Endotoxin Molecule Research Articles

Intestinal microbiota as a fundamental basis for homeostasis, general pathology and aging

Intestinal microbiota is a kind of satellite organ that performs digestive and protective functions, a supplier of molecules vital for homeostasis processes, involved in metabolic processes and determining the activity level of adaptive systems, including immunity. Immunity works both for and against the host, which is predetermined by the very nature of adaptive immunity and its interaction with the innate link of the immune system. The key element of this interaction is endotoxin molecules, or lipopolysaccharides, the concentration of which in the general bloodstream determines the activity level of adaptive (mediated by innate) immunity, which operates in a stochastic mode. This provides both antitumor protection and self-destruction of the body based on autoimmune damage. Over the past 35 years, there has been a powerful breakthrough in the field of understanding the mechanisms of interaction between the intestinal microbiota and the host organism. Interesting data have been obtained and published that have not yet been fully systematized and understood. The methodology for studying the biological role of lipopolysaccharides in clinical settings developed by Russian scientists was based on the ability of agents for reducing their blood levels to increase the effectiveness of the treatment and preventive process. In particular, it made it possible to establish the involvement of the lipopolysaccharide factor in the pathogenesis of a number of diseases. The phenomenon of systemic endotoxinemia discovered by Russian researchers is a process of controlling the activity of adaptive systems (including the immune system) with the participation of the hypothalamic-pituitary-adrenal system by means of intestinal endotoxins. We see the following issues for wide discussion in the clinical community: (1) determining the directions for finding agents for normalizing systemic endotoxinemia indicators as the basis of preventive medicine, including pro- and prebiotics, entero- and hemosorbents, hepatoprotectors, immunopreparations, chaotropic effects (plasmapheresis, blood irradiation); (2) clinical and experimental models for studying diseases associated with intestinal microbiota; (3) creation of a research protocol to establish the age range of integral indicators of systemic endotoxinemia (the level of lipopolysaccharides and the activity of antiendotoxin immunity that resists endotoxin aggression); (4) ways to understand the mechanisms of development of endotoxin tolerance accompanying aging and its overcoming.

A Surface-Enhanced Raman Spectroscopy-Based Biosensor for the Detection of Biological Macromolecules: The Case of the Lipopolysaccharide Endotoxin Molecules.

The development of sensitive methods for the detection of endotoxin molecules, such as lipopolysaccharides (LPS), is essential for food safety and health control. Conventional analytical methods used for LPS detection are based on the pyrogen test, plating and culture-based methods, and the limulus amoebocyte lysate method (LAL). Alternatively, the development of reliable biosensors for LPS detection would be highly desirable to solve some critical issues, such as high cost and a long turnaround time. In this work, we present a label-free Surface-Enhanced Raman Spectroscopy (SERS)-based method for LPS detection in its free form. The proposed method combines the benefits of plasmonic enhancement with the selectivity provided by a specific anti-lipid A antibody (Ab). A high-enhancing nanostructured silver substrate was coated with Ab. The presence of LPS was quantitatively monitored by analyzing the changes in the Ab spectra obtained in the absence and presence of LPS. A limit of detection (LOD) and quantification (LOQ) of 12 ng/mL and 41 ng/mL were estimated, respectively. Importantly, the proposed technology could be easily expanded for the determination of other biological macromolecules.

Design of AgNPs doped chitosan/sodium lignin sulfonate/polypyrrole films with antibacterial and endotoxin adsorption functions

There is an urgent need to develop materials to prevent bacterial infection and the deleterious effects of endotoxins. In this study, we introduce a one-step electrodeposition method to prepare films composed of chitosan/Ag/polypyrrole and layer-by-layer self-assembly to introduce lignin sulphonate (LS) to obtain chitosan/Ag/polypyrrole/LS films. Antibacterial effects against both E. coli and S. aureus are shown by bacterial growth profiles and observation of bacteriostatic zones. Meanwhile, the addition of self-assembled LS improved the antibacterial effect of the film. For E. coli, the inhibition zone diameter was 0.93 cm, while for S. aureus, the inhibition zone diameter was 0.72 cm. Rapid and efficient endotoxin adsorption effects were shown whereby the electrostatic interactions between chitosan and endotoxin molecules played a major role. After adsorption for 1 h, in initial concentration of 1 EU/mL endotoxin solution, the adsorption efficiency could reach up to 85 %, while in initial concentration of 5 EU/mL endotoxin solution, the adsorption efficiency could reach up to 87.6 %. The results suggest chitosan/Ag/polypyrrole/LS films for their capability as a new type of antibacterial film with intrinsic endotoxin adsorption activity.

