Endotoxin-neutralizing Protein Research Articles

Bactericidal/Permeability-Increasing Protein (rBPI 21 ) and Fluoroquinolone Mitigate Radiation-Induced Bone Marrow Aplasia and Death

Identification of safe, effective treatments to mitigate toxicity after extensive radiation exposure has proven challenging. Only a limited number of candidate approaches have emerged, and the U.S. Food and Drug Administration has yet to approve any agent for a mass-casualty radiation disaster. Because patients undergoing hematopoietic stem cell transplantation undergo radiation treatment that produces toxicities similar to radiation-disaster exposure, we studied patients early after such treatment to identify new approaches to this problem. Patients rapidly developed endotoxemia and reduced plasma bactericidal/permeability-increasing protein (BPI), a potent endotoxin-neutralizing protein, in association with neutropenia. We hypothesized that a treatment supplying similar endotoxin-neutralizing activity might replace the BPI deficit and mitigate radiation toxicity and tested this idea in mice. A single 7-Gy radiation dose, which killed 95% of the mice by 30 days, was followed 24 hours later by twice-daily, subcutaneous injections of the recombinant BPI fragment rBPI21 or vehicle alone for 14 or 30 days, with or without an oral fluoroquinolone antibiotic with broad-spectrum antibacterial activity, including that against endotoxin-bearing Gram-negative bacteria. Compared to either fluoroquinolone alone or vehicle plus fluoroquinolone, the combined rBPI21 plus fluoroquinolone treatment improved survival, accelerated hematopoietic recovery, and promoted expansion of stem and progenitor cells. The observed efficacy of rBPI21 plus fluoroquinolone initiated 24 hours after lethal irradiation, combined with their established favorable bioactivity and safety profiles in critically ill humans, suggests the potential clinical use of this radiation mitigation strategy and supports its further evaluation.

Determinants of the Proinflammatory Action of Ambient Particulate Matter in Immortalized Murine Macrophages

BackgroundProximity to traffic-related pollution has been associated with poor respiratory health in adults and children.ObjectivesWe wished to test the hypothesis that particulate matter (PM) from high-traffic sites would display an enhanced capacity to elicit inflammation.MethodsWe examined the inflammatory potential of coarse [2.5–10 μm in aerodynamic diameter (PM2.5–10)] and fine [0.1–2.5 μm in aerodynamic diameter (PM0.1–2.5)] PM collected from nine sites throughout Europe with contrasting traffic contributions. We incubated murine monocytic-macrophagic RAW264.7 cells with PM samples from these sites (20 or 60 μg/cm2) and quantified their capacity to stimulate the release of arachidonic acid (AA) or the production of interleukin-6 and tumor necrosis factor-α (TNFα) as measures of their inflammatory potential. Responses were then related to PM composition: metals, hydrocarbons, anions/cations, and endotoxin content.ResultsInflammatory responses to ambient PM varied markedly on an equal mass basis, with PM2.5–10 displaying the largest signals and contrasts among sites. Notably, we found no evidence of enhanced inflammatory potential at high-traffic sites and observed some of the largest responses at sites distant from traffic. Correlation analyses indicated that much of the sample-to-sample contrast in the proinflammatory response was related to the content of endotoxin and transition metals (especially iron and copper) in PM2.5–10. Use of the metal chelator diethylene triamine pentaacetic acid inhibited AA release, whereas recombinant endotoxin-neutralizing protein partially inhibited TNFα production, demonstrating that different PM components triggered inflammatory responses through separate pathways.ConclusionsWe found no evidence that PM collected from sites in close proximity to traffic sources displayed enhanced proinflammatory activity in RAW264.7 cells.

Electronic tongue for simultaneous detection of endotoxins and other contaminants of microbiological origin

Endotoxins, also referred to as pyrogens, are lipopolysaccharides (LPS) present in the outer membrane of Gram-negative bacteria, and represent one of the most dangerous microbiological contaminants in water for hemodialysis and intravenous infusion. A method is presented for the simultaneous detection of endotoxins and other bacterial lysis contaminating species in purified water for parenteral formulations. The technique used is electrochemical impedance spectroscopy, with data interpretation using principal component analysis (PCA), cluster analysis (CA), and multivariate discriminant analysis (MDA). Two types of electrode surfaces were modified with LPS recognition agents: (i) a 37 amino acids fragment of a 18 kDa cationic antimicrobial protein (CAP18F) that has LPS binding activity; (ii) the highly selective endotoxin neutralizing protein (ENP). Statistical multivariate analysis of the impedance spectral data allowed the detection of endotoxin at, and below, the threshold pharmaceutical regulatory level. Discrimination of LPS from samples containing proteins, nucleic acids, phospholipids or their mixtures was achieved. These results open a new route to a practical instrumental method capable of detecting and discriminating LPS from other potential pro-inflammatory species of microbiological origin, such as nucleic acids.

Size-partitioning of an urban aerosol to identify particle determinants involved in the proinflammatory response induced in airway epithelial cells

BackgroundThe contribution of air particles in human cardio-respiratory diseases has been enlightened by several epidemiological studies. However the respective involvement of coarse, fine and ultrafine particles in health effects is still unclear. The aim of the present study is to determine which size fraction from a chemically characterized background aerosol has the most important short term biological effect and to decipher the determinants of such a behaviour.ResultsAmbient aerosols were collected at an urban background site in Paris using four 13-stage low pressure cascade impactors running in parallel (winter and summer 2005) in order to separate four size-classes (PM0.03–0.17 (defined here as ultrafine particles), PM0.17–1 (fine), PM1–2.5(intermediate) and PM2.5–10 (coarse)). Accordingly, their chemical composition and their pro-inflammatory potential on human airway epithelial cells were investigated. Considering isomass exposures (same particle concentrations for each size fractions) the pro-inflammatory response characterized by Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) release was found to decrease with aerosol size with no seasonal dependency. When cells were exposed to isovolume of particle suspensions in order to respect the particle proportions observed in ambient air, the GM-CSF release was maximal with the fine fraction. In presence of a recombinant endotoxin neutralizing protein, the GM-CSF release induced by particles is reduced for all size-fractions, with exception of the ultra-fine fraction which response is not modified. The different aerosol size-fractions were found to display important chemical differences related to the various contributing primary and secondary sources and aerosol age. The GM-CSF release was correlated to the organic component of the aerosols and especially its water soluble fraction. Finally, Cytochrome P450 1A1 activity that reflects PAH bioavailability varied as a function of the season: it was maximal for the fine fraction in winter and for the ultrafine fraction in summer.ConclusionIn the frame of future regulations, a particular attention should thus be paid to the ultrafine/fine (here referred to as PM1) fraction due to their overwhelming anthropogenic origin and predominance in the urban aerosol and their pro-inflammatory potential.

Early Deficiency of Endogenous Proteins Inhibiting LPS-Induced TNF-α Production Correlates with Acute Graft vs Host Disease (aGVHD) after Myeloablative Stem Cell Transplantation (SCT).

Background: SCT is an important treatment modality complicated by a number of immune-mediated toxicities, including aGVHD. In animal SCT models, intestinal translocation of endotoxin (LPS) induces production of TNF-α contributing to development of aGVHD. We tested the hypothesis that in human SCT, myeloablative chemoradiotherapy regimens contribute to the early development of both endotoxemia and a profound deficiency in host defense molecules that normally serve to inhibit endotoxin signaling. In aggregate, these effects may potentiate endotoxin-directed innate immunity and contribute to development of aGVHD and other regimen related toxicities.Methods: Peripheral blood was obtained from 30 patients (43% < 21 yrs, 60% male) at baseline prior to the start of conditioning, Days 0 (Day of SCT), 7, 14, 21, and 28 post-SCT). A single baseline sample was obtained from each sibling SCT donor. Plasma was analyzed for endotoxin (LPS) and endotoxin-interactive proteins bactericidal/permeability-increasing protein (BPI, a potent endotoxin-neutralizing protein of human neutrophils), the acute phase reactants LPS-binding protein (LBP) and soluble CD14 (sCD14), as well as soluble MD-2 (sMD-2). In addition, plasma concentrations of Regulated upon Activation Normal T Cell Expressed and Secreted (RANTES) and monocyte chemoattractant protein-1 (MCP-1), both chemokines that inhibit Toll-like receptor-4 (TLR4)-mediated TNF and IL-6 production, were determined. Monocyte surface expression of LPS-receptor components membrane CD14 (mCD14) and TLR4 was measured by flow cytometry.Findings: Nearly 90% of patients had endotoxemia at some time point. Maximal fever incidence and nadir ANC were observed D7 after these myeloablative SCT. Endotoxemia was accompanied by a profound deficiency in plasma BPI concentrations which fell >10-fold from baseline to D7 (p < 0.0001) where it was undetectable in 20/30 patients. RANTES levels were also very reduced at D7 (p < 0.0001). At Day 0, monocyte surface expression of mCD14 fell and TLR4 increased (p < 0.05), a pattern consistent with exposure of monocytes to endotoxin early after conditioning. Subsequently, levels of endotoxin-modulating proteins LBP (D7–14) and sCD14 (D7, 21, and 28) increased (p < 0.05). Low plasma concentrations of the endotoxin-neutralizing protein BPI (baseline) and of RANTES (donor, baseline, and D0) significantly correlated with subsequent risk of aGVHD (p < 0.05).Interpretation: During myeloablative SCT, endotoxemia occurs in the context of deficient circulating levels of BPI and RANTES. These deficiencies likely contribute to greater endotoxin-TLR4 signalling and resulting production of TNF-α (and other cytokines) and may therefore contribute to consequent aGVHD. Therefore, therapies aimed at enhancing endotoxin-neutralization, such as replenishment of deficient endotoxin antagonists, may be effective approaches to reduce toxicities associated with myeloablative HSCT, including aGVHD. We have therefore initiated a multi-institutional clinical trial to explore this hypothesis by administering recombinant BPI (opebecan, XOMA (US) LLC) to patients undergoing myeloablative SCT.

Capacitive biosensor for detection of endotoxin

A capacitive biosensor for the detection of bacterial endotoxin has been developed. Endotoxin-neutralizing protein derived from American horseshoe crab was immobilized to a self-assembled thiol layer on a biosensor transducer (Au). Upon injection of a sample containing endotoxin, a decrease in the observed capacitive signal was registered. Endotoxin could be determined under optimum conditions with a detection limit of 1.0 x 10(-13) M and linearity ranging from 1.0 x 10(-13) to 1.0 x 10(-10) M. Good agreement was achieved when applying endotoxin preparations purified from an Escherichia coli cultivation to the capacitive biosensor system, utilizing the conventional method for quantitative endotoxin determination, the Limulus amebocyte lysate test as a reference. The capacitive biosensor method was statistically tested with the Wilcoxon signed rank test, which proved the system is acceptable for the quantitative analysis of bacterial endotoxin (P<0.05).

Endotoxin detection in a competitive electrochemical assay: Synthesis of a suitable endotoxin conjugate

A biotin–lipopolysaccharide (biotin–LPS) conjugate was synthesized from LPS smooth from Salmonella minnesota, yielding a conjugate with a biotin/LPS ratio equal to 1:1 and endotoxic activity of 0.08 EU ng −1. The conjugate was used in an amperometric competitive assay to determine endotoxins with endotoxin-neutralizing protein (ENP) as the recognition element. The assay is performed on a modified electrode, involving the covalent binding of carboxymethyl dextran (CMDex) to a cystamine-modified gold electrode and then the covalent binding of the recognition protein, ENP, to CMDex. The assay is carried out by incubating the modified electrode in an LPS sample to which biotin–LPS was added. Both species compete for the recognition sites on the modified surface. After the incubation stage and a careful rinsing, the electrode is immersed in a solution containing neutravidin–horseradish peroxidase conjugate (N-HRP), which binds to the sites containing biotin–LPS on the electrode. The system is rinsed and a current signal is generated by the addition of hydrogen peroxide and a redox mediator. The assay is able to detect LPS from Salmonella minnesota at concentrations as low as 0.1 ng ml −1, equivalent to 0.07 EU ml −1.

