lipid-a and Shock--Septic

Septic shock is caused by Gram-negative bacterial infection. Lipopolysaccharide (LPS) is the bioactive molecule present on the outer membrane of the Gram-negative bacteria. It is generally thought that LPS interacts with sensors on the host cell membrane to activate the intracellular signaling pathway resulting in the overproduction of cytokines such as TNF-alpha. This causes inflammation and ultimately, septic shock. Lipid A is the pharmacophore of the LPS molecule. Thus, developing bio-molecules which are capable of binding LPS at high affinity, especially to the lipid A moiety is an efficient way to neutralize the LPS toxicity. Factor C, a serine protease in the horseshoe crab ameobocytes, is sensitive to trace levels of LPS. We have derived Sushi peptides from the LPS-binding domains of Factor C. Our earlier study showed that the Sushi peptides inhibit LPS-induced septic shock in mice. Here, we demonstrate that the molecular interaction between LPS and Sushi 1 peptide is supported by the hydrophobic interaction between the lipid tail of LPS and Sushi 1 peptide. Furthermore, in the presence of LPS, the peptide transitions from a random structure into an alpha-helical conformation and it disrupts LPS aggregates, hence, neutralizing the LPS toxicity.

Topics: Animals; Arthropod Proteins; Binding Sites; Enzyme Precursors; Gram-Negative Bacterial Infections; Humans; Lipid A; Peptides; Protein Binding; Protein Structure, Secondary; Pseudomonas; Pseudomonas Infections; Serine Endopeptidases; Shock, Septic; Structure-Activity Relationship

Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins.

Topics: Acute-Phase Proteins; Bacteremia; Carrier Proteins; CD18 Antigens; Cytokines; Humans; Immune Tolerance; Kupffer Cells; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Lipoproteins; Membrane Glycoproteins; Shock, Septic

Bacterial lipopolysaccharide (LPS), the major structural component of the outer wall of Gram-negative bacteria, is a potent activator of macrophages. Activated macrophages produce a variety of inflammatory cytokines. Excessive production of cytokines in response to LPS is regarded as the cause of septic shock. On the other hand, macrophages exposed to suboptimal doses of LPS are rendered tolerant to subsequent exposure to LPS and manifest a profoundly altered response to LPS. Increasing evidence suggests that monocytic cells from patients with sepsis and septic shock survivors have characteristics of LPS tolerance. Thus, an understanding of the molecular mechanisms underlying activation and deactivation of macrophages in response to LPS is important for the development of therapeutics for septic shock and the treatment of septic shock survivors. Over the past several years, significant progress has been made in identifying and characterizing several key molecules and signal pathways involved in the regulation of macrophage functions by LPS. In this paper, we summarize the current findings of the functions of the LPS receptor complex, which is composed of CD14, Toll-like receptor 4 (TLR4), and myeloid differentiation protein-2 (MD-2), and the signal pathways of this LPS receptor complex with regard to both activation and deactivation of macrophages by LPS. In addition, recent therapeutic approaches for septic shock targeting the LPS receptor complex are described.

Topics: Animals; Antigens, Surface; Humans; Immune Tolerance; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Lymphocyte Antigen 96; Macrophage Activation; Membrane Glycoproteins; Receptors, Cell Surface; Shock, Septic; Toll-Like Receptor 4; Toll-Like Receptors

The ability of a host to sense invasion by a pathogenic organism, and to respond appropriately to control infection, is paramount to survival. To that end, an array of receptors and binding proteins has evolved as part of the innate immune system to detect Gram-negative bacteria. This article reviews the role of CD14, other LPS binding proteins, and the Toll family of receptors in the innate recognition of bacterial lipopolysaccharide.

Topics: Animals; CD11 Antigens; CD18 Antigens; Drosophila Proteins; Endotoxins; Humans; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Membrane Glycoproteins; Mice; Mice, Knockout; Receptors, Cell Surface; Shock, Septic; Signal Transduction; Toll-Like Receptors

Therapy for Gram-negative sepsis remains unsatisfactory despite a concerted effort to develop new treatments for this common, life-threatening syndrome. Current research continues on several fronts to improve the treatment options available to clinicians in the management of these critically ill patients. Recently, a greater understanding of the complex molecular basis of endotoxin-mediated pathophysiological effects in humans has generated a number of novel therapeutic agents for sepsis. Several of these treatment strategies have already entered clinical trials and it is hoped that some of these therapies will become widely available in the near future. In this review, the current status of the most promising new antiendotoxin agents is summarised, and the major obstacles to the successful clinical development of these therapies are described. New antiendotoxin therapies include those which interrupt the synthesis of endotoxin, bind and neutralise its activity, prevent endotoxin interactions with host effector cells and interfere with endotoxin-mediated signal transduction pathways. Potential therapeutic strategies involving these agents consist of endotoxin analogues, antibodies, subunit vaccines, binding columns, recombinant human proteins and small molecule inhibitors of endotoxin synthesis and intracellular signalling. The pitfalls of previous antiendotoxin clinical investigations and the perils of future clinical trial designs are discussed in the context of unmet needs and realistic expectations for success. While considerable progress has been made, effective and new treatments for Gram-negative bacterial sepsis continues to elude us at the present time. This has been to the detriment of patients, investigators and pharmaceutical companies alike. It will require focused efforts by basic scientists, continued support by industry and enlightened study designs by clinical investigators to successfully develop antiendotoxin in therapies for use in septic patients in the future.

Topics: Anti-Bacterial Agents; Antibodies; Antigen-Antibody Complex; Antimicrobial Cationic Peptides; Bacterial Vaccines; Blood Proteins; Endotoxemia; Endotoxins; Gram-Negative Bacteria; Humans; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Membrane Proteins; Recombinant Proteins; Shock, Septic

The activation of leukocytes by bacterial cell wall lipopolysaccharide (LPS) contributes to the pathogenesis of septic shock. It is well established that, in the presence of plasma LPS-binding protein (LBP), LPS binds with high affinity to CD14. The binding of LPS to CD14 has been associated with the activation of cells, although available evidence indicates that CD14 itself does not transduce intracellular signalling. The physiological function of this interaction is to promote host defense mechanisms of cells to combat the infection and clear LPS from the circulation. At higher concentrations of LPS, however, the activation of cells can take place in the absence of LBP and CD14, presumably through a distinct low-affinity signalling LPS receptor. On the evidence published by us and others, we propose that in neutrophils, and possibly other leukocytes, L-selectin can act as a low-affinity LPS receptor.

Topics: Animals; Bacteremia; Carbohydrate Sequence; Humans; L-Selectin; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Models, Biological; Molecular Sequence Data; Shock, Septic

Topics: Antibodies, Monoclonal; Bacterial Toxins; Cytokines; Endotoxins; Lipid A; Lipopolysaccharides; Shock, Septic

Topics: Anti-Bacterial Agents; Anti-Inflammatory Agents; Child, Preschool; Diagnosis, Differential; Female; Humans; Inflammation Mediators; Interleukins; Lipid A; Naloxone; Narcotic Antagonists; Nitric Oxide; Receptors, Immunologic; Shock, Septic; Tumor Necrosis Factor-alpha

Septic shock remains a major cause of morbidity and mortality, especially in the intensive care setting. A vast array of treatment strategies is under investigation; despite success in animal models, no effective adjunctive therapy has yet been approved for clinical use. This paper reviews the development of experimental therapies for sepsis and discusses those treatments that show promise for application in humans. Approaches to treatment fall into three broad categories: strategies directed against bacterial components, those directed against host-derived inflammatory mediators, and those designed to limit tissue damage. Because septic shock is a dynamic and evolving condition, different strategies may be needed at different stages in the pathogenesis of sepsis. Through carefully performed trials and thoughtful selection of combination therapy aimed at different points in the pathological process, it may be possible in the future to modify the course of this serious condition.

Topics: Acute-Phase Proteins; Animals; Antibodies, Bacterial; Bacteremia; Carrier Proteins; Combined Modality Therapy; Cytokines; Endotoxins; Humans; Lipid A; Lipopolysaccharides; Membrane Glycoproteins; Shock, Septic

Endotoxin (lipopolysaccharide, LPS), a constituent of the outer membrane of the cell wall of gram-negative bacteria, exerts a wide variety of biological effects in humans. This review focuses on the molecular mechanisms underlying these activities and discusses structure-function relationships of the endotoxin molecule, its interaction with humoral and cellular receptors involved in cell activation, and transmembrane and intracellular signal transduction pathways.

Topics: Animals; Carbohydrate Sequence; Endotoxins; Humans; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Membrane Proteins; Molecular Sequence Data; Shock, Septic; Signal Transduction

Topics: Animals; Bacterial Infections; Clinical Trials as Topic; Humans; Lipid A; Mice; Safety; Sepsis; Shock, Septic; Structure-Activity Relationship

The literature concerning human responses to bacterial endotoxin, in particular to the purified lipopolysaccharide derivative, has been critically reviewed and summarized. Papers selected for review are those that reported studies of human subjects in a normal state of health administered defined doses of either intravenous or aerosol inhaled material. Emphasis was placed on cardiovascular, pulmonary, metabolic, and inflammatory parameters. The information detailed here can be applied to understanding the pathophysiology of human consequences to spontaneous, clinical diseases induced by this highly inflammatory and ubiquitous substance.

Topics: Adrenocorticotropic Hormone; Aerosols; Animals; Asthma; Blood Coagulation; Bronchoalveolar Lavage Fluid; Cytokines; Endotoxins; Hemodynamics; Humans; Infusions, Intravenous; Interleukin-1; Lipid A; Lipopolysaccharides; Lung; Lymphocyte Subsets; Occupational Diseases; Permeability; Respiration; Shock, Septic; Sleep; Tumor Necrosis Factor-alpha

Gram-negative shock is thought to result primarily from the effects of endotoxin, a component of the bacterial outer membrane. Accordingly, therapies aimed at inhibiting, neutralizing, or clearing endotoxin have been extensively explored. Despite over 30 years of research, no antiendotoxin approach to the treatment of human septic shock is of proven benefit. In recent randomized clinical trials of monoclonal antibodies against endotoxin, therapeutic efficacy was not convincingly demonstrated. This result, however, does not eliminate the possibility that other antiendotoxin therapies may be effective. The antibodies used in these clinical trials do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. Newer agents with well-defined mechanisms of antiendotoxin activity may help clarify the role of endotoxin in septic shock and prove useful therapy for some patients.