Outstanding Contributions of LAL Technology to Pharmaceutical and Medical Science: Review of Methods, Progress, Challenges, and Future Perspectives in Early Detection and Management of Bacterial Infections and Invasive Fungal Diseases.

The blue blood of the horseshoe crab is a natural, irreplaceable, and precious resource that is highly valued by the biomedical industry. The Limulus amebocyte lysate (LAL) obtained from horseshoe crab blood cells functions as a surprisingly sophisticated sensing system that allows for the extremely sensitive detection of bacterial and fungal cell-wall components. Notably, LAL tests have markedly contributed to the quality control of pharmaceutical drugs and medical devices as successful alternatives to the rabbit pyrogen test. Furthermore, LAL-based endotoxin and (1→3)-β-D-glucan (β-glucan) assay techniques are expected to have optimal use as effective biomarkers, serving as adjuncts in the diagnosis of bacterial sepsis and fungal infections. The innovative β-glucan assay has substantially contributed to the early diagnosis and management of invasive fungal diseases; however, the clinical significance of the endotoxin assay remains unclear and is challenging to elucidate. Many obstacles need to be overcome to enhance the analytical sensitivity and clinical performance of the LAL assay in detecting circulating levels of endotoxin in human blood. Additionally, there are complex interactions between endotoxin molecules and blood components that are attributable to the unique physicochemical properties of lipopolysaccharide (LPS). In this regard, while exploring the potential of new LPS-sensing technologies, a novel platform for the ultrasensitive detection of blood endotoxin will enable a reappraisal of the LAL assay for the highly sensitive and reliable detection of endotoxemia.

Bacterial Endotoxin Testing-Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine Oxide.

For release of parenteral drug products, bacterial endotoxin testing is one of a panel of necessary tests. In order to ensure the validity of such tests, various controls are performed, including demonstration of compendial method suitability or method qualification. In addition to compendial suitability testing, quality control (QC) sample hold-time studies are requested by authorities like the Food and Drug Administration (FDA) as described in “Guidance for Industry: Pyrogen and Endotoxins Testing.” It is requested to be determine whether the ability to detect endotoxins can be affected by storage and handling of the sample to be tested. To accomplish these studies, endotoxin is introduced or spiked into the undiluted product and held for a certain period of time in process-representative containers. This time period reflects procedural maximum QC sample hold time from sampling until analysis. Inadequate detection of endotoxin can be caused by adsorption of endotoxin to container surfaces or molecular masking effects, in which the binding sites on the endotoxin molecules are prevented from triggering the enzymatic cascade necessary in the assay, are obscured. The endotoxin may form macromolecular structures, such as sheets or blebs, or the binding sites may otherwise be rendered unavailable due to the sample matrix composition. In either case, the endotoxin assay may yield falsely low results if and when masking occurs. In this work, the QC sample hold times of different in-process controls within the production process of a biopharmaceutical product were analyzed. One out of eight different samples showed a strong masking of endotoxin. Analysis of the sample composition revealed that either kifunensine, mycophenolic acid (MPA), or lauryl-N, N-dimethylamine oxide (LDAO) was responsible for masking. Further analysis clearly identified LDAO as the root cause for masking. A novel one-step mechanism for LDAO-induced endotoxin masking is proposed. The principle is similar to an already-proposed two-step mechanism for endotoxin masking, but the LDAO case combines these two steps: the disturbance of the salt bridges and hydrophobic interactions with LPS in one molecule. These molecular interactions occur quickly when both endotoxin and LDAO are present in the same matrix. Thus, depending on the masking agents, low endotoxin recovery (LER) can occur regardless of the QC sample hold duration.

Toll-like receptor 4 agonist and antagonist lipopolysaccharides modify innate immune response in rat brain circumventricular organs

BackgroundThe circumventricular organs (CVOs) are blood-brain-barrier missing structures whose activation through lipopolysaccharide (LPS) is a starting point for TLR-driven (Toll-like receptors) neuroinflammation. The aim of this study was to evaluate in the CVO area postrema (AP), subfornical organ (SFO), and median eminence (ME), the inflammatory response to two TLR4 agonists: LPS from Escherichia coli (EC-LPS), the strongest endotoxin molecule described, and LPS from Porphyromonas gingivalis (PG-LPS), a pathogenic bacteria present in the periodontium related to neuroinflammation in neurodegenerative/psychiatric diseases. The response to LPS from the cyanobacteria Rhodobacter sphaeroides (RS-LPS), a TLR4 antagonist with an interesting anti-inflammatory potential, was also assessed.MethodsLPSs were intraperitoneally administered to Wistar rats and, as indicatives of neuroinflammation in CVOs, the cellular localization of the nuclear factor NF-κB was studied by immunofluorescence, and microglia morphology was quantified by fractal and skeleton analysis.ResultsData showed that EC-LPS increased NF-κB nuclear translocation in the three CVOs studied and PG-LPS only induced NF-κB nuclear translocation in the ME. RS-LPS showed no difference in NF-κB nuclear translocation compared to control. Microglia in the three CVOs showed an ameboid-shape after EC-LPS exposure, whereas PG-LPS only elicited a mild tendency to induce an ameboid shape. On the other hand, RS-LPS produced a markedly elongated morphology described as “rod” microglia in the three CVOs.ConclusionsIn conclusion, at the doses tested, EC-LPS induces a stronger neuroinflammatory response than PG-LPS in CVOs, which might be related to their different potency as TLR4 agonists. The non-reduction of basal NF-κB activation and induction of rod microglia by RS-LPS, a cell morphology only present in severe brain injury and infections, suggests that this molecule must be carefully studied before being proposed as an anti-inflammatory treatment for neuroinflammation related to neurodegenerative/psychiatric diseases.

Analysis of Endotoxin Adsorption in Two Swedish Patients with Septic Shock

Background: Lipopolysaccharide (endotoxin) from the outer Gram-negative bacterial wall can induce a harmful immunologic response, involving hemodynamic deprivation, and is one important motor driving the septic cascade. The positively charged poly-imine ethylene layer on the oXiris membrane is capable of adsorbing negatively charged endotoxin molecules and removing them from the blood compartment. Endotoxin is detrimental and should be removed from blood. Summary: The adsorbable endotoxin fraction in blood arises from a tight balance between seeding from an infectious focus and removal by an overwhelmed immune system. The net sum of remaining endotoxin in blood is available for an adsorption process in the oXiris filter. Endotoxin data from 2 patients with severe Gram-negative septic shock and endotoxemia in this case series, speaks for a considerable share of the adsorption of the oXiris filter in the endotoxin net removal over time. Key Messages: Analysis of combined in vitro and in vivo data speaks for an effect of the oXiris filter in lowering endotoxin.

Mechanism of Low Endotoxin Recovery Caused by a Solution Containing a Chelating Agent and a Detergent

Mechanism of Low Endotoxin Recovery (LER) was investigated by observing the change in particle sizes and activity of endotoxin in LER solutions containing sodium citrate and polysorbate 20. Interestingly, endotoxin aggregates were not dispersed to be smaller particles under LER conditions according to the decease of the activity. This observation was different from previous predictions of the mechanism. Endotoxin aggregates were observed by Dynamic Light Scattering, and activity was measured by the Limulus amebocyte lysate test. Particles with sizes similar to endotoxin aggregates were always observed despite different remaining activity of spiked endotoxin. This observation differed from previously proposed mechanisms for LER that dispersed endotoxin aggregates were smaller in sizes. Based on the findings, a new mechanism of LER is proposed. Purified endotoxin forms non-lamellar cubic structures in water, and the endotoxin aggregates are reinforced by divalent cations. When purified endotoxin is added to LER solutions, divalent cations are removed from outer layer of endotoxin by the chelating agent, and the loss of divalent cations weakens the outside layer of the endotoxin aggregates. The endotoxin aggregation structure is maintained at low temperature, but endotoxin molecules on the surface of the endotoxin aggregates are gradually replaced with detergent molecules at higher temperature. This replacement reduces the surface area of the endotoxin, and cause reduction of activity of the endotoxin aggregates. The apparent sizes of the aggregates are not changed after the replacement.

Investigation of the kinetics and mechanism of low endotoxin recovery in a matrix for biopharmaceutical drug products

The inability to detect endotoxin added to undiluted drug samples has been called: Low Endotoxin Recovery (LER). The phenomenon has caused concerns amongst drug manufacturing quality control scientists in that manufactured solutions contaminated with endotoxin could show false-negative results via routine Limulus-based tests. The time-dependent appearance of LER has been analyzed in detail to provide a better understanding of the mechanism. The assumption has been that the root-cause of LER involves the interplay of endotoxin with surfactants and results in aggregate structures that are complexed with surfactants. The endotoxin molecules when complexed with surfactants are not accessible for Limulus-based detection. The results demonstrate a predominant role of complex-forming agents. It was shown that although the presence of surfactants is a strong prerequisite for masking, it does not determine the kinetics of endotoxin masking. Interestingly, the endotoxin concentration itself had no substantial impact on LER kinetics. By adjusting the ratios of complex-forming constituents, including surfactant, chelator and endotoxin, and by testing the order in which the constituents are added, a new model for simulating masking kinetics has been determined. Our work provides for the first time a model to simulate masking kinetics of endotoxin which lends a better understanding of LER.

Sensitive Monitoring of Enterobacterial Contamination of Food Using Self-Propelled Janus Microsensors.

Food poisoning caused by bacteria is a major cause of disease and death worldwide. Herein we describe the use of Janus micromotors as mobile sensors for the detection of toxins released by enterobacteria as indicators of food contamination. The micromotors are prepared by a Pickering emulsion approach and rely on the simultaneous encapsulation of platinum nanoparticles for enhanced bubble-propulsion and receptor-functionalized quantum dots (QDs) for selective binding with the 3-deoxy-d-manno-oct-2-ulosonic acid target in the endotoxin molecule. Lipopolysaccharides (LPS) from Salmonella enterica were used as target endotoxins, which upon interaction with the QDs induce a rapid quenching of the native fluorescence of the micromotors in a concentration-dependent manner. The micromotor assay can readily detect concentrations as low as 0.07 ng mL-1 of endotoxin, which is far below the level considered toxic to humans (275 μg mL-1). Micromotors have been successfully applied for the detection of Salmonella toxin in food samples in 15 min compared with several hours required by the existing Gold Standard method. Such ultrafast and reliable approach holds considerable promise for food contamination screening while awaiting the results of bacterial cultures in a myriad of food safety and security defense applications.

Changes in the levels of liver HSP70, plasma nitric oxide, and the antioxidative system in an experimentally induced endotoxemia mouse model and the role of reduced glutathione

Endotoxin molecules in lipopolysaccharides are among most important molecules that initiate a cascade of events in sepsis/endotoxemia. Lipopolysaccharide exposure may result in strong immune responses, disrupt the intracellular oxidant/antioxidant balance, and cause excessive reactive oxygen species generation. The purpose of the study was to examine if reduced glutathione (GSH) has a protective role against lipopolysaccharides. The effects of lipopolysaccharide (LPS) or GSH alone or in combination on the levels of the plasma antioxidant system, NO, and liver HSP70 were investigated. A total of 100 Swiss albino mice were divided into 4 groups as Group I (control), Group II (20 μg/kg LPS), Group III (10 mg/kg GSH), and Group IV (20 μg/kg LPS + 10 mg/kg GSH). Blood and liver samples both pre- and post-LPS and/or GSH injections after 1, 3, and 6 h were collected. Total antioxidant capacity was demonstrated with a reduction in response to lipopolysaccharide. Total oxidant capacity was higher after the injection of lipopolysaccharide alone or in combination with GSH. NO levels were elevated in response to lipopolysaccharide. The liver HSP70 level was determined to be higher in the lipopolysaccharide-treated group. These results indicate that exogenously administered GSH may have regulatory effects on liver HSP70 and plasma NO levels, and GSH treatment might have beneficial effects on antioxidant status by inhibiting the increase of oxidant molecules in endotoxemia-induced mice.

A non-chromatographic method for the removal of endotoxins from bacteriophages.

The Ff filamentous bacteriophages show potential as a new class of therapeutics, displaying utility in materials science as well as pharmaceutical applications. These phages are produced by the infection of E. coli, a Gram-negative bacterium which unavoidably sheds endotoxins into the extracellular space during growth. Since endotoxin molecules are highly immunoreactive, separation from the phage product is of critical importance, particularly those developed for human therapeutic use. The properties of M13, one of the Ff group, present a purification challenge chiefly because the standard scalable method for endotoxin removal from proteins-anion exchange chromatography-is not applicable due to pI similarity between the particles. This article examines the potential of polyethylene glycol (PEG)-NaCl precipitation as a scalable method for the separation of endotoxins from phage M13. Precipitation of M13 by 2% (w/v) PEG 6 000, 500 mM NaCl reduced endotoxin contamination of the phage product by 88%, but additional precipitation rounds did not maintain this proportional decrease. Dynamic light scattering was subsequently used to determine the effectiveness of a detergent to disassociate endotoxin molecules from M13. As a result, PEG-NaCl precipitation was supplemented with up to 2% (v/v) Triton X-100 to improve separation. A 5.7 log10 reduction in endotoxin concentration was achieved over three rounds of precipitation whilst retaining over 97% of the phage. This method compares favorably with the well-known ATPS (Triton X-114) technique for endotoxin removal from protein solutions.

Endotoxin inactivation via steam‐heat treatment in dilute simethicone emulsions used in biopharmaceutical processes

Simethicone emulsion is used to regulate foaming in cell culture operations in biopharmaceutical processes. It is also a potential source of endotoxin contamination. The inactivation of endotoxins in dilute simethicone emulsions was assessed as a function of time at different steam temperatures using a Limulus amebocyte lysate kinetic chromogenic technique. Endotoxin inactivation from steam-heat treatment was fit to a four-parameter double exponential decay model, which indicated that endotoxin inactivation was biphasic, consisting of fast and slow regimes. In the fast regime, temperature-related effects were dominant. Transitioning into the slow regime, the observed temperature dependence diminished, and concentration-related effects became increasingly significant. The change in the Gibbs free energy moving through the transition state indicated that a large energy barrier must be overcome for endotoxin inactivation to occur. The corresponding Arrhenius pre-exponential factor was >>10(12) s(-1) suggesting that endotoxins in aqueous solution exist as aggregates. The disorder associated with the endotoxin inactivation reaction pathway was assessed via the change in entropy moving through the transition state. This quantity was positive indicating that endotoxin inactivation may result from hydrolysis of individual endotoxin molecules, which perturbs the conformation of endotoxin aggregates, thereby modulating the biological activity observed. Steam-heat treatment decreased endotoxin levels by 1-2 logarithm (log) reduction (LRV), which may be practically relevant depending on incoming raw material endotoxin levels. Antifoam efficiency and cell culture performance were negligibly impacted following steam-heat treatment. The results from this study show that steam-heat treatment is a viable endotoxin control strategy that can be implemented to support large-scale biopharmaceutical manufacturing.

Endotoxin Molecule Lipopolysaccharide-Induced Zebrafish Inflammation Model: A Novel Screening Method for Anti-Inflammatory Drugs

Lipopolysaccharide (LPS), an endotoxin molecule, has been used to induce inflammatory responses. In this study, LPS was used to establish an in vivo inflammation model in zebrafish for drug screening. We present an experimental method that conveniently and rapidly assesses the anti-inflammatory properties of drugs. The yolks of 3-day post-fertilization (dpf) larvae were injected with 0.5 mg/mL LPS to induce fatal inflammation. After LPS stimulation, macrophages were tracked by NR and SB staining and neutrophil migration was observed using the MPO:GFP line. Larval mortality was used as the primary end-point. Expression levels of key cytokines involved in the inflammatory response including IL-1β, IL-6, and TNF-α, were measured using quantitative reverse transcription polymerase chain reaction (RT-PCR). Macrophages and neutrophils were both recruited to the LPS-injected site during the inflammatory response. Mortality was increased by LPS in a dose-dependent manner within 48 h. Analyses of IL-1β, IL-6, and TNF-α expression levels revealed the upregulation of the inflammatory response in the LPS-injected larvae. Further, the anti-inflammatory activity of chlorogenic acid (CA) was evaluated in this zebrafish model to screen for anti-inflammatory drugs. A preliminary result showed that CA revealed a similar effect as the corticosteroid dexamethasone (DEX), which was used as a positive control, by inhibiting macrophage and neutrophil recruitment to the LPS site and improving survival. Our results suggest that this zebrafish screening model could be applied to study inflammation-mediated diseases. Moreover, the Traditional Chinese Medicine CA displays potential anti-inflammatory activity.

Continuous renal replacement therapy with the adsorbent membrane oXiris in septic patients: a clinical experience

Renal failure is an important complication of sepsis and CRRT with adsorbing membranes may be useful in this clinical setting [1]. The aims of the study in septic/septic shock patients are to evaluate: the safety of a new hemofilter membrane oXiris with adsorbing properties and anti-endotoxin activity; the renal and hemodynamic response; and the changes of endotoxin and proinflammatory molecules.

Influence of droplet size, pH and ionic strength on endotoxin-triggered ordering transitions in liquid crystalline droplets.

We report an investigation of ordering transitions that are induced in water-dispersed, micrometer-sized droplets of a thermotropic liquid crystal (LC) by the bacterial lipopolysaccharide endotoxin. We reveal that the ordering transitions induced by endotoxin - from a bipolar state of the droplets to a radial state - are strongly dependent on the size of the LC droplets. Specifically, as the diameters of the LC droplets increase from 2 μm to above 10 μm (in phosphate buffered saline with an ionic strength of 90 mM and a pH of 7.2), we measured the percentage of droplets exhibiting a radial configuration in the presence of 100 pg/mL endotoxin to decrease from 98 ± 1 % to 3 ± 2 %. In addition, we measured a decrease in either the ionic strength or pH of the aqueous phase to reduce the percentage of droplets exhibiting a radial configuration in the presence of endotoxin. These results, when interpreted within the context of a simple thermodynamic model that incorporates the contributions of elasticity and surface anchoring to the free energies of the LC droplets, lead us to conclude that (i) the elastic constant K24 plays a central role in determining the size-dependent response of the LC droplets to endotoxin, and (ii) endotoxin-triggered ordering transitions occur only under solution conditions (pH, ionic strength) where the combined contributions of elasticity and surface anchoring to the free energies of the bipolar and radial configurations of the LC droplets are similar in magnitude. Our analysis also suggests that the presence of endotoxin perturbs the free energies of the LC droplets by ~10-17 J/droplet, which is comparable to the standard free energy of self-association of ~103 endotoxin molecules. These results, when combined with prior reports of localization of endotoxin at the center of LC droplets, are consistent with the hypothesis that self-assembly of endotoxin within micrometer-sized LC droplets provides the driving force for the ordering transitions. Overall, these results advance our understanding of ordering transitions triggered by the interactions of analytes with LC droplets and, more broadly, provide guidance to the design of LC droplet systems as the basis of stimuli-responsive soft materials.

C1 Inhibitor Suppresses the Endotoxic Activity of a Wide Range of Lipopolysaccharides and Interacts With Live Gram-negative Bacteria

Human C1 inhibitor (C1INH) prevents endotoxin shock via a direct interaction with Gram-negative bacterial lipopolysaccharide (LPS) and improves survival in animal models of sepsis. In this report, we further characterize the interaction of C1INH with LPS and whole live bacteria. We investigate C1INH interactions with LPS from five different strains of Gram-negative enteric bacteria known to participate in the pathogenesis of human sepsis. Treatment with C1INH improved survival in mice with endotoxin shock induced by LPS from Salmonella enterica serovar typhimurium as previously shown, as well as LPS from Escherichia coli O55:B5 and Pseudomonas aeruginosa, and a trend to improved survival was observed when Klebsiella pneumoniae and Serratia marcescens LPS were used. Enzyme-linked immunosorbent assay and native polyacrylamide gel electrophