) used in the Laboratory of Enteric Pathogens (Health Protection Agency, London, UK) phage-typing scheme have not previously been characterized in detail. We have examined the adsorption properties of the phages with respect to a number of S. enterica serovars and defined phage morphology with electron microscopy. PFGE was used to estimate overall genome size and banding patterns generated by electrophoresis following restriction endonuclease digestion of the genome with HindIII were compared. PCR amplification and sequencing of selected genes was performed. The 16 phages comprise three morphotypes, Podoviridae (SETP1, 8, 10, 14, 15 and 16), Siphoviridae (SETP3, 5, 7, 11, 12 and 13) and Myoviridae (SETP2, 4, 6 and 9). All Podoviridae and Siphoviridae, but not Myoviridae, adsorbed to the O12 lipopolysaccharide antigen of Salmonella serogroups B (4,12) and D(1) (9,12). The genome sizes for the Podoviridae and Siphoviridae (PFGE-A) were approximately 42 kb. The genome size for Myoviridae SETP2, 4 and 9 was 36.5 kb, and for myovirus SETP6 was 27 kb. HindIII digestion of phage DNA produced 9 distinct patterns of 8 to 11 bands. Relationships between phages based on digest patterns were consistent with those defined by morphology. The Podoviridae had homologues of several P22 genes while the Siphoviridae had homologues of several genes present in the sequenced siphovirus SETP3 (EF177456). This study represents an initial step in characterizing the molecular basis that underlies the widely used S. Enteritidis typing scheme.

Reduced lipopolysaccharide O antigen expression, increased acid susceptibility and multicellular behaviour in anEscherichia coliisolate after long-termin vitroexposure to formic acid

Organic acids are being used in animal husbandry due to their positive effects on overall health and growth rate in animal husbandry. However, little is known about the long-term effects of organic acids on the normal microbiota. In this work, an Escherichia coli strain isolated from a healthy pig was passed weekly for 20 months in medium with and without formic acid (FA) to investigate the effects of long-term exposure to organic acids. Passages were made using buffered medium at pH 5.25 and 6.1 with and without FA. Colonies showing reduced O antigen lipopolysaccharide production and increased susceptibility to FA were isolated after 12 months exposure to FA at pH 5.25 and after 20 months exposure to FA at pH 6.1. Adaptation to acid and bacterial growth was strongly affected by FA at pH 5.25. Also, after 2 months of serial passages, we observed colonies exhibiting multicellular behaviour, with production of fimbriae and an extracellular matrix. We conclude that long-term exposure to FA of an E. coli isolate from the normal intestinal microbiota is associated with various changes in the bacterial cell surface involving reduced O antigen expression and increased extracellular matrix production together with increased susceptibility to FA. Key words: Biofilm, cellulose, curli fimbriae, organic acid, swine

Antigen-specific B memory cell responses to lipopolysaccharide (LPS) and invasion plasmid antigen (Ipa) B elicited in volunteers vaccinated with live-attenuated Shigella flexneri 2a vaccine candidates

We evaluated B memory responses in healthy adult volunteers who received one oral dose of live-attenuated Shigella flexneri 2a vaccine. LPS-specific B M cells increased from a median of 0 at baseline to 20 spot forming cells (SFC)/10 6 expanded cells following vaccination ( p = 0.008). A strong correlation was found between post-vaccination anti-LPS B M cell counts and peak serum anti-LPS IgG titers ( rs = 0.95, p = 0.0003). Increases in B M specific for IpaB approaching significance were also observed. In sum, oral vaccination with live-attenuated S. flexneri 2a elicits B M cells to LPS and IpaB, suggesting that B M responses to Shigella antigens should be further studied as a suitable surrogate of protection in shigellosis.

Salmonella Enteritidis experimental infection in chickens: Effects of challenge dose on serum immunoglobulin G antibody response