Topics: Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Endotoxins; Humans; Lipid A; Polymyxin B; Protein Binding; Shock, Septic

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antimicrobial Cationic Peptides; Bacteremia; Blood Proteins; Cell Adhesion Molecules; Child; Cytokines; Endotoxins; Genetic Therapy; Humans; Immunotherapy, Adoptive; Lipid A; Membrane Proteins; Multiple Organ Failure; Neutrophils; Shock, Septic

Topics: Animals; Antibodies; Bacterial Toxins; Carbohydrate Sequence; Clinical Trials as Topic; Endotoxins; Hemodynamics; Humans; Lipid A; Lipopolysaccharides; Molecular Sequence Data; Molecular Structure; Shock, Septic

Topics: Antibodies, Bacterial; Antigens, Bacterial; Antitoxins; Bacterial Infections; Blood Donors; Carbohydrate Sequence; Endotoxins; Enzyme-Linked Immunosorbent Assay; Humans; Immunotherapy; Lipid A; Lipopolysaccharides; Molecular Sequence Data; Pseudomonas aeruginosa; Shock, Septic; Tumor Necrosis Factor-alpha

Topics: Cell Wall; Endotoxins; Escherichia coli; Gram-Negative Bacteria; Humans; Limulus Test; Lipid A; Lipopolysaccharides; Molecular Sequence Data; Salmonella; Shock, Septic; Tumor Necrosis Factor-alpha

Topics: Cardiovascular System; Endotoxins; Humans; Lipid A; Shock, Septic

Topics: Acute Kidney Injury; Animals; Anti-Bacterial Agents; Disease Models, Animal; Endotoxins; Glomerular Filtration Rate; Glycerol; Humans; Kidney; Lipid A; Renal Circulation; Shock, Septic; Vasoconstriction

Topics: Animals; Bacterial Toxins; Cobalt Radioisotopes; Endotoxins; Humans; Immunity, Innate; Lipid A; Neoplasms; Shock, Septic

Topics: Antibodies, Bacterial; Antibodies, Monoclonal; Clinical Trials as Topic; Endotoxins; Gram-Negative Bacteria; Humans; Immunization; Lipid A; Lipopolysaccharides; Sepsis; Shock, Septic

The protective effect of high-titer anti-lipid A hyperimmune globulin with respect to the course of the disease and the mortality rate was studied in patients with septicemia verified by positive blood cultures. Six patients were treated with anti-lipid A in an open study. Dramatic improvement in fever curves and clinical condition in some of the patients encouraged us to start a randomized double blind study. So far, 17 patients have entered the study, 16 of whom were evaluable. Immediately after a positive blood culture was found, patients received either high doses of anti-lipid A or placebo (saline solution) on two subsequent days. Before and after each infusion blood samples were taken in order to assess serum bactericidal activity and anti-lipid A titers. Because of the still small numbers of patients the results of both studies were summarized. In all patients treated with anti-lipid A clear-cut increases in anti-lipid A titers were shown. Patients with repeated gram-negative infections showed higher median anti-lipid A titers than patients without such a history. The patients treated with anti-lipid A immune globulin ran a significantly milder course than the placebo group. The severe signs of septic shock were reversed in seven of 15 patients on anti-lipid A compared to two of seven patients treated with placebo. In the anti-lipid A-treated group, three of 15 patients died, and in the placebo group two of seven. This difference is not statistically significant.

Topics: Antibody Formation; Antitoxins; Clinical Trials as Topic; Double-Blind Method; Humans; Immunoglobulin G; Lipid A; Random Allocation; Shock, Septic

Endotoxin research in recent years at the molecular level has required chemically synthesized lipid A without contamination by other bioactive components. Total synthesis of Escherichia coli-type lipid A was achieved in the 1980s by the challenging spirits of the scientists at Osaka University, Japan. They clarified the role of lipid A in the immunological activities of endotoxin in collaboration with Japanese and German researchers, based on the friendships that existed between them. This article introduces the great contributions made by three generations of professors, Tetsuo Shiba, Shoichi Kusumoto, and Koichi Fukase, at the Laboratory of Natural Product Chemistry at Osaka University, to the study over four decades of endotoxin.

Topics: Chemistry, Organic; Escherichia coli; History, 20th Century; History, 21st Century; International Cooperation; Japan; Lipid A; Shock, Septic

Monophosphoryl lipid A (MPLA), a nontoxic TLR4 ligand derived from lipopolysaccharide (LPS), is used clinically as an adjuvant in cancer, hepatitis, and malaria vaccines and in allergen-specific immunotherapy. Nevertheless, its cell-activating effects have not been analyzed in a comprehensive direct comparison including a wide range of different immune cells. Therefore, the objective of this study was the side-by-side comparison of the immune-modulating properties of MPLA and LPS on different immune cells.. Immune-activating properties of MPLA and LPS were compared in human monocytes and mast cells (MCs), a mouse endotoxin shock model (ESM), and mouse bone marrow (BM)-derived myeloid dendritic cells (mDCs), T cells (TCs), B cells, and MCs.. In a mouse in vivo ESM and a human ex vivo monocyte activation test (MAT), MPLA induced the same cytokine secretion pattern as LPS (ESM: IL-6, IL-12, TNF-α; MAT: IL-1β, IL-6, TNF-α), albeit at lower levels. Mouse mDCs and ex vivo isolated B cells stimulated with MPLA required a higher threshold to induce TRIF-dependent cytokine secretion (IL-1β, IL-6, IL-10, and TNF-α) than did LPS-stimulated cells. In mDC:DO11.10 CD4 TC cocultures, stimulation with MPLA, but not with LPS, resulted in enhanced OVA-specific IL-4 and IL-5 secretion from DO11.10 CD4 TCs. Unexpectedly, in both human and mouse MCs, MPLA, unlike LPS, did not elicit secretion of pro-inflammatory cytokines.. Compared to LPS, MPLA induced a qualitatively similar, but less potent pro-inflammatory immune response, but was unable to activate human or mouse MCs.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Antigens; B-Lymphocytes; Cytokines; Dendritic Cells; Disease Models, Animal; Epithelial Cells; Humans; Inflammation Mediators; Lipid A; Lipopolysaccharides; Mast Cells; Mice; Mice, Knockout; Monocytes; Shock, Septic; Th2 Cells

Preconditioning with low doses of monophosphoryl lipid A (MPL) has been shown to induce endotoxin tolerance and to reduce the metabolic and hemodynamic consequences of endotoxin shock. However, no data are available about the effects of endotoxin preconditioning on cerebral metabolism during endotoxemia. The study was designed to determine the effects of endotoxin preconditioning with MPL on cerebral metabolism via microdialysis compared to muscle tissue metabolism during experimental endotoxemia.. In a controlled animal study, continuous endotoxin infusion (1μg/kg b.w. per h) was administrated to 7 female mixed-breed pigs after pretreatment with MPL in incremental doses of endotoxin during days 5-2 before the experiments. In the control group, 7 animals received a saline pretreatment. In addition to hemodynamic monitoring and blood gas analyses, interstitial lactate, pyruvate, glucose and glycerol concentrations in muscle and cerebral tissue were measured using in vivo microdialysis.. There were no significant differences between the two groups with respect to hemodynamic parameters, while mixed venous oxygen saturation (SvO2), arterial blood pH and mean pulmonary arterial pressure (MPAP) were significantly higher in the preconditioned group. Cerebral perfusion pressure (CPP) and brain tissue oxygen pressure (ptiO2) values stayed stable throughout the experiment with no inter-group differences. While interstitial concentrations of lactate and glycerol as well as the lactate/pyruvate (LP) and the lactate/glucose (LG) ratio in muscle tissues were significantly increased in control animals compared to those who had been pretreated with MPL; the results of cerebral microdialysis showed no significant changes in interstitial lactate or glycerol levels in both groups. However, the lactate/glucose (LG) ratio in the control group showed a significantly higher increase than in the preconditioned group.. Preconditioning with low doses of MPL ameliorates the negative metabolic effects of endotoxin shock in muscle tissue. With regard to cerebral metabolism, the present study suggests that MLP preconditioning provides moderate advantages, at least in an experimental model of endotoxin shock.

Topics: Animals; Cerebrum; Disease Models, Animal; Endotoxemia; Endotoxins; Female; Lipid A; Microdialysis; Shock, Septic; Swine

Topics: Animals; Caspases; Caspases, Initiator; Immunity, Innate; Inflammasomes; Lipid A; Macrophages; Shock, Septic; Toll-Like Receptor 4

Inflammatory caspases, such as caspase-1 and -11, mediate innate immune detection of pathogens. Caspase-11 induces pyroptosis, a form of programmed cell death, and specifically defends against bacterial pathogens that invade the cytosol. During endotoxemia, however, excessive caspase-11 activation causes shock. We report that contamination of the cytoplasm by lipopolysaccharide (LPS) is the signal that triggers caspase-11 activation in mice. Specifically, caspase-11 responds to penta- and hexa-acylated lipid A, whereas tetra-acylated lipid A is not detected, providing a mechanism of evasion for cytosol-invasive Francisella. Priming the caspase-11 pathway in vivo resulted in extreme sensitivity to subsequent LPS challenge in both wild-type and Tlr4-deficient mice, whereas Casp11-deficient mice were relatively resistant. Together, our data reveal a new pathway for detecting cytoplasmic LPS.

Topics: Animals; Apoptosis Regulatory Proteins; Calcium-Binding Proteins; Caspases; Caspases, Initiator; Cross-Priming; Enzyme Activation; Francisella; Gram-Negative Bacterial Infections; Lipid A; Mice; Mice, Inbred C57BL; Poly I-C; Salmonella; Salmonella Infections; Shock, Septic; Toll-Like Receptor 4

Topics: Animals; Caspases; Immunity, Innate; Inflammasomes; Lipid A; Macrophages; Shock, Septic; Toll-Like Receptor 4

New glycolipids and a benzylammonium lipid were rationally designed by varying the chemical structure of a D-glucose-derived hit compound active as lipid A antagonist. We report the synthesis of these compounds, their in vitro activity as lipid A antagonists on HEK cells, and the capacity to inhibit LPS-induced septic shock in vivo. The lack of toxicity and the good in vivo activity suggest the use of some compounds of the panel as hits for antisepsis drug development.

Topics: Anti-Infective Agents; Benzylammonium Compounds; Cell Line; Drug Design; Glycolipids; Humans; Lipid A; Lipids; Sepsis; Shock, Septic; Structure-Activity Relationship

Galectin-3 is a beta-galactoside-binding lectin that plays an important role in inflammatory diseases. It also interacts with the surface carbohydrates of many pathogens, including LPS. However, its role in infection is not fully understood. Data presented herein demonstrate for the first time that galectin-3 is a negative regulator of LPS-induced inflammation. Galectin-3 is constitutively produced by macrophages and directly binds to LPS. Galectin-3-deficient macrophages had markedly elevated LPS-induced signaling and inflammatory cytokine production compared with wild-type cells, which was specifically inhibited by the addition of recombinant galectin-3 protein. In contrast, blocking galectin-3 binding sites by using a neutralizing Ab or its ligand, beta-lactose, enhanced LPS-induced inflammatory cytokine expression by wild-type macrophages. In vivo, mice lacking galectin-3 were more susceptible to LPS shock associated with excessive induction of inflammatory cytokines and NO production. However, these changes conferred greater resistance to Salmonella infection. Thus, galectin-3 is a previously unrecognized, naturally occurring, negative regulator of LPS function, which protects the host from endotoxin shock but, conversely, favors Salmonella survival.

