Search

Infections are a significant cause of morbidity and mortality in patients with chronic lymphocytic leukemia (CLL). The pathogenesis of infections is multifactorial and includes hypogammaglobulinemia, conventional therapy with alkylating drugs, and recently, purine analogs and mAb-associated T cells. Patients without these risk factors also suffer from infections, although the mechanism remains unknown. In a cohort of 70 patients with CLL, we demonstrated that their monocytes were locked into a refractory state and were unable to mount a classic inflammatory response to pathogens. In addition, they exhibited the primary features of endotoxin tolerance, including low cytokine production, high phagocytic activity, and impaired Ag presentation. The involvement of miR-146a in this phenomenon was suspected. We found miR-146a target genes, such as IRAK1 and TRAF6, were manifestly downregulated. Our study provides a new explanation for infections in patients with CLL and describes a cross-tolerance between endotoxins and tumors., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Endotoxin tolerance in macrophages is a key regulatory mechanism to limit the innate immune response to infection or injury. Long considered a state of unresponsiveness to Toll-like receptor activation, tolerance is now recognized as a state of altered responsiveness to infection or injury. Endotoxin tolerance leads to a shift away from a pro-inflammatory response toward a response with key anti-inflammatory and pro-resolution features. Advances in our understanding of Toll-like receptor function have identified a number of molecular mechanisms that promote tolerance, but how these are integrated to achieve gene-specific regulation is an important outstanding question. The potential to harness the mechanisms of endotoxin tolerance to promote the resolution of chronic inflammation warrants the continued investigation of this fundamental feature of innate immunity. This review focuses on the endotoxin tolerant state, our understanding of the underlying molecular mechanisms, and the clinical significance of endotoxin tolerance.

Prior exposure to lipopolysaccharides (LPS) induces a state of cell resistance to subsequent LPS restimulation, known as endotoxin tolerance, mainly by repressing the expression of pro-inflammatory cytokines. We established an endotoxin tolerance model in human monocytes Endotoxin-tolerant cells showed a decrease in IκBα degradation and diminished expression of Tumor necrosis factor (TNF) (both messenger RNA [mRNA] and protein content). The myeloid differentiation factor 88 (MyD88)/MyD88 splice variant (MyD88s) ratio, an indirect way to test the Toll-like receptor 4 (TLR4) MyD88-dependent signaling cascade, did not change in endotoxin-tolerant cells when compared to LPS-stimulated or -unstimulated ones. Remarkably, cell population analysis indicated a significant increase of the CD14+ CD16+ subset only under the endotoxin-tolerant condition. Furthermore, endotoxin-tolerant cells produced higher amounts of C-X-C motif chemokine 10 (CXCL10), a typical MyD88-independent cytokine., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Two novel surface plasmon resonance immunosensors were fabricated for detection of the Bacillus thuringiensis Cry1Ab protein and to demonstrate their performance in analyzing Cry1Ab protein in crop samples. Sensor 2 was modified by 1,6-hexanedithiol, Au/Ag alloy nanoparticles, 3-mercaptopropionic acid, and protein A (or not [sensor 1]), with Cry1Ab monoclonal antibody. As a result, both of the immunosensors exhibited satisfactory linear responses in the Cry1Ab protein concentration ranges of 10 to 500ngml(-1) and 8 to 1000ngml(-1), and the detection limits were 5.0 and 4.8ngml(-1), respectively. The immunosensors possessed good specificity and acceptable reproducibility. In addition, crop samples could be analyzed after a simple treatment. The transgenic crops could be easily identified from the conventional ones by the two immunosensors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The lipopolysaccharide (LPS) of Acinetobacter baumannii is a potent stimulator of proinflammatory cytokines, such as interleukin-6 (IL-6). The 3-O-deacylase (PagL)-modifying enzyme that removes the 3-O-linked acyl chain from the disaccharide backbone of lipid A provides the opportunity to develop a new therapeutic compound that could reduce detrimental inflammatory responses. The plasmid pMMB66EH-PagL obtained by recombinant DNA technology was electroporated into A. baumannii ATCC 19606. Compared with wild-type LPS, outer membrane vesicles and inactivated whole cells of engineered bacteria had a statistically significant decreased ability to produce IL-6. Structural analysis of lipid A by negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that the profile of lipid A fractions under PagL expression was changed. Taken together, our data showed that recombinant penta-acylated lipid A had less immunoreactivity and that the tetra-acylated version of lipid A with TLR4 antagonist activity decreased the induction of IL-6 production in the murine macrophage cell line J774 A.1., (© 2015 S. Karger AG, Basel.)

The variable domain of the heavy-chain-only antibody (VHH) or nanobody (Nb), derived from camelids, begins to play an important role on the detection of protein markers. In this study, we constructed a phage-displayed library of VHHs against Cry1Fa by immunizing a healthy Bactrian camel with Cry1Fa toxin. After a series of bio-panning and screening by phage display technology, three anti-Cry1Fa nanobodies (Nbs) with great difference in complementarity determining region 3 (CDR3) were obtained and they were highly specific to Cry1Fa as well as showed full of activity when exposed to 70 °C for 3 h. Through modifying Nbs with Horseradish Peroxidase (HRP) and biotin, two Nbs which can recognize the different epitopes of Cry1Fa were determined and they were used to establish a novel sandwich immune ELISA based on biotin-SA interaction for Cry1Fa detection. The immunoassay exhibited a linear range from 1 to 100 ng/mL with a detection limit of 0.88 ng/mL. The recoveries from spiked corn and soybean samples were ranged from 83.33 to 117.17%, with a coefficient of variation (C.V) less than 6.0%. All together, the proposed immunoassay will be a promising way for sensitive and accurate determination of Cry1Fa toxin., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002-2 ng/ml). We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

