Journal of endotoxin research最新文献

We live in a world populated by an enormous number of micro-organisms. This necessitates the existence of highly effective mechanisms to control microbial growth. Through many research efforts, a chemical defense system based on the production of antimicrobial proteins (AMPs) has been identified. AMPs are endogenous, small proteins exhibiting antimicrobial activity against a wide variety of micro-organisms. The wide distribution of these molecules in the plant and animal kingdom reflects their biological significance. Various human AMPs show a potent effect on pathogenic micro-organisms including antibiotic-resistant bacteria. Thus, there is great interest in understanding the role of AMPs within innate immunity and evaluating their use and/or specific induction to fend off infections. In this review, we provide an overview of the characteristics of human AMPs and discuss examples where AMPs may be involved in the pathogenesis of infectious and inflammatory diseases.

Meningococcal endotoxin is the major contributor to the pathogenesis of fulminant sepsis and meningitis of meningococcal disease and is a potent activator of the MyD88-dependent and MyD88-independent pathways via the MD-2/TLR4 receptor. To understand better the biological properties of meningococcal endotoxin that initiates these events, the physicochemical structure of Neisseria meningitidis lipopoly(oligo)saccharide (LOS) of the serogroup B wild-type strain NMB (NeuNAc-Gal beta-GlcNAc-Gal beta-Glc beta-Hep2(GlcNAc,Glc alpha)PEA-Kdo2-lipid A, 1,4'-bisphosphorylated +/- PEA, PEtN) and the genetically-defined mutants (gmhB, Kdo2 -lipid A; kdtA, meningococcal lipid A; gmhB-lpxL1, Kdo2penta-acylated lipid A and NMB-lpx1, penta-acylated meningococcal LOS) were assessed in relation to bioactivity. Confirming previous work, Kdo2lipid A was the minimal structure required for optimal activation of the MD-2/TLR4 pathway of human macrophages. Meningococcal lipid A alone was a very weak agonist in stimulating human macrophages, even at high doses. Penta-acylated LOS structures demonstrated a moderate reduction in TLR4/MyD88-dependent signaling and a dramatic decrease in TLR4-TRIF-dependent signaling. For a better understanding of these results, we have performed an analysis of physicochemical parameters of the LOS structures such as the gel-to-liquid crystalline phase transition of the acyl chains, the inclination angle of the diglucosamine backbone with respect to the membrane surface, and the aggregate structure, and have found a very significant correlation of these parameters with biological activities extending our concept of endotoxicity.

Severe injury and infection are associated with autonomic dysfunction. Diminished heart rate variability (HRV) is also observed as a component of autonomic dysfunction and is induced by endotoxin administration to healthy subjects. It is established that low-dose glucocorticoid administration diminishes the systemic inflammatory manifestations of endotoxinemia but the influence of this anti-inflammatory intervention on overall autonomic dysfunction and HRV responses to endotoxin is unknown. This study was designed to assess the influence of a low-dose hydrocortisone infusion upon endotoxin-elicited systemic inflammatory responses including phenotypic features, cytokine production, and parameters of HRV. Of 19 subjects studied, nine received a continuous infusion of hydrocortisone (3 microg/kg/min continuously over 6 h) prior to intravenous administration of Escherichia coli endotoxin (2 ng/kg, CC-RE, Lot #2) while 10 healthy subjects received only the endotoxin after a 6-h period of saline control infusion. Serial determinations of vital signs, heart rate variability assessments, and cytokine levels were obtained over the subsequent 24 h. Prior cortisol infusion diminished the peak TNF-alpha (P < 0.01) and IL-6 (P < 0.0001) responses after endotoxin challenge, as compared to saline infusion controls and diminished the peak core temperature response to endotoxin (P < 0.01). In contrast to the influence of cortisol on the above parameters of systemic inflammation, the significant endotoxin-induced decreases in HRV time and frequency domains were not influenced by prior hydrocortisone treatment. Hence, alterations in autonomic dysfunction occur despite hydrocortisone attenuation of other traditional systemic manifestations of endotoxinemia. The maintenance or restoration of autonomic balance is not influenced by glucocorticoid administration.

The aim of this study was to evaluate and compare the effectiveness of N-acetylcysteine (NAC) and liposomally-encapsulated NAC (L-NAC) in ameliorating the hepatotoxic effects of lipopolysaccharide (LPS). LPS, a major cell wall molecule of Gram-negative bacteria and the principal initiator of septic shock, causes liver injury in vivo that is dependent on neutrophils, platelets, and several inflammatory mediators, including tumour necrosis factor-alpha (TNF-alpha). Male Sprague-Dawley rats were pretreated intravenously with saline, plain liposomes (dipalmitoylphosphatidylcholine [DPPC]), NAC (25 mg/kg body weight), or L-NAC (25 mg/kg NAC body weight) and 4 h later were challenged intravenously with LPS (Escherichia coli O111:B4, 1.0 mg/kg body weight); animals were killed 20 h post-LPS challenge. Hepatic cell injury was evaluated by measuring the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in plasma. LPS-induced activation of the inflammatory response was evaluated by measuring the levels of myeloperoxidase activity and chloramine concentration in liver homogenates as well as TNF-alpha levels in plasma. The hepatic levels of lipid peroxidation products and non-protein thiols (NPSH) were used to assess the extent of involvement of oxidative stress mechanisms. In general, challenge of animals with LPS resulted in hepatic injuries, activation of the inflammatory response, decreases in NPSH levels and increases in the levels of lipid peroxidation products (malondialdehyde and 4-hydroxyalkenals). Pretreatment of animals with NAC or empty liposomes did not have any significant protective effect against LPS-induced hepatotoxicity. On the other hand, pretreatment of animals with an equivalent dose of L-NAC conferred protection against the liver injuries induced following LPS challenge. These data suggest that NAC when delivered as a liposomal formulation is a potentially more effective prophylactic pharmacological agent in alleviating LPS-induced liver injuries.