Topics: Animals; Cells, Cultured; Down-Regulation; Female; Galectin 3; Gene Expression Regulation; Immunity, Innate; Inflammation Mediators; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Salmonella Infections, Animal; Shock, Septic; Toll-Like Receptor 4

Lipopolysaccharide (LPS) endotoxin is the bacterial product responsible for the clinical syndrome of Gram-negative septicemia and endotoxic shock. During sepsis, microbial antigens, such as LPS, activate monocytes and macrophages to produce several pro-inflammatory cytokines, among which tumor necrosis factor-alpha (TNF-alpha) appears to be very important for the development of endotoxic shock. The endotoxic properties of LPS principally reside in the lipid A (LIP A) component, which is the primary immunostimulatory center of Gram-negative bacteria. In recent years there has been a continuous effort to identify molecules able to antagonize the deleterious effects of endotoxic shock. In this study we show that a novel LIP A fraction from the LPS of Halomonas magadiensis (Hm), a Gram-negative extremophilic and alkaliphilic bacterium, significantly inhibits the synthesis of TNF-alpha by human monocytes activated by Escherichia coli LPS. LIP A from Hm exerts these effects by interfering with E. coli LPS for activation of Toll-like receptor 4 expressed in human cells. This result defines Hm LIP A as a novel class of LPS antagonist whose structural features could be utilized for the design of compounds for the treatment of Gram-negative sepsis.

Topics: Cell Line; Drug Antagonism; Escherichia coli; Halomonas; Humans; Lipid A; Macrophage Activation; Monocytes; Shock, Septic; Species Specificity; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

A novel nontoxic lipopolysaccharide (LPS) was purified from Acidiphilium strain GS18h/ATCC55963. The chemical composition of the lipid A part of this LPS is distinctly different from that of known lipid A molecules. The LPS was investigated to determine its capacity to provide protection against toxic LPS or endotoxic shock, as has been reported for other nontoxic LPSs (Rhodobacter sphaeroides and Rhodobacter capsulatus), and also the extent and type of immunomodulatory response in terms of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-beta), and IL-6 release as well as NO secretion by stimulated monocyte-macrophage systems. This study demonstrates clearly that mice immunized or primed with this LPS are fully protected against challenge with toxic Escherichia coli LPS. Unlike most of the extensively studied nontoxic LPSs, this LPS induced reactive nitrogen intermediates and released TNF-alpha, IL-beta and IL-6 in both mouse and human monocyte-macrophage systems. However, the extent of the cytokine and lymphokine releasing response was well below the range of the toxic LPS, for example that of E. coli. Owing to its capacity to provide immunostimulation of the host without causing any lethality to ensure protection against endotoxic shock, this LPS appears to have potential therapeutic value.

Topics: Acidiphilium; Animals; Copper; Escherichia coli; Immune Tolerance; Interleukins; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mining; Shock, Septic; Soil Microbiology; Tumor Necrosis Factor-alpha

E5564, a lipid A analogue, is a potent antagonist of lipopolysaccharide (LPS). Clinically, E5564 was developed as a possible therapy for treatment of sepsis and septic shock. Surface plasmon resonance (SPR) analysis indicates that E5564 binds to LPS binding protein (LBP), in a manner similar to LPS. Gel-filtration radioactive chromatograms of [(14)C]-E5564 in plasma revealed that E5564 initially distributes to the lipoprotein fractions, separated from high-density lipoprotein (HDL); the bound fraction is then released and binds to HDL. Similar results were obtained by heparin-manganese precipitation. At doses of E5564 relevant to its clinical use (i.e. 6 microg/ml), antibodies against LBP did not influence either the distribution of E5564 to non-HDL lipoprotein fractions or the transfer of E5564 from non-HDLs to HDL. Under these conditions, transfer of E5564 to HDL occurs similarly in the plasma of LBP knockout (KO) mice as in the plasma from wild-type mice. In addition, plasma clearance of E5564 in LBP KO mice is similar to that of wildtype mice. Thus, LBP binds E5564 in a manner similar to LPS, but does not play a role in E5564 redistribution/binding to lipoprotein and plasma clearance.

Topics: Acute-Phase Proteins; Animals; Antibody Formation; Anticoagulants; Carrier Proteins; Chemical Precipitation; Drug Interactions; Female; Heparin; Lipid A; Lipopolysaccharides; Manganese; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Shock, Septic

Alpha-D-glucopyranose,3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl]-6-O-methyl-2-[[(11Z)-1-oxo-11-octadecenyl]amino]-4-O-phosphono-beta-D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-1-(dihydrogen phosphate), tetrasodium salt (E5564) is a second-generation synthetic lipodisaccharide designed to antagonize the toxic effects of endotoxin, a major immunostimulatory component of the outer cell membrane of Gram negative bacteria. In vitro, E5564 dose dependently (nanomolar concentrations) inhibited lipopolysaccharide (LPS)-mediated activation of primary cultures of human myeloid cells and mouse tissue culture macrophage cell lines as well as human or animal whole blood as measured by production of tumor necrosis factor-alpha and other cytokines. E5564 also blocked the ability of Gram negative bacteria to stimulate human cytokine production in whole blood. In vivo, E5564 blocked induction of LPS-induced cytokines and LPS or bacterial-induced lethality in primed mice. E5564 was devoid of agonistic activity when tested both in vitro and in vivo and has no antagonistic activity against Gram positive-mediated cellular activation at concentrations up to 1 microM. E5564 blocked LPS-mediated activation of nuclear factor-kappaB in toll-like receptor 4/MD-2-transfected cells. In a mouse macrophage cell line, activity of E5564 was independent of serum, suggesting that E5564 exerts its activity through the cell surface receptor(s) for LPS, without the need for serum LPS transfer proteins. Similar to (6-O-[2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-endoyloxydecl]-2-[3-oxo-tetradecanoylamino]-beta-O-phosphono-alpha-D-glucopyranose tetrasodium salt (E5531), another lipid A-like antagonist, E5564 associates with plasma lipoproteins, causing low concentrations of E5564 to be quantitatively inactivated in a dose- and time-dependent manner. However, compared with E5531, E5564 is a more potent inhibitor of cytokine generation, and higher doses retain activity for durations likely sufficient to permit clinical application. These results indicate that E5564 is a potent antagonist of LPS and lacks agonistic activity in human and animal model systems, making it a potentially effective therapeutic agent for treatment of disease states caused by endotoxin.

Topics: Animals; Blood; Cells, Cultured; Cytokines; Disease Models, Animal; Drosophila Proteins; Drug Interactions; Endotoxins; Escherichia coli; Guinea Pigs; Humans; Lipid A; Lipopolysaccharides; Macrophages; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Monocytes; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface; Shock, Septic; Time Factors; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Preconditioning pigs with low doses of monophosphoryl lipid A (MPL), a non toxic derivate of lipid A, has been shown to induce endotoxin hyporesponsiveness and to reduce the metabolic and hemodynamic consequences of endotoxin shock. However, the mechanism is presently unclear. This study was designed to elucidate the effects of pretreatment with MPL on tissue metabolism in different organs by in vivo microdialysis of interstitial fluid.. In a controlled animal study at the university research laboratory, seven female mixed-breed pigs were exposed to an endotoxin infusion (1 microg/kg b.w. per h) after pretreatment with MPL in incremental doses of endotoxin during days 5-2 before the experiments. Seven animals receiving a saline pretreatment served as a control group. Hemodynamic variables and blood gas analyses including blood lactate were determined every 30 min until the animals died. Interstitial lactate and glycerol levels were measured in muscle, subcutaneous tissue and liver using in vivo microdialysis.. Survival time was significantly prolonged after MPL preconditioning (8.95 (7.5-9.1) h vs. 5.35 (5.0-5.6) h, P<0.05). Hemodynamic parameters were not significantly different between the treatment and control groups, while mixed venous saturation (81% (70-93%) vs. 30% (22-48%)) and arterial blood pH (7.39 (7.33-7.44) vs. 7.21 (7.1-7.25)) and pO(2) were significantly higher in the preconditioned group (P<0.05). The interstitial concentrations of lactate and glycerol in all investigated tissues were significantly higher in control animals than the those who had been pretreated with MPL (P<0.05).. Preconditioning with low doses of monosphosphoryl lipid A attenuates the negative effects of endotoxemia on tissue metabolism, probably by reducing O(2)-consumption. These changes may be subtle and, hence, only fully detectable by monitoring tissue metabolism.

Topics: Animals; Endotoxins; Female; Glycerol; Lactic Acid; Lipid A; Liver; Microdialysis; Muscle, Skeletal; Shock, Septic; Subcutaneous Tissue; Swine; Time Factors

Topics: Animals; Antibodies; Diphosphonates; Escherichia coli; Glycoconjugates; Glycolipids; Hemocyanins; Lipid A; Mice; Molecular Mimicry; Shock, Septic; Tumor Necrosis Factor-alpha; Vaccination; Vaccines, Synthetic

Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signaling in a variety of cell types. MD-2 is associated with the extracellular domain of TLR4 and augments TLR4-dependent LPS responses in vitro. We show here that MD-2(-/-) mice do not respond to LPS, do survive endotoxic shock but are susceptible to Salmonella typhimurium infection. We found that in MD-2(-/-) embryonic fibroblasts, TLR4 was not able to reach the plasma membrane and predominantly resided in the Golgi apparatus, whereas TLR4 was distributed at the leading edge surface of cells in wild-type embryonic fibroblasts. Thus, MD-2 is essential for correct intracellular distribution and LPS-recognition of TLR4.

