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.

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.

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.

Decreased heart rate variability (HRV) in critically ill patients indicates a poor prognosis. In heart failure patients, there is an elevated sympathetic tone, reflected by a dominance of sympathetic parameters in HRV, whereas in critically ill patients sympathetic and parasympathetic modulation of heart rate is attenuated despite increased catecholamine blood levels. Thus, autonomic dysfunction in the critically ill cannot be causally related to an impairment at the level of neural transmission, but may be due to a derangement of signal transduction at the effector cell level. On the basis of our working hypothesis that endotoxin may be involved in this blunting of effector cell response to nerval input, we studied the spontaneous beating of cardiomyocytes under the influence of endotoxin. Applying the clinically established indices of HRV to the analysis of beating rate variability (BRV) of neonatal rat cardiomyocytes in serum-free medium, a narrowing of their BRV by endotoxin is demonstrated. We propose that the narrowing of HRV in critically ill patients does not only reflect the altered input from the central or peripheral neurons, but rather a remodeling of the cardiac pacemaker cells by endotoxin and inflammatory mediators.

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.

Infectious diseases have a major impact on both the development and the severity of asthma. The rise in incidence of asthma in industrialized countries over the last decades has been attributed to increased hygiene standards as well as the concomitant usage of antibiotics, which together lower the incidence of infections. Although this point of view is supported by both clinical studies and experimental approaches in mice, an increasing body of evidence suggests that certain infectious diseases may predispose for the development of asthma, thus challenging the ;hygiene hypothesis' in its classical form. Toll-like receptors (TLRs) are centrally involved in orchestrating immune responses towards various micro-organisms. Because of this, it is tempting to speculate that signaling through TLRs may be involved in mechanisms provoking Th1- or Th2-biased immune responses and may, therefore, be an important factor in either preventing or promoting allergic airway disease. This review summarizes clinical and experimental data from mouse models focused on the impact of TLR-signaling on allergic asthma.

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.

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.

The evolution of Bordetella pertussis and Bordetella parapertussis from Bordetella bronchiseptica involved changes in host range and pathogenicity. Recent data suggest that the human-adapted Bordetella modified their interaction with host immune systems to effect these changes and that decreased stimulation of Toll-like receptor 4 (TLR4) by lipid A is central to this. We discuss Bordetella lipid A structure and genetics within the context of evolution and host immunity.

Lipopolysaccharide (LPS) antagonists inhibit the response of inflammatory cells to LPS, presumably by competitive inhibition, and may be of therapeutic value in the treatment of endotoxemia and sepsis. The inhibitory effects of some LPS antagonists are restricted to certain host species, however, as the same molecules can have significant endotoxic activity in other species. This species-specific recognition appears to be mediated by Toll-like receptor 4 (TLR4) and/or MD-2. We have shown previously that LPS from Rhodobacter sphaeroides (RsLPS) is an LPS antagonist in human cells but an agonist (or LPS mimetic) in equine cells. In the present study, HEK293 cells were transfected with combinations of human and equine CD14, TLR4 and MD-2, and incubated with either RsLPS or with LPS from Escherichia coli as an endotoxin control. NF-kappaB activation was measured in a dual luciferase assay as an indicator of cellular activation. Our results indicate that E. colic LPS activated NF-kappaB in cells transfected with all combinations of the three receptor proteins, whereas RsLPS activated NF-kappaB only in cells expressing the single combination of equine TLR4 and equine MD-2. We conclude that the TLR4/MD-2 complex is responsible for recognition of RsLPS as an agonist in equine cells.

Diabetic subjects are susceptible to atherosclerosis. It has been postulated that inflammation plays a crucial role in atherogenesis. Since previous studies suggested persistent low-grade infection by Gram-negative bacteria such as Chlamydia spp. and/or periodontal infection is associated with increased atherogenesis among diabetic subjects, we hypothesized that macrophages under hyperglycemia respond to lipopolysaccharide (LPS) challenge in a more exaggerated manner than under normal glucose conditions. Therefore, we examined cytokine productivity and associated signal transduction molecules in LPS-stimulated the monocytic cell line THP-1, under conditions of hyperglycemia. Differentiated THP-1 cells were cultured under normal and high glucose conditions without fetal bovine serum, and were stimulated with Escherichia coli LPS in the presence of LPS binding protein. Following stimulation, activated signal transduction molecules were detected by protein microarray and confirmed thereafter. Results indicated that c-jun N-terminal kinase (JNK) was highly-phosphorylated at high glucose concentrations, and this was confirmed by Western-immunoblotting. Tumor necrosis factor-alpha and monocyte chemo-attractant protein-1 production were significantly enhanced under these conditions. SP600125, a selective inhibitor of JNK, dose-dependently suppressed the production of these cytokine. Therefore, we suggest that this may be one of the mechanisms by which sub-clinical infection by Gram-negative bacteria promotes atherosclerosis in diabetic subjects.

Peptostreptococcus micros is a Gram-positive anaerobic bacterium associated with periodontitis, a chronic inflammatory disease affecting tooth-supporting tissues. In the present study, we investigated the response of human macrophages to stimulation with a cell wall preparation from P. micros. In addition, the effect of the preparation on the phosphorylation of macrophage kinases was studied. The preparation, which was non-toxic for macrophages, significantly increased the secretion of the pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6. It also increased the secretion of two potent chemokines IL-8 and, to a lesser extent, RANTES. Lastly, stimulation of macrophages by the P. micros cell wall preparation induced a significant increase in MMP-9 secretion but had no effect on the production of prostaglandin E2. The phosphorylation of macrophage kinases, including cAMP-dependent protein-serine kinase (PKA) catalytic subunit beta, G protein-coupled receptor-serine kinase 2, mitogen-activated protein-serine kinase p38 alpha (p38a MAPK), extracellular regulated protein-serine kinase 2 (ERK2) and Jun N-terminus protein-serine kinases (JNK), increased following stimulation with cell wall. In summary, our study showed that the P. micros cell wall preparation induced intracellular signaling pathways, leading to an increased production of pro-inflammatory cytokines, chemokines and MMP-9 by macrophages.

The course of every infection is different. The same pathogen can lead to subclinical, mild, severe or lethal infections in individuals. But is this just chance or determined by individual differences--on the side of the host as well as on the side of the pathogen? If so, we might need to consider these variations for treatment decisions. Indeed, we now understand that genetic polymorphisms and health status represent inborn and acquired risk factors. Similarly, pathogens impress with an increasing number of already identified virulence factors and host response modifiers. The emerging, more complex, view of the factors determining course and outcome of infections promises to enable more tailored and thus, hopefully, more effective treatment decisions.

Partial structures of peptidoglycan were chemically synthesized for elucidation of their precise biological activities. By using an efficient synthetic strategy, mono-, di-, tetra- and octasaccharide fragments of peptidoglycan were synthesized in good yields. The biological activity of synthetic fragments of peptidoglycan was evaluated by induction of TNF-alpha from human monocytes, and TLR2 and NOD2 dependencies by using transfected HEK293 cells, respectively. We revealed that TLR2 was not stimulated by the series of synthetic peptidoglycan partial structures, whereas NOD2 recognizes the partial structures containing the MDP moiety. We also synthesized potent NOD1 agonists, which showed several hundred-fold stronger activity than gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP). Interaction of PGRPs with synthetic peptidoglycan fragments is also described.