Bacterial endotoxin as inhibitor of the enzymatic activity of human thrombin

Endotoxemia caused by bacterial lipopolysaccharides (LPS) deleteriously affects many aspects of hemostasis. Much of this effect is well characterized as being secondary to the LPS-mediated inflammatory response, but direct effects of LPS on coagulation factors may also contribute to disregulation of the hemostatic process. Spectrophotometric assays were used to investigate the effects of LPS from different bacteria on thrombin and plasmin activities. We found that enzymatic activity of purified thrombin, but not plasmin, decreases in the presence of endotoxin. LPS-mediated inhibition of thrombin activity can be reversed by plasma gelsolin and recombinant endotoxin-neutralizing protein. Preincubation of thrombin with LPS before platelet activation results in inhibition of aggregation and secretion. Additionally, a decrease of elastic shear moduli of fibrin gels was observed when their formation was induced with thrombin preincubated with LPS or when LPS was present in fibrinogen solutions during fibrin gel formation. When added to platelet-rich plasma, after activation with collagen, LPS-inhibited thrombin activity. LPS-mediated inhibition of thrombin activity may contribute to the hemostasis dysfunctions observed during endotoxemia.

Use of an Antimicrobial Protein for Endotoxin Detection in a Competitive Electrochemical Assay

Endotoxins, also referred to as pyrogens, are part of the cellular walls of Gram-negative bacteria and are capable of inducing fever when entering into the blood stream. Current methods of detection involve the use of either amoebocytes from horseshoe crab that clots upon exposure to endotoxin or a live assay on rabbits. In this work, the detection of endotoxins by an electrochemical competitive assay is presented. Two configurations of modified electrodes were constructed using a recombinant endotoxin neutralizing protein (ENP) as recognition element. A modified lipopolysaccharide with horseradish peroxidase was used for the competitive assay. Modified electrodes constructed by electrostatic interaction of ENP and an electroactive polymer can detect the presence of endotoxins in concentrations as low as 0.2EU mL(-1), below the limit imposed for water in injectable drugs in the American Pharmacopoeia. Modified electrodes constructed by covalent linking of ENP to a carboxymethyl dextran matrix bound to the electrode show a better dynamic range but a higher detection limit.

Biophysical characterization of the interaction of Limulus polyphemus endotoxin neutralizing protein with lipopolysaccharide.

Endotoxin-neutralizing protein (ENP) of the horseshoe crab is one of the most potent neutralizers of endotoxins [bacterial lipopolysaccharide (LPS)]. Here, we report on the interaction of LPS with recombinant ENP using a variety of physical and biological techniques. In biological assays (Limulus amebocyte lysate and tumour necrosis factor-alpha induction in human mononuclear cells), ENP causes a strong reduction of the immunostimulatory ability of LPS in a dose-dependent manner. Concomitantly, the accessible negative surface charges of LPS and lipid A (zeta potential) are neutralized and even converted into positive values. The gel to liquid crystalline phase transitions of LPS and lipid A shift to higher temperatures indicative of a rigidification of the acyl chains, however, the only slight enhancement of the transition enthalpy indicates that the hydrophobic moiety is not strongly disturbed. The aggregate structure of lipid A is converted from a cubic into a multilamellar phase upon ENP binding, whereas the secondary structure of ENP does not change due to the interaction with LPS. ENP contains a hydrophobic binding site to which the dye 1-anilino-8-sulfonic acid binds at a K(d) of 19 micro m, which is displaced by LPS. Because lipopolysaccharide-binding protein (LBP) is not able to bind to LPS when ENP and LPS are preincubated, tight binding of ENP to LPS can be deduced with a K(d) in the low nonomolar range. Importantly, ENP is able to incorporate by itself into target phospholipid liposomes, and is also able to mediate the intercalation of LPS into the liposomes thus acting as a transport protein in a manner similar to LBP. Thus, LPS-ENP complexes might enter target membranes of immunocompetent cells, but are not able to activate due to the ability of ENP to change LPS aggregates from an active into an inactive form.