Topics: Animals; Antigens, Surface; B-Lymphocytes; Cells, Cultured; Dendritic Cells; Disease Models, Animal; Drosophila Proteins; Female; Interleukin-12; Interleukin-6; Intracellular Fluid; Lipid A; Lipopolysaccharide Receptors; Lymphocyte Antigen 96; Macrophages; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptors, Cell Surface; Salmonella Infections; Salmonella typhimurium; Shock, Septic; Spleen; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Toll-like receptors 2 and 4 (TLR2 and TLR4) have been found to transduce signals of peptidoglycan (PGN) and lipopolysaccharide (LPS), respectively, for NF-kappa B activation. However, little is known about the expression and regulation of the TLR2 gene in monocytes/macrophages in response to the two typical bacterial products. We show in the present study that both PGN and a high concentration of LPS increase TLR2 gene expression in macrophage-like cells, 1 alpha,25-dihydroxyvitamin D(3)-differentiated human HL60 and mouse RAW264.7 cells, and human monocytes in a dose- and time-dependent manner. Actinomycin D and pyrrolidine dithiocarbamate inhibition of gene transcription and NF-kappa B activation, respectively, blocks LPS- and PGN-elevated TLR2 mRNA in monocytic cells. The LPS-induced increase in TLR2 mRNA in monocytic cells is abolished by polymyxin B pretreatment and is observed in peripheral blood mononuclear cells from pigs subjected to endotoxic shock. Further, high concentrations of LPS and synthetic lipid A increase TLR2 mRNA expression in peritoneal macrophages from both TLR4-deficient C3H/HeJ mice and normal C3H/HeN mice, a process that constitutes induction of TLR4-independent TLR2 expression. These findings demonstrate that TLR2 gene expression is upregulated in macrophage responses to PGN and to high concentrations of LPS in vitro and in vivo and correlates with NF-kappa B activation.

Topics: Animals; Drosophila Proteins; Gene Expression Regulation; HL-60 Cells; Humans; Lipid A; Lipopolysaccharides; Macrophages; Membrane Glycoproteins; Mice; Mice, Inbred C3H; NF-kappa B; Peptidoglycan; Receptors, Cell Surface; Shock, Septic; Swine; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors; Transcription, Genetic; Up-Regulation

Porphyromonas gingivalis strain 381 lipid A showed lower activity in inducing interleukin (IL)-1alpha and IL-1beta production and cytokine mRNA expression than synthetic Escherichia coli lipid A (compound 506) in alveolar macrophages of C57BL/6 mice. Both the lipid As induced tumor necrosis factor alpha in alveolar macrophages and IL-6 in peritoneal macrophages. A calmodulin (CaM) antagonist, W-7, inhibited IL-1beta production and its mRNA expression induced by P. gingivalis lipid A but not compound 506 in alveolar macrophages. A CaM kinase activator reduced the induction of IL-1beta in the serum of mice when administered with compound 506, and protected the mice against the lethal toxicity. The modulation of a variety of intracellular enzymes including the CaM kinase may result in clinical control of endotoxic sepsis.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cytokines; Endotoxins; Enzyme Activators; Enzyme Inhibitors; Escherichia coli; Ionophores; Lipid A; Macrophage Activation; Macrophages; Male; Mice; Mice, Inbred C57BL; Porphyromonas gingivalis; Protein Kinase C; Shock, Septic; Sulfonamides

Mortality due to gram-negative septic shock remains high despite advances in medical care. Induction of endotoxin tolerance might be a new treatment strategy to prevent septic shock in the newborn. The present study was performed to show that an injection in pregnant rats of monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS), induces tolerance to Salmonella enteritidis LPS and tumor necrosis factor alpha (TNF-alpha) in their offspring. MPL at a dose of 2 mg/kg was injected into pregnant rats on the 19th day of gestation. Their 0-day-old offspring later received an intraperitoneal injection of S. enteritidis LPS or TNF-alpha. Newborn rats of MPL-treated dams exhibited a higher survival rate, absence of lactacidemia and lower plasma TNF-alpha concentration in response to S. enteritidis LPS when compared to the newborn rats of saline-treated dams. Newborn rats of MPL-treated dams were more tolerant to TNF-alpha than those of saline-treated dams. MPL injection into pregnant rats did not increase plasma endotoxin concentration in the fetuses, suggesting no placental passage took place, but it did increase plasma TNF-alpha concentration. We concluded that an injection of MPL into pregnant rats induced tolerance to LPS in their offspring, which might be due to TNF-alpha-induced TNF-alpha tolerance.

Topics: Adjuvants, Immunologic; Animals; Animals, Newborn; Endotoxins; Female; Fetal Blood; Interleukin-10; Lactic Acid; Lipid A; Maternal-Fetal Exchange; Pregnancy; Preventive Medicine; Rats; Rats, Sprague-Dawley; Shock, Septic; Tumor Necrosis Factor-alpha

Systemically administered tumor-targeted Salmonella has been developed as an anticancer agent, although its use could be limited by the potential induction of tumor necrosis factor alpha (TNFalpha)-mediated septic shock stimulated by lipid A. Genetic modifications of tumor-targeting Salmonella that alter lipid A and increase safety must, however, retain the useful properties of this bacteria. We report here that disruption of the Salmonella msbB gene reduces TNFalpha induction and increases the LD50 of this pathogenic bacteria by 10,000-fold. Notwithstanding this enormous difference, Salmonella retains its tumor-targeting properties, exhibiting tumor accumulation ratios in excess of 1000:1 compared with normal tissues. Administration of this bacteria to mice bearing melanoma results in tumors that are less than 6% the size of tumors in untreated controls at day 18. Thus, the antitumor activity previously demonstrated using tumor-targeting Salmonella with normal lipid A is retained. Lipid modification of tumor-specific bacterial vectors provides a means for reducing septic shock and further suggests that the antitumor activity of these bacteria may be independent of TNFalpha.

Topics: Acyltransferases; Animals; Bacterial Proteins; Cell Survival; Escherichia coli Proteins; Humans; Lipid A; Liver; Macrophages; Melanoma, Experimental; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Respiration; Salmonella; Salmonella Infections, Animal; Sequence Deletion; Shock, Septic; Skin Neoplasms; Swine; Tumor Necrosis Factor-alpha; Virulence

MyD88 is a general adaptor protein that plays an important role in the Toll/IL-1 receptor family signalings. Recently, Toll-like receptors 2 and 4 (TLR2 and TLR4) have been suggested to be the signaling receptors for lipopolysaccharide (LPS). In this study, we demonstrate that MyD88 knockout mice lack the ability to respond to LPS as measured by shock response, B cell proliferative response, and secretion of cytokines by macrophages and embryonic fibroblasts. However, activation of neither NF-kappaB nor the mitogen-activated protein (MAP) kinase family is abolished in MyD88 knockout mice. These findings demonstrate that signaling via MyD88 is essential for LPS response, but the inability of MyD88 knockout mice to induce LPS-dependent gene expression cannot simply be attributed to lack of the activation of MAP kinases and NF-kappaB.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antigens, Differentiation; B-Lymphocytes; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cytokines; Drosophila Proteins; Enzyme Activation; Histocompatibility Antigens Class II; Immune Tolerance; Immunity, Innate; Interleukin-1 Receptor-Associated Kinases; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Lymphocyte Activation; Macrophages, Peritoneal; Membrane Glycoproteins; Mice; Mice, Knockout; Myeloid Differentiation Factor 88; Protein Biosynthesis; Protein Kinases; Proteinase Inhibitory Proteins, Secretory; Proteins; Receptors, Cell Surface; Receptors, Immunologic; Receptors, Interleukin-1; Shock, Septic; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors

The lipid A component of lipopolysaccharide (LPS) derived from Escherichia coli has been implicated as a significant mediator in the development of circulatory and metabolic dysfunction and lethality associated with sepsis. A synthetic peptide corresponding to amino acid residues 20 through 44 of the neutrophil-derived 37-kDa cationic antimicrobial protein (CAP37 P(20-44)) possesses lipid A binding characteristics which may be useful in attenuating in vivo responses induced during circumstances of endotoxemia, including sepsis. The E. coli LPS to be used in the in vivo study was shown to be attenuated by CAP37 P(20-44) in a dose-dependent manner in the in vitro reaction with Limulus amoebocyte lysate. Intravenous infusion of CAP37 P(20-44) (1.5 or 3.0 mg/kg of body weight) with E. coli LPS (250 microg/kg over 30 min) into conscious, unrestrained rats prevented LPS-induced hyperdynamic and hypodynamic circulatory shock, hyperlactacidemia, and leukopenia in a dose-related fashion. CAP37 P(20-44) (0.2, 1.0, and 5.0 mg/kg) administered intravenously to conscious, actinomycin D-sensitized rats following a lethal dose of LPS neutralized LPS toxicity, resulting in dose-dependent 7-day survival rates of 30, 50, and 80%, respectively. CAP37 P(20-44) (5.0 mg/kg) significantly inhibited the endotoxin-induced increase in circulating tumor necrosis factor alpha in sensitized rats. These data demonstrate that CAP37 P(20-44) has the capacity to abolish in vivo biological responses to LPS that are relevant to human sepsis and to significantly neutralize the toxicity of circulating E. coli LPS.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Blood Proteins; Carrier Proteins; Dose-Response Relationship, Drug; Endotoxemia; Leukopenia; Lipid A; Male; Molecular Sequence Data; Peptides; Rats; Rats, Sprague-Dawley; Shock, Septic; Tumor Necrosis Factor-alpha

Lipopolysaccharides from Gram-negative bacteria interact with the mammalian immune system to trigger a cascade of physiological events leading to a shock syndrome which results in the death in over 70% of cases of severe shock. It is known that the supramolecular structures of lipopolysaccharide aggregates are critical contributors to their biological activities. Despite this, the molecular basis for the formation if the regular hexagonal plates and arrays observed in lipopolysaccharide films and suspensions is unknown. Since these structures are two dimensional, it is unlikely that X-ray crystallographic methods will shed much light on their detailed structure. Knowing this structure is important since it is becoming increasingly likely that the insertion of the lipopolysaccharide hydrocarbon chains in the target host cell membrane may be involved in triggering host responses. This work describes the three-dimensional structure of the lipopolysaccharide lipid A moiety. The structure was obtained by a combination of molecular mechanics calculations and nuclear magnetic resonance spectroscopy. This involved calculation of the dihedral angle between the two glucosamine residues of the lipid A molecule from coupling constants and measuring critical interresidue NOE values. The study also takes into account information from X-ray powder diffraction and electron microscopy studies.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Escherichia coli; Humans; Lipid A; Lipopolysaccharides; Magnetic Resonance Spectroscopy; Microscopy, Electron; Models, Molecular; Molecular Sequence Data; Molecular Structure; Shock, Septic

Bacterial endotoxin (lipopolysaccharide [LPS]) causes severe damage to the host organism as a result of excessive release of inflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), from mononuclear phagocytes during gram-negative bacterial infection. We evaluated the ability of a novel synthetic lipid A analog with low endotoxicity, DT-5461, to antagonize LPS-induced IL-1 and TNF-alpha production in cells of monocyte/macrophage lineage and examined the protective effect of DT-5461 against lethal endotoxic shock in mice. The IL-1- or TNF-alpha-inducing activity of DT-5461 is 100,000 to 10,000 times less active than that of Escherichia coli LPS (EcLPS) or synthetic lipid A. DT-5461 significantly inhibited EcLPS-induced IL-1 and TNF-alpha release when murine peritoneal macrophages were incubated with DT-5461 2 h prior to EcLPS stimulation at the same concentration (1 microgram/ml). The antagonistic effect of DT-5461 on the production of IL-1 and TNF-alpha induced by EcLPS occurred in a concentration-dependent manner. DT-5461 also inhibited IL-1 and TNF-alpha induction when murine peritoneal macrophages were stimulated by LPS from Salmonella typhimurium or synthetic lipid A, as well as by EcLPS, but not by muramyl dipeptides. This indicated that DT-5461 specifically antagonized the action of LPS. DT-5461 also antagonized EcLPS-mediated activation of human peripheral blood monocytes. DT-5461 blocked the binding of fluorescein isothiocyanate-labelled LPS to murine peritoneal macrophages as well as it did the binding of EcLPS and synthetic lipid A, i.e., in a concentration-dependent fashion. Injection of DT-5461 2 h before EcLPS challenge prevented the production of serum IL-1 and TNF-alpha in D-galactosamine-treated mice. Furthermore, this treatment modality protected mice against LPS-induced lethal toxicity. This study suggests that DT-5461 possesses a potent LPS antagonistic effect and may be useful in a protective strategy against lethal endotoxemia caused by gram-negative bacterial infection.

