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CD14, a glycosylphosphatidylinositol-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, is a receptor for lipopolysaccharide (LPS). CD14 binding of LPS is enhanced by serum proteins, especially lipopolysaccharide binding protein. The serum-dependent binding of LPS to CD14 stimulates macrophages to make cytokines, which can cause septic shock in humans and animals. Here, we identify a region in human CD14 which is important in serum-dependent LPS binding and LPS-induced cellular activation. Four small regions (4-5 amino acids long) within the N-terminal 65 amino acids of CD14 were deleted singly or in combination. The deletion mutants were stably expressed in Chinese hamster ovary (CHO) cells. The mutants were characterized in three assays: reactivity with anti-CD14 monoclonal antibody, serum-dependent LPS binding, and LPS-induced activation of NF-kappa B. Some of the mutants selectively lost reactivity with the anti-CD14 monoclonal antibody that inhibited serum-dependent LPS binding and cellular activation. All of the mutants bound much less LPS than wild type CD14 in the presence of serum. None of the mutants bound more LPS than control CD14-CHO cells in the absence of serum. CD14-CHO cells respond to LPS by activation of NF-kappa B. All of the deletion mutants were less active LPS receptors than wild type CD14-CHO cells. The delta AVEVE mutant, the delta DDED and delta PQPD double mutant, and the delta DDED, delta PQPD, delta AVEVE, and delta DPRQY quadruple deletion mutants were essentially inactive LPS receptors in CHO cells. These studies suggest that the 65 N-terminal amino acids of CD14 are critical for serum-dependent binding of LPS to CD14 and subsequent signal transduction in CHO cells.

Outer membrane protein F, a major component of the Escherichia coli outer membrane, was crystallized for the first time in lipidic mesophase of monoolein in novel space groups, P1 and H32. Due to ease of its purification and crystallization OmpF can be used as a benchmark protein for establishing membrane protein crystallization in meso, as a “membrane lyzozyme”. The packing of porin trimers in the crystals of space group H32 is similar to natural outer membranes, providing the first high-resolution insight into the close to native packing of OmpF. Surprisingly, interaction between trimers is mediated exclusively by lipids, without direct protein–protein contacts. Multiple ordered lipids are observed and many of them occupy identical positions independently of the space group, identifying preferential interaction sites of lipid acyl chains. Presence of ordered aliphatic chains close to a positively charged area on the porin surface suggests a position for a lipopolysaccharide binding site on the surface of the major E. coli porins.

Antimicrobial cationic peptides have been discovered in many different organisms and often possess a broad range of activity. In this study, we investigated the mechanisms of actions of melittin and two synthetic peptides, CEME (a cecropin-melittin hybrid) and CEMA, against gram-negative bacteria. CEMA was produced by recombinant DNA procedures and is an analog of CEME with a modified C terminus resulting in two additional positive charges. All three peptides showed good antimicrobial activity against four different gram-negative bacteria, but only CEMA was able to somewhat augment the activity of some conventional antibiotics in synergy studies. Studies using the bacteria Pseudomonas aeruginosa and Enterobacter cloacae showed that the peptides all possessed the ability to permeabilize bacterial outer membranes to the hydrophobic fluorophor 1-N-phenylnaphthylamine and the protein lysozyme, with CEMA being the most active. CEMA also had the strongest relative binding affinity for bacterial endotoxin (lipopolysaccharide). These data collectively indicated that these peptides all cross the outer membrane by the self-promoted uptake pathway and that CEMA is the peptide most effective at accessing this pathway.

A method is described in these investigations for the semi-synthetic production of polymyxin-B conjugated ovalbumin in the form of polymyxin-B.Sulfo-SMCC.ovalbumin (PSO). The heterobifunctional "cross-linking" agent, Sulfo-SMCC was first reacted with polymyxin-B to produce a relatively pure reactive intermediate in the form of polymyxin-B.Sulfo-SMCC. Highly purified ovalbumin was then combined with the polymyxin-B.Sulfo-SMCC reactive intermediate and contaminants removed from the final PSO end product by exhaustive microdialysis. Purity of PSO was established with by high-performance cellulose acetate electrophoresis (HPCAE), and high-performance thin layer chromatography (HPTLC) analyses. Verification of polymyxin-B.Sulfo-SMCC.ovalbumin binding avidity for lipopolysaccharide (LPS) was determined by DotBLot analysis applying fluorescein isothiocyanate labeled E. coli (055:B5) LPS fractions (FITC-LPS). Efficacy of PSO to inhibit in vitro LPS-induced synthesis of tumor necrosis factor-alpha (TNF-alpha) was assessed with a tissue culture based biological assay system. In this context, semi-synthetic conjugates of PSO (0.349 microgram/ml) effectively inhibited Salmonella minnesota (RS) LPS (2.5 ng/ml well) induced TNF-alpha synthesis and corresponding cytoprotection (100%) to WEHI 164 clone 13 cell populations.

Mononuclear phagocytes in distinct differentiation stages and cultured under different conditions were tested for their sensitivity towards lipopolysaccharide (LPS), using procoagulant activity (PCA) expression and tumor necrosis factor (TNF) production as indices. The response of mature monocyte-derived macrophages differed from that of freshly isolated monocytes 1) by higher levels of constitutive PCA, 2) by responding to approximately 1,000-fold lower concentrations of LPS with PCA and TNF production, and 3) by a faster rise in PCA and TNF production. Due to the high constitutive level of PCA expression, the PCA stimulation index for LPS was low in macrophages when compared with that in monocytes. Thus, during differentiation to macrophages, human monocytes acquire increased sensitivity to LPS (2 orders of magnitude more sensitive than a sensitive turbidimetric Limulus amoebocyte lysate assay). This exquisite sensitivity to LPS is expressed regardless of whether LPS is offered in the presence or absence of lipopolysaccharide binding protein-containing serum. This points to as yet uncharacterized pathways of high affinity interaction between LPS and macrophages.

We have previously established that lipopolysaccharide (LPS) induces the expression of new specific LPS-binding sites (LpsR) in mouse bone marrow cells (BMC). We now show that exposure of human BMC to LPS elicits the production of both CD14 molecules (detectable with monoclonal antibody My4) and LpsR (detectable with fluorescein isothiocyanate-LPS). Pretreatment of stimulated human BMC with My4 inhibited the binding of fluorescein isothiocyanate-LPS. The stimulation of human BMC, but not mouse BMC, required the presence of serum. Other characteristics of mouse and human BMC examined were very similar. Their inducible LpsR interacted with the lipid moieties of LPS and Leishmania donovani lipophosphoglycan and with a soluble preparation of peptidoglycan. Moreover, mouse and human LpsR were susceptible to treatment with a phosphatidylinositol-specific phospholipase C (PI-PLC), thus suggesting that both are PI-anchored CD14 molecules. Neither LpsR appeared able to interact with a synthetic LPS antagonist (compound PPDm2) structurally related to the lipid region of LPS. However, PPDm2 blocked LPS-induced expression of LpsR in both BMC. Furthermore, in both species, pretreatment of BMC with PI-PLC did not prevent the cells from expressing LpsR in response to LPS. The results support the hypothesis that the elicited LpsR of mouse and human BMC is an inducible form of CD14, whereas the putative "signaling LPS receptor" of these cells is not CD14 or any other PI-anchored molecule.

The 23-kDa recombinant amino-terminal bactericidal/permeability increasing protein fragment (rBPI23) has all of the antibacterial and antiendotoxin properties of the holoprotein. In the current studies, we have identified multiple active domains within rBPI23 with chemical and proteolytic cleavage fragments and with synthetic overlapping peptides. We also demonstrate a novel, high affinity heparin binding property for rBPI23, in addition to its established bactericidal and lipopolysaccharide binding properties. Cleavage fragments and synthetic, overlapping peptides of rBPI23 were analyzed for inhibition of the lipopolysaccharide-induced Limulus amebocyte lysate reaction, for bactericidal activity, and for heparin binding. Three separate, active domains were identified in amino acid regions 17-45, 65-99, and 142-169. A single synthetic peptide (85-99) was bactericidal. These results indicate that rBPI23 is comprised of three separate functional domains which contribute to the high affinity interaction of rBPI23 with Gram-negative bacteria. The individual activity of each domain and the cooperative interaction among domains provide the basis for developing rBPI23 analogues with increased biologic efficacy.

C-type lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-type lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-type lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-type lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of lectin.

The cell surface protein CD14 binds bacterial lipopolysaccharide (LPS) in the presence of the serum protein, LPS-binding protein (LBP). This interaction is important for LPS-induced activation of mammalian myeloid cells. We performed quantitative studies of 3H-labeled LPS binding to human CD14 expressed on Chinese hamster ovary cells and on a human macrophage cell line (THP-1). At the concentrations studied (20-100 nM) LPS binding required the expression of CD14 and could be inhibited by a subset of anti-CD14 monoclonal antibodies. LBP was required for LPS binding to CD14. The binding occurred within 10 min and was relatively unaffected by temperature over the range of 4-37 degrees C. Quantitative binding assays were performed at 10 degrees C, or at 37 degrees C, using Chinese hamster ovary cells depleted of ATP. In both cases, 75-90% of the LPS could be released by treatment with phosphatidylinositol-specific phospholipase C, suggesting that it remains associated with the glycosyl phosphatidylinositol-anchored CD14. The apparent dissociation constant of recombinant human CD14 expressed on Chinese hamster ovary cells for LPS at 10 degrees C was 2.74 (+/- 0.99) x 10(-8) M; the apparent dissociation constant of CD14 expressed on THP-1 cells at 10 degrees C was 4.89 (+/- 1.42) x 10(-8) M. In both cell lines, at saturating LPS concentrations, the molar ratio of LPS bound per surface CD14 was approximately 20:1. At 37 degrees C the apparent dissociation constant of recombinant human CD14 for LPS at 37 degrees C was 2.7 (+/- 1.2) x 10(-8) M, and the molar ratio of LPS bound per surface CD14 was approximately 8:1. Although the difference in molar ratio of LPS bound per surface CD14 at the two temperatures is difficult to interpret, it is clear that at both temperatures the molar ratio is not 1:1. The basis of this phenomenon is unclear, but may involve the repeated leucine-rich motifs, which are found within CD14.

Bacterial endotoxin (LPS) can induce a tolerance to its own effects in vitro, in macrophages. To achieve a better understanding of the mechanism of this type of desensitization, we studied the regulation of the production of interleukin 1, interleukin 6, and tumor necrosis factor alpha by mouse peritoneal macrophages in response to LPS, after a preliminary exposure to LPS or to structurally related synthetic lipids. The concomitant down-regulation of LPS-binding sites was also analyzed. Complete desensitization of macrophages by LPS treatment was accompanied by a decrease of up to 70% in the number of LPS-binding sites. Preexposure of the cells to synthetic lipids also could induce down-regulation of LPS-binding sites and desensitization for cytokine production. However, there was no correlation between the structures that induce desensitization and those that elicit a down-regulation of LPS receptors. Furthermore, some synthetic compounds induced in the cells a restricted state of tolerance, characterized by a lack of secretion of only one cytokine in response to LPS. The results show that down-regulation of LPS receptors is not a prerequisite for the induction of LPS tolerance, and that different LPS substructures can down-regulate differently the various responses of the cells to LPS, thus suggesting that different pathways are operative for induction of endotoxin tolerance.

CAP37 (cationic antimicrobial protein of molecular mass 37 kDa) is a multifunctional protein isolated from the granules of human neutrophils. It is antibiotic and chemotactic and binds lipopolysaccharide. A synthetic peptide, amino acid sequence NQGRHFCGGALIHARFVMTAASCFQ, based on residues 20-44 of CAP37 protein mimics its antibiotic and lipopolysaccharide binding action. Peptide 20-44, at the concentrations tested, has antibacterial activity against Salmonella typhimurium, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. The bactericidal action of the peptide was pH dependent, with maximum activity at pH 5.0 and pH 5.5 and decreased activity at pH 7.0. Various truncations, substitutions, and other modifications in the sequence deteriorate its activity. Free sulfhydryl groups and/or disulfide bridge formation are required for optimum antibiotic activity, since substitution of serines for, or alkylation of, cysteine residues 26 and 42 eliminates bactericidal activity. Evidently amino acids 20-44 represent an important, perhaps principal, antibacterial domain of CAP37. This peptide should provide new insight into the mechanism of antimicrobial activity of CAP37 and may serve as a model for new, useful, synthetic antibiotics.

Limulus amebocyte lysate, obtained from horseshoe crab (Limulus polyphemus) blood cells, contains a coagulation system which is activated by bacterial lipopolysaccharide (LPS). A chromatographic fraction of Limulus lysate, containing the endotoxin-sensitive factor(s) which initiates the coagulation cascade, was studied. We utilized a photoreactive, cleavable, radiolabeled derivative of Salmonella minnesota LPS, LPS-(p-azidosalicylamido)-1,3'-dithiopropionamide (LPS-ASD), to identify LPS-binding proteins. The lysate fraction was incubated with LPS-ASD, and LPS-binding proteins were identified by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. An 82-kDa protein, a major protein component of this fraction from Limulus lysate, was identified as a LPS-binding protein in a majority of lysates. Incubation of whole Limulus lysate with antiserum to this protein resulted in enhanced sensitivity of the lysate to LPS, suggesting that this 82-kDa protein is a negative regulator of coagulation. A minor 50-kDa protein component of lysate also was identified as a LPS-binding protein and is a candidate for the LPS-sensitive coagulation protein in L. polyphemus.

1. 1. Haemolymph from the brown shrimp (Penaeus californiensis Holmes) agglutinates erythrocytes of different vertebrate species. 2. 2. A fraction containing haemagglutinating activity was isolated by affinity chromatography. The corresponding protein showed a molecular weight of 175 kDa, formed by four subunits. 3. 3. The haemagglutinin is inhibited by GalNAc, NANA, fetuin and bacterial lipopolysaccharide. 4. 4. Here we demonstrate the ability of this agglutinin to react with different Vibrio strains, and the inhibitory capacity of LPS in this reaction.

Bactericidal/permeability-increasing protein (BPI) is a natural constituent of human neutrophils. Recombinant BPI has been shown to bind to bacterial lipopolysaccharide (LPS), and to neutralize the ability of LPS to stimulate inflammatory cells in vitro and in vivo. BPI shares sequence homology and immunocrossreactivity with another endogenous LPS binding protein, lipopolysaccharide binding protein (LBP). Despite the homology, these proteins have opposite effects on LPS. LBP mediates cell activation by low, otherwise nonstimulatory concentrations, while BPI neutralizes LPS bioactivity. Exogenous LPS binding proteins in the form of monoclonal antibodies have been developed with the goal of generating antiendotoxin therapeutics to treat gram-negative sepsis and related syndromes. Here we show that LPS-binding and neutralizing properties of BPI compare favorably with two monoclonal antibodies tested, HA-1A and XMMEN-OE5. BPI also competes effectively with LBP for LPS. Thus, BPI may represent an endogenous LPS-regulatory molecule suitable for use as a potent antiendotoxin therapeutic.

We determined toll-like receptor expression in normal human urothelium and functional responses in normal human urothelial cell cultures to bacterial lipopolysaccharide via toll-like receptor-4 and to flagellin via toll-like receptor-5.Toll-like receptor protein expression was examined immunohistochemically. Toll-like receptor transcript expression was determined in freshly isolated urothelium, and in proliferating and differentiated normal human urothelial cultured cells. Lipopolysaccharide binding was assessed by flow cytometry. Functional responses of proliferating and differentiated normal human urothelial cells to lipopolysaccharide and flagellin were determined by interleukin-6 and 8 secretion, and transcription factor activation. Polymyxin B and siRNA were used to confirm the specificity of toll-like receptor-4 and 5 responses, respectively. Western blot detection of phosphorylated IκB was used to confirm toll-like receptor-4 results.Human urothelium expressed transcripts for toll-like receptor-4 and 5. Although bladder cancer derived T24 cells responded to lipopolysaccharide, there was no lipopolysaccharide binding to normal human urothelial cells and no functional response of proliferative or differentiated normal human urothelial cells even in the presence of exogenous CD14 and MD-2 accessory proteins. In contrast, flagellin evoked a toll-like receptor-5 mediated response in proliferating but not in differentiated normal human urothelial cells, which was abrogated by toll-like receptor-5 specific siRNA.Results suggest that human urothelium may mediate a host response to uropathogenic Escherichia coli through the detection of flagellin. The absent constitutive toll-like receptor-4 response may reflect an adaptation of urothelium toward sustaining barrier function and limiting inflammation to soluble bacterial products.

Fluorescence assays employing semi-synthetic or commercial dansyl-polymyxin B, have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary γ-amines on diaminobutyric-acid residues of polymyxin B are potentially derivatized with dansyl-cholride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra- dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]1polymyxinB3. The affinity of polymyxin for purified Gram-negative LPS, and whole bacterial cells was investigated. The affinity of dansyl[Lys]1polymyxinB3 for LPS was comparable to polymyxin B and colistin, and considerably greater (kd < 1 μM) than for whole cells (kd ~6 to 12 μM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]1polymyxinB3 to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]1polymyxinB3-LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]1polymyxinB3-LPS complex due to steric hindrance from the dansyl[Lys]1 fluorophore; this corresponded with diminished antibacterial activity (MIC ≥ 16 μg/mL). Dansyl[Lys]1polymyxinB3 may prove useful as a screening tool for drug development.

Lipopolysaccharide (LPS)-binding proteins interact with LPS in human serum and mediate various immune responses. We describe a high-throughput LPS-binding protein profiling platform for discovering unknown LPS-binding proteins and potential inflammatory mediators. As a pull-down method, the LPS molecules were immobilized onto epoxy beads and then directly incubated with human serum to screen LPS-binding proteins. Through the "untargeted" mass spectrometric approach, potential LPS-binding proteins which elicit various immune responses in human serum were identified by a highly sensitive LTQ Orbitrap Hybrid Fourier Transform Mass Spectrometer (LTQ Orbitrap FT MS). Therefore, this mass spectrometry (MS)-based profiling method is straightforward for screening unknown LPS-binding proteins and provides physiologically relevant binding partners in human serum.

Lipid-binding properties and/or involvement with host defense are often found in allergen proteins, implying that these intrinsic biological functions likely contribute to the allergenicity of allergens. The group 2 major mite allergens, Der f 2 and Der p 2, show structural homology with MD-2, the lipopolysaccharide (LPS)-binding component of the Toll-like receptor (TLR) 4 signalling complex. Elucidation of the ligand-binding properties of group 2 mite allergens and identification of interaction sites by structural studies are important to explore the relationship between allergenicity and biological function. Here, we report a ligand-fishing approach in which His-tagged Der f 2 was incubated with sonicated stable isotope-labelled Escherichia coli as a potential ligand source, followed by isolation of Der f 2-bound material by a HisTrap column and NMR analysis. We found that Der f 2 binds to LPS with a nanomolar affinity and, using fluorescence and gel filtration assays that LPS binds to Der f 2 in a molar ratio of 1 : 1. We mapped the LPS-binding interface of Der f 2 by NMR perturbation studies, which suggested that LPS binds Der f 2 between the two large beta-sheets, similar to its binding to MD-2, the LPS-binding component of the innate immunity receptor TLR4.

