Localization of lipopolysaccharide from Escherichia Coli into human atherosclerotic plaque

Experimental studies showed that gut-derived lipopolysaccharide (LPS) is pro-atherogenic, however, its relationship with human atherosclerosis is still to be defined. We investigate if gut-derived LPS from Escherichia Coli localizes in human carotid plaque and its potential role as pro-inflammatory molecule in the atherosclerotic lesion. LPS from Escherichia Coli and Toll-like receptor 4 (TLR4) were studied in specimens from carotid and thyroid arteries of 10 patients undergoing endarterectomy and 15 controls matched for demographic and clinical characteristics. Blood LPS were significantly higher in patients compared to controls. Immunochemistry analysis revealed positivity for antibodies against LPS and TLR4 coincidentally with positivity for CD68 only in the atherosclerotic plaque of carotid arteries but not in thyroid arteries; the positivity for LPS and TLR4 was greater in the area with activated macrophages. LPS concentration similar to that detected in atherosclerotic plaque resulted in a dose-dependent TLR4-mediated Nox2 up-regulation by human monocytes. These data provide the first evidence that LPS from Escherichia Coli localizes in human plaque and may contribute to atherosclerotic damage via TLR4-mediated oxidative stress

Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary cells in the presence of adenovirus

Endotoxin (lipopolysaccharide, LPS) is commonly found as a contaminant in plasmid DNA preparations. We demonstrate here that the quantities of LPS typically contaminating DNA preparations can generate a toxicity to primary cells (primary human skin fibroblasts, primary human melanoma cells) in the presence of entry-competent adenovirus particles. Toxicity can be observed with as little as 100 ng/ml free LPS or 100 pg/ml LPS when the LPS is assembled into polylysine/adenovirus complexes. Simple and effective methods of removing the contaminating LPS using either a polymyxin B resin or Triton X-114 extraction are described. Treatment of DNA samples to remove LPS eliminates the toxicity to primary cells

Expression of \u3cem\u3eRhizobium leguminosarum\u3c/em\u3e CFN42 Genes for Lipopolysaccharide in Strains Derived from Different \u3cem\u3eR. leguminosarum\u3c/em\u3e Soil Isolates

Two mutant derivatives of Rhizobium leguminosarum ANU843 defective in lipopolysaccharide (LPS) were isolated. The LPS of both mutants lacked O antigen and some sugar residues of the LPS core oligosaccharides. Genetic regions previously cloned from another Rhizobium leguminosarum wild-type isolate, strain CFN42, were used to complement these mutants. One mutant was complemented to give LPS that was apparently identical to the LPS of strain ANU843 in antigenicity, electrophoretic mobility, and sugar composition. The other mutant was complemented by a second CFN42 lps genetic region. In this case the resulting LPS contained O-antigen sugars characteristic of donor strain CFN42 and reacted weakly with antiserum against CFN42 cells, but did not react detectably with antiserum against ANU843 cells. Therefore, one of the CFN42 lps genetic regions specifies a function that is conserved between the two R. leguminosarum wild-type isolates, whereas the other region, at least in part, specifies a strain-specific LPS structure. Transfer of these two genetic regions into wild-type strains derived from R. leguminosarum ANU843 and 128C53 gave results consistent with this conclusion. The mutants derived from strain ANU843 elicited incompletely developed clover nodules that exhibited low bacterial populations and very low nitrogenase activity. Both mutants elicited normally developed, nitrogen-fixing clover nodules when they carried CFN42 lps DNA that permitted synthesis of O-antigen-containing LPS, regardless of whether the O antigen was the one originally made by strain ANU843

Enhancement of platelet response to immune complexes and IgG aggregates by lipid A-rich bacterial lipopolysaccharides.

The effect of the common lipid moiety of bacterial LPS on secretion from washed human platelets has been studied. The lipid A-rich LPS of S. minnesota R595 and a lipid A preparation both potentiated platelet serotonin secretion in response to IgG aggregates or immune complexes up to 50-fold but had little effect in the absence of IgG. Lipid A has been shown to bind immune aggregates, raising the possibility that its mechanism of action involved effective enlargement or insolubilization of the aggregates. IgG aggregates of dimer to tetramer size were shown to be platelet simuli, equivalent on a weight basis to larger soluble aggregates. The effect of both sizes of aggregates on platelets were equally enhanced by the LPS, indicating that increased size of aggregates alone could not account for the effect of LPS. Similarly, because lipid A-rich LPS enhanced platelet response to already insoluble immune complexes, its mechanism of action cannot simply be insolubilization of immune aggregates. These LPS did not enhance platelet stimulation by antiplatelet antibody, monosodium urate crystals, or thrombin and only slightly enhanced stimulation by insoluble human skin collagen. This indicates some stimulus specificity in the ability of LPS to increase platelet secretion. The enhancement of cell response to immune complexes by the common lipid region of LPS may represent a mechanism for the diverse effects of LPS in vivo and in vitro