Topics: Animals; Disaccharides; Female; Galactosamine; Humans; In Vitro Techniques; Interleukin-1; Lipid A; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Monocytes; Shock, Septic; Tumor Necrosis Factor-alpha

Lipopolysaccharide (LPS) is implicated in much of the pathophysiology associated with gram-negative septic shock. One approach to this serious clinical problem is to develop new drugs that antagonize the action of toxic LPS. A model system to study LPS action and test for potential antagonists is readily provided by LPS regulation of the kappa gene in the murine B-cell line 70Z/3. Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA) effectively blocked toxic LPS induction of kappa light-chain immunoglobulin expression in 70Z/3 cells. Induction of kappa expression by LPS is dependent on the activation of at least two transcription factors, Oct-2 and NF-kappa B. RsDPLA completely repressed the long-term activation of NF-kappa B observed after 24 h of Salmonella typhosa LPS treatment and antagonized activation of oct-2 mRNA expression. However, RsDPLA was not an inert competitor of LPS. RsDPLA alone strongly activated NF-kappa B binding activity by 30 min but not beyond 9 h of treatment. It also induced a small increase in oct-2 mRNA levels. RsDPLA is not simply a weak agonist; we found no graded increase in kappa expression with increasing RsDPLA concentrations up to 50 micrograms/ml. The NF-kappa B complexes activated by RsDPLA and S. typhosa LPS were both composed of the p50-p65 heterodimer. These results suggest that the physiological LPS receptor(s) on B cells transmits qualitatively different signals depending on the nature of the binding ligand and that the fatty acyl groups of LPS play an important role in activating signal transduction.

Topics: Animals; B-Lymphocytes; Base Sequence; DNA-Binding Proteins; Dose-Response Relationship, Drug; Gene Expression Regulation; Immunoglobulin kappa-Chains; Immunoglobulin M; Lipid A; Lipopolysaccharides; Lymphocyte Activation; Mice; Molecular Sequence Data; NF-kappa B; Octamer Transcription Factor-2; Rhodobacter sphaeroides; Salmonella typhi; Shock, Septic; Transcription Factors; Tumor Cells, Cultured

Topics: Bacteremia; Carbohydrate Sequence; Chemical Phenomena; Chemistry, Physical; Cytokines; Female; Humans; Lipid A; Lipopolysaccharides; Male; Meningitis, Meningococcal; Meningococcal Infections; Molecular Sequence Data; Molecular Structure; Neisseria meningitidis; Shock, Septic

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Arthropod Proteins; Binding Sites; Binding, Competitive; Humans; In Vitro Techniques; Invertebrate Hormones; Limulus Test; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Molecular Sequence Data; Protein Binding; Recombinant Proteins; Shock, Septic

1. Mabs with a high affinity for free lipid A do not bind when it is covalently linked, i.e. in the form of LPS. 2. Lipid A-binding Mabs may be divided into three categories: I. Monoreactive Mabs that bind to the hydrophillic backbone of lipid A II. Polyreactive Kdo Mabs III. Polyreactive Mabs that bind by hydrophobic interactions 3. rBPI23 binds either free or covalently linked lipid A.

Topics: Animals; Antibodies, Monoclonal; Antimicrobial Cationic Peptides; Binding Sites; Blood Proteins; Gram-Negative Bacterial Infections; Humans; In Vitro Techniques; Ligands; Lipid A; Lipopolysaccharides; Membrane Proteins; Protein Binding; Protein Folding; Recombinant Proteins; Shock, Septic

Lipid As from non-toxic bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides have been shown to antagonize the immunostimulatory effects of lipid A and LPS from pathogenic bacteria. We have biologically characterized a series of synthetic LPS antagonists including the proposed structures of the lipid A and R. sphaeroides containing fatty acid side chains ester-linked to the disaccharide backbone, as well as an analog of R. capsulatus lipid A containing ether-linked alkyloxy side chains (E5531). In vitro assays utilizing LPS-stimulated human monocytes or whole blood demonstrated that low nanomolar concentrations of E5531 inhibited cellular activation as indicated by decreased release of the cytokines TNF-a, and interleukins-1, 6, and 8. E5531 also inhibited LPS-induced release of cytokines and nitric oxide from murine macrophages. Synthetic antagonists at up to 100 microM were devoid of agonistic activity in murine and human in vitro systems. In vivo, E5531 blocked induction of TNF-a by LPS and reduced LPS-induced lethality in mice. These in vitro and in vivo results indicate that E5531 may have clinical therapeutic utility as an antagonist of endotoxin-mediated morbidity and mortality.

Topics: Animals; Carbohydrate Sequence; Disease Models, Animal; Endotoxins; Humans; In Vitro Techniques; Lipid A; Macrophages; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Monocytes; Nitric Oxide; Shock, Septic; Tumor Necrosis Factor-alpha

The ability of monophosphoryl lipid A (MLA) to provide prophylactic protection against septic shock was evaluated in a mouse model of induced endotoxin hypersensitivity. Treatments of hypersensitized animals with low doses of MLA attenuated endotoxin lethality and endotoxin-mediated liver damage. These effects were related to the ability of MLA to suppress accumulation of TNF-alpha and IFN-gamma in the bloodstream of animals. MLA treatments had only a modest effect in suppressing the accumulation of nitrate in the bloodstream. This implied that MLA did not suppress induction of macrophage and hepatocyte nitric oxide synthetases that contribute to antimicrobial defense and protect against endotoxin-mediated liver damage. The MLA treatments did not appear to compromise inflammatory defenses against local infection since locally recruited leukocytes remained responsive to endotoxin after hypersensitivity had been attenuated. In agreement with these findings, other studies have shown that the induction of endotoxin tolerance by MLA parallels the induction of resistance of animals to lethal challenges with either Gram negative or Gram positive bacteria. As predicted from preclinical studies, human trials of the clinical form of MLA (MPL-immunostimulant) have confirmed that MLA could attenuate systemic responses to endotoxin in normal volunteers, including the attenuation of blood cytokine accumulation and attenuation of symptomatic responses.

Topics: Adjuvants, Immunologic; Animals; Disease Models, Animal; Female; Humans; Interferon-gamma; Lipid A; Lipopolysaccharides; Liver; Macrophages, Peritoneal; Mice; Mice, Inbred ICR; Nitric Acid; Sepsis; Shock, Septic; Tumor Necrosis Factor-alpha

Lipopolysaccharide (LPS)-binding protein (LBP) has been shown to regulate the response of monocytes to LPS in vitro. In a previous study, polyclonal anti-LBP IgGs were found to protect D-galactosamine-sensitized mice against a lethal endotoxemic shock induced by a low challenge of LPS or lipid A when administered simultaneously with endotoxin. In the present study, we investigated the mode of action of these anti-LBP IgGs. In vitro, we demonstrated that they interfere with LPS binding to monocytes or polymorphonuclear cells in different ways: by the mere prevention of binding of LPS to LBP thus preventing the binding of LPS to CD14, or by reacting with LPS-LBP complexes thus mediating their binding to complement or Fc receptors on monocytes and on polymorphonuclear cells. In vivo, we demonstrated that anti-LBP IgGs afforded protection against lethal endotoxemic shock by one of two mechanisms. First, LBP blockade by pretreatment with anti-LBP IgG allowed protection against a low dose of LPS (100 ng). This protection occurred despite LPS levels in blood similar to those in control mice but in the absence of detectable tumor necrosis factor (TNF). This demonstrated that anti-LBP IgG could block the LBP-mediated TNF release upon LPS challenge. In contrast, anti-LBP IgG did not afford protection in mice not sensitized with D-galactosamine and challenged with high-dose LPS (1 mg), confirming that LPS at high concentrations could stimulate cells independently of the LBP pathway. Second, anti-LBP treatment administered simultaneously with LPS challenge protected mice against both low and high doses of LPS. Unlike after pretreatment with anti-LBP IgG, this protection was accompanied by a decrease of circulating LPS, suggesting that anti-LBP IgG in these conditions facilitated clearance of LPS probably by clearing LPS-LBP complexes. These data and the fact that LBP binds to all LPS through lipid A suggest that antibody directed to LBP could be a candidate for therapeutic strategies in endotoxemic shock.