Background: Genetic variants in endotoxin signaling pathway are important in modulating the effect of environmental endotoxin on asthma and atopic phenotypes. Our objective was to determine the single nucleotide polymorphisms (SNPs) in the endotoxin signaling pathway that may influence in vitro IgE synthesis and to investigate the relationship between these variants and endotoxin exposure in relation to the development of asthma and atopy in a birth cohort., Methods: Peripheral blood mononuclear cells from 45 children with asthma were stimulated with 2 and 200 ng/ml lipopolysaccharide in vitro and IgE was measured in the culture supernatants. Children were genotyped for 121 SNPs from 30 genes in the endotoxin signaling pathway. Variants with a dose-response IgE production in relation to lipopolysaccharide (LPS) were selected for replication in a population-based birth cohort, in which we investigated the interaction between these SNPs and endotoxin exposure in relation to airway hyper-responsiveness, wheeze, and atopic sensitization., Results: Twenty-one SNPs in nine genes (CD14, TLR4, IRF3, TRAF-6, TIRAP, TRIF, IKK-1, ST-2, SOCS1) were found to modulate the effect of endotoxin on in vitro IgE synthesis, with six displaying high linkage disequilibrium. Of the remaining 15 SNPs, for seven we found significant relationships between genotype and endotoxin exposure in the genetic association study in relation to symptomatic airway hyper-responsiveness (CD14-rs2915863 and rs2569191, TRIF-rs4807000), current wheeze (ST-2-rs17639215, IKK-1-rs2230804, and TRIF-rs4807000), and atopy (CD14-rs2915863 and rs2569192, TRAF-6-rs5030411, and IKK-1-rs2230804)., Conclusions: Variants in the endotoxin signaling pathway are important determinants of asthma and atopy. The genotype effect is a function of the environmental endotoxin exposure., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Little is known about exposure to mouse allergen (Mus m 1) and allergic rhinitis (AR)., Objective: To evaluate the association between mouse allergen exposure and AR in children., Methods: We examined the relation between mouse allergen level in house dust and AR in 511 children aged 6 to 14 years in San Juan, Puerto Rico. Study participants were chosen from randomly selected households using a multistage probability sample design. The study protocol included questionnaires, allergy skin testing, and collection of blood and dust samples. AR was defined as current rhinitis symptoms and skin test reactivity to at least one allergen., Results: In the multivariate analyses, mouse allergen level was associated with a 25% decreased odds of AR in participating children (95% confidence interval, 0.62-0.92). Although endotoxin and mouse allergen levels were significantly correlated (r = 0.184, P < .001), the observed inverse association between Mus m 1 and AR was not explained by levels of endotoxin or other markers of microbial or fungal exposure (peptidoglycan and glucan)., Conclusion: Mouse allergen exposure is associated with decreased odds of AR in Puerto Rican school-aged children., (Copyright © 2014 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Vizantin has immunostimulating properties and anticancer activity. In this study, we investigated the molecular mechanism of immune activation by vizantin. THP-1 cells treated with small interfering RNA for TLR-4 abolished vizantin-induced macrophage activation processes such as chemokine release. In addition, compared with wild-type mice, the release of MIP-1β induced by vizantin in vivo was significantly decreased in TLR-4 knockout mice, but not in TLR-2 knockout mice. Vizantin induced the release of IL-8 when HEK293T cells were transiently cotransfected with TLR-4 and MD-2, but not when they were transfected with TLR-4 or MD-2 alone or with TLR-2 or TLR-2/MD-2. A dipyrromethene boron difluoride-conjugated vizantin colocalized with TLR-4/MD-2, but not with TLR-4 or MD-2 alone. A pull-down assay with vizantin-coated magnetic beads showed that vizantin bound to TLR-4/MD-2 in extracts from HEK293T cells expressing both TLR-4 and MD-2. Furthermore, vizantin blocked the LPS-induced release of TNF-α and IL-1β and inhibited death in mice. We also performed in silico docking simulation analysis of vizantin and MD-2 based on the structure of MD-2 complexed with the LPS antagonist E5564; the results suggested that vizantin could bind to the active pocket of MD-2. Our observations show that vizantin specifically binds to the TLR-4/MD-2 complex and that the vizantin receptor is identical to the LPS receptor. We conclude that vizantin could be an effective adjuvant and a therapeutic agent in the treatment of infectious diseases and the endotoxin shock caused by LPS., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Asthma and allergy may develop as a result of interactions between environmental factors and the genetic characteristics of an individual. This review aims to summarize the available evidence for, and potential effects of, an interaction between polymorphisms of the CD14 gene and exposure to microbes on the risk of asthma and allergic diseases. We searched PubMed, MEDLINE and Global Health databases, finding 12 articles which met inclusion criteria. Most studies reported a significant interaction between CD14 polymorphisms and microbial exposure. When stratified by age at microbial exposure (early life vs adult life), there was evidence of a protective effect of gene-environment interaction against atopy in children, but not adults. We also found different effects of interaction depending on the type of microbial exposures. There was no strong evidence for asthma and eczema. Future studies should consider a three-way interaction between CD14 gene polymorphisms, microbial exposures and the age of exposure., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background & Aims: Patients with non-alcoholic steatohepatitis (NASH) have increased mortality, including from infections. We, therefore, tested in a rodent model of steatohepatitis whether the hepatic acute phase response is intact., Methods: Steatohepatitis was induced in rats by feeding a high-fat, high-cholesterol diet for 4 (early) and 16 weeks (advanced NASH). 2 h after low-dose LPS (0.5 mg/kg i.p.), we measured the serum concentrations of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6). We also measured liver mRNA's and the serum concentrations of acute phase proteins 24 h after LPS., Results: Non-alcoholic steatohepatitis in itself increased the liver mRNA levels of TNF-α and IL-6 and also the liver mRNA and serum levels of the acute phase proteins. The exposure to LPS increased serum TNF-α in both early and advanced NASH and more so than in the control rats. However, the increases in acute phase protein genes in liver tissue and proteins in the blood were lower than in the control rats., Conclusion: In rats with early or advanced experimental NASH, LPS despite an increased interleukin release resulted in a blunted acute phase protein response. This tachyphylaxis may be part of the mechanism for the increased infection susceptibility of patients with NASH. We speculate that the steatosis-related interleukin release desensitises the signalling pathway leading to acute phase protein synthesis., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Mechanism-based sepsis treatments are unavailable, and their incidence is rising worldwide. Deaths occur during the early acute phase of hyperinflammation or subsequent postacute hypoinflammatory phase with sustained organ failure. The acute sepsis phase shifts rapidly, and multiple attempts to treat early excessive inflammation have uniformly failed. We reported in a sepsis cell model and human sepsis blood leukocytes that nuclear NAD+ sensor SIRT1 deacetylase remodels chromatin at specific gene sets to switch the acute-phase proinflammatory response to hypoinflammatory. Importantly, SIRT1 chromatin reprogramming is reversible, suggesting that inhibition of SIRT1 might reverse postacute-phase hypoinflammation. We tested this concept in septic mice, using the highly specific SIRT1 inhibitor EX-527, a small molecule that closes the NAD+ binding site of SIRT1. Strikingly, when administered 24 h after sepsis, all treated animals survived, whereas only 40% of untreated mice survived. EX-527 treatment reversed the inability of leukocytes to adhere at the small intestine MVI, reversed in vivo endotoxin tolerance, increased leukocyte accumulation in peritoneum, and improved peritoneal bacterial clearance. Mechanistically, the SIRT1 inhibitor restored repressed endothelial E-selectin and ICAM-1 expression and PSGL-1 expression on the neutrophils. Systemic benefits of EX-527 treatment included stabilized blood pressure, improved microvascular blood flow, and a shift toward proimmune macrophages in spleen and bone marrow. Our findings reveal that modifying the SIRT1 NAD+ axis may provide a novel way to treat sepsis in its hypoinflammatory phase., (© 2014 Society for Leukocyte Biology.)

