The N-termini of bacterial lipoproteins are acylated with a (S)-(2,3-bisacyloxypropyl)cysteinyl residue. Lipopeptides derived from lipoproteins activate innate immune responses by engaging Toll-like receptor 2 (TLR2), and are highly immunostimulatory and yet without apparent toxicity in animal models. The lipopeptides may therefore be useful as potential immunotherapeutic agents. Previous structure-activity relationships in such lipopeptides have largely been obtained using murine cells and it is now clear that significant species-specific differences exist between human and murine TLR responses. We have examined in detail the role of the highly conserved Cys residue as well as the geometry and stereochemistry of the Cys-Ser dipeptide unit. (R)-diacylthioglycerol analogues are maximally active in reporter gene assays using human TLR2. The Cys-Ser dipeptide unit represents the minimal part-structure, but its stereochemistry was found not to be a critical determinant of activity. The thioether bridge between the diacyl and dipeptide units is crucial, and replacement by an oxoether bridge results in a dramatic decrease in activity.

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The role of lipopolysaccharide (LPS) in the pathogenesis of Gram-negative septic shock is well established. The corresponding proinflammatory and immunostimulatory molecule(s) on the Gram-positive bacteria is less well understood, and its identification and characterization would be a key prerequisite in designing specific sequestrants of the Gram-positive endotoxin(s). We report in this paper the comparison of NF-kappaB-, cytokine- and chemokine-inducing activities of the TLR2 ligands, lipoteichoic acid (LTA), peptidoglycan (PGN), and lipopeptides, to LPS, a prototype TLR4 agonist, in murine macrophage cell-lines as well as in human blood. In murine cells, di- and triacyl liopopeptides are equipotent in their NF-kappaB inducing activity relative to LPS, but elicit much lower proinflammatory cytokines. However, both LPS and the lipopeptides potently induce the secretion of a pattern of chemokines that is suggestive of the engagement of a TLR4-independent TRIF pathway. In human blood, although the lipopeptides induce p38 MAP kinase phosphorylation and CD11b upregulation in granulocytes at ng/ml concentrations, they do not elicit proinflammatory cytokine production even at very high doses; LTA, however, activates neutrophils and induces cytokine secretion, although its potency is considerably lower than that of LPS, presumably due to its binding to plasma proteins. We conclude that, in human blood, the pattern of immunostimulation and proinflammatory mediator production elicited by LTA parallels that of LPS.

ABSTRACT␣7 nicotinic acetylcholine receptors (nAChRs) have been a puzzle since their discovery in brain and non-neuronal tissues. Maximal transient probability of an ␣7 nAChR being open with rapid agonist applications is only 0.002. The concentration dependence of ␣7 responses measured from transfected cells and Xenopus laevis oocytes shows the same disparity in potency estimations for peak currents and net charge, despite being studied at 1000-fold different time scales. In both cases the EC 50 was approximately 10-fold lower for net charge than for peak currents. The equivalence of the data obtained at such disparate time scales indicates that desensitization of ␣7 is nearly instantaneous. At high levels of agonist occupancy, the receptor is preferentially converted to a ligand-bound nonconducting state, which can be destabilized by the positive allosteric modulator N-(5-chloro-2,4-dimethoxyphenyl)-NЈ-(5-methyl-3-isoxazolyl)-urea (PNU-120596). Such currents can be sufficiently large to be cytotoxic to the ␣7-expressing cells. Both the potentiating effect of PNU-120596 and the associated cytotoxicity have a high temperature dependence that can be compensated for by serum factors. Therefore, despite reduced potentiation at body temperatures, use of type II positive allosteric modulators may put cells that naturally express high levels of ␣7 nAChRs, such as neurons in the hippocampus and hypothalamus, at risk. With a low intrinsic open probability and high propensity toward the induction of nonconducting ligand-bound states, it is likely that the well documented regulation of signal transduction pathways by ␣7 nAChRs in cells such as those that regulate inflammation may be independent of ion channel activation and associated with the nonconducting conformational states.

