Endotoxin [lipopolysaccharide (LPS)], the major antigen of the outer membrane of Gram-negative bacteria, consists of a variable-size carbohydrate chain that is covalently linked to N,O-acylated beta-1,6-D-glucosamine disaccharide 1,4'-bisphosphate (lipid A). The toxic activity of LPS resides in the lipid A structure. The structural features of synthetic peptides that bind to lipid A with high affinity, detoxify LPS in vitro, and prevent LPS-induced cytokine release and lethality in vivo were defined. The binding thermodynamics were comparable to that of an antigen-antibody reaction. Such synthetic peptides may provide a strategy for prophylaxis and treatment of LPS-mediated diseases.

A synthetic peptide, KFFKFFKFFK [corrected], consisting of cationic lysine residues and hydrophobic phenylalanine residues was found to sensitize gram-negative bacteria to hydrophobic and amphipathic antibiotics. At a concentration of 3 micrograms/ml, it decreased the MIC of rifampin for smooth, encapsulated Escherichia coli by a factor of 300. Other susceptible bacterial species included Enterobacter cloacae, Klebsiella pneumoniae, and Salmonella typhimurium, but Pseudomonas aeruginosa was resistant. Similar results were obtained with another synthetic peptide, IKFLKFLKFLK [corrected]. The fractional inhibitory concentration indices for the synergism of these peptides with rifampin, erythromycin, fusidic acid, and novobiocin were very close to those determined for the previously characterized potent outer-membrane-disorganizing agents polymyxin B nonapeptide and deacylpolymyxin B. KFFKFFKFFK [corrected] had direct activity against the gram-positive organism Micrococcus strain ML36, was strongly hemolytic, and was as active on polymyxin-resistant E. coli mutants as on their parent. These three attributes made KFFKFFKFFK [corrected] different from polymyxin derivatives and similar to cationic detergents, such as cetylpyridinium chloride. However, whereas the MIC of cetylpyridinium chloride for E. coli is low (0.5 to 4 micrograms/ml), that of KFFKFFKFFK [corrected] is much higher (30 to 100 micrograms/ml). Other groups of synthetic peptides studied included polymyxin-like peptides with an intrachain disulfide bridge. Their synergism with antibiotics was less marked. Still other peptides, including KEKEKEKEKE and KKKKKKFLFL, lacked any synergism with the probe antibiotics.

Neutralization and sequestration of bacterial lipopolysaccharide which plays a key role in gram-negative sepsis is required to block the progression of sepsis at early stages in addition to destroying bacteria. Many of the host defense peptides which have antimicrobial activity are also able to bind to and neutralize LPS, however, these two activities do not necessarily correlate. Due to its toxicity application of polymyxin B as the prototype of LPS neutralizing peptide is limited to topical applications and extracorporeal removal of endotoxin. Development of novel endotoxin neutralizing peptides without the toxicity of polymyxin B have been based on the natural host defense peptides, fragments of LPS binding proteins and engineered peptides. Neutralization of LPS can be achieved through several different peptide fold motifs, which are reviewed in this article. Endogenous host defense peptides, fragments of endotoxin-binding proteins and synthetic anti-endotoxin peptides fold into alpha-helical, beta-hairpin, extended and compact conformations without regular secondary structure. In animal models many of the peptides have demonstrated good in vitro and in vivo endotoxin neutralizing activity but up to now none of the peptides has been approved for clinical application with an anti-endotoxin indication. Recent developments include preparation of novel types of endotoxin neutralizing compounds such as peptides modified by lipophilic moieties and non-peptidic molecules, particularly lipopolyamines and on the other hand additional medical applications such as extracorporeal endotoxin removal, targeting to inflammation sites or endotoxoid based vaccines.

Lipopolysaccharides (LPS), or endotoxins, are major structural and functional components of the outer membrane of gram-negative bacteria (24). These complex macromolecules exhibit a variety of toxic and proinflammatory activities that are associated with the lipid A moiety and are causally related to the pathogenesis of gram-negative sepsis and septic shock (17, 18).Many of the local and systemic pathophysiologic phenomena produced by LPS in the exposed host result from the ability of LPS to activate host inflammatory cells (7), including monocytes, macrophages, and polymorphonuclear leukocytes. Recent attention has focused on putative LPS receptors found on the surfaces of these cells, the relation of these receptors to LPS-induced signal transduction, and the role of each in the development of proinflammatory responses.Membrane-bound CD14 (mCD14), a glycosyl phosphatidylinositol-anchored protein expressed on myeloid cells, is the best characterized LPS receptor identified to date (9, 33, 37). mCD14 appears to be part of a multicomponent LPS receptor functionally linked to the initiation of intracellular signaling events related to LPS-induced cell activation (29). The signaling unit of the LPS receptor is comprised of members of the Toll-like receptor family of transmembrane proteins characterized by their amphiphilic properties and leucine-rich repeats (31, 36). Serum-associated LPS-binding protein (LBP), which forms complexes with LPS through high-affinity attachment to the lipid A moiety, catalyzes LPS recognition by mCD14, resulting in the generation of LPS-induced proinflammatory signals (12,14).Recent experiments have attempted to define the roles of mCD14 and LBP in LPS-related septic events as well as the possible protective or therapeutic activities of proteins, including antibodies, that neutralize LPS by interrupting its proinflammatory interactions with mCD14 and LBP.We previously showed that LPS-specific monoclonal antibodies (MAbs) are capable of neutralizing cytokine-and transcription factor-inducing activities of LPS by inhibiting the binding of LPS to mCD14 expressed on human peripheral blood monocytes (PBMC) and on CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) (20,21).Polymyxin B (PMB), a cationic, cyclic peptide antibiotic, inhibits biological activities of LPS through high-affinity bind-* Corresponding author. Mailing address:

The in vivo immunizing potency of diphtheria toxoid and formalin-treated corss-reacting material (CRM197, a nontoxic mutant protein) was compared in guinea pigs. Major antigenic differences between the two untreated proteins were also tested in rats. The results showed that diphtheria toxoid and CRM197 were equally effective immunogens, but only if the latter was treated with formalin in the same concentration (0.7% vol/vol) was that of the toxoid. Formalin treatment rendered the antigens more resistant to enzymatic proteolysis by trypsin in vitro.