Endotoxin tolerance (ET) is a state of reduced responsiveness to endotoxin stimulation after a primary bacterial insult. This phenomenon has been described in several pathologies, including sepsis, in which an endotoxin challenge results in reduced cytokine production. In this study, we show that the NFκ L chain enhancer of activated B cells 2 (NFκB2)/p100 was overexpressed and accumulated in a well-established in vitro human monocyte model of ET. The p100 accumulation in these cells inversely correlated with the inflammatory response after LPS stimulation. Knocking down NFκB2/p100 using small interfering RNA in human monocytes further indicated that p100 expression is a crucial factor in the progression of ET. The monocytes derived from patients with sepsis had high levels of p100, and a downregulation of NFκB2/p100 in these septic monocytes reversed their ET status., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Background: We investigated the interaction between genetic variants in endotoxin signalling pathway and domestic endotoxin exposure in relation to asthma presence, and amongst children with asthma, we explored the association of these genetic variants and endotoxin exposure with hospital admissions due to asthma exacerbations., Methods: In a case-control study, we analysed data from 824 children (417 asthmatics, 407 controls; age 5-18 yr). Amongst asthmatics, we extracted data on hospitalization for asthma exacerbation from medical records. Endotoxin exposure was measured in dust samples collected from homes. We included 26 single-nucleotide polymorphisms (SNPs) in the final analysis (5 CD14, 7LY96 and 14 TLR4)., Results: Two variants remained significantly associated with hospital admissions with asthma exacerbations after correction for multiple testing: for CD14 SNP rs5744455, carriers of T allele had decreased risk of repeated hospital admissions compared with homozygotes for C allele [OR (95% CI), 0.42 (0.25-0.88), p = 0.01, False Discovery Rate (FDR) p = 0.02]; for LY96 SNP rs17226566, C-allele carriers were at a lower risk of hospital admissions compared with T-allele homozygotes [0.59 (0.38-0.90), p = 0.01, FDR p = 0.04]. We observed two interactions between SNPs in CD14 and LY96 with environmental endotoxin exposure in relation to hospital admissions due to asthma exacerbation which remained significant after correction for multiple testing (CD14 SNPs rs2915863 and LY96 SNP rs17226566)., Conclusion: Amongst children with asthma, genetic variants in CD14 and LY96 may increase the risk of hospital admissions with acute exacerbations. Polymorphisms in endotoxin pathway interact with domestic endotoxin exposure in further modification of the risk of hospitalization., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Nasopharyngeal carriage of Streptococcus pneumoniae (pneumococcus) plays an important role in the development of invasive diseases, and is also critically involved in setting up respiratory bacterial and viral infections. We previously reported that pneumococcus, one of the commonly carried bacteria in the nasopharynx, regulates non-typeable Haemophilus influenzae-induced inflammation by upregulating the expression of Toll-like receptor 2 (TLR2). However, the underlying molecular mechanisms by which TLR2 expression is regulated during pneumococcal infections have not yet been well characterized. TBX21 is an important transcription factor of adaptive immunity, but there is an increasing body of evidence pointing to a role in regulating innate immunity. The expression of TBX21 was reported in epithelial cells, but the expression and role of TBX21 in respiratory epithelium, especially for regulating TLR2, has not yet been studied. In this study, we found that pneumococcus upregulates TBX21 expression in the respiratory epithelium. The effect of pneumococcus on TBX21 expression was dependent on its cytoplasmic toxin, pneumolysin. In addition, epithelial TBX21 expression was not regulated by the gram-negative bacterium non-typeable Haemophilus influenzae, peptidoglycan or endotoxin. Deficiency of TBX21 in mice or knocking down TBX21 in epithelial cells suppressed pneumococcus-induced TLR2 expression, but not that of TLR4 or TLR9. These results indicate that the adaptive immune regulator TBX21 participates in regulating innate immune responses, through regulation of TLR2 expression during pneumococcal infections., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Autoimmune uveitis is an organ-specific disorder characterized by irreversible lesions to the eye that predominantly affect people in their most productive years and is among the leading causes of visual deficit and blindness. Currently available therapies are effective in the treatment of a wide spectrum of uveitis, but are often associated with severe side effects. Here, we review ongoing research with promising immunomodulatory therapeutic strategies, describing their specific features, interactions and the responses triggered by the targeted immune molecules that aim to minimize clinical complications and the likelihood of disease relapse. We first review the main features of the disease, diagnostic tools, and traditional forms of therapy, as well as the animal models predominantly used to understand the pathogenesis and test the novel intervention approaches aiming to control the acute immune and inflammatory responses and to dampen chronic responses. Both exploratory research and clinical trials have targeted either the blockade of effector pathways or of their companion co-stimulatory molecules. Examples of targets are T cell receptors (CD3), their co-stimulatory receptors (CD28, CTLA-4) and corresponding ligands (B7-1 and B7-2, also known as CD80 and CD86), and cytokines like IL-2 and their receptors. Here, we summarize the available evidence on effectiveness of these treatments in human and experimental uveitis and highlight a novel CD28 antagonist monovalent Fab' antibody, FR104, which has shown preclinical efficacy suppressing effector T cells while enhancing regulatory T cell function and immune tolerance in a humanized graft-versus-host disease (GVHD) mice model and is currently being tested in a mouse autoimmune uveitis model with encouraging results., (Copyright © 2014. Published by Elsevier B.V.)

Hepatic ischemia/reperfusion (I/R) injury is an unavoidable consequence of major liver surgery, especially in liver transplantation with bowel congestion, during which endotoxemia is often evident. The inflammatory response aggravated by endotoxin after I/R contributes to liver dysfunction and failure. The purpose of the present study was to investigate the protective effect of butyrate, a naturally occurring four-carbon fatty acid in the body and a dietary component of foods such as cheese and butter, on hepatic injury complicated by enterogenous endotoxin, as well as to examine the underlying mechanisms involved. SD rats were subjected to a total hepatic ischemia for 30 min after pretreatment with either vehicle or butyrate, followed by 6 h and 24 h of reperfusion. Butyrate preconditioning markedly improved hepatic function and histology, as indicated by reduced transaminase levels and ameliorated tissue pathological changes. The inflammatory factors levels, macrophages activation, TLR4 expression, and neutrophil infiltration in live were attenuated by butyrate. Butyrate also maintained the intestinal barrier structures, reversed the aberrant expression of ZO-1, and decreased the endotoxin translocation. We conclude that butyrate inhibition of endotoxin translocation, macrophages activation, inflammatory factors production, and neutrophil infiltration is involved in the alleviation of total hepatic I/R liver injury in rats. This suggests that butyrate should potentially be utilized in liver transplantation.