Topics: Acute-Phase Proteins; Animals; Biomarkers; Carrier Proteins; Endotoxins; Female; Galactosamine; Immunoglobulin G; Lipid A; Lipopolysaccharides; Membrane Glycoproteins; Mice; Mice, Inbred Strains; Monocytes; Neutrophils; Shock, Septic; Tumor Necrosis Factor-alpha

The major objective of the present study was to determine the effects of a partial structure of the lipid A moiety of gram-negative lipopolysaccharide, monophosphoryl lipid A (MLA), on endotoxin-induced mortality and disseminated intravascular coagulation (DIC) in rats. A second objective was to examine the role of polymorphonuclear neutrophil invasion to visceral organs, including lung, liver, heart, and kidney in the pathogenesis of the compromised multiorgan function which occurs in endotoxic shock. Finally, a third aim was to determine if the potential protective effects of MLA might be mediated via inhibiting neutrophil invasion to various visceral organs. Male Sprague-Dawley rats (220-260 g) were fasted over night and used the following day. In control rats, endotoxin (S. abortus equi LPS, 15 mg/kg, i.v.) produced a 89% mortality at 48 hr following its administration, and gross pathological and laboratory signs of DIC at 3 hr after injection. The latter included increased serum fibrin(ogen) degradation products (FDP, 24.00 +/- 7.81 vs. 0 micrograms/ml, P < .05), prothrombin time (PT, 16.20 +/- 1.12 vs. 13.03 +/- 0.20 sec, P < .05), and activated partial thromboplastin time (APTT, 32.70 +/- 3.83 vs. 20.11 +/- 0.60 sec, P < .05), and decreased plasma fibrinogen (233.2 +/- 41.6 vs. 406.3 +/- 23.2 mg/dl, P < .05) as well as evidence of gross visceral hemorrhage. Pretreatment with MLA (5 mg/kg) for 24 hr produced a marked reduction in endotoxin-induced mortality at 48 hr (0% versus 89% in controls) and inhibited all of the manifestations of DIC produced by endotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Disseminated Intravascular Coagulation; Endotoxins; Lipid A; Male; Neutrophil Activation; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Shock, Septic

Protective effects of SDZ MRL 953, a monosaccharidic lipid A analog with a reduced toxicity, were investigated in models of experimental septic shock caused by injections of LPS, and inoculations of heat-killed or live bacteria. Female B6D2F1 mice were challenged with a combination of galactosamine (800 mg/kg) plus various doses of heat-killed isolates of Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, and Staphylococcus aureus or LPS from Salmonella abortus equi. In some experiments, isolates of living bacteria at sublethal inocula were also combined with galactosamine. More than 90% of the animals died within 24 hr when the challenge was performed either simultaneously with or up to 4 hr after an intraperitoneal administration of galactosamine. No death was observed when galactosamine was omitted or administered after the microbial or LPS challenge. Pretreatment of the animals with SDZ MRL 953 (1-10 mg/kg) rendered the animals resistant to the lethal effects of both bacterial and LPS challenge in a time- and dose-dependent manner. The levels of TNF-alpha in control mice rose to greater than 600 pg/ml 2 hr postbacterial or LPS challenge, but were below detection in animals pretreated with SDZ MRL 953. Protection against both the infection and the toxicity of heat-killed bacteria or LPS was also achieved when murine anti-TNF-alpha monoclonal antibody was administered prophylactically. Together, these data suggest that SDZ MRL 953 enhances the resistance of mice against the toxicity of heat-killed gram-negative bacteria and S. aureus, and attenuates host responses to living bacteria which may lead to irreversible shock and death.

Topics: Animals; Anti-Bacterial Agents; Bacteria; Bacterial Physiological Phenomena; Female; Galactosamine; Hot Temperature; Immune System; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Neutropenia; Shock, Septic; Survival Analysis; Tumor Necrosis Factor-alpha; Vaccines, Inactivated

To study the toxicology and pharmacology of the endotoxin-neutralizing agent, bactericidal/permeability-increasing protein.. Prospective, randomized, placebo-controlled laboratory study.. Academic research laboratory.. CD-1 mice (n = 259); Sprague Dawley rats (n = 26); New Zealand White rabbits (n = 19).. Pharmacokinetics of intravenously injected bactericidal/permeability-increasing protein was assessed in mice. Toxicology was tested in mice and rats. Efficacy of intravenously administered bactericidal/permeability-increasing protein as an endotoxin-neutralizing agent was tested in mice, rats, and rabbits.. Administration of a single 10-mg/kg bolus injection of bactericidal/permeability-increasing protein resulted in no alterations in hematologic, renal, or hepatic function, activity level, or weight gain in animals observed over a 7-day study period. A single bolus injection (10 mg/kg) of bactericidal/permeability-increasing protein protected 15 of 16 mice from a lethal endotoxin challenge (mortality rate 1/16 [6.25%]) compared with a 100% (16/16) mortality rate in the saline-treated controls (p < .001). Bactericidal/permeability-increasing protein administered up to 1 hr after endotoxin provided significant protection against lethal endotoxin challenge. Furthermore, bactericidal/permeability-increasing protein reduced the induration and dermal necrosis observed in the localized dermal Shwartzman reaction.. Bactericidal/permeability-increasing protein is a potent antiendotoxin that neutralizes endotoxin in vivo and prevents mortality in animal models of lethal endotoxemia.

Topics: Animals; Antimicrobial Cationic Peptides; Blood Proteins; Endotoxins; Female; Humans; Injections, Intravenous; Lipid A; Membrane Proteins; Mice; Neutrophils; Rabbits; Random Allocation; Rats; Rats, Sprague-Dawley; Shock, Septic

Endotoxemia induced by gram-negative bacteria leads to endotoxic shock pathogenetically stemming from the integral component of the bacterial wall--lipid A. The study made to define the ability of lipid A monoclonal antibodies to correct hemodynamic disturbances due to endotoxemia in dog experiments showed the efficacy of the antibodies administration. ReLPS isolated from Salmonella Minnesota was used as an antigen. Administration of the complex monoclonal antibodies-endotoxin caused no hemodynamic impairment.

Topics: Animals; Antibodies, Monoclonal; Dogs; Drug Evaluation, Preclinical; Gram-Negative Bacterial Infections; Hemodynamics; Immunization; Immunoglobulin M; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Salmonella; Shock, Septic

Despite the use of modern broad spectrum antibiotics nosocomial infections are an unsolved problem, especially in the field of neonatal intensive care (preterm babies and newborns). In patients with septic shock human monoclonal antibodies in combination with appropriate antibiotics have proven effective and compatible for children older than one year. So far, there have been no reports in the literature on the application of such kind of antiendotoxin immunotherapy for pre-term babies and newborns. We describe the effectiveness of monoclonal antibodies in two newborns. Already 12 respectively 16 hours after application of the human monoclonal IgM antibodies (Centoxin) and appropriate antibiotics, the clinical condition of our patients stabilized. Consecutively further clinical symptoms improved rapidly.

Topics: Anti-Bacterial Agents; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibody Specificity; Combined Modality Therapy; Cross Infection; Female; Hirschsprung Disease; Humans; Infant, Newborn; Infant, Premature, Diseases; Lipid A; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Pseudomonas Infections; Shock, Septic; Surgical Wound Infection

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bacterial Infections; Cross Infection; Endotoxins; Humans; Immunization, Passive; Incidence; Lipid A; Shock, Septic

Early septic shock is characterized by fever, increased cardiac output, decreased systemic vascular resistance, and dilation of higher-order arterioles in peripheral tissues, such as skeletal muscle. We used a rat model of low-dose lipopolysaccharide (LPS) "septic" shock to investigate the potential benefit of an anti-lipid A monoclonal antibody preparation (E5) on macro- and microcirculatory function. Twenty-five male Sprague-Dawley rats were anesthetized and instrumented for measurement of arterial pressure (AP), heart rate (HR), and cardiac output (CO). The left cremaster muscle of each rat was prepared for in vivo video microscopic examination of changes in third-order arteriolar (A3) diameter and erythrocyte velocity. Rats were randomly assigned to two groups: Group I (n = 13) received E5 vehicle and 200 micrograms/kg Escherichia coli LPS; Group II (n = 12) received 2 mg/kg E5 iv prior to LPS administration. All variables were recorded at 15-min intervals for 30 min prior to and 150 min following LPS. Microcirculatory recordings were restricted to those rats where arteriolar diameters were 20-40 microns and vessels displayed obvious vasomotion (n = 7/group). Infusion of LPS caused no significant change in AP, an increase in CO by 105 min, an increase in HR by 75 min, an increase in diameter by 75 min, and a decrease in velocity by 165 min (P < 0.01). Pretreatment with E5 inhibited the A3 vasodilation but did not affect the macrocirculatory changes. These data suggest a potential therapeutic role for E5 in ameliorating LPS-induced changes in skeletal muscle microcirculation.

Topics: Animals; Antibodies, Monoclonal; Lipid A; Lipopolysaccharides; Male; Microcirculation; Rats; Rats, Sprague-Dawley; Shock, Septic

To examine the hemodynamic effects of diphosphoryl lipid A from Rhodopseudomonas sphaeroides and to examine the ability of this substance to induce tolerance to endotoxic shock.. Randomized, prospective, controlled study comparing the hemodynamic actions of R. sphaeroides diphosphoryl lipid A to those effects of lipopolysaccharide form Salmonella minnesota, followed by a prospective, randomized, controlled study comparing pretreatment with R. sphaeroides diphosphoryl lipid A and phosphate-buffered saline in the induction of tolerance to endotoxic shock.. Laboratory of the Section of Critical Care Medicine at a University Hospital.. Male Sprague-Dawley rats.. Eight rats were randomized to receive intravenous R. sphaeroides diphosphoryl lipid A, 0.5 mg/100 g body weight or S. minnesota lipopolysaccharide, 0.5 mg/100 g body weight. Ten rats were then randomized to receive R. sphaeroides diphosphoryl lipid A, 0.5 mg/100 g body weight, or phosphate-buffered saline intravenously 48 hrs before receiving S. minnesota lipopolysaccharide, 5 mg/100 g body weight, by intravenous infusion.. Cardiac index was significantly decreased from baseline in rats treated with lipopolysaccharide; there was no significant change in the R. sphaeroides diphosphoryl lipid A group. Peak circulating tumor necrosis factor (TNF) concentrations in the lipopolysaccharide-treated rats were higher than in R. sphaeroides diphosphoryl lipid A-treated rats (3.1 +/- 1.0 vs. 1.5 +/- 0.4 ng/mL). R. sphaeroides diphosphoryl lipid A significantly attenuated lipopolysaccharide-induced changes in mean arterial pressure and cardiac index. At baseline, there was no significant difference in serum TNF concentrations between rats pretreated with R. sphaeroides diphosphoryl lipid A and those rats pretreated with phosphate-buffered saline. TNF levels peaked at 1 hr post-lipopolysaccharide infusion at 4.3 +/- 0.6 ng/mL in the phosphate-buffered saline group and at 2.0 +/- 0.5 ng/mL in the R. sphaeroides diphosphoryl lipid A group (p < .02). Four of five rats pretreated with R. sphaeroides diphosphoryl lipid A survived endotoxic shock, whereas none of the phosphate-buffered saline-pretreated rats survived (p = .05).. These observations are consistent with previous reports of the limited toxic effects of R. sphaeroides diphosphoryl lipid A and suggest that this molecule retains the ability to induce tolerance to endotoxic shock.