This study on the structure-activity relationship of polymyxin B, a cyclic peptide antibiotic, used sixteen synthetic polymyxin B(3) analogs including alanine scanning analogs to elucidate the contribution of the side chains to antimicrobial activity and lipopolysaccharide (LPS) binding. Of these analogs, [Ala(5)]-polymyxin B(3) showed greatly reduced antimicrobial activity against Escherichia coli (E. coli), Salmonella Typhimurium (S. Typhimurium) and Pseudomonas aeruginosa (P. aeruginosa) with MIC values of 4-16 nmol/ml, suggesting that the Dab (alpha,gamma-diaminobutyric acid) residue at position 5 is the most important residue contributing to bactericidal activity. The antibacterial contribution of Dab when located within the lactam ring (positions 5, 8 and 9) was greater than when located outside the ring (positions 1 and 3). [D-Ala(6)]-, [L-Phe(6)]-, [Ala(7)]-, and [Gly(7)]-polymyxin B(3) analogs retained potent antimicrobial activity, indicating that neither the reduction of hydrophobic character of the D-Phe(6)-Leu(7) region nor the D-configuration at position 6 is indispensable for antimicrobial activity. LPS binding studies showed that decreased hydrophobicity of the lactam ring had little effect, but the N(gamma)-amino function of the Dab residues at position 1, 3, 5, 8 and 9 greatly affected LPS binding, with the contribution of Dab(5) being the most significant.

A hemagglutinin was isolated from hemocytes of the ascidian, Halocynthia roretzi, by a procedure including extraction and ion-exchange chromatography on CM-cellulose. The molecular weight of the hemagglutinin was estimated to be 120,000 by gel filtration. It was resistant to acid treatment but sensitive to alkali or heat treatment. The hemagglutinating activity was inhibited by heparin, chondroitin sulfate, and lipopolysaccharide (LPS), but not by mono- and disaccharides such as N-acetyl-galactosamine, galactose, and melibiose. The hemagglutinin showed binding ability to heparin and LPS, as demonstrated by heparin-Sepharose chromatography and centrifugation experiments, respectively. It was also found that the hemagglutinin can bind to various bacteria such as Escherichia coli, Bacillus subtilis, Vibrio anguillarum, Pseudomonas perfectomarinus, Achromobacter aquamarinus, and Alteromonas putrefaciens, and can agglutinate all of them.

Lipopolysaccharide-(LPS) binding proteins present on murine-lymphocyte and macrophage-like cell lines were identified by a ligand-blotting method and subsequent immunological detection of bound LPS. Membrane proteins of the murine-pre-B-cell line 70Z/3 were separated by SDS-PAGE, transferred electrophoretically onto nitrocellulose, and the blot was incubated with LPS of the Salmonella minnesota Re-mutant R595 (mRe-LPS). LPS bound to proteins on nitrocellulose was immunologically detected by anti-mRe-LPS antibodies; LPS was associated with one of the membrane proteins of 70Z/3 cells. This protein was 40 kDa under reducing and 45 kDa under non-reducing conditions, respectively. Treatment of 70Z/3 cells with pronase led to the disappearance of the LPS-binding protein indicating its surface location. Excess free lipid A, which represents the biologically active region of LPS, inhibited the binding of mRe-LPS to the protein. This LPS-binding protein was also identified on the pre-B-cell line CYG8, the B-cell line CYG101 and the murine-T-cell line BW5147. It was, however, not detectable on the B-cell line CYG34 and the myeloma-cell line P3-X63-Ag8.653. No other LPS-binding protein could be detected on these cell lines. In the murine-macrophage-like cell line J774.1, two LPS-binding proteins, one of 40 kDa and one of 80 kDa, were detected. These results indicate that mRe-LPS is specifically bound to a 40-kDa protein of lymphocytes, whereas in the case of macrophages it is associated with two LPS-binding proteins of 40 and 80 kDa.

Summary Lipopolysaccharide binding sites of guinea pig peritoneal macrophages were demonstrated by means of immunogold technique. Resident peritoneal macrophages show a strong specific binding of bacterial lipopolysaccharides from E. coli to cell surface structures.

BACKGROUND: Due to increasing antibiotics resistance, antimicrobial peptides (AMPs) are receiving increased attention. Pseudomonas aeruginosa is a major pathogen in this context, involved, e.g., in keratitis and wound infections. Novel bactericidal agents against this pathogen are therefore needed. METHODS: Bactericidal potency was monitored by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was probed by hemolysis. Mechanistic information was obtained from assays on peptide-induced vesicle disruption and lipopolysaccharide binding. RESULTS: End-tagging by hydrophobic amino acids yields increased potency of AMPs against P. aeruginosa, irrespective of bacterial proteinase production. Exemplifying this by two peptides from kininogen, GKHKNKGKKNGKHNGWK and KNKGKKNGKH, potency increased with tag length, correlating to more efficient bacterial wall and vesicle rupture, and to more pronounced P. aeruginosa lipopolysaccharide binding. End-tag effects remained at high electrolyte concentration and in the presence of plasma or anionic macromolecular scavengers. The tagged peptides displayed stability against P. aeruginosa elastase, and were potent ex vivo, both in a contact lens model and in a skin wound model. GENERAL SIGNIFICANCE: End-tagging, without need for post-peptide synthesis modification, may be employed to enhance AMP potency against P. aeruginosa at maintained limited toxicity.

Successful embryonic development of parasitoid wasps in lepidopteran hosts is achieved through co-injection of polydna viruses whose gene products are thought to target the immune responses of the host. One gene product of the endosymbiont bracovirus of the parasitic wasp Cotesia rubecula, CrV1, has been reported to inhibit the immune responses of its endoparasitized lepidopteran host through interference with the haematocyte cytoskeletal structure. Here we establish that CcV1, the Cotesia congregata bracovirus orthologue of CrV1, is also uptaken by lepidopteran haemocytes and haemocyte-like established cell lines, but we also report on a different function of CcV1, which is highly relevant to the inhibition of the host immune responses and is based on its direct interaction with the pattern recognition molecule hemolin. Recombinant CcV1 inhibits hemolin functions, such as lipopolysaccharide binding and bacterial agglutination as well as bacterial phagocytosis by haemocytes and haemocyte-like cell lines, producing functional phenotypes equivalent to those observed to arise from RNAi-based inhibition of hemolin gene expression. Finally, we show that CcV1 and hemolin colocalize on the membrane surface of hemolin-expressing cells, a finding suggesting that CcV1 may be uptaken by haemocytes and inhibit haemocyte function as a result of its interaction with membrane-anchored hemolin.

PURPOSE OF REVIEW The intention of this article is to review endotoxin, host response to endotoxin, clinical significance of endotoxemia, past failed therapies targeting endotoxin, current therapeutic efforts in this area and the authors' opinion on the future of such therapy. RECENT FINDINGS Endotoxin or lipopolysaccharide is implicated in the activation of cytokine release with the potential to lead to severe sepsis. Therapies targeting endotoxin are very appealing and remain a matter of study and debate. Antiendotoxin antibody studies did not show consistent benefit to warrant its approval for use. Lipid A analog, phospholipid emulsion, and ethyl pyruvate are currently being evaluated for potential clinical use. Polymyxin B as an antiendotoxin strategy has an unacceptable toxicity profile for routine use as an intravenous agent and its use in plasmapheris is too cumbersome. Curcumin and lipopolysaccharide binding peptides, although having a potentially desirable effect on ameliorating endotoxin toxicity, remain to be shown effective in clinical trials. The development of a vaccine against endotoxin carries promise. SUMMARY The benefits of therapies targeting endotoxin remain to be elucidated. Clinical trials targeting populations with documented endotoxemia are more likely to provide an adequate test of this therapeutic approach. Prophylaxis of high-risk populations should also be considered.

A radioiodinated, photoactivatable derivative of Salmonella minnesota Re595 lipopolysaccharide (LPS) was used to label LPS-binding proteins in 70Z/3 cells. The labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. 125I-Labeled-2-(p-azidosalycylamido)1,3'-dithiopropionamide S. minnesota Re595 LPS (125I-ASD-Re595) labeled a limited number of proteins. The most prominent of these had a apparent molecular mass of 18 kDa. Less prominent labeling of 25- and 28-kDa proteins was also seen. Labeling was saturated by 5 micrograms/ml 125I-ASD-Re595 and was inhibited by a 10-100-fold excess of unlabeled LPS or lipid A. Labeling was maximal within 30 min at 37 degrees C; much less labeling occurred at lower temperatures. The proteins labeled with 125I-ASD-Re595 appear to be on the surface of the cell, since they can be digested by trypsin and were found in the membrane fraction of the cell but not in the cytosol. Studies with competitive inhibitors suggested that the proteins bind to the lipid A region of the LPS molecule. Biologically inactive lipid A analogs were poor inhibitors of labeling, suggesting that the LPS-binding proteins could discriminate between active lipid A and inactive analogs. These studies suggest that the 18- and 25-kDa proteins bind specifically to the lipid A region of the LPS molecule and should be considered as candidates for a functional LPS receptor.

An invading pathogen must be held in check by the innate immune system until a specific immune response is mounted. Nonclonal pattern recognition receptors like CD14 or lipopolysaccharide (LPS) binding protein (LBP) recognize ubiquitous pathogen-associated molecular patterns, e.g. LPS. LBP mediates the binding of minute amounts of LPS to membrane-bound CD14 (mCD14) triggering a proinflammatory response of macrophages, which is crucial for keeping an infection under control. Moreover, in vitro mCD14 and LBP are involved in recognition and phagocytosis of heat-killed bacteria. Living Salmonella typhimurium or Escherichia coli depend on the presence of LBP to induce the generation of reactive oxygen species in human or murine macrophages. Using LBP-deficient mice it could be demonstrated that LBP is essential to control low dose (100 CFU S. typhimurium) infection. Therefore, LPS binding proteins play a pivotal role in physiology as well as pathophysiology of Gram-negative infection.

Plasma kallikrein kinin system (KKS) activation along with its cellular receptors expression are increased after injury and in patients with septic shock, hypotensive bacteremia and rhesus monkey infected with Salmonella typhimurium. KKS signaling cascade is activated by activated factor XII (FXIIa, Hageman factor)- and prolylcarboxypeptidase (PRCP)-dependent pathways on endothelial cells. Among the many entities that comprise the KKS, high molecular weight kininogen (HK), a bradykinin precursor, is critical in the assembly and activation of this system. HK is primarily expressed in the liver and secreted into the bloodstream. The activation of the KKS influences the permeability of the endothelium by liberating bradykinin (BK) from HK. BK is a potent inflammatory peptide which stimulates constitutive bradykinin B2 and inducible B1 receptors to release nitric oxide and prostacyclin. Regardless of the triggers, PK can only be activated on HK bound to the artificial negatively charged or to cell membrane surfaces. Since LPS has a negatively charged moiety and the ability to induce inflammatory responses in human, we determined the interaction between LPS and HK. HKH19 (HK cell binding site) and heparin inhibited LPS binding to HK with IC(50)s of 15nM and 20 microg/ml, respectively. C1-inhibitor and N-acetylglucosamine glycan inhibited LPS binding to HK with IC(50)s of about 10 microg/ml and 10mM, respectively. This novel study underscores the implication of HK in infection. We propose that HKH19, heparin, and C1-inhibitor present therapeutic potential for the treatment of sepsis and hypotensive bacteremia.

KS Sruthy,1 Aishwarya Nair,1 Sherine Sonia Cubelio,2 IS Bright Singh,3 Rosamma Philip11Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, 2Centre for Marine Living Resources and Ecology, Ministry of Earth Sciences, Government of India, 3National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Kochi, Kerala, IndiaAbstract: Antimicrobial peptides are small cationic, gene-encoded, amphipathic, host defense peptides with a ubiquitous distribution in all living kingdoms. They are

Introduction: Gut derived lipopolysaccharide (LPS) is important factor in development of single and multiple organ failure during severe acute pancreatitis (SAP). Aims: To evaluate the effects of enteral administration of colistin (CS) on preventing LPS absorption from gut during SAP. Materials & methods: In 200 rats SAP was induced by L-arginine injection. Enteral administration of 2 mg/kg of CS every 6 h performed in CS high dose (CHD) group (n 1⁄4 10), 1 mg/kg every 12 h in CS low dose (CLD) group (n1⁄4 10), 1 mg/kg included in chitosan nanoparticles every 12 h in CS nanoparticles (CNP) group (n 1⁄4 10). Levels of LPS, sCD14, TNFa, morphological changes in pancreas, other internal organs and intestinal microbiota have been studied during 7 days. Results: CS administration caused reduction of LPS levels in faeces for 100-1000 times in all treated groups, but in mucus such events appeared only in CNP group. LPS concentrations in portal and system blood were similar to sham control level in CNP and elevated 2 and 4-5 times in CHD and CLD animals. Administration of CS reduced amounts of sCD14 and TNFa in all treatment cases, but only in CNP group there levels were closed to sham animals. Tissue injuries were much lesser in case of CNP group. Conclusion: Selective binding of gut-derived LPS during SAP by enteral administration of CS effectively decreases tissue injury in case it is incorporated to mucoadhesive nanoparticle delivery system or applied in high dose regimen. Abstracts / Pancreatolog S2

Objective: Lipopolysaccharide (LPS), a potent and pleiotropic stimulator of immune cells, binds to neutrophils via CD14, but less densely than to monocytes. The present study was designed to investigate whether cytokines modulate LPS binding to neutrophils via CD14.¶Methods: Neutrophils were cultured with LPS after pretreatment with cytokines, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-1 alpha (IL-1α), or granulocyte-colony stimulating factor (G-CSF). Binding of LPS and CD14 expression on neutrophils were analyzed by flow cytometry, using anti-LPS and anti-CD14 monoclonal antibodies (mAb).¶Results: LPS alone showed only slight binding to neutrophils, but pretreatment with IFN-γ or TNF-α before LPS exposure markedly increased LPS binding and CD14 expression on the surfaces of neutrophils. The dramatic increase in LPS binding was not seen with IL-1α or G-CSF. Anti-CD14 blocking mAb completely inhibited the binding effect.¶Conclusions: These results demonstrate that IFN-γ and TNF-α enhance LPS binding to neutrophils via CD14, suggesting that the priming effect of cytokines on neutrophils is important for LPS binding.

Apolipophorin III (apoLp-III) is a prototypical apolipoprotein used for structure-function studies. Besides its crucial role in lipid transport, apoLp-III is able to associate with fungal and bacterial membranes and stimulate cellular immune responses. We recently demonstrated binding interaction of apoLp-III of the greater wax moth, Galleria mellonella, with lipopolysaccharides (LPS). In the present study, the requirement of helix bundle opening for LPS binding interaction was investigated. Using site-directed mutagenesis, two cysteine residues were introduced in close spatial proximity (P5C/A135C). When the helix bundle was locked by disulfide bond formation, the tethered helix bundle failed to associate with LPS. In contrast, the mutant protein regained its ability to bind upon reduction with dithiothreitol. Thus, helix bundle opening is a critical event in apoLp-III binding interaction with LPS. This mechanism implies that the hydrophobic interior of the protein interacts directly with LPS, analogous to that observed for lipid interaction.

Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are an integral part of the outer leaflet of the outer-membrane of Gram-negative bacteria. Lipopolysaccharides play a pivotal role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient, worldwide. The sequestration of circulatory endotoxin may be a viable therapeutic strategy for the prophylaxis and treatment of Gram-negative sepsis. We have earlier shown that the pharmacophore necessary for small molecules to bind LPS involves two protonatable cationic functions separated by about 15 A, permitting the simultaneous interaction with the negatively charged phosphates on lipid A, the toxically active center of endotoxin. In this report, screening of a multi-thousand membered polyamine library through the combined use of computational and bioassay-guided screens resulted in the discovery of two novel classes of LPS-binding agents. These are represented by the 1) spermine sulfonamides and 2) C-aryl-substituted spermine analogs. We present the selection approach, screening results, computational multivariate analyses and initial structure-activity relationship evaluation herein.

Item does not contain fulltext The interaction of lipopolysaccharide with CD14 plays a key role in signaling that activates an early defense against pathogens but also contributes to the development of sepsis and septic shock. Here we have mapped the entire 356-amino-acid protein with synthetic 20-amino-acid peptides and have identified a new lipopolysaccharide-binding domain with a strong LPS-neutralizing activity. Moreover, analysis of the structure-activity relationship of this peptide, which corresponds to amino acids 81-100 of human CD14, revealed that leucines 87, 91, and 94 are essential for these activities. The functional relevance of these residues was confirmed by cellular expression of mutant CD14 proteins that are no longer able to bind LPS. Furthermore, the peptide provided a basis for the generation of highly soluble analogues with stronger lipopolysaccharide-neutralizing activity.

Prior to being recognized by the cell surface Toll-like receptor 4/MD-2 complex, lipopolysaccharide (LPS) in the bacterial outer membrane has to be processed by LPS-binding protein and CD14. CD14 forms a complex with monomeric LPS extracted by LPS-binding protein and transfers LPS to the cell surface signaling complex. In a previous study, we prepared a functional recombinant MD-2 using a bacterial expression system. We expressed the recombinant protein inEscherichia colias a fusion protein with thioredoxin and demonstrated specific binding to LPS. In this study, we prepared recombinant CD14 fusion proteins using the same approach. Specific binding of LPS was demonstrated with a recombinant protein containing 151 amino-terminal residues. The region contained a hydrophilic region and the first three leucine-rich repeats (LRRs). The LRRs appeared to contribute to the binding because removal of the region resulted in a reduction in the binding function. LPS binding to the recombinant MD-2 was resistant to detergents. On the other hand, the binding to CD14 was prevented in the presence of low concentrations of detergents. In the case of human MD-2, the secondary myristoyl chain of LPS added by LpxM was required for the binding. A nonpathogenic penta-acyl LPS mutant lacking the myristoyl chain did not bind to MD-2 but did so normally to CD14. The broader LPS-binding spectrum of CD14 may allow recognition of multiple pathogens, and the lower affinity for LPS binding of CD14 allows transmission of captured materials to MD-2.

Lipopolysaccharide, the endotoxin of Gram-negative bacteria, induces extensive immune responses that can lead to fatal septic shock syndrome. The core receptors recognizing lipopolysaccharide are CD14, TLR4, and MD-2. CD14 binds to lipopolysaccharide and presents it to the TLR4/MD-2 complex, which initiates intracellular signaling. In addition to lipopolysaccharide, CD14 is capable of recognizing a few other microbial and cellular products. Here, we present the first crystal structure of CD14 to 2.5 angstroms resolution. A large hydrophobic pocket was found on the NH2-terminal side of the horseshoe-like structure. Previously identified regions involved in lipopolysaccharide binding map to the rim and bottom of the pocket indicating that the pocket is the main component of the lipopolysaccharide-binding site. Mutations that interfere with lipopolysaccharide signaling but not with lipopolysaccharide binding are also clustered in a separate area near the pocket. Ligand diversity of CD14 could be explained by the generous size of the pocket, the considerable flexibility of the rim of the pocket, and the multiplicity of grooves available for ligand binding.

A new role of apolipophorin III (apoLp-III) as an immune activator has emerged recently. To gain insight into this novel function, the interaction of apoLp-III with lipopoly-saccharide (LPS) was investigated. ApoLp-III fromGalleria mellonellawas incubated with LPS fromEscherichia coliO55:B5, and analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE). Protein staining showed that apoLp-III mobility was significantly reduced. In addition, silver and LPS fluorescent staining demonstrated that LPS mobility was increased upon incubation with apoLp-III. This result suggests association of apoLp-III with LPS. Sodium dodecyl sulfate (SDS) PAGE analysis showed decreased apoLp-III mobility upon LPS addition, indicative of LPS apoLp-III interaction in the presence of SDS. The unique tyrosine residue that resides in apoLp-III was used to provide additional evidence for LPS binding interaction. In the absence of LPS, apoLp-III tyrosine fluorescence was relatively low. However, LPS addition resulted in a progressive increase in the fluorescence intensity, indicating tertiary rearrangement in the environment of tyrosine 142 upon LPS interaction. Other well-characterized apoLp-IIIs were also examined for LPS binding.Manduca sexta,Bombyx moriandLocusta migratoriaapoLp-III were all able to interact with LPS. The ability of apoLp-III to form complexes with LPS supports the proposed role of apoLp-III in innate immunity.

Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, can be beneficial to the host by activating the innate immune system, or harmful, by inducing inflammation, disseminated intravascular coagulation, multiple organ failure, shock and often death. On the bacteria, and in host biological fluids and cells, LPS is never free but constantly attached to cognate-binding proteins. Understanding how LPS is transported and further recognized by sensors able to deliver a signal, or by inactivating molecules able to neutralize its biological effects, is an important goal. This review describes the large panel of peptides and proteins reported to associate with LPS, and provides information on their origin, their structure and the location of amino acid residues involved in their interaction with LPS. A better understanding of the mode of recognition of LPS by cognate proteins prompted many laboratories to design on a rational basis synthetic molecules which can be used to detect low amounts of endotoxin, or to act as efficient blockers of in vitro and in vivo responses to LPS.

Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are outer-membrane constituents of Gram-negative bacteria. Lipopolysaccharides play a key role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient. Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time. We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS, and have shown using animal models of sepsis that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy. Assays reported previously in the literature do not lend themselves well to the rapid screening of large numbers of structurally diverse compounds. In this report, we describe a highly sensitive and robust fluorescent displacement assay using BODIPY TR cadaverine (BC), which binds specifically to the toxic center of LPS, lipid A, and is competitively displaced by compounds displaying an affinity for lipid A. The assay clearly discriminates subtle differences in the binding of polymyxin B, and its nonapeptide derivative, with LPS. The spectral properties of the BODIPY fluorophore are ideally suited for screening diverse structural classes of compounds, including those with conjugated aromatic groups, or with chromophores in the 260-500 nm range. The fluorescent probe: LPS complex is stable under physiologically relevant salt concentrations, resulting in the rapid rejection of spurious binders interacting via non-specific electrostatic interactions, and, therefore, in greatly improved dispersion of ED(50)values.

In both humans and mice, a deficiency of surfactant protein B (SP-B) is associated with a decreased concentration of mature SP-C and accumulation of a larger SP-C peptide, denoted SP-C(i), which is not observed under normal conditions. Isolation of hydrophobic polypeptides from the lungs of children who died with two different SP-B mutations yielded pure SP-C(i) and showed only trace amounts of mature SP-C. Determination of the SP-C(i) covalent structure revealed a 12-residue N-terminal peptide segment, followed by a 35-residue segment that is identical to mature SP-C. The SP-C(i) structure determined herein is similar to that of a proposed late intermediate in the processing of proSP-C, suggesting that SP-C(i) is the immediate precursor of SP-C. In bronchoalveolar lavage fluid from transgenic mice with a focal deficiency of SP-B, SP-C(i) was detected in the biophysically active, large aggregate fraction and was associated with membrane structures that are typical for a large aggregate surfactant. However, unlike SP-C, SP-C(i) exhibited a very poor ability to promote phospholipid adsorption, gave high surface tension during cyclic film compression, and did not bind lipopolysaccharide in vitro. SP-C(i) is thus capable of associating with surfactant lipids, but its N-terminal dodecapeptide segment must be proteolytically removed to generate a biologically functional peptide. The results of this study indicate that the early postnatal fatal respiratory distress seen in SP-B-deficient children is combined with the near absence of active variants of SP-C.

Circulating lipoproteins are thought to play an important role in the detoxification of lipopolysaccharide (LPS) by binding the bioactive lipid A portion of LPS to the lipoprotein surface. It has been assumed that hypocholesterolemia contributes to inflammation during critical illness by impairing LPS neutralization. We tested whether critical illness impaired LPS binding to lipoproteins and found, to the contrary, that LPS binding was enhanced and that LPS binding to the lipoprotein classes correlated with their phospholipid content. Whereas low serum cholesterol was almost entirely due to the loss of esterified cholesterol (a lipoprotein core component), phospholipids (the major lipoprotein surface lipid) were maintained at near normal levels and were increased in a hypertriglyceridemic subset of septic patients. The levels of phospholipids found in the LDL and VLDL fractions varied inversely with those in the HDL fraction, and LPS bound predominantly to lipoproteins in the LDL and VLDL fractions when HDL levels were low. Lipoproteins isolated from the serum of septic patients neutralized the bioactivity of the LPS that had bound to them. Our results show that the host response to acute inflammation and infection tends to maintain lipoprotein phospholipid levels and that, despite hypocholesterolemia and reduced HDL levels, circulating lipoproteins maintain their ability to bind and neutralize an important bacterial agonist, LPS.

Unlike soluble and membrane forms of lipopolysaccharide (LPS)-binding proteins, intracellular LPS-binding molecules are poorly documented. We looked for such molecules in a murine lung epithelial cell line. Two proteins with LPS-binding activity were isolated and unambiguously identified as histones H2A.1 and H4 by mass spectrometry. Synthetic peptides representing partial structures indicated that the LPS binding site is located in the C-terminal moiety of the histones. Extending the study, we found that histones H1, H2A, H2B, H3, and H4 from calf thymus are all able to bind LPS. Bindings were specific, and affinities, determined by isothermal titration calorimetry, were (except for H4) higher than that of the LPS-binding antibiotic polymyxin B. In the presence of H2A the binding of LPS to the macrophage cell line RAW 264.7, and the LPS-induced production of TNF-alpha and nitric oxide by these cells, were markedly reduced. Histones may thus represent a new class of intracellular and extracellular LPS sensors.

BACKGROUND/AIM—Components of the tear fluid contribute to the biochemical defence system of the eye. To reveal whether the immune mediator and lipopolysaccharide binding galectin-3 is present in tears, tear samples were collected from eyes in healthy and pathological states. Investigation of expression of galectin-3 and galectin-3 reactive glycoligands in normal human conjunctival and corneal epithelia was also initiated as a step to understand the role of galectin-3 in ocular surface pathology. METHODS—Immunoblot analysis using either a rabbit polyclonal or a mouse monoclonal antibody against galectin-3 was employed to detect galectin-3 in tear fluid. Galectin-3 expression in tissue specimens was detected by immunocytochemistry employing A1D6 mouse monoclonal antibody, and galectin-3 reactive glycoligands were visualised by lectin histochemistry using labelled galectin-3. RESULTS—Galectin-3 was found only in tears from patients with ocular surface disorders. It was expressed in normal corneal and conjunctival epithelia but not in lacrimal glands. Inflammatory leucocytes and goblet cells found in galectin-3 containing tear fluid also expressed galectin-3. Galectin-3 binding sites were detected on the surface of conjunctival and corneal epithelial cells co-localising with desmoglein. CONCLUSIONS—This study revealed expression of galectin-3 in tear fluid obtained from patients with eye diseases. The role of this endogenous lectin (produced by inflammatory as well as epithelial cells) in antimicrobial action and inflammation modulation could be expected.