Salmonella enterica serovar Enteritidis is a food borne pathogen of humans causing food-poisoning and sometimes deaths. In order to control egg-borne transmission of Salmonella Enteritidis to humans, prompt and accurate detection of infected poultry flocks is essential. This paper examined the effects of challenge dose ofSalmonella Enteritidis on detection of specific immunoglobulin G (IgG) antibody in serum of experimentally infected chickens. An indirect ELISA technique based on lipopolysaccharide (LPS) antigen of Salmonella Enteritidis was used. Two groups of specific-pathogen-free chickens were infected orally with 108 and 104 colony forming units (cfu) of Salmonella Enteritidis. Serum samples were collected prior to challenge and at five subsequent weekly intervals. Levels of serum IgG antibody response observed in the chickens infected with 108 cfu of Salmonella Enteritidis were significantly stronger (P<0.05) than those of the 104 cfu group. Although considerable IgG response was seen with most of the chickens by 2 weeks post-infection, highest group mean optical density values of 2.177 and 0.984 were observed at 5 weeks for 108 and 104 cfu Salmonella Enteritidis infected groups, respectively. Chickens exposed to few Salmonella colonies during infection do not produce high IgG antibody and may continue to contaminate the flock. Key words: Salmonella Enteritidis, immunoglobulin G, serum, chickens, antibodies.

Inhibition of Salmonella enterica Serovar Typhimurium Motility and Entry into Epithelial Cells by a Protective Antilipopolysaccharide Monoclonal Immunoglobulin A Antibody

Secretory immunoglobulin A (SIgA) antibodies directed against the O antigen of lipopolysaccharide (LPS) are the primary determinants of mucosal immunity to gram-negative enteric pathogens. However, the underlying mechanisms by which these antibodies interfere with bacterial colonization and invasion of intestinal epithelial cells are not well understood. In this study, we report that Sal4, a protective, anti-O5-specific monoclonal IgA, is a potent inhibitor of Salmonella enterica serovar Typhimurium flagellum-based motility. Using video light microscopy, we observed that Sal4 completely and virtually instantaneously "paralyzed" laboratory and clinical strains of serovar Typhimurium. Sal4-mediated motility arrest preceded and occurred independently of agglutination. Polyclonal anti-LPS IgG antibodies and F(ab)(2) fragments were as potent as was Sal4 at impeding bacterial motility, whereas monovalent Fab fragments were 5- to 10-fold less effective. To determine whether motility arrest can fully account for Sal4's protective capacity in vitro, we performed epithelial cell infection assays in which the requirement for flagellar motility in adherence and invasion was bypassed by centrifugation. Under these conditions, Sal4-treated serovar Typhimurium cells remained noninvasive, revealing that the monoclonal IgA, in addition to interfering with motility, has an effect on bacterial uptake into epithelial cells. Sal4 did not, however, inhibit bacterial uptake into mouse macrophages, indicating that the antibody interferes specifically with Salmonella pathogenicity island 1 (SPI-1)-dependent, but not SPI-1-independent, entry into host cells. These results reveal a previously unrecognized capacity of SIgA to "disarm" microbial pathogens on mucosal surfaces and prevent colonization and invasion of the intestinal epithelium.

Multiplex pathogen detection based on spatially addressable microarrays of barcoded resins

Suspension microsphere immunoassays are rapidly gaining recognition in antigen identification and infectious disease biodetection due to their simplicity, versatility and high-throughput multiplex screening. We demonstrate a multiplex assay based on antibody-functionalized barcoded resins (BCRs) to identify pathogen antigens in complex biological fluids. The binding event of a particular antibody on given bead (fluorescence) and the identification of the specific pathogen agent (vibrational fingerprint of the bead) can be achieved in a dispersive Raman system by exciting the sample with two different laser lines. Anthrax protective antigen, Franciscella tularensis lipopolysaccharide and CD14 antigens were accurately identified and quantified in tetraplex assays with a detection limit of 1 ng/mL. The rapid, versatile and simple analysis enabled by the BCRs demonstrates their potential for multiplex antigen detection and identification in a reconfigurable microarray format.

Kinetics of the Immune Response Associated with Tularemia: Comparison of an Enzyme-Linked Immunosorbent Assay, a Tube Agglutination Test, and a Novel Whole-Blood Lymphocyte Stimulation Test

We have developed and evaluated a novel and simplified whole-blood lymphocyte stimulation assay that focuses on the measurement of gamma interferon after 24 h of stimulation with whole-cell tularemia antigen and a tularemia enzyme-linked immunosorbent assay (ELISA) based on highly purified lipopolysaccharide antigen. Comparison of the kinetics of the two assays and those of the traditional tube agglutination test shows that the cellular immune response can be detected earlier by the lymphocyte stimulation assay. This test already shows a high proportion of positive results during the first week after the onset of the disease, may be applicable in everyday laboratory practice, and has the potential of changing routine diagnostics for tularemia. The new ELISA has a high sensitivity and becomes positive to a high degree during the second week of disease.