Binge or moderate alcohol exposure impairs host defense and increases susceptibility to infection because of compromised innate immune responses. However, there is a lack of consensus on the molecular mechanism by which alcohol mediates this immunosuppression. In this study, we show that cellular stress proteins HSF1 and hsp70 play a mechanistic role in alcohol-mediated inhibition of the TLR4/MyD88 pathway. Alcohol exposure induced transcription factor HSF1 mRNA expression and DNA binding activity in primary human monocytes and murine macrophages. Furthermore, HSF1 target gene hsp70 mRNA and protein are upregulated by alcohol in monocytes. In vitro pre-exposure to moderate alcohol reduced subsequent LPS-induced NF-κB promoter activity and downstream TNF-α, IL-6 and IL-1β production in monocytes and macrophages, exhibiting endotoxin tolerance. Mechanistic analysis demonstrates that alcohol-induced HSF1 binds to the TNF-α promoter in macrophages at early time points, exerting transrepression and decreased TNF-α expression. Furthermore, association of hsp70 with NF-κB subunit p50 in alcohol-treated macrophages correlates with reduced NF-κB activation at later time points. Hsp70 overexpression in macrophages was sufficient to block LPS-induced NF-κB promoter activity, suggesting alcohol-mediated immunosuppression by hsp70. The direct crosstalk of hsp70 and HSF1 was further confirmed by the loss of alcohol-mediated endotoxin tolerance in hsp70- and HSF1-silenced macrophages. Our data suggest that alcohol-mediated activation of HSF1 and induction of hsp70 inhibit TLR4-MyD88 signaling and are required for alcohol-induced endotoxin tolerance. Using stress proteins as direct drug targets would be clinically relevant in alcohol abuse treatment and may serve to provide a better understanding of alcohol-mediated immunosuppression., (Copyright © 2014 by The American Association of Immunologists, Inc.)

A major focus of work in our laboratory concerns the molecular mechanisms and structural bases of Gram-negative bacterial endotoxin recognition by host (e.g., human) endotoxin-recognition proteins that mediate and/or regulate activation of Toll-like receptor (TLR) 4. Here, we review studies of wild-type and variant monomeric endotoxin.MD-2 complexes first produced and characterized in our laboratories. These purified complexes have provided unique experimental reagents, revealing both quantitative and qualitative determinants of TLR4 activation and antagonism. This review is dedicated to the memory of Dr. Theresa L. Gioannini (1949-2014) who played a central role in many of the studies and discoveries that are reviewed.

The primary objective of this study was to determine the role of β2 integrin α-subunit (CD11b) in the mechanism of human polymorphonuclear leukocyte (PML) priming by S or Re endotoxin glycoforms from Escherichia coli for fMLP-induced respiratory burst. Similar priming activity of S and Re endotoxin glycoforms for fMLP-induced reactive oxygen species (ROS) generation from primed PML was found. Anti-CD11b antibodies (clone ICRF 44) as well as isotype-matched immunoglobulin G1 (clone MOPC-21) do not influence the fMLP-induced ROS generation from unprimed PML. Antibodies against CD11b do not change fMLP-induced ROS generation from endotoxin-primed PML as well. The involvement of different isoforms of Fcγ receptors in fMLP-induced ROS generation from activated PML is proposed.

Sepsis and septic shock remain important medical problems with high mortality rates. Today's treatment is based mainly on using antibiotics to target the bacteria, without addressing the systemic inflammatory response, which is a major contributor to mortality in sepsis. Therefore, novel treatment options are urgently needed to counteract these complex sepsis pathologies. Heparin cofactor II (HCII) has recently been shown to be protective against Gram-negative infections. The antimicrobial effects were mapped to helices A and D of the molecule. Here we show that KYE28, a 28 amino acid long peptide representing helix D of HCII, is antimicrobial against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida albicans. Moreover, KYE28 binds to LPS and thereby reduces LPS-induced pro-inflammatory responses by decreasing NF-κB/AP-1 activation in vitro. In mouse models of LPS-induced shock, KYE28 significantly enhanced survival by dampening the pro-inflammatory cytokine response. Finally, in an invasive Pseudomonas infection model, the peptide inhibited bacterial growth and reduced the pro-inflammatory response, which lead to a significant reduction of mortality. In summary, the peptide KYE28, by simultaneously targeting bacteria and LPS-induced pro-inflammatory responses represents a novel therapeutic candidate for invasive infections.

Toll-like receptor (TLR) signaling plays a fundamental role in the induction and progression of autoimmune disease. In the present study, we showed that lipopolysaccharide (LPS), a TLR4 ligand, functions as an antagonist of peroxisome proliferator-activated receptor alpha (PPARα), a nuclear transcription factor. Using endotoxin induced uveitis (EIU) as a model, we found that TLR was negatively regulated by PPARα. Our data revealed that treatment with the PPARα agonist fenofibrate dramatically prevented LPS-induced uveitis and inhibited TLR/ Nuclear factor-kappaB (NF-κB) signaling during inflammation. Evaluation of the severity of anterior uveitis further showed that PPARα agonist treatment significantly decreased inflammatory cell infiltration, total protein concentration, vessel density, inflammatory cytokine production, and clinical scores in the anterior section of the eye during EIU. Moreover, fenofibrate administration recovered retinal function and decreased the production of inflammatory cytokines, retinal vascular leukostasis, and inflammatory cell infiltration into the posterior section of the eyes during EIU. In vitro studies further showed that down-regulation or deletion of PPARα led to increased TLR4 levels and the activation of NF-κB signaling in RPE cells and also blocked the anti-inflammatory effects of fenofibrate. Furthermore, activation or up-regulation of PPARα decreased TLR4 levels and inhibited the NF-κB signaling pathway induced by LPS in RPE cells. In TLR4-expressing reporter cells, activation or up-regulation of PPARα partially inhibited the activation of NF-κB and also decreased TLR4 transcriptional activity. In conclusion, the activation of PPARα represents a novel therapeutic strategy for human uveitis, as PPARα negatively regulates TLR4 activity and therefore exerts anti-inflammatory actions., (Published by Elsevier B.V.)

Objectives: Transcompartmental signaling during early inflammation may lead to propagation of disease to other organs. The time course and the mechanisms involved are still poorly understood. We aimed at comparing acute transcompartmental inflammatory responses in humans following lipopolysaccharide-induced pulmonary and systemic inflammation., Design: Randomized, double-blind, placebo-controlled, crossover study., Setting Icu Subjects: Healthy male volunteers., Interventions: Fifteen volunteers (mean age, 23; SD, 2 yr) received Escherichia coli endotoxin (lipopolysaccharide, 4 ng/kg) IV or endobronchially on two different study days. Groups were evaluated by bronchoalveolar lavage at baseline (0 hr) and 2, 4, 6, 8, or 24 hours postchallenge. Cardiorespiratory variables were continuously recorded throughout the study day, and plasma and bronchoalveolar lavage fluid markers of inflammation were measured., Measurements and Main Results: IV endotoxin elicited a systemic inflammatory response with a time-dependent increase and peak in tumor necrosis factor-α, interleukin-6, and leukocyte counts (all p < 0.001). Furthermore, a delayed (6-8 hr) increase in bronchoalveolar lavage fluid interleukin-6 concentration (p < 0.001) and alveolar leukocyte count (p = 0.03) and a minor increase in bronchoalveolar lavage fluid tumor necrosis factor-α were observed (p = 0.06). Endobronchial endotoxin was followed by progressive alveolar neutrocytosis and increased bronchoalveolar lavage fluid tumor necrosis factor-α, interleukin-6, and albumin (all p < 0.001); a systemic inflammatory response was observed after 2-4 hours, with no change in plasma tumor necrosis factor-α., Conclusions: Acute lung or systemic inflammation in humans is followed by a transcompartmental proinflammatory response, the degree and differential kinetics of which suggests that the propagation of inflammation may depend on the primary site of injury.