Characterization of the Lipopolysaccharide from a \u3cem\u3eRhizobium phaseoli\u3c/em\u3e Mutant that is Defective in Infection Thread Development

The lipopolysaccharide (LPS) from a Rhizobium phaseoli mutant, CE109, was isolated and compared with that of its wild-type parent, CE3. A previous report has shown that the mutant is defective in infection thread development, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it has an altered LPS (K. D. Noel, K. A. VandenBosch, and B. Kulpaca, J. Bacteriol. 168:1392-1462, 1986). Mild acid hydrolysis of the CE3 LPS released a polysaccharide and an oligosaccharide, PS1 and PS2, respectively. Mild acid hydrolysis of CE109 LPS released only an oligosaccharide. Chemical and immunochemical analyses showed that CE3-PS1 is the antigenic O chain of this strain and that CE109 LPS does not contain any of the major sugar components of CE3-PS1. CE109 oligosaccharide was identical in composition to CE3-PS2. The lipid A\u27s from both strains were very similar in composition, with only minor quantitative variations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CE3 and CE109 LPSs showed that CE3 LPS separated into two bands, LPS I and LPS II, while CE109 had two bands which migrated to positions similar to that of LPS II. Immunoblotting with anti-CE3 antiserum showed that LPS I contains the antigenic O chain of CE3, PS1. Anti-CE109 antiserum interacted strongly with both CE109 LPS bands and CE3 LPS II and interacted weakly with CE3 LPS I. Mild-acid hydrolysis of CE3 LPS I, extracted from the polyacrylamide gel, showed that it contained both PS1 and PS2. The results in this report showed that CE109 LPS consists of only the lipid A core and is missing the antigenic O chain

Downregulation of Tumor Necrosis Factor Expression in the Human Mono-Mac-6 Cell Line

Mono-Mac-6 cells, but not U937 cells, can be Induced to rapidly express tumor necrosis factor (TNF) mRNA and protein when triggered with Ilpopolysaccharlde (LPS) at 1 pg/mI. Preincubatlon of the cells for 3 d with low amounts of LPS (10 ng/mI) results In nearly complete suppression of TNF secretion. This downreguiatlon appears to occur at the pretranslational level since specIfIc mRNA is virtually undetectable under these conditions. By contrast, the same prelncubatlon with 10 ng/mI LPS results in enhanced phagocytosls (28.6-67.2% for Staphylococcus aureus), demonstrating that not all monocyte functions are suppressed. While these results show that only stringent exclusion of LPS from culture media allows for Induction of TNF In the Mono-Mac-6 cell line, the pronounced effect of LPS preincubatlon may also provide a suitable model with which to study the mechanisms of LPS-lnduced desensitizatIon

Learning and Skills Council and Learning Partnerships national policy framework : final draft

"This framework outlines the strategic role of LPs and offers guidance on the relationships between the LSC and LPs. It is intended to provide a focus and a steer for both the local LSCs and LPs on both the context and future delivery" -- page 2

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

EMPIRICAL STUDY ON THE INFLUENCE OF PROCUREMENT METHODS ON LAST PLANNER® SYSTEM IMPLEMENTATION

Previous studies have examined various factors that influence the implementation of the Last Planner System (LPS) in construction projects. However, there is limited documented evidence on the influence of procurement methods on the implementation of the LPS. The aim of this study, therefore; is to understand the influence of some selected procurement methods on the implementation of the LPS using case study approach. Three in-depth case studies were conducted on building and highways projects in the UK. The projects were managed with the LPS principles with dissimilar procurement methods. In addition to document analysis and physical observation, 28 in-depth-interviews were conducted. The investigation shows that the prevailing traditional mindset exhibited by the designers in the traditional design bid build (DBB) influences the quality of promises and commitments that could be made during the lookahead planning. From the study, it seems no single procurement method is a sure way to the full application of the LPS process on a project. The study observes that irrespective of the procurement route used, a mindset change towards collaboration among the different stakeholders on the project is fundamental to successful LPS implementation. For instance, on projects where DBB was used and the subcontractors were in framework agreement, the LPS implementation worked well among the subcontractors. The study recommends that the procurement approach to be used on LPS projects should not be too firm, but lithe enough to integrate collaborative working among the different stakeholders on the project for a smooth workflow