Curcumin, a polyphenolic antioxidant, attenuates chronic fatigue syndrome in murine water immersion stress model

Chronic fatigue syndrome, infection and oxidative stress are interrelated in epidemiological case studies. However, data demonstrating scientific validation of epidemiological claims regarding effectiveness of nutritional supplements for chronic fatigue syndrome are lacking. This study is designed to evaluate the effect of natural polyphenol, curcumin, in a mouse model of immunologically induced fatigue, where purified lipopolysaccharide (LPS) and Brucella abortus (BA) antigens were used as immunogens. The assessment of chronic fatigue syndrome was based on chronic water-immersion stress test for 10 min daily for 19 days and the immobility time was taken as the marker of fatigue. Mice challenged with LPS or BA for 19 days showed significant increase in the immobility time and hyperalgesia on day 19, as well as marked increase in serum tumor necrosis factor-alpha (TNF- α) levels. Concurrent treatment with curcumin resulted in significantly decreased immobility time as well as hyperalgesia. There was significant attenuation of oxidative stress as well as TNF- α levels. These findings strongly suggest that during immunological activation, there is significant increase in oxidative stress and curcumin can be a valuable option in the treatment of chronic fatigue syndrome.

Characterization of Two β-1,3-Glucosyltransferases fromEscherichia coliSerotypes O56 and O152

The O antigens of outer membrane-bound lipopolysaccharides (LPS) in gram-negative bacteria are oligosaccharides consisting of repeating units with various structures and antigenicities. The O56 and O152 antigens of Escherichia coli both contain a Glc-beta1-3-GlcNAc linkage within the repeating unit. We have cloned and identified the genes (wfaP in O56 and wfgD in O152) within the two O-antigen gene clusters that encode glucosyltransferases involved in the synthesis of this linkage. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-alpha-PO3-PO3-(CH2)11-O-phenyl] was used as an acceptor and UDP-Glc as a donor substrate to demonstrate that both wfgD and wfaP encode glucosyltransferases. Enzyme products from both glucosyltransferases were isolated by high-pressure liquid chromatography and analyzed by nuclear magnetic resonance. The spectra showed the expected Glc-beta1-3-GlcNAc linkage in the products, confirming that both WfaP and WfgD are forms of UDP-Glc: GlcNAc-pyrophosphate-lipid beta-1,3-glucosyltransferases. Both WfaP and WfgD have a DxD sequence, which is proposed to interact with phosphate groups of the nucleotide donor through the coordination of a metal cation, and a short hydrophobic sequence at the C terminus that may help to associate the enzymes with the inner membrane. We showed that the enzymes have similar properties and substrate recognition. They both require a divalent cation (Mn2+ or Mg2+) for activity, are deactivated by detergents, have a broad pH optimum, and require the pyrophosphate-sugar linkage in the acceptor substrate for full activity. Substrates lacking phosphate or pyrophosphate linked to GlcNAc were inactive. The length of the aliphatic chain of acceptor substrates also contributes to the activity.

Lymphangitis in aPortuguese Patient Infected withRickettsia sibirica

Lymphangitis in a<b />Portuguese Patient Infected with<i>Rickettsia sibirica</i>

Validation of a second generation competitive enzyme immunoassay (CELISA) for the diagnosis of brucellosis in various species of domestic animals

A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed to eliminate reagent variables in the assay. This assay was different from earlier CELISA formats in that it used recombinant protein A and protein G immunoglobulin receptors (PAG), labelled with horseradish peroxidase, thus eliminating the requirement for polyclonal anti-mouse-enzyme conjugate for detection. This allowed standardization of the assay. The CELISA uses a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3. This antibody did not react with PAG. This monoclonal antibody was used to compete with antibody in the bovine test serum to the smooth lipopolysaccharide (SLPS) antigen. Reaction of bovine antibody was then measured directly with the PAG enzyme conjugate. In this case, development of colour in the reaction indicated a positive reaction. The performance characteristics of the new CELISA, sensitivity, specificity and exclusion of antibody of B. abortus S19 vaccinated animals, were very similar to those of the classical CELISA and to the indirect enzyme immunoassay (IELISA) when using sera deemed positive by isolation of the bacterium, either from individual animals or from some animals on the premises. All sera were tested by the buffered antigen plate agglutination test (BPAT) and the complement fixation test (CFT). Only samples positive on both BPAT and CFT were considered as positive and only samples negative on both tests were used considered negative. Sufficient samples from cattle, swine, sheep and goats to validate the test were included based on OIE guidelines suggesting inclusion of a minimum of 300 positive and 1000 negative samples.