Topics: Animals; Disease Models, Animal; Drug Evaluation, Preclinical; Gram-Negative Bacterial Infections; Hemodynamics; Infusions, Intravenous; Lipid A; Lipopolysaccharides; Male; Random Allocation; Rats; Rats, Sprague-Dawley; Rhodobacter sphaeroides; Salmonella; Shock, Septic; Survival Rate; Time Factors; Tumor Necrosis Factor-alpha

Three murine hybridomas secreting IgM monoclonal antibodies (MAbs) to lipid A (LA) of Salmonella minnesota R595 were generated. These MAbs serologically cross-reacted with LA and lipopolysaccharide (LPS) of unrelated gram-negative bacterial species. All three MAbs significantly suppressed the ability of LA and LPS from various gram-negative bacteria to induce tumor necrosis factor (TNF)-alpha (36%-67%) and interleukin-1 (30%-98%) in murine peritoneal macrophages and to stimulate B lymphocytes (37%-78%). Lipid A-induced TNF alpha production was also suppressed in mice (86%-88%). All three antibodies protected adrenalectomized mice against lethal shock induced by LA of S. minnesota R595. Optimal protection was achieved with one of the antibodies (MLA-1), if it was administered 2 h before injection of lipid A, and full protection persisted < or = 24 h. Moreover, MLA-1 was able to protect adrenalized or D(+)-galactosamine-sensitized mice against lethal shock induced by LPS derived from various gram-negative bacteria. This cross-protection could be predicted on the basis of serologic cross-reactivity and cross-neutralization by MLA-1 of the bioactivity of the heterologous LA or LPS in vitro.

Topics: Animals; Antibodies, Monoclonal; B-Lymphocytes; Cell Division; Cross Reactions; Cytokines; Female; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Neutralization Tests; Shock, Septic; Tumor Necrosis Factor-alpha

The sensitivity (LD100) of mice to lipopolysaccharide (LPS) endotoxin and to its toxic moiety, lipid A (LA), increased 500-fold after adrenalectomy (ADX). Inhibition of glucocorticoid synthesis in intact mice by metyrapone had a similar, though less dramatic, sensitizing effect to LPS. In ADX mice, the serum level of tumor necrosis factor-alpha (TNF) was 40-60 times higher than that in controls at 2 h after LPS/LA treatment. In intact mice the serum corticosterone level fell 1 h after lipid A injection to below detectable levels, which was followed by a brisk increase reaching the peak level of 48-50 micrograms/100 ml at 2 h. Both TNF production and the lethal effect of PLS/LA could be inhibited in ADX mice by glucocorticoid treatment. Plasma prolactin was increased significantly 1 h after endotoxin administration in both intact and ADX animals.

Topics: Adrenalectomy; Animals; Corticosterone; Dexamethasone; Disease Susceptibility; Dose-Response Relationship, Drug; Female; Lipid A; Lipopolysaccharides; Metyrapone; Mice; Mice, Inbred BALB C; Pituitary-Adrenal System; Prolactin; Shock, Septic; Tumor Necrosis Factor-alpha

Monophosphoryl lipid A (MPL) is a nontoxic lipid A derivative that maintains many of the beneficial immunomodulatory activities of the parent lipopolysaccharide molecule, including the induction of tolerance to endotoxin. The hemodynamic effects of Salmonella minnesota MPL (300 mg/kg) and S. minnesota lipopolysaccharide (300 micrograms/kg) were compared in 20 minipigs. Decreases in cardiac output and arterial pressure and increases in pulmonary artery pressure and lactic acidosis were significantly greater in animals treated with lipopolysaccharide. These changes were associated with peak tumor necrosis factor (TNF) levels of 1373 +/- 79 U/ml in animals treated with LPS and 157 +/- 31 U/ml in animals treated with MPL. Ten minipigs were subsequently randomized to receive S. minnesota MPL (30 micrograms/kg) or diluent intravenously 48 hours before receiving S. minnesota lipopolysaccharide (300 micrograms/kg IV). MPL significantly attenuated lipopolysaccharide-induced decreases in mean arterial pressure, cardiac index, stroke volume index, and mixed venous oxygen saturation. At baseline, no significant difference could be seen in TNF levels between diluent and MPL pigs. TNF levels peaked 2 hours after LPS infusion at 1190 +/- 156 U/ml in diluent pigs and at 539 +/- 126 U/ml in MPL pigs (p less than 0.05). Each of the pigs pretreated with MPL survived endotoxic shock, whereas only one of the five diluent pigs survived. These observations are consistent with the induction of endotoxin tolerance by pretreatment with MPL.

Topics: Animals; Blood Cell Count; Endotoxins; Hemodynamics; Lipid A; Salmonella; Shock, Septic; Swine; Swine, Miniature; Time Factors; Tumor Necrosis Factor-alpha

Monophosphoryl lipid A (MLA), a substructure of bacterial lipopolysaccharide (LPS), is being developed as a prophylactic for sepsis and septic shock. In the present study it was shown that MLA induced a rapid accumulation of IFN-gamma in mice that correlated with an in vivo priming of macrophages. Primed macrophages could be induced in vitro to synthesize nitric oxide, a key mediator of macrophage cytotoxicity. Due to its rapid clearance, MLA was not present in circulation at the time when IFN-gamma accumulated, suggesting that MLA could not synergize with IFN-gamma to systemically activate macrophages in vivo. MLA treatment tolerized mice against the IFN-gamma response--ie., treatment of mice with MLA on day 1 blocked LPS from inducing IFN-gamma on days 2-4. The significance of these results in relation to MLA's ability to enhance non-specific resistance and block LPS lethality in animals is discussed.

Topics: Animals; Cells, Cultured; Female; Interferon-gamma; Lipid A; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Mice, Inbred ICR; Nitrites; Salmonella; Sepsis; Shock, Septic

The newborn is very susceptible to gram-negative sepsis/septic shock. The mortality of newborn endotoxic shock continues to be high. Since lipid A is responsible for the toxic effects of lipopolysaccharide, anti-lipid A antibodies may prevent endotoxic shock in the newborn. This study showed that both anti-lipid A monoclonal IgG (A78S1) and anti-lipid A monoclonal IgM (A523) decreased the mortality of endotoxic shock in 10 day old rats. Prophylactic administration of A78S1 and A523 to the pregnant rat decreased the mortality of endotoxic shock in their 0-day-old offspring. Prophylaxis was due to transplacental passage of A78S1 treatment. The mechanism of prophylaxis remains unclear in A523 treatment.

Topics: Animals; Animals, Newborn; Antibodies, Monoclonal; Female; Immunization, Passive; Immunoglobulin G; Immunoglobulin M; Lipid A; Pregnancy; Rats; Rats, Inbred Strains; Shock, Septic

Monophosphoryl lipid A (MPL) is a nontoxic derivative of lipid A. In this study, the induction of tolerance by MPL to the hemodynamic effects of lethal endotoxemia was tested. Ten Sprague-Dawley rats were received either Salmonella minnesota MPL 0.5 mg/100 g intravenously (i.v.) or equivalent volume of diluent (control) i.v. on day zero. On day 3 S. minnesota endotoxin (LPS) 5.0 mg/100 g was administered i.v. Cardiac output (CO), arterial lactate (L), and central venous oxygen saturation (SvO2) were measured before and 3 and 6 hr after LPS administration. Survival was determined at 72 hr. At 6 hr, CO was 217 +/- 11 ml/kg/min in controls, and 435 +/- 28 ml/kg/min in the MPL animals (P less than 0.01). Arterial lactate was 2.6 +/- 0.3 mmol/liter in controls and 1.3 +/- 0.2 mmol/liter in MPL animals at 6 hr (P less than 0.05). The controls died 7.5 +/- 1.3 hr after LPS administration, whereas all the MPL-pretreated animals survived. These data indicate that MPL induces tolerance to the acute hemodynamic effects of LPS and enhances survival from lethal endotoxemia.

Topics: Animals; Cardiac Output; Hemodynamics; Lactates; Lactic Acid; Lipid A; Lipopolysaccharides; Oxygen; Rats; Rats, Inbred Strains; Salmonella; Shock, Septic

LPS is the major surface glycolipid on gram-negative bacteria. In this work, we have idiotypically characterized the antibody response against LPS in different species. To do this, we have produced mAb against LPS. Binding of many of these antibodies to LPS could be inhibited by LPS and lipid A, indicating that the monoclonals are specific for lipid A, the toxic moiety of the LPS molecule. One anti-lipid A antibody, IC9, proved protective against gram-negative bacteremia and endotoxic shock in murine protection models. We generated anti-idiotypic antibodies against IC9. The binding of several of these anti-Id to IC9 was specifically inhibited by lipid A. We used these anti-Id to characterize the anti-LPS response, and the results revealed that the IC9 Id is conserved in different species. The importance of an interspecies cross-reactive Id in the response to endotoxin and its relevance in vaccine development for septic shock are discussed.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibody Specificity; Bacterial Vaccines; Binding Sites, Antibody; Chickens; Cross Reactions; Immunization, Passive; Immunoglobulin Idiotypes; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred Strains; Rabbits; Salmonella Infections, Animal; Salmonella typhimurium; Shock, Septic

Topics: Animals; Antibodies, Monoclonal; Blood Pressure; Cardiovascular Diseases; Dogs; Enzyme-Linked Immunosorbent Assay; Hemodynamics; Immunoglobulin M; Iodine Radioisotopes; Lipid A; Mice; Mice, Inbred BALB C; Salmonella; Shock, Septic

Topics: Antibodies, Monoclonal; Enzyme-Linked Immunosorbent Assay; Humans; Immunodiffusion; Lipid A; Lipopolysaccharides; Shock, Septic

Monoclonal antibodies to the lipopolysaccharide (LPS) core region were produced by immunising mice with Escherichia coli strain J5 (chemotype Rc). One of these bound to the deepest part of the core, i.e., Lipid A, and reacted with other heat-killed but not live gram-negative bacilli, including E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Eight other monoclonal antibodies, binding to the terminal glucose residue of Rc LPS, reacted with live cells of E. coli strains only. Thus, the O antigen does not necessarily render the core inaccessible to antibody. However, despite binding to live bacteria, these monoclonal antibodies neither enhanced phagocytic killing, nor protected mice from dying from gram-negative infection or endotoxaemia. It is concluded that antibodies reacting with the most immunodominant parts of the J5 core are not protective.