To determine whether low molecular weight heparin (LMWH) is able to reduce pulmonary inflammation and improve the survival in rats with endotoxin-induced acute lung injury (ALI). Rat ALI model was reproduced by injection of lipopolysaccharide (LPS) into tail vein. Rats were divided randomly into three groups: control group, ALI group, LMWH-treated group. Blood was collected and lung tissue was harvested at the designated time points for analysis. The lung specimens were harvested for morphological studies, streptavidin-peroxidase immunohistochemistry examination. Lung tissue edema was evaluated by tissue water content. The levels of lung tissue myeloperoxidase (MPO) were determined. Meanwhile, the nuclear factor-kappa B (NF-κB) activation, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) levels and high mobility group box 1 (HMGB1) and intercellular adhesion molecule-1 (ICAM-1) protein levels in the lung were studied. In survival studies, a separate group of rats were treated with LMWH or sterile saline after LPS administration. Then, the mortality was recorded. Treatment with LMWH after ALI was associated with a reduction in the severity of LPS-induced lung injury. Treatment with LMWH significantly decreased the expression of TNF-α, IL-1β, HMGB1 and ICAM-1 in the lung of ALI rats. Similarly, treatment with LMWH dramatically diminished LPS-induced neutrophil sequestration and markedly reduced the enhanced lung permeability. In the present study, LMWH administration inhibited the nuclear translocation of NF-κB in the lung. Survival was significantly higher among the LMWH-treated group compared with the ALI group. These data suggest that LMWH attenuates inflammation and prevents lethality in endotoxemic rats.

We previously reported that jellyfish collagen stimulates both the acquired and innate immune responses. In the acquired immune response, jellyfish collagen enhanced immunoglobulin production by lymphocytes in vitro and in vivo. Meanwhile, in the innate immune response jellyfish collagen promoted cytokine production and phagocytotic activity of macrophages. The facts that jellyfish collagen plays several potential roles in stimulating cytokine production by macrophages have further attracted us to uncover its mechanisms. We herein describe that the cytokine production-stimulating activity of jellyfish collagen was canceled by a Toll-like receptor 4 (TLR4) inhibitor. Moreover, jellyfish collagen stimulated phosphorylation of inhibitor of κBα (IκBα), promoted the translocation of nucleus factor-κB (NF-κB), and activated c-Jun N-terminal kinase (JNK). A JNK inhibitor also abrogated the cytokine production-stimulating activity of jellyfish collagen. These results suggest that jellyfish collagen may facilitate cytokine production by macrophages through activation of NF-κB and JNK via the TLR4 signaling pathways., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this article, we report that retreatment of human monocytic THP-1 cells and primary monocytes with pathogenic Neisseria or with purified lipooligosaccharides (LOS) after previous exposure to LOS induced immune tolerance, as evidenced by reduced TNF-α and IL-1β cytokine expression. LOS that we have previously shown to vary in their potential to activate TLR4 signaling, which was correlated with differences in levels of lipid A phosphorylation, had similarly variable ability to induce tolerance. Efficacy for induction of tolerance was proportional to the level of lipid A phosphorylation, as LOS from meningococcal strain 89I with the highest degree of phosphorylation was the most tolerogenic following retreatment with LOS or whole bacteria, compared with LOS from gonococcal strains 1291 and GC56 with reduced levels of phosphorylation. Hydrogen fluoride treatment of 89I LOS to remove phosphates rendered the LOS nontolerogenic. Tolerance induced by the more highly inflammatory meningococcal LOS was correlated with significantly greater downregulation of p38 activation, greater induction of the expression of A20 and of microRNA-146a, and greater reductions in IL-1R-associated kinase 1 and TRAF6 levels following LOS retreatment of cells. The role of miR-146a in regulation of induction of TNF-α was confirmed by transfecting cells with an inhibitor and a mimic of miR-146a. Our results provide a mechanistic framework for understanding the variable pathophysiology of meningococcal and gonococcal infections given that after an initial exposure, greater upregulation of microRNA-146a by more highly inflammatory LOS conversely leads to the suppression of immune responses, which would be expected to facilitate bacterial survival and dissemination.

Background: In most children with asthma and atopy, onset of disease occurs early in life, indicating a crucial role of in utero and early childhood environment. However, only a small part of this burden of disease established early in life has been explained., Objective: To examine the effects of early environmental exposures on the development of asthma and atopy within the setting of an affluent urban population., Methods: The authors followed 526 German children from birth to 5 years of age. Parental interviews in pregnancy and then yearly assessed the health of the child and environmental characteristics. Endotoxin and allergens in house dust were measured at 3 months. Atopic sensitization was assessed at 1 and 5 years., Results: In atopic mothers, acute atopic symptoms during pregnancy were associated with increased risk of early atopic dermatitis (adjusted odds ratio [aOR] 1.74, 95% confidence interval [CI] 1.00-3.02) and allergic rhinitis at 5 years (aOR 2.11, 95% CI 1.01-4.41). Further, maternal illnesses during pregnancy (ie, repeated common colds) increased the risk of asthma at 5 years (aOR 2.31, 95% CI 1.12-4.78). Endotoxin in the child's mattress was inversely associated with atopic sensitization (aOR 0.79, 95% CI 0.64-0.97) and asthma (aOR 0.71, 95% CI 0.55-0.93). A contrasting effect of early endotoxin and mite exposure was observed for mite sensitization: mite exposure increased the risk of mite sensitization at 5 years (aOR 1.30, 95% CI 1.11-1.53), whereas endotoxin exposure was inversely associated with mite sensitization (aOR 0.73, 95% CI 0.57-0.95)., Conclusion: Factors affecting the in utero environment, such as maternal atopy and infections, and bacterial exposure in pregnancy or early life may act as immunomodulators enhancing or inhibiting the development of asthma and atopy in childhood., (Copyright © 2014 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Histologic chorioamnionitis, frequently associated with preterm births and adverse outcomes, results in prolonged exposure of preterm fetuses to infectious agents and pro-inflammatory mediators, such as LPS. Endotoxin tolerance-type effects were demonstrated in fetal sheep following repetitive systemic or intra-amniotic (i.a.) exposures to LPS, suggesting that i.a. LPS exposure would cause endotoxin tolerance to a postnatal systemic dose of LPS in preterm sheep. In this study, randomized pregnant ewes received either two i.a. injections of LPS or saline prior to preterm delivery. Following operative delivery, the lambs were treated with surfactant, ventilated, and randomized to receive either i.v. LPS or saline at 30  min of age. Physiologic variables and indicators of systemic and lung inflammation were measured. Intravenous LPS decreased blood neutrophils and platelets values following i.a. saline compared to that after i.a. LPS. Intra-amniotic LPS prevented blood pressure from decreasing following the i.v. LPS, but also caused an increased oxygen index. Intra-amniotic LPS did not cause endotoxin tolerance as assessed by cytokine expression in the liver, lung or plasma, but increased myeloperoxidase-positive cells in the lung. The different compartments of exposure to LPS (i.a. vs i.v.) are unique to the fetal to newborn transition. Intra-amniotic LPS incompletely tolerized fetal lambs to postnatal i.v. LPS.

Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a hyporesponsive state to subsequent challenge, which is termed endotoxin tolerance. In this experiment, we studied the cytokine production in THP-1 cells upon single or repeated Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) or Escherichia coli (E. coli) LPS stimulation by ELISA. In addition, the protein expression profiles of Toll-like receptor 2 (TLR2), TLR4, IL-1 receptor-associated kinase 4 (IRAK4) and IRAK-M and the gene expression changes of Toll-interacting protein (Tollip) and suppressor of cytokine-signaling-1 (SOCS1) were explored to identify possible mechanisms for changes in cytokine secretion. After repeated stimulation with P. gingivalis LPS or E. coli LPS, secretions of TNF-α and IL-1β were decreased significantly compared with those following single challenge, while the levels of IL-10 were increased (p < 0.05). Only comparable levels of IL-8 were confirmed in P. gingivalis LPS-tolerized cells (p > 0.05). In addition, severe downregulation of TLR2 was detected in THP-1 cells retreated with P. gingivalis LPS, and the reduction of TLR4 expression was observed in cells restimulated with E. coli LPS (p < 0.05). Precondition with P. gingivalis LPS or E. coli LPS also led to an enhancement of IRAK-M and SOCS1, while maintaining the expressions of IRAK4 and Tollip. This pattern of cytokine production indicates the different effects of endotoxin tolerance triggered by P. gingivalis LPS and E. coli LPS, which might contribute to limiting inflammatory damage. Moreover, TLR2, TLR4, IRAK-M, and SOCS1 might play important roles in developing tolerance.

Background: It is considered that the pathogenesis is closely related to an excessive production of pro-inflammatory cytokines caused by bacterial toxins and an imbalance between pro-inflammatory and anti-inflammatory mediators., Materials and Methods: This work investigates the effect of electro-acupuncturing (EA), at Zusanli point (ST36) on plasma cytokine release and organ dysfunction and their mechanism in conscious rats with endotoxin challenge., Results: EA at Zusanli points obviously lowered the elevated levels of plasma TNF-α, and attenuated changes in parameters relevant to various organ functions at 2 h after LPS challenge. α-BGT injection or bilateral cervical vagotomy could weaken or eliminate the effects of EA, and further aggravated the elevated levels of pro-inflammatory cytokines and organ dysfunction., Conclusion: The results suggested that EA at Zusanli points significantly reduced the release of pro-inflammatory cytokines and organ dysfunction after LPS challenge by activating cholinergic anti-inflammatory pathway.

E. coli O111 strains are responsible for outbreaks of blood diarrhea and hemolytic uremic syndrome throughout the world. Because of their phenotypic variability, the development of a vaccine against these strains which targets an antigen that is common to all of them is quite a challenge. Previous results have indicated, however, that O111 LPS is such a candidate, but its toxicity makes LPS forbidden for human use. To overcome this problem, O111 polysaccharides were conjugated either to cytochrome C or to EtxB (a recombinant B subunit of LT) as carrier proteins. The O111-cytochrome C conjugate was incorporated in silica SBA-15 nanoparticles and administered subcutaneously in rabbits, while the O111-EtxB conjugate was incorporated in Vaxcine(TM), an oil-based delivery system, and administered orally in mice. The results showed that one year post-vaccination, the conjugate incorporated in silica SBA-15 generated antibodies in rabbits able to inhibit the adhesion of all categories of O111 E. coli to epithelial cells. Importantly, mice immunized orally with the O111-EtxB conjugate in Vaxcine(TM) generated systemic and mucosal humoral responses against all categories of O111 E. coli as well as antibodies able to inhibit the toxic effect of LT in vitro. In summary, the results obtained by using 2 different approaches indicate that a vaccine that targets the O111 antigen has the potential to prevent diarrhea induced by O111 E. coli strains regardless their mechanism of virulence. They also suggest that a conjugated vaccine that uses EtxB as a carrier protein has potential to combat diarrhea induced by ETEC.

Endotoxin, one of the principal components on the outer membrane of Gram-negative bacteria, is considered a key and early component in the pathogenesis of sepsis. Polymyxin B bound to polystyrene fibers (PMX) is a medical device capable of removing circulating endotoxin by adsorption. The most comprehensive analysis to date of clinical experience with this device remains a meta-analysis of 28 studies between 1998 and 2006. This showed that PMX hemoperfusion was associated with improved blood pressure and a reduction in dopamine dose, improved PaO2/FiO2 ratio and lower mortality. Since this meta-analysis, over 50 additional studies on PMX have been published. The majority are observational, with small sample sizes. Notable among the newer studies is the increasing interest in the use of PMX therapy in interstitial pneumonias and idiopathic pulmonary fibrosis, as well as in longer treatment duration and earlier initiation of PMX therapy in an attempt to further improve clinical outcomes. These observational data highlight important aspects of PMX therapy worthy of more rigorous investigation in future studies., (© 2014 S. Karger AG, Basel.)

This study examined the effect of feeding yeast cell wall (YCW) products on the metabolic responses of newly-received feedlot cattle to an endotoxin challenge. Heifers were separated into treatment groups receiving either a Control diet, YCW-A or YCW-C, and were fed for 52 d. Heifers were weighed on d 0, 14, 36, 38 and 52. On d 37 heifers were challenged i.v. with LPS [0.5 µg/kg body weight (BW)] and blood samples were collected relative to LPS challenge. Heifer BW increased from d 0 to 36 and from d 38 to 52, but was not affected by treatment. Post-LPS, glucose concentrations increased and were less in YCW-A than Control and YCW-C heifers. Pre-LPS, insulin concentrations were greater in YCW-A and YCW-C than Control heifers. Post-LPS, insulin concentrations increased with YCW-C having greater insulin than Control heifers. Pre-LPS, NEFA concentrations tended to be less in YCW-C than Control heifers. Post-LPS non-esterified fatty acids (NEFA) concentrations were less in YCW-C than Control and YCW-A heifers. Post-LPS, blood urea nitrogen (BUN) concentrations were greater in YCW-A than Control and YCW-C. These data indicate, based on NEFA and BUN data, that certain YCW products can enhance energy metabolism during an immune challenge without causing lipolysis or muscle catabolism.

Endotoxin is a potent mediator of a broad range of patho-physiological effects in humans. It is present in all Gram negative (GN) bacteria. It would be expected that anti-endotoxin therapies, whether antibody based or not, would have an important adjuvant therapeutic role along with antibiotics and other supportive therapies for GN infections. Indeed there is an extensive literature relating to both pre-clinical and clinical studies of anti-endotoxin antibodies. However, the extent of disconnect between the generally successful pre-clinical studies versus the failures of the numerous large clinical trials of antibody based and other anti-endotoxin therapies is under-appreciated and unexplained. Seeking a reconciliation of this disconnect is not an abstract academic question as clinical trials of interventions to reduce levels of endotoxemia levels are ongoing. The aim of this review is to examine new insights into the complex relationship between endotoxemia and sepsis in an attempt to bridge this disconnect. Several new factors to consider in this reappraisal include the frequency and types of GN bacteremia and the underlying mortality risk in the various study populations. For a range of reasons, endotoxemia can no longer be considered as a single entity. There are old clinical trials which warrant a re-appraisal in light of these recent advances in the understanding of the structure-function relationship of endotoxin. Fundamentally however, the disconnect not only remains, it has enlarged.