Just as the prescient comment by Gaston Ramon was relegated to the last footnote of his 1926 paper, 1 so has research on the mechanisms of action of adjuvants, until recently, languished as parenthetical annotations and addenda in the archives of immunology and vaccine development. Ramon defined immunological adjuvants as "substances used in combination with a specific antigen that produced a more robust immune response than the antigen alone." Interestingly enough, he was referring to his empirical findings that the addition of bread crumbs, tapioca, saponin and 'starch oil' to antigenic preparations greatly enhanced antibody responses to diphtheria or tetanus. 2 A year later, the adjuvanticity of aluminum salts (primarily phosphate and hydroxide) was discovered by Glenny and coworkers. 3 In the 83 years that have elapsed, the repertoire of investigational adjuvants has grown to encompass a very wide range of materials, 4 but aluminum salt-based mineral salts (generically, and incorrectly, termed "alum") have remained the only adjuvants currently approved by the FDA. Aluminum salts have enjoyed a good safety record, but they are weak adjuvants for antibody induction and induce a T helper-2 (T H 2)-skewed, rather than a T helper-1 (T H 1) response. 5,6 Furthermore, not only are aluminum salts ineffective at inducing cytotoxic T lymphocyte (CTL) or mucosal IgA antibody responses, but also have a propensity to induce IgE responses, which have been associated with allergic reactions in some subjects. 5,6 Very recent reports implicate the Nalp3 inflammasome, a component of the innate immune response, as the effector limb of alum-associated adjuvanticity. [7][8][9] In 1962, Dresser observed that injection of purified soluble proteins not only failed to stimulate an immune response, but tolerized animals unless a bacterial extract was admixed with the protein immunogen. 10 This led him to redefine adjuvanticity as "a property of a substance which can act as a physiological switch, directing at least some immunologically competent cells to respond by making antibody rather than by becoming immunologically paralyzed by the antigen," 11 confirming Johnson's earlier observations that lipopolysaccharide (LPS) from Gram-negative bacteria exerted potent adjuvant properties, 12 and perhaps paved the way for the subsequent discovery of the wide range of microorganism-derived adjuvants. 13 TLRs are pattern recognition receptors present on diverse cell types that recognize specific molecular patterns present in molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs). 14,15 There are 10 TLRs in the human genome;

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Lipopolysaccharide (LPS), or endotoxin, a structural component of gram-negative bacterial outer membranes, plays a key role in the pathogenesis of septic shock, a syndrome of severe systemic inflammation which leads to multiple-system organ failure. Despite advances in antimicrobial chemotherapy, sepsis continues to be the commonest cause of death in the critically ill patient. This is attributable to the lack of therapeutic options that aim at limiting the exposure to the toxin and the prevention of subsequent downstream inflammatory processes. Polymyxin B (PMB), a peptide antibiotic, is a prototype small molecule that binds and neutralizes LPS toxicity. However, the antibiotic is too toxic for systemic use as an LPS sequestrant. Based on a nuclear magnetic resonance-derived model of polymyxin B-LPS complex, we had earlier identified the pharmacophore necessary for optimal recognition and neutralization of the toxin. Iterative cycles of pharmacophore-based ligand design and evaluation have yielded a synthetically easily accessible N 1 ,mono-alkyl-mono-homologated spermine derivative, DS-96. We have found that DS-96 binds LPS and neutralizes its toxicity with a potency indistinguishable from that of PMB in a wide range of in vitro assays, affords complete protection in a murine model of LPS-induced lethality, and is apparently nontoxic in vertebrate animal models.Endotoxin, or lipopolysaccharide (LPS), a structural component of the outer membrane of most gram-negative bacteria (31), plays a pivotal role in septic shock, a syndrome of systemic toxicity which occurs frequently as a sequel to serious systemic gram-negative infections (23). The activation by LPS of the innate immune response, mediated via toll-like receptor 4 (TLR4) (39), leads to a dysregulated production of numerous inflammatory mediators, including tumor necrosis factor alpha (TNF-␣), interleukin-1␤ (IL-1␤), and IL-6 (11), gamma interferon (IFN-␥), and IL-12, which appears to be inadequately compensated for by the production of anti-inflammatory cytokines, such as IL-10 and transforming growth factor ␤ (6). The resultant systemic inflammatory response progresses to the frequently fatal syndrome of multiple-system organ failure (3). Despite continuing advances in antimicrobial chemotherapy, the incidence of sepsis has risen almost threefold from 1979 through 2000 (25), emphasizing an urgent, unmet need to develop therapeutic options specifically targeting the pathophysiology of sepsis.The toxicity of LPS resides in its structurally highly conserved glycolipid component called lipid A (22), which is composed of a hydrophilic, bis-phosphorylated diglucosamine backbone, and a hydrophobic domain comprised of acyl chains in amide and ester linkages (14). Polymyxin B (PMB) is a membrane-active peptide antibiotic (37) known to sequester LPS and abrogate its toxicity (12, 16). The otoand nephrotoxicity of PMB limit its systemic use and have led to the development of an extracorporeal hemoperfusion cartridge based on PMB covalently immobilized on a polysty...

T lymphocytes constitute a major effector cell population in autoimmune type 1 diabetes. Despite essential functions of mitochondria in regulating activation, proliferation, and apoptosis of T cells, little is known regarding T cell metabolism in the progression of human type 1 diabetes. In this study, we report, using two independent cohorts, that T cells from patients with type 1 diabetes exhibited mitochondrial inner-membrane hyperpolarization (MHP). Increased MHP was a general phenotype observed in T cell subsets irrespective of prior antigen exposure, and was not correlated with HbA1C levels, subject age, or duration of diabetes. Elevated T cell MHP was not detected in subjects with type 2 diabetes. T cell MHP was associated with increased activation-induced IFNγ production, and activation-induced IFNγ was linked to mitochondria-specific ROS production. T cells from subjects with type 1 diabetes also exhibited lower intracellular ATP levels. In conclusion, intrinsic mitochondrial dysfunction observed in type 1 diabetes alters mitochondrial ATP and IFNγ production; the latter is correlated with ROS generation. These changes impact T cell bioenergetics and function.