Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. It occurs with a prevalence of up to 25%, of which 10-20% cases progress to nonalcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. The histopathology of NASH is characterized by neutrophilic infiltration, and endotoxins from gram-negative rods have been postulated as a contributing factor. Elevations in endotoxin levels in the blood can be classified as intestinal and hepatic factors. In recent years, leaky gut syndrome, which is characterized by impaired intestinal barrier function, has become a significant issue. A leaky gut may prompt intestinal bacteria dysbiosis and increase the amount of endotoxin that enters the liver from the portal vein. These contribute to persistent chronic inflammation and progressive liver damage. In addition, hepatic factors suggest that liver damage can be induced by low-dose endotoxins, which does not occur in healthy individuals. In particular, increased expression of CD14, an endotoxin co-receptor in the liver, may result in leptin-induced endotoxin hyper-responsiveness in obese individuals. Thus, elevated blood endotoxin levels contribute to the progression of NASH. The current therapeutic targets for NASH treat steatosis and liver inflammation and fibrosis. While many clinical trials are underway, no studies have been performed on therapeutic agents that target the intestinal barrier. Recently, a randomized placebo-controlled trial examined the role of the intestinal barrier in patients with NAFLD. To our knowledge, this study was the first of its kind and study suggested that the intestinal barrier may be a novel target in the future treatment of NAFLD.

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Bacterial lipopolysaccharides (LPS), also known as endotoxins, are major structural components of the outer membrane of Gram-negative bacteria that serve as a barrier and protective shield between them and their surrounding environment. LPS is considered to be a major virulence factor as it strongly stimulates the secretion of pro-inflammatory cytokines which mediate the host immune response and culminating in septic shock. Quantitative structure-activity relationship studies of the LPS neutralization activities of anti-endotoxins were performed using charge and quantum chemical descriptors. Artificial neural network implementing the back-propagation algorithm was selected for the multivariate analysis. The predicted activities from leave-one-out cross-validation were well correlated with the experimental values as observed from the correlation coefficient and root mean square error of 0.930 and 0.162, respectively. Similarly, the external testing set also yielded good predictivity with correlation coefficient and root mean square error of 0.983 and 0.130. The model holds great potential for the rational design of novel and robust compounds with enhanced neutralization activity.

Increasingly, feed additives for livestock, such as amino acids and vitamins, are being produced by Gram-negative bacteria, particularly Escherichia coli. The potential therefore exists for animals, consumers and workers to be exposed to possibly harmful amounts of endotoxin from these products. The aim of this review was to assess the extent of the risk from endotoxins in feed additives and to calculate how such risk can be assessed from the properties of the additive. Livestock are frequently exposed to a relatively high content of endotoxin in the diet: no additional hazard to livestock would be anticipated if the endotoxin concentration of the feed additive falls in the same range as feedstuffs. Consumer exposure will be unaffected by the consumption of food derived from animals receiving endotoxin-containing feed, because the small concentrations of endotoxin absorbed do not accumulate in edible tissues. In contrast, workers processing a dusty additive may be exposed to hazardous amounts of endotoxin even if the endotoxin concentration of the product is low. A calculation method is proposed to compare the potential risk to the worker, based on the dusting potential, the endotoxin concentration and technical guidance of the European Food Safety Authority, with national exposure limits.

Bacterial lipopolysaccharides (LPS), potent inducers of inflammation, have been associated with chronic metabolic disturbances. Obesity is linked to dyslipidemia, increased body adiposity, and endotoxemia. We investigated the cross-sectional relationships between serum LPS activity and body adiposity as well as inflammation in 242 subjects with type 1 diabetes. Body fat distribution was measured by DXA and serum LPS activity by the limulus amebocyte lysate end-point assay. Since no interaction between visceral fat mass and sex was observed, data were pooled for the subsequent analyses. LPS was independently associated with visceral fat mass, when adjusted for traditional risk factors (age, sex, kidney status, hsCRP, insulin sensitivity). In the multivariate analysis, serum LPS activity and triglyceride concentrations had a joint effect on visceral fat mass, independent of these factors alone. A combination of high LPS and high hsCRP concentrations was also observed in those with the largest visceral fat mass. In conclusion, high serum LPS activity levels were associated with visceral fat mass in subjects with type 1 diabetes strengthening its role in the development of central obesity, inflammation and insulin resistance.

Endotoxins (lipopolysaccharides, LPS) are one of the strongest immunostimulators in nature, responsible for beneficial effects at low, and pathophysiological effects at high concentrations, the latter frequently leading to sepsis and septic shock associated with high mortality in critical care settings. There are no drugs specifically targeting the pathophysiology of sepsis, and new therapeutic agents are therefore urgently needed. The lipopolyamines are a novel class of small molecules designed to sequester and neutralize LPS. To understand the mechanisms underlying the binding and neutralization of LPS toxicity, we have performed detailed biophysical analyses of the interactions of LPS with candidate lipopolyamines which differ in their potencies of LPS neutralization. We examined gel-to-liquid crystalline phase behavior of LPS and of its supramolecular aggregate structures in the absence and presence of lipopolyamines, the ability of such compounds to incorporate into different membrane systems, and the thermodynamics of the LPS:lipopolyamine binding. We have found that the mechanisms which govern the inactivation process of LPS obey similar rules as found for other active endotoxin neutralizers such as certain antimicrobial peptides.

Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment.

In the track of new biopesticides, four genes namely cytA, cytB, cytC and cytD encoding proteins homologous to Bacillus thuringiensis (Bt) Cyt toxins have been identified in the plant pathogenic bacteria Dickeya dadantii genome. Here we show that three Cyt-like δ-endotoxins from D. dadantii (CytA, CytB and CytC) are toxic to the pathogen of the pea aphid Acyrthosiphon pisum in terms of both mortality and growth rate. The phylogenetic analysis of the comprehensive set of Cyt toxins available in genomic databases shows that the whole family is of limited taxonomic occurrence, though in quite diverse microbial taxa. From a structure-function perspective the 3D structure of CytC and its backbone dynamics in solution have been determined by NMR. CytC adopts a cytolysin fold, structurally classified as a Cyt2-like protein. Moreover, the identification of a putative lipid binding pocket in CytC structure, which has been probably maintained in most members of the Cyt-toxin family, could support the importance of this lipid binding cavity for the mechanism of action of the whole family. This integrative approach provided significant insights into the evolutionary and functional history of D. dadantii Cyt toxins, which appears to be interesting leads for biopesticides.

System dynamics is a powerful tool that allows modeling of complex and highly networked systems such as those found in the human immune system. We have developed a model that reproduces how the exposure of human monocytes to lipopolysaccharides (LPSs) induces an inflammatory state characterized by high production of tumor necrosis factor alpha (TNFα), which is rapidly modulated to enter into a tolerant state, known as endotoxin tolerance (ET). The model contains two subsystems with a total of six states, seven flows, two auxiliary variables, and 14 parameters that interact through six differential and nine algebraic equations. The parameters were estimated and optimized to obtain a model that fits the experimental data obtained from human monocytes treated with various LPS doses. In contrast to publications on other animal models, stimulation of human monocytes with super-low-dose LPSs did not alter the response to a second LPSs challenge, neither inducing ET, nor enhancing the inflammatory response. Moreover, the model confirms the low production of TNFα and increased levels of C-C motif ligand 2 when monocytes exhibit a tolerant state similar to that of patients with sepsis. At present, the model can help us better understand the ET response and might offer new insights on sepsis diagnostics and prognosis by examining the monocyte response to endotoxins in patients with sepsis.

Colchicine is an anti-inflammatory drug with a narrow therapeutic index. Its binding to tubulin prevents microtubule polymerization; however, little is known about how depolymerization of microtubules interferes with the phagocytosis function of Kupffer cells (KC). Here, we applied functional intravital imaging techniques to investigate the influence of microtubule disruption by colchicine on KC morphology, as well as its capacity to clear foreign particles and bacterial lipopolysaccharide (LPS) in anesthetized mice. Intravital imaging of KC in healthy mice showed the typical elongated morphology, localization at the luminal side of the sinusoidal endothelial cells, and moving cell protrusions. In contrast, at colchicine doses of 1 mg/kg and higher (intraperitoneal), KC appeared roundish with strongly reduced protrusions and motility. To study the functional consequences of these alterations, we analyzed the capacity of KC to phagocytose fluorescent nanospheres (100 nm-size) and LPS. After tail vein injection, the nanospheres formed aggregates of up to ~ 5 µm moving along the sinusoidal bloodstream. In controls, the nanosphere aggregates were rapidly captured by the Kupffer cell protrusions, followed by an internalization process that lasted up to 10 min. Similar capture events and internalization processes were observed after the administration of fluorescently labeled LPS. In contrast, capture and internalization of both nanospheres and LPS by KC were strongly reduced in colchicine-treated mice. Reduced phagocytosis of LPS was accompanied by aggravated production of inflammatory cytokines. Since 0.4 mg/kg colchicine in mice has been reported to be bio-equivalent to human therapeutic doses, the here-observed adverse effects on KC occurred at doses only slightly above those used clinically, and may be critical for patients with endotoxemia due to a leaky gut-blood barrier.

Lipopolysaccharides (LPS) are highly toxic compounds, even at a trace amount. When recombinant proteins are produced in E. coli, it is inevitable that LPS contaminates. However, LPS removal is still technically challenging and costly due to the high degree of solubility in a wide range of solvents. In this study, we explored the possibility of using the N-terminal region containing cysteine-rich, EGF-like, and sushi1-3 domains (CES3) of Factor C from the horseshoe crab Carcinoscorpius rotundicauda to develop a platform to remove LPS from recombinant proteins. We expressed CES3 as part of a recombinant protein, BiP:NT:CBM3:SUMO:CES3:His:HDEL, in Nicotiana benthamiana and found that purified or microcrystalline cellulose (MCC) bead-immobilised CES3 showed strong binding to LPS-containing E. coli. To produce CES3:CBM3 in an LPS-free environment, we generated Arabidopsis transgenic plants harbouring a recombinant gene, BiP:NT:SUMO:CES3:CBM3:HDEL, and found that transgenic plants mainly produce CES3:CBM3:His:HDEL, a truncated version of BiP:NT:SUMO:CES3:CBM3:HDEL via endogenous protease-mediated proteolytic processing in vivo. CES3:CBM3:HDEL purified from Arabidopsis plant extracts and immobilised onto MCC beads removed LPS contamination from protein samples. We propose that the CES3:CBM3 fusion protein produced in plants and immobilised on MCC beads can be a robust and easy platform for LPS removal from recombinant proteins.

Coxiella burnetii is the etiological agent of Q fever found worldwide. The microorganism has like other Gram-negative bacteria a lipopolysaccharide (LPS, endotoxin) in its outer membrane, which is important for the pathogenicity of the bacteria. In order to understand the biological activity of LPS, a detailed physico-chemical analysis of LPS is of utmost importance.

A newly developed semi-automatic synthetic luminescence substrate (SALS) method for measuring endotoxin was compared with the existing turbidimetric kinetic assay (TKA) using leukocyte-rich plasma to verify its usefulness. As a result, the endotoxin levels by this method were higher than that by the existing assay in most specimens, and the time required for measurement was much shorter. In addition, the leukocyte-rich plasma endotoxin level minus the plasma endotoxin levels were named leukocyte-associated endotoxin, and these levels per one leukocyte were compared. As a result, those levels were highly correlated with the endotoxin measurement levels of leukocyte-rich plasma. The correlation coefficient of SALS method was superior to the existing TKA method, the endotoxin level by this method may be close to true endotoxin levels.

The capability of pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) for the direct analysis of endotoxins is demonstrated in this research article using the lipopolysaccharides of Pseudomonas aeruginosa 10. Analytical methods based on evolved gas analysis-MS, single-shot (SS) Py-GC-MS, and multishot heart cut Py-GC-MS were investigated. Among the various methods developed, the SS Py-GC-MS method shows superior potential for identifying bacterial endotoxins effectively through their biomarkers. The results obtained were validated with conventional mass spectral analysis after hydrolysis. The method was also evaluated for its robustness based on quality control criteria indicated by the U.S. EPA Method 8270D. When applied onto endotoxins of different Gram-negative bacteria, this method produced vastly distinct pyrograms. The results show that rapid and sensitive direct detection of endotoxins is possible with the Py-GC-MS method developed.

Endotoxins are common contaminants in allergen preparations and affect antigen-specific cellular responses. Distinct effects of endotoxin on cells in human umbilical cord and adult blood are poorly defined.

P. aeruginosa, a highly virulent Gram-negative bacterium, can cause severe nosocomial infections, and it has developed resistance against most antibiotics. New therapeutic strategies are urgently needed to treat such bacterial infection and reduce its toxicity caused by endotoxin (lipopolysaccharide, LPS). Neutrophils have been proven to be able to target inflammation site and neutrophil membrane receptors such as Toll-like receptor-4 (TLR4) and CD14, and exhibit specific affinity to LPS. However, antibacterial delivery system based on the unique properties of neutrophils has not been reported.

The Toll-like receptor 4/MD-2 receptor complex recognizes endotoxin, a Gram-negative bacterial cell envelope component. Recognition of the most potent hexaacylated form of endotoxin is mediated by the sixth acyl chain that protrudes from the MD-2 hydrophobic pocket and bridges TLR4/MD-2 to the neighboring TLR4 ectodomain, driving receptor dimerization via hydrophobic interactions. In hypoacylated endotoxins all acyl chains could be accommodated within the binding pocket of the human hMD-2. Nevertheless, tetra- and pentaacylated endotoxins activate the TLR4/MD-2 receptor of several species. We observed that amino acid residues 82 and 122, located at the entrance to the endotoxin binding site of MD-2, have major influence on the species-specific endotoxin recognition. We show that substitution of hMD-2 residue V82 with an amino acid residue with a bulkier hydrophobic side chain enables activation of TLR4/MD-2 by pentaacylated and tetraacylated endotoxins. Interaction of the lipid A phosphate group with the amino acid residue 122 of MD-2 facilitates the appropriate positioning of the hypoacylated endotoxin. Moreover, mouse TLR4 contributes to the agonistic effect of pentaacylated msbB endotoxin. We propose a molecular model that explains how the molecular differences between the murine or equine MD-2, which both have sufficiently large hydrophobic pockets to accommodate all five or four acyl chains, influence the positioning of endotoxin so that one of the acyl chains remains outside the pocket and enables hydrophobic interactions with TLR4, leading to receptor activation.

Protein nanocages represent an emerging candidate among nanoscaled delivery systems. Indeed, they display unique features that proved to be very interesting from the nanotechnological point of view such as uniform structure, stability in biological fluids, suitability for surface modification to insert targeting moieties and loading with different drugs and dyes. However, one of the main concerns regards the production as recombinant proteins in E. coli, which leads to a product with high endotoxin contamination, resulting in nanocage immunogenicity and pyrogenicity. Indeed, a main challenge in the development of protein-based nanoparticles is finding effective procedures to remove endotoxins without affecting protein stability, since every intravenous injectable formulation that should be assessed in preclinical and clinical phase studies should display endotoxins concentration below the admitted limit of 5 EU/kg. Different strategies could be employed to achieve such a result, either by using affinity chromatography or detergents. However, these strategies are not applicable to protein nanocages as such and require implementations. Here we propose a combined protocol to remove bacterial endotoxins from nanocages of human H-ferritin, which is one of the most studied and most promising protein-based drug delivery systems. This protocol couples the affinity purification with the Endotrap HD resin to a treatment with Triton X-114. Exploiting this protocol, we were able to obtain excellent levels of purity maintaining good protein recovery rates, without affecting nanocage interactions with target cells. Indeed, binding assay and confocal microscopy experiments confirm that purified H-ferritin retains its capability to specifically recognize cancer cells. This procedure allowed to obtain injectable formulations, which is preliminary to move to a clinical trial.

African Dust Events (ADE) are a seasonal phenomenon that has been suggested to exacerbate respiratory and proinflammatory diseases in Puerto Rico (PR). Increases in PM10 concentration and the effects of biological endotoxins (ENX) are critical factors to consider during these storms. ENX promote proinflammatory responses in lungs of susceptible individuals through activation of the Toll-like receptors (TLR2/4) signaling pathways. The objective of the study was to evaluate the toxicological and proinflammatory responses stimulated by ADE PM10 ENX reaching PR using human bronchial epithelial cells. PM10 organic extracts from a rural and urban site in PR (March 2004) were obtained from ADE and non-ADE and compared. A retrospective data analysis (PM10 concentration, aerosol images, and pediatric asthma claims) was performed from 2000 to 2012 with particular emphasis in 2004 to classify PM samples. Urban extracts were highly toxic, proinflammatory (IL-6/IL-8 secretion), and induced higher TLR4 expression and NF-κB activation compared to rural extracts. ENX were found to contribute to cytotoxicity and inflammatory responses provoked by urban ADE PM10 exposure suggesting a synergistic potency of local and natural ENX incoming from ADE. The contribution of ADE PM10 ENX is valuable in order to understand interactions and action mechanisms of airborne pollutants as asthma triggers in PR.

The article presents a new approach in the purification of chitosan (CS) hydrogel in order to remove a significant amount of endotoxins without changing its molecular weight and viscosity. Two variants of the method used to purify CS hydrogels from endotoxins were investigated using the PyroGene rFC Enzymatic Cascade assay kit. The effect of the CS purification method was assessed in terms of changes in the dynamic viscosity of its hydrogels, the molecular weight of the polymer, microbiological purity after refrigerated storage and cytotoxicity against L929 cells based on the ISO 10993-5:2009(E) standard. The proposed purification method 1 (M1) allows for the removal of significant amounts of endotoxins: 87.9-97.6% in relation to their initial concentration in the CS hydrogel without affecting the solution viscosity. Moreover, the final solutions were sterile and microbiologically stable during storage. The M1 purification method did not change the morphology of the L929 cells.