Syk kinase is best known as a critical component of immunoreceptor signaling in leukocytes. Activation of Syk following cross-linking of Fcgamma and Fcepsilon receptors on macrophages, mast cells, and other cells induces various inflammatory events. We hypothesized that Syk is involved in inflammatory responses induced by the lipopolysaccharide (LPS). We studied the role of Syk using its inhibition by antisense oligonucleotides, or small interfering RNA. Our data demonstrated that in vivo inhibition of Syk caused down-regulation of LPS-induced responses in rat alveolar macrophages. In in vitro experiments, inhibition of Syk in rat peritoneal macrophages, as well as in human myelomonocyte cell line THP-1 also caused a decrease in LPS-induced cytokine release. Our data support the hypothesis that, in macrophages, Syk is involved in LPS-induced intracellular signaling pathways leading to the release of pro-inflammatory mediators. Understanding the role of Syk in LPS-induced signaling may help in developing new therapeutic tools for inflammatory disorders.

The monocyte activation test (MAT) has been introduced as an alternative for the detection of pyrogens in pharmaceuticals with the rabbit pyrogen test or the Limulus amebocyte lysate (LAL) test. The basis of the MAT is that pyrogens, via Toll-like receptors (TLRs) expressed on monocytes, stimulate cytokine production. Here, we report that, at concentrations that did not induce whole blood cytokine production when tested separately, (1-->3)-beta-D-glucans powerfully co-stimulated cytokine production (IL-6/IL-8) induced by ligands for TLR1/2, TLR2/6, TLR4, and TLR5. Experiments were performed to investigate the involvement of particular (1-->3)-beta-D-glucan receptors such as dectin-1. Spleen tyrosine kinase (Syk) inhibition attenuated the potentiating effects of (1-->3)-beta-D-glucans on TLR-induced cytokine production, suggesting that dectin-1 was involved. However, experiments with low molecular (1-->3)-beta-D-glucans such as laminarin argued against the involvement of dectin-1 in the co-stimulatory effects of (1-->3)-beta-D-glucans. Thus, although the receptors involved in the co-stimulatory actions of (1-->3)-beta-D-glucans on TLR-induced cytokine production are yet to be elucidated, it is clear that (1-->3)-beta-D-glucans may greatly affect MAT results and, when undetected in pharmaceuticals, may give rise to serious side-effects in patients co-exposed to other elicitors of innate immunity, such as during infections.

Toll-like receptors (TLRs) initiate and maintain host defenses and inflammation, and directly contribute to diseases such as atherosclerosis. It is not completely understood in what cell types proatherogenic TLR-induced signaling arises and, particularly, there is uncertainty regarding the potential functional role of TLR2 in endothelial cells (ECs). We determined TLR2 and TLR4 gene expression in four different human and two different murine primary ECs using gene array analysis, RT-PCR, and flow cytometry and confirmed these data by functional studies by stimulating ECs with the corresponding TLR ligands. TLR4 was expressed in all human and murine ECs and these cells responded to stimulation with LPS. Faint expression of TLR2 was observed in human ECs, whereas murine ECs express considerable amounts of TLR2 mRNA. Human ECs failed to respond to TLR2 ligands while murine ECs responded to TLR2 ligands. Furthermore, in murine ECs, TLR2 was located on the cell surface while in human ECs, TLR2 was sequestered in intracellular compartments. After IFN-gamma or IL-1beta stimulation, TLR2 translocated to the cell surface of human ECs. In conclusion, TLR2 is expressed intracellularly in human ECs and, therefore, TLR2 ligands are inaccessible to the receptor. Murine ECs express membrane TLR2 and respond to TLR2 ligands, but human ECs normally will not respond unless they are first primed with inflammatory stimulation, which appears to trigger translocation of TLR2 to the cell surface.

This article is a review of studies in which endotoxin has been administered to human subjects for experimental purposes. Data are presented in tabular form so the reader can better appreciate the objectives of individual studies. Although the original intention was to focus on the adverse events associated with these studies, unexpected serious adverse events rarely have been reported.

Activated neutrophils contribute to the development of preterm delivery. Because of its ability to suppress inflammation, bikunin, a Kunitz-type protease inhibitor, is currently in clinical trials. To investigate the molecular mechanism of this inhibition, we analyzed the effect of bikunin on pro-inflammatory cytokine production and nuclear factor-kappaB (NF-kappaB) activation in mouse neutrophils stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Here, we show that bikunin: (i) blocks LPS-induced secretion of pro-inflammatory cytokines, including TNF-alpha and IL-1beta, in a dose-dependent manner; (ii) has an inhibitory effect on cytokine production at a concentration of 0.2 microM, reaching 65% inhibition at the highest doses of bikunin tested (5 microM); (iii) has the suppressive capacity of ERK1/2 and p38 signaling pathways; and (iv) inhibited sequentially the LPS-induced phosphorylation of IkappaB-alpha, degradation of IkappaB-alpha, and nuclear translocation of NF-kappaB. When the MAPK data are analyzed, a significant decrease in phosphorylation is not seen at 0.2 microM bikunin but is at 1.0 microM dosing. Bikunin can inhibit LPS-induced neutrophil activation and cytokine release, although it is unlikely that it works primarily through the inhibition of MAPK phosphorylation. These data suggest that such effects are important in vivo and play a major contributory role in abrogation of neutrophil-mediated inflammatory responses, such as preterm delivery.