Drosophila Toll functions in embryonic development and innate immunity and is activated by an endogenous ligand, Spätzle (Spz). The related Toll-like receptors in vertebrates also function in immunity but are activated directly by pathogen-associated molecules such as bacterial endotoxin. Here, we present the crystal structure at 2.35-Å resolution of dimeric Spz bound to a Toll ectodomain encompassing the first 13 leucine-rich repeats. The cystine knot of Spz binds the concave face of the Toll leucine-rich repeat solenoid in an area delineated by N-linked glycans and induces a conformational change. Mutagenesis studies confirm that the interface observed in the crystal structure is relevant for signaling. The asymmetric binding mode of Spz to Toll is similar to that of nerve growth factor (NGF) in complex with the p75 neurotrophin receptor but is distinct from that of microbial ligands bound to the Toll-like receptors. Overall, this study indicates an allosteric signaling mechanism for Toll in which ligand binding to the N terminus induces a conformational change that couples to homodimerization of juxtamembrane structures in the Toll ectodomain C terminus.

Campylobacter jejuni is the most common bacterial cause of human gastroenteritis and often precedes development of Guillain-Barré syndrome (GBS), a life-threatening paralytic disease. The incorporation of the carbohydrate sialic acid into C. jejuni lipooligosaccharides (LOS) is associated with increased severity of gastroenteritis and with induction of GBS; however, the underlying mechanisms remain completely unknown. In this study, we demonstrate that sialic acids in C. jejuni endotoxin enhance the rapid production of IFN-β and TNF-α by human dendritic cells (DCs). Using neutralizing Abs and receptors it was shown that these DC-derived cytokines promote the proliferation of human mucosal B cells in a T cell-independent manner. The production of both IFN-β and TNF-α by DCs in response to LOS requires CD14, and the amplified response of DCs to sialylated C. jejuni LOS is CD14 dependent. Together, these results indicate that sialylation of C. jejuni LOS increases DC activation and promotes subsequent B cell responses through CD14-driven production of IFN-β and TNF-α. This enhanced DC/B cell response may explain the increased pathogenicity of sialylated C. jejuni and may be key to the initiation of B cell-mediated autoimmunity in GBS.

Lipopolysaccharide (LPS, endotoxin) is a structural component of the gram-negative outer membrane. The lipid A moiety of LPS binds to the LPS receptor complex expressed by leukocytes, endothelial cells, and parenchymal cells and is the primary component of gram-negative bacteria that is recognized by the immune system. Activation of the LPS receptor complex by native lipid A induces robust cytokine production, leukocyte activation, and inflammation, which is beneficial for clearing bacterial infections at the local level but can cause severe systemic inflammation and shock at higher challenge doses. Interestingly, prior exposure to LPS renders the host resistant to shock caused by subsequent LPS challenge, a phenomenon known as endotoxin tolerance. Treatment with lipid A has also been shown to augment the host response to infection and to serve as a potent vaccine adjuvant. However, the adverse effects associated with the pronounced inflammatory response limit the use of native lipid A as a clinical immunomodulator. More recently, analogs of lipid A have been developed that possess attenuated proinflammatory activity but retain attractive immunomodulatory properties. The lipid A analog monophosphoryl lipid A exhibits approximately 1/1,000th of the toxicity of native lipid A but retains potent immunoadjuvant activity. As such, monophosphoryl lipid A is currently used as an adjuvant in several human vaccine preparations. Because of the potency of lipid A analogs as immunoadjuvants, numerous laboratories are actively working to identify and develop new lipid A mimetics and to optimize their efficacy and safety. Based on those characteristics, lipid A analogs represent an attractive family of immunomodulators.

Endotoxin tolerance was first described in a study that exposed animals to a sublethal dose of bacterial endotoxin. The animals subsequently survived a lethal injection of endotoxin. This refractory state is associated with the innate immune system and, in particular, with monocytes and macrophages, which act as the main participants. Several mechanisms are involved in the control of endotoxin tolerance; however, a full understanding of this phenomenon remains elusive. A number of recent reports indicate that clinical examples of endotoxin tolerance include not only sepsis but also diseases such as cystic fibrosis and acute coronary syndrome. In these pathologies, the risk of new infections correlates with a refractory state. This review integrates the molecular basis and clinical implications of endotoxin tolerance in various pathologies.

Background: Knowledge of the inhalable particulate matter components responsible for health effects is important for developing targeted regulation., Objectives: In a double-blind randomised cross-over trial of controlled human exposures to concentrated ambient particles (CAPs) and their endotoxin and (1→3)-β-D-glucan components, we evaluated acute inflammatory responses., Methods: 35 healthy adults were exposed to five 130-min exposures at rest: (1) fine CAPs (~250 µg/m(3)); (2) coarse CAPs (200 µg/m(3)); (3) second coarse CAPs (~200 µg/m(3)); (4) filtered air; and (5) medical air. Induced sputum cell counts were measured at screening and 24 h postexposure. Venous blood total leucocytes, neutrophils, interleukin-6 and high-sensitivity C reactive protein (CRP) were measured pre-exposure, 3 and 24 h postexposure., Results: Relative to filtered air, an increase in blood leucocytes 24 h (but not 3 h) postexposure was significantly associated with coarse (estimate=0.44×10(9) cells/L (95% CI 0.01 to 0.88); n=132) and fine CAPs (0.68×10(9) cells /L (95% CI 0.19 to 1.17); n=132), but not medical air. Similar associations were found with neutrophil responses. An interquartile increase in endotoxin (5.4 ng/m(3)) was significantly associated with increased blood leucocytes 3 h postexposure (0.27×10(9) cells/L (95% CI 0.03 to 0.51); n=98) and 24 h postexposure (0.37×10(9) cells/L (95% CI 0.12 to 0.63); n=98). This endotoxin effect did not differ by particle size. There were no associations with glucan concentrations or interleukin-6, CRP or sputum responses., Conclusions: In healthy adults, controlled coarse and fine ambient particle exposures independently induced acute systemic inflammatory responses. Endotoxin contributes to the inflammatory role of particle air pollution.

Periodontal disease (PD) is a chronic inflammatory and alveolar bone destructive disease triggered by oral biofilm-producing microorganisms, such as Aggregatibacter actinomycetemcomitans. The levels of the phospholipid platelet-activating factor (PAF) in the saliva, gingival crevicular fluid, and periodontal tissues are significantly increased during inflammatory conditions, such as PD, but the exact mechanism that links PAF to alveolar bone resorption is not well understood. In the current study, alveolar bone resorption was induced by experimental PD through the oral inoculation of A. actinomycetemcomitans in wild-type (WT) and PAF receptor knockout (Pafr(-/-)) mice. In vitro experiments using A. actinomycetemcomitans lipopolysaccharide (LPS)-stimulated RAW 264.7 cells treated with a PAF receptor antagonist (UK74505) were also performed. The expression of lyso-PAF acetyltransferase in periodontal tissues was significantly increased 3 h after A. actinomycetemcomitans LPS injection in mice. WT and Pafr(-/-) mice that were subjected to oral inoculation of A. actinomycetemcomitans presented neutrophil accumulation and increased levels of CXCL-1 and tumor necrosis factor alpha (TNF-α) in periodontal tissues. However, Pafr(-/-) mice presented less alveolar bone loss than WT mice. The in vitro blockade of the PAF receptor impaired the resorptive activity of A. actinomycetemcomitans LPS-activated osteoclasts. In conclusion, this study shows for the first time that the blockade of PAF receptor may contribute to the progression of PD triggered by A. actinomycetemcomitans by directly affecting the differentiation and activity of osteoclasts.

Background: The response to endotoxin (LPS), and subsequent signal transduction lead to the production of cytokines such as tumor necrosis factor-α (TNF-α) by innate immune cells. Cells or organisms pretreated with endotoxin enter into a transient state of hyporesponsiveness, referred to as endotoxin tolerance (ET) which represents a particular case of negative preconditioning. Despite recent progress in understanding the molecular basis of ET, there is no consensus yet on the primary mechanism responsible for ET and for the more complex cases of cross tolerance. In this study, we examined the consequences of the macromolecular crowding (MMC) and of fractal-like kinetics (FLK) of intracellular enzymatic reactions on the LPS signaling machinery. We hypothesized that this particular type of enzyme kinetics may explain the development of ET phenomenon., Method: Our aim in the present study was to characterize the chemical kinetics framework in ET and determine whether fractal-like kinetics explains, at least in part, ET. We developed an ordinary differential equations (ODE) mathematical model that took into account the links between the MMC and the LPS signaling machinery leading to ET. We proposed that the intracellular fractal environment (MMC) contributes to ET and developed two mathematical models of enzyme kinetics: one based on Kopelman's fractal-like kinetics framework and the other based on Savageau's power law model., Results: Kopelman's model provides a good image of the potential influence of a fractal intracellular environment (MMC) on ET. The Savageau power law model also partially explains ET. The computer simulations supported the hypothesis that MMC and FLK may play a role in ET., Conclusion: The model highlights the links between the organization of the intracellular environment, MMC and the LPS signaling machinery leading to ET. Our FLK-based model does not minimize the role of the numerous negative regulatory factors. It simply draws attention to the fact that macromolecular crowding can contribute significantly to the induction of ET by imposing geometric constrains and a particular chemical kinetic for the intracellular reactions.

Autotoxic production of proinflammatory mediators during early sepsis induces excessive inflammation, and their later suppression may limit the immune response. We previously reported that sepsis differentially represses transcription and translation of tumor necrosis factor alpha (TNF-α) and interleukin 1β (IL-1β) to reprogram sepsis inflammation. This switch is gene specific and plays a crucial role in the clinically relevant syndrome of endotoxin adaptation/tolerance, multiorgan failure, and poor sepsis outcome. To further define the mechanisms responsible for translation disruption that follows inflammation induction, we used THP-1 human promonocytes as a model of Toll-like receptor 4 (TLR4) responses found in sepsis. We showed that phosphorylation-dependent activation of p38 mitogen-activated protein kinase (MAPK) and translation disruption of TNF-α and IL-6 follow increased MAPK phosphatase 1 (MKP-1) expression and that MKP-1 knockdown rephosphorylates p38 and restores the capacity to translate TNF-α and IL-6 mRNAs. We also observed that the RNA-binding protein motif 4 (RBM4), a p38 MAPK target, accumulates in an unphosphorylated form in the cytosol in endotoxin-adapted cells, suggesting that dephosphorylated RBM4 may function as a translational repressor. Moreover, MKP-1 knockdown promotes RBM4 phosphorylation, blocks its transfer from the nucleus to the cytosol, and reverses translation repression. We also found that microRNA 146a (miR-146a) knockdown prevents and miR-146a transfection induces MKP-1 expression, which lead to increases or decreases in TNF-α and IL-6 translation, respectively. We conclude that a TLR4-, miR-146a-, p38 MAPK-, and MKP-1-dependent autoregulatory pathway regulates the translation of proinflammatory genes during the acute inflammatory response by spatially and temporally modifying the phosphorylation state of RBM4 translational repressor protein.

Within hours after its initiation, the severe systemic inflammatory response of sepsis shifts to an adaptive anti-inflammatory state with coincident immunosuppression. This anti-inflammatory phenotype is characterized by diminished proinflammatory cytokine gene expression in response to toll-like receptor (TLR) stimulation with bacterial endotoxin/lipopolysaccharide (LPS), also known as endotoxin tolerance/adaptation. Our and other studies have established that gene-specific reprogramming following TLR4 responses independently represses transcription and translation of proinflammatory genes such as tumor necrosis factor alpha (TNFα). We also previously demonstrated that TNFα and interleukin (IL)-6 mRNA translation is repressed in endotoxin-adapted THP-1 human monocytes by an miRNA-based mechanism involving the argonaute family protein argonaute 2 (Ago2). Here, we further define the molecular nature of reprogramming translation by showing that TLR4-induced microRNA-146 promotes a feed-forward loop that modifies the subcellular localization of the RNA-binding protein RBM4 (RNA-binding motif protein 4) and promotes its interaction with Ago2. This interaction results in the assembly of a translation-repressor complex that disrupts TNFα and IL-6 cytokine synthesis in endotoxin-adapted THP-1 monocytes. This novel molecular path prevents the phosphorylation of RBM4 on serine-309 by p38 MAPK (mitogen-activated protein kinase), which leads to RBM4 accumulation in the cytosol and interaction with Ago2. We further find that microRNA-146a knockdown by antagomirs or protein phosphatase inhibition by okadaic acid increases p38 MAPK phosphorylation and results in RBM4 serine-309 phosphorylation and nuclear relocalization, which disrupts RBM4 and Ago2 interactions and restores TLR4-dependent synthesis of TNFα and IL-6. We conclude that miR-146a has a diverse and critical role in limiting an excessive acute inflammatory reaction.

Background: Crohn's disease and ulcerative colitis are the major types of chronic inflammatory bowel disease occurring in the colon and small intestine. A growing body of research has proposed that probiotics are able to attenuate the inflammatory symptoms of these diseases in vitro and in vivo. However, the mechanism of probiotic actions remains unclear., Results: Our results suggested Lactobacillus plantarum MYL26 inhibited inflammation in Caco-2 cells through regulation of gene expressions of TOLLIP, SOCS1, SOCS3, and IκBα, rather than SHIP-1 and IRAK-3., Conclusions: We proposed that live/ heat-killed Lactobacillus plantarum MYL26 and bacterial cell wall extract treatments impaired TLR4-NFκb signal transduction through Tollip, SOCS-1 and SOCS-3 activation, thus inducing LPS tolerance. Our findings suggest that either heat-killed probiotics or probiotic cell wall extracts are able to attenuate inflammation through pathways similar to that of live bacteria.