A Critical Involvement of Oxidative Stress in Acute Alcohol-Induced Hepatic TNF-α Production

Tumor necrosis factor-alpha (TNF-alpha) production is a critical factor in the pathogenesis of alcoholic liver injury. Both oxidative stress and endotoxin have been implicated in the process of alcohol-induced TNF-alpha production. However, a cause-and-effect relationship between these factors has not been fully defined. The present study was undertaken to determine the mediators of acute alcohol-induced TNF-alpha production using a mouse model of acute alcohol hepatotoxicity. Alcohol administration via gavage at a dose of 6 g/kg to 129/Sv mice induced hepatic TNF-alpha production in Kupffer cells as demonstrated by measuring protein levels, immunohistochemical localization, and mRNA expression. Alcohol intoxication caused liver injury in association with increases in plasma endotoxin and hepatic lipid peroxidation. Treatment with an endotoxin neutralizing protein significantly suppressed alcohol-induced elevation of plasma endotoxin, hepatic lipid peroxidation, and inhibited TNF-alpha production. Treatment with antioxidants, N-ACETYL-L-CYSTEINE, or dimethylsulfoxide, failed to attenuate plasma endotoxin elevation, but significantly inhibited alcohol-induced hepatic lipid peroxidation, TNF-alpha production and steatosis. All treatments prevented alcohol-induced necrotic cell death in the liver. This study thus systemically dissected the relationship among plasma endotoxin elevation, hepatic oxidative stress, and TNF-alpha production following acute alcohol administration, and the results demonstrate that oxidative stress mediates endotoxin-induced hepatic TNF-alpha production in acute alcohol intoxication.

Bactericidal/permeability-increasing protein is expressed by human dermal fibroblasts and upregulated by interleukin 4.

The bactericidal/permeability-increasing protein (BPI) is an antibiotic- and endotoxin-neutralizing protein of granulocytes and epithelial cells. Constitutive expression of BPI, which increases upon interleukin 4 stimulation, by human dermal fibroblast was demonstrated, suggesting an important role of BPI in gram-negative bacterial clearance and a dampened response to endotoxin in the skin.

Proinflammatory and cytotoxic effects of Mexico City air pollution particulate matter in vitro are dependent on particle size and composition.

Exposure to urban airborne particulate matter (PM) is associated with adverse health effects. We previously reported that the cytotoxic and proinflammatory effects of Mexico City PM10 (less than or equal to 10 micro m mean aerodynamic diameter) are determined by transition metals and endotoxins associated with these particles. However, PM2.5 (less than or equal to 2.5 micro m mean aerodynamic diameter) could be more important as a human health risk because this smaller PM has the potential to reach the distal lung after inhalation. In this study, we compared the cytotoxic and proinflammatory effects of Mexico City PM10 with those of PM2.5 using the murine monocytic J774A.1 cell line in vitro. PMs were collected from the northern zone or the southeastern zone of Mexico City. Elemental composition and bacterial endotoxin on PMs were measured. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) production by J774A.1 cells was measured in the presence or absence of recombinant endotoxin-neutralizing protein (rENP). Both northern and southeastern PMs contained endotoxin and a variety of transition metals. Southeastern PM10 contained the highest endotoxin levels, 2-fold higher than that in northern PM10. Northern and southeastern PM2.5 contained the lowest endotoxin levels. Accordingly, southeastern PM10 was the most potent in causing secretion of the proinflammatory cytokines TNF-alpha and IL-6. All PM2.5 and PM10 samples caused cytotoxicity, but northern PMs were the most toxic. Cytokine secretion induced by southeastern PM10 was reduced 50-75% by rENP. These results indicate major differences in PM10 and PM2.5. PM2.5 induces cytotoxicity in vitro through an endotoxin-independent mechanism that is likely mediated by transition metals. In contrast, PM10 with relatively high levels of endotoxin induces proinflammatory cytokine release via an endotoxin-dependent mechanism.

Reactions of macrophages exposed to particles

This study describes experiments on cytotoxic effects and the production of oxidative radicals and the proinflammatory cytokine tumor growth factor alpha (TNF α) in a cell line of rat lung macrophages exposed to aqueous extracts from ambient air particles <10 μm (PM 10) collected on Teflon filters. The particles were collected during the four seasons at two urban sites, one rural site, and one alpine site in Switzerland. Cytotoxic effects, determined as a reduction in the metabolic activity, were found in particle extracts from all sites and seasons. Taking together the data from all sites and seasons, a dose–response function was observed between the particle mass on the filter and toxicity ( r 2=0.633, linear regression). The release of the pro-inflammatory cytokine TNFα as well as of oxidative radicals was most pronounced in particles collected in spring–summer and autumn. While at Montana (alpine), the stimulation of the cells was positively correlated with the particle mass on the filters, this correlation was negative at the urban sites Zürich and Lugano. It is interpreted that at high PM 10 levels, as in these cities, macrophages are inhibited by increasing air pollution due to toxic effects. Cytotoxic effects and the release of oxidative radicals could be inhibited when the extracts were treated with an endotoxin-neutralizing protein. This suggests that endotoxin, a cell-wall constituent of gram-negative bacteria, is one of the factors which modulates macrophage activity. All together, the experiments indicate that in the PM 10 fraction, water-soluble macrophage-toxic and macrophage-stimulating compounds are present. The data offer an explanation for at least some of the known harmful effects of PM 10, and confirm endotoxin as a possible reactant.

Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification

Production of recombinant Limulus endotoxin neutralizing protein (rENP) was attained with the GS115 methylotrophic strain of Pichia pastoris transformed with a plasmid, bearing multiple ENP gene copies. The synthetic gene for Limulus ENP was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter. Clones containing a single enp insert were used to construct cassettes bearing 2 and 3 tandem copies of enp. These were then integrated at the HIS locus of P. pastoris GS115 ( his4). Clones were chosen for their ability to produce rENP upon methanol induction in shaker flasks, and then the 1x, 2x, and 3x- enp strains were analyzed by Southern blot for the presence of the ENP gene(s). Isolate 3x5q, containing a 3x- enp cassette, was the best producer of rENP. Under optimal conditions this strain grown in a fed-batch mode produced yields of >500 mg rENP/L with an average of 5.46 mg rENP/g DCW. Purification of rENP from the clarified broth resulted in a yield of 35% and a purity of >86%. Glycosylated rENP, the main contaminant, was removed with a concanavalin-A column and characterized. The pure rENP neutralized lipopolysaccharide and had the mass, amino-acid composition and N-terminal sequence expected from the cloned gene.

Involvement of lipopolysaccharide binding protein, CD14, and Toll-like receptors in the initiation of innate immune responses by Treponema glycolipids.

Culture supernatants from Treponema maltophilum associated with periodontitis in humans and Treponema brennaborense found in a bovine cattle disease accompanied with cachexia caused a dose-dependent TNF-alpha synthesis in human monocytes increasing with culture time. This activity could be reduced significantly by blocking the CD14-part of the LPS receptor using the My 4 mAb and by polymyxin B. In the murine macrophage cell line RAW 264.7, Treponema culture supernatants induced TNF-alpha secretion in a LPS binding protein (LBP)-dependent fashion. To enrich for active compounds, supernatants were extracted with butanol, while whole cells were extracted using a phenol/water method resulting in recovery of material exhibiting a similar activity profile. An LPS-LBP binding competition assay revealed an interaction of the treponeme phenol/water extracts with LBP, while precipitation studies implied an affinity to polymyxin B and endotoxin neutralizing protein. Macrophages obtained from C3H/HeJ mice carrying a Toll-like receptor (TLR)-4 mutation were stimulated with treponeme extracts for NO release to assess the role of TLRs in cell activation. Furthermore, NF-kappaB translocation in TLR-2-negative Chinese hamster ovary (CHO) cells was studied. We found that phenol/water-extracts of the two strains use TLRs differently with T. brennaborense-stimulating cells in a TLR-4-dependent fashion, while T. maltophilum-mediated activation apparently involved TLR-2. These results indicate the presence of a novel class of glycolipids in Treponema initiating inflammatory responses involving LBP, CD14, and TLRs.

Reversible binding of heparin to the loop peptide of endotoxin neutralizing protein.

Endotoxin neutralizing protein (ENP) from Limulus polyphemus is an amphipathic, 11.8 kDa protein with an isoelectric point of 10.2. ENP neutralizes lipopolysaccharide (LPS) and possesses antibacterial activity against Gram-negative bacteria. Heparin binds to ENP and blocks its LPS-neutralizing activity. The relative blocking activity of heparin is equal to low molecular weight heparin and polyanetholsulfonic acid > heparan sulfate > chondroitin sulfate A > chondroitin sulfate C. Endoproteinase Glu-C hydrolysis of recombinant ENP results in four major peptides, three of which are seen following separation on reversed phase HPLC. Heparin binds to the loop peptide (31-72), which includes the heparin binding consensus sequence XBBXBX between the two cysteine residues of ENP. When heparin is added to the digest and then applied to a C18 column, the loop peptide is bound; however, it dissociates and elutes with either 5 M NaCl or 0.1 M sodium phosphate, demonstrating reversible binding to heparin. LPS and lipid A both bind to the loop peptide and remove it from digests of ENP; however, neither complex could be dissociated by salt or sodium phosphate. Heparin, LPS, and lipid A individually bind to the same site on ENP.

Induction of the lung myofibroblast PDGF receptor system by urban ambient particles from Mexico City.

Platelet-derived growth factor (PDGF) and its receptor system regulate mesenchymal cell proliferation. We recently reported that emission-source fly-ash particles and asbestos fibers induce the PDGF alpha-receptor through a macrophage-dependent pathway, and upregulation of this receptor greatly enhances the mitogenic response of lung myofibroblasts to PDGF (Lindroos and colleagues, Am. J. Respir. Cell Mol. Biol. 1997;16:283-292). In the present study we investigated the effect of particulate matter <= 10 micrometers in size (PM10) from the southern, central, and northern regions of Mexico City on PDGF receptor induction and compared these urban, ambient particles with Mt. St. Helen's volcanic ash particles as a negative control. All Mexico City PM10 samples, but not volcanic ash, stimulated rat alveolar macrophages to secrete a soluble, upregulatory factor(s) for the PDGF alpha-receptor on early passage rat lung myofibroblasts. The macrophage-derived upregulatory activity was blocked by the interleukin (IL)-1 receptor antagonist. The ability of PM10 to stimulate IL-1beta release was blocked in part by a recombinant endotoxin neutralizing protein (rENP). Lipopolysaccharide/endotoxin (LPS) and vanadium, both constituents that were present within these PM10 samples, also stimulated macrophages to secrete factor(s) that upregulated PDGF-Ralpha on lung myofibroblasts. Direct exposure of myofibroblasts to PM10 also elicited upregulation of the PDGF alpha-receptor, and this effect was blocked by rENP and mimicked by LPS, but not vanadium. These findings suggest that PM10 particles induce expression of the PDGF receptor system through macrophage-dependent and -independent mechanisms involving endotoxin and metals.

Endotoxin-neutralizing protein protects against endotoxin-induced endothelial barrier dysfunction.

Bacterial lipopolysaccharide induces tyrosine phosphorylation of paxillin, actin reorganization, and opening of the transendothelial paracellular pathway through which macromoles flux. In this study, lipid A was shown to be the bioactive portion of the lipopolysaccharide molecule responsible for changes in endothelial barrier function. We then studied whether endotoxin-neutralizing protein, a recombinant peptide that is derived from Limulus antilipopolysaccharide factor and targets lipid A, could block the effects of lipopolysaccharide on protein tyrosine phosphorylation, actin organization, and movement of 14C-bovine serum albumin across bovine pulmonary artery endothelial cell monolayers. In the presence of serum, a 6-h exposure to lipopolysaccharide (10 ng/ml) increased transendothelial 14C-albumin flux compared to the simultaneous media control. Coadministration of endotoxin-neutralizing protein (> or =10 ng/ml) with lipopolysaccharide (10 ng/ml) protected against lipopolysaccharide-induced barrier dysfunction. This protection was dose dependent, conferring total protection at endotoxin-neutralizing protein/lipopolysaccharide ratios of > or =10:1. Similarly, endotoxin-neutralizing protein was capable of blocking the lipopolysaccharide-induced endothelial cell responses that are prerequisite to barrier dysfunction, including tyrosine phosphorylation of paxillin and actin depolymerization. Finally, endotoxin-neutralizing protein cross-protected against lipopolysaccharide derived from diverse gram-negative bacteria. Thus, endotoxin-neutralizing protein offers a novel therapeutic intervention for the vascular endothelial dysfunction of gram-negative sepsis and its attendant endotoxemia.

5594113 Endotoxin binding and neutralizing protein and uses thereof : Wainwright Norman R; Novitsky Thomas Falmouth, MA, United States Assigned to Associates of Cape Cod Inc

5594113 Endotoxin binding and neutralizing protein and uses thereof : Wainwright Norman R; Novitsky Thomas Falmouth, MA, United States Assigned to Associates of Cape Cod Inc