Topics: Animals; Antibodies, Monoclonal; Antigens, Bacterial; Bacterial Infections; Dose-Response Relationship, Immunologic; Enzyme-Linked Immunosorbent Assay; Epitopes; Escherichia coli; Female; Gram-Negative Bacteria; Hybridomas; Lipid A; Lipopolysaccharides; Mice; Phagocytosis; Shock, Septic

The effect of the i.v. administration of endotoxin (LPS), diphosphoryl lipid A, and the non-toxic derivative monophosphoryl lipid A (MPL), on the production of serum cachectin (TNF), IFN-gamma, and the appearance of endotoxin shock have been measured in mice primed with Listeria monocytogenes. All three of the lipid A varieties induced the production of TNF in a dose-dependent manner. Although comparable peak levels of TNF were produced (5 X 10(5) to 1 X 10(6) U/ml), treatments with LPS or diphosphoryl lipid A were lethal whereas those with MPL were not. A study following TNF in the mouse sera for up to 24 h after treatment with the lipid A types showed that serum levels of TNF peaked 90 min after the treatment, and that TNF levels induced by LPS treatment were maintained for several hours longer than those induced by lipid A or MPL. All three molecules also resulted in the production of IFN-gamma in the serum, which peaked at 4 to 5 h after treatment. After 90 min there were no significant differences in the levels of serum IFN-gamma in any of the groups of treated animals. However, as was observed with the TNF, the levels of IFN in animals treated with LPS persisted longer than those induced by MPL or lipid A. These results suggest that the non-toxic MPL as well as the toxic forms of lipid A can induce the production of TNF by macrophages. Furthermore, although it is essential, TNF alone is not necessarily sufficient to induce septic shock in mice.

Topics: Adjuvants, Immunologic; Animals; Humans; Immune Sera; Injections, Intravenous; Interferon-gamma; Lethal Dose 50; Lipid A; Lipopolysaccharides; Listeria monocytogenes; Macrophages; Mice; Mice, Inbred C3H; Mice, Inbred ICR; Shock, Septic; Tumor Necrosis Factor-alpha

Topics: Humans; In Vitro Techniques; Interleukin-1; Lipid A; Lipopolysaccharides; Monocytes; Renal Dialysis; Shock, Septic

The effects of lipid X and 3-aza-lipid X on in vitro neutrophil function were related to their ability to inhibit the toxicity of endotoxin in galactosamine-sensitized mice. In vitro, lipid X and 3-aza-lipid X (100 ng/ml) blocked completely endotoxin (100 ng/ml)-enhanced neutrophil aggregation, superoxide anion generation, and release of beta-glucuronidase in response to a chemotactic tripeptide, f-met-leu-phe (10(-7) M). In vivo, lipid X at 250 micrograms/mouse (but not 3-aza-lipid X at a similar dose) protected groups of 10 mice from an otherwise lethal dose of endotoxin in galactosamine-sensitized mice when it was administered IV 4 hr or 2 hr before endotoxin challenge. The minimum effective dose of lipid X that could protect 50% of the challenged mice was calculated to be 715 micrograms/kg. However, lipid X failed to suppress neutrophil infiltration into the lungs. The ability of lipid X to inhibit endotoxin-induced neutrophil responses and to protect against lethal endotoxemia may be due to induction of early phase tolerance to endotoxin by the compound.

Topics: Animals; Cell Aggregation; Galactosamine; Glucuronidase; Glycolipids; Humans; In Vitro Techniques; Lipid A; Lipopolysaccharides; Lung Diseases; Male; Mice; Mice, Inbred C57BL; Neutrophils; Shock, Septic; Superoxides

The in vivo cardiovascular effect of intravenous administration of monophosphoryl lipid A (mp-lipid A) and diphosphoryl lipid A (dp-lipid A) in awake New Zealand white rabbits was investigated. Observed changes were evaluated in comparison to a control group and an endotoxin-treated group. Rabbits given lipid A showed a significant depression in cardiac index (p less than .025), mean arterial pressure (p less than .025, dp-lipid A only), arterial carbon dioxide tension (p less than .025), and total leukocyte count (p less than .05) compared to controls. Animals receiving lipid A tended to respond overall in a manner closely matching that of the endotoxin group. Dosages of lipid A given were approximately 3.5 times larger than the endotoxin dosages with respect to actual number of molecules administered (1.25-2.0 times larger by mass). These results indicate that lipid A is active in producing the cardiovascular and leukopenic effects characteristic of experimental septic shock.

Topics: Animals; Cardiac Output; Endotoxins; Female; Hemodynamics; Infusions, Intravenous; Lipid A; Rabbits; Shock, Septic

Lipid X, the major biosynthetic precursor of lipid A, has recently been described. Although lipid X is a mitogen and coagulates the Limulus amebocyte lysate, we found that it is not lethal for mice, even when given in large doses (2 X 10(6) micrograms/kg). Furthermore, lipid X was found to give partial protection against a 100% lethal dose of endotoxin, even if the lipid X was given as late as 6 h after endotoxin challenge.

Topics: Animals; Drug Administration Schedule; Endotoxins; Escherichia coli; Glycolipids; Lipid A; Mice; Shock, Septic; Structure-Activity Relationship

An early hypotensive phase was induced in rats by different strains of Escherichia coli and cell wall fractions to study the role of the bacterial surface structure, the complement system, histamine, and serotonin in induction of hypotension. E. coli strains with only core glycolipid (E. coli strain J5) or with intact lipopolysaccharide O antigens on their surface induced hypotension and thrombopenia within 5 min after intravenous administration. This response was reduced by prior decomplementation of the rats and by methysergide, a serotonin antagonist. Two K antigen-positive strains induced no hypotension except after removal of K antigen. The isolated lipopolysaccharide fractions and the lipid A subfractions, but not the polysaccharide subfractions, were also able to induce hypotension. Thus the core glycolipid structure, by interactions that involve platelets and the complement system, is mainly responsible for induction of an early hypotensive phase in rats, and K antigens interfere with this response.

Topics: Animals; Antigens, Bacterial; Antigens, Surface; Complement System Proteins; Escherichia coli; Glycolipids; Histamine; Hypotension; Lipid A; Lipopolysaccharides; Male; Rats; Rats, Inbred Strains; Serotonin; Shock, Septic; Species Specificity

Hepatic uptake of bacterial lipopolysaccharides (LPS) in defined salt forms and free lipid A was studied in C3H mice. Extracts of 14C-labeled and unlabeled LPS from Salmonella abortus equi and lipid A from Salmonella minnesota R 595 (Re) were administered intravenously in doses sufficient to induce endotoxic shock. Sixty minutes after administration of 14C-LPS, 40% of the total activity was found in the liver tissue, 10% was in the isolated nonparenchymal cells, and only 1% was in the isolated hepatocytes. However, at this time only one third of the hepatocytes could be isolated; the other two thirds were obviously damaged. After 240 minutes, 55% of the total activity was measured in the liver tissue. The nonparenchymal cells had 8% of the activity, and all hepatocytes were damaged. By use of immunofluorescence, LPS S abortus equi was localized in sinusoidal cells 5 to 10 minutes after administration, and LPS S minnesota R 595 and lipid A were found in both nonparenchymal and parenchymal liver cells. All toxins were localized in both cell populations 60 and 240 minutes after injection. After application of LPS or lipid A, the third complement component (C3) was detectable in sinusoidal cells. In decomplemented mice the hepatic deposits of LPS and lipid A were unaffected, without demonstration of C3. The data indicate that LPS and lipid A interact in vivo with Kupffer cells and hepatocytes. Hepatic clearance of endotoxin seems to be independent of complement.

Topics: Animals; Complement System Proteins; Female; Lipid A; Lipopolysaccharides; Liver; Male; Mice; Mice, Inbred C3H; Shock, Septic; Staphylococcus

Lipopolysaccharide (LPS) produced time- and dose-dependent bovine endothelial cell injury in vitro that was manifested initially by cell detachment from culture substrate with subsequent cell lysis. Bovine endothelial cell injury was observed with LPS derived from Salmonella minnesota R595, a LPS comprised only of lipid A and a trisaccharide core, as well as intact LPS preparations derived Escherichia coli and S. typhosa. LPS-mediated bovine endothelial cell detachment was prevented by incubation at 4 degrees C but was not prevented by indomethacin, lidocaine, chlorpromazine or trifluoperazine, methylprednisolone or p-bromophenacyl bromide, protease inhibitors, and catalase or superoxide dismutase. Of note, LPS-mediated injury was markedly enhanced by cycloheximide. Although augmented by serum, LPS-mediated bovine endothelial cell detachment was observed in C8-deficient serum and also in serum-free medium at higher LPS concentrations. Bovine aortic, pulmonary artery, mesenteric artery, and mesenteric vein endothelial cells were all sensitive to LPS at a concentration of 1 microgram/ml. In contrast, bovine aortic smooth muscle, human umbilical vein, goat aortic, and canine vena cava endothelial cells were unaffected by LPS at a concentration of 100 micrograms/ml. We conclude that the lipid A moiety of LPS mediates direct, complement-independent endothelial cell cytotoxicity and that this injury is not prevented by inhibitors of protein and prostaglandin synthesis, oxygen radical production, protease and phospholipase activity, and cytoskeletal function. Importantly, this direct LPS-mediated cytotoxic effect is dependent on the species from which the endothelial cells are derived.

Topics: Animals; Blood Vessels; Cattle; Dose-Response Relationship, Drug; Endothelium; Goats; Humans; Lipid A; Lipopolysaccharides; Shock, Septic; Species Specificity; Temperature

Galanos-type endotoxin obtained from the heptose-less mutant of Salmonella typhimurium was converted to Lipid A by two cycles of treatment with sodium acetate, pH 4.5, at 100 degrees and separated on a DEAE-cellulose column into several fractions (Fractions III to VII). Tumor regression studies with strain 2 guinea pigs and syngeneic line 10 hepatocellular carcinoma showed that all fractions were effective when combined with trehalose dimycolates and an additional tumor regression factor (previously designated ACP) and incorporated into oil droplets (78 to 100% cures). A low polar fraction (Fraction IV) was relatively nontoxic [the medium lethal dose for 11-day-old chick embryos inoculated i.v. (CELD50) was more than 10 micrograms] and nonpyrogenic [the dose estimated to give a fever index (area under fever curve) of 40 sq cm in rabbits when 1 hr and 1 degrees are plotted as 1 (FI40) was 5 micrograms] as compared to the unfractionated Lipid A (CELD50 of 0.0546 micrograms; FI40 of 0.046 micrograms). All other fractions were toxic and pyrogenic and caused severe endotoxic shocks when combined with N-acetylmuramyl-L-seryl-D-isoglutamine and injected i.v. into guinea pigs. Fraction IV plus N-acetylmuramyl-L-seryl-D-isoglutamine did not cause endotoxic shock. The phosphate content of Fraction IV was about one-half of that detected in the toxic fractions.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Animals; Cell Line; Chromatography, DEAE-Cellulose; Endotoxins; Guinea Pigs; Injections, Intravenous; Lipid A; Lipopolysaccharides; Liver Neoplasms, Experimental; Salmonella typhimurium; Shock, Septic; Trehalose

Topics: Antibodies; Humans; Lipid A; Lipopolysaccharides; Sepsis; Shock, Septic