Many naturally occurring peptides exhibit lipopolysaccharide binding properties. In this work we describe the endotoxin binding properties of a series of 23-residue peptides based on the sequence corresponding to the antisense strand of the magainin gene. Biochemical and biophysical characterization of these peptides reveals that they have the tendency to perturb both the inner and outer membranes of test pathogens. Structurally these peptides are amphiphilic and adopt helical conformations in membranes. Three of the seven peptides tested have high affinities for endotoxin that approach the values shown by polymyxin B, a cyclic cationic acylated decapeptide, which is used clinically in treating extreme cases of sepsis. The kinetic parameters obtained using stopped-flow methods and $BIA-core^{TM}$ analysis, when considered in conjunction with the isothermal titration calorimetry-derived thermodynamic parameters, allow us to highlight the key structural features essential for lipopolysaccharide (LPS) recognition by these peptides. The studies stress the role of ionic forces in the initial recognition of LPS. The fortification of the strength of these ionic charges increases affinity for LPS, whereas the hydrophobic residues involved in interactions are more amenable to disruptions in contiguity. Peptides that improve these features further are expected to perform better as endotoxin-neutralizing agents.

Induction of resting B cell growth and differentiation requires a complex series of temporally coordinated signals that are initiated on contact with activated helper T cells. These signals complement one another, each rendering the B cell susceptible to factors supporting progressive activation. Here, we demonstrate that soluble CD14 (sCD14) bypasses the physiological sequelae of events that limit B cell activation. B cell growth and differentiationin vitrois induced by both native and recombinant forms of sCD14 at nanomolar concentrations. sCD14-mediated cellular activation does not require membrane CD14 expression, depends on a region of CD14 that is not involved in lipopolysaccharide binding, and requires functional Toll-like receptor 4. Consistent with biological activity of sCD14in vitro, its administration to neonatal mice enhances Ig secretion. The results presented establish sCD14 as a naturally occurring soluble B cell mitogen of mammalian origin.

This study was performed to examine the prevalence and clinical correlates of bactericidal/permeability-increasing protein anti-neutrophil cytoplasmic antibodies (BPI-ANCA) in pediatric cystic fibrosis (CF) patients and to elucidate their possible role in CF pulmonary pathology. Sera of 27 CF patients were tested for ANCA by indirect immunofluorescence (IFT) and by enzyme-linked immunosorbent assay (ELISA) for ANCA sub-specificities. BPI-ANCA were examined by using standard ELISA for BPI, lipopolysaccharide-binding protein (LBP), and BPI/LBP fusion proteins to epitope map the main binding sites and look for cross-reactivity with LBP. Pulmonary function and serum concentrations of total immunoglobulin G (IgG) were measured and infections were diagnosed. In addition, release of reactive oxygen species (ROS) by neutrophil granulocytes was measured after stimulation with monoclonal BPI-ANCA. Using IFT, two patients showed atypical ANCA staining, six patients exhibited perinuclear ANCA staining, and no cytoplasmic ANCA staining was detected. Of 27 patients, 13 (48%) were BPI-ANCA (IgG) positive, and three were also immunoglobulin A (IgA) BPI-ANCA positive; one patient had ANCA against lactoferrin; and no proteinase 3 ANCA was detected in any of the patients. All BPI-ANCA bound to the C-terminal region of the molecule; none bound to the N-terminus or to LBP. There was no significant correlation between clinical data and the occurrence of BPI-ANCA in this cross-sectional study. Release of ROS from granulocytes was induced by monoclonal BPI-ANCA. Activation of neutrophils and possible modulation of BPI-mediated opsonophagocytosis and disposal of Gram-negative bacteria and lipopolysaccharides by BPI-ANCA raise the possibility that they contribute to pulmonary pathology in pediatric CF patients but intervention longitudinal studies in large groups of patients are needed to establish a causative association.

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Lactoferrin (LFR) plays an important role in the anti-microbial defense through iron binding, lipopolysaccharide binding and immunomodulation. In this study, we demonstrate that bovine LFR specifically inhibits the hemolytic activity of listeriolysin O (LLO) produced by Listeria monocytogenes. The hemolytic activity of LLO was completely inhibited in the presence of bovine LFR that was highly purified on two cation-exchange columns, whereas that of streptolysin O or perfringolysin O was not inhibited at all. A rabbit anti-LFR antibody canceled this inhibitory activity of bovine LFR. Although human transferrin exhibits 62% amino acid identity with bovine LFR, human apo-transferrin could not inhibit LLO-induced hemolysis. An increase in the concentration of FeCl3 or the Fe3+-saturation of bovine LFR, however, slightly reduced its inhibition of the hemolysis. The inhibitory activity of bovine LFR was dependent on pH, since it was observed under neutral and alkali conditions, but not under acidic conditions. These results suggest that the inhibition of LLO-induced hemolysis by bovine LFR is influenced by pH and iron ions, both of which may lead to conformational changes of LFR.

The purpose of this study was to determine whether antiendotoxin agents exhibit molecular homology within their lipopolysaccharide (LPS) binding domains, suggesting a common mechanism of action. We hypothesized that the presence of positively charged basic amino acids or a paucity of negatively charged acidic amino acids, or both, would be a critical characteristic of that portion of the molecule that binds to the highly negatively charged deep core/lipid A (DCLA) region of LPS.We analyzed the amino acid sequences of the variable light (VL) and heavy (VH) chain complementarity-determining regions (CDRs) of anti-DCLA monoclonal antibodies (mAbs) 1B6, 5A5, and 7C5 and compared them with (1) the CDRs of three irrelevant control mAbs and (2) the LPS binding region of bactericidal permeability-increasing protein (BPI). We purified and amplified the specific nucleotide sequences of the variable regions using reverse transcriptase polymerase chain reaction. DNA was sequenced by dideoxy termination, and protein sequences were deduced and analyzed. The percentages of acidic, basic, polar, and hydrophobic amino acids within VH and VL chain CDRs were determined.We identified a paucity of negatively charged acidic amino acids exclusively within VL chain CDRs of anti-DCLA mAbs (P0.005). Although increased, the number of positively charged basic residues was not statistically significantly different; neither was the number of polar or hydrophobic amino acids. Conclusions. Our data suggest that the near absence of negatively charged acidic residues is critical for LPS binding. This characteristic appears to reside exclusively in the VL chain CDRs of anti-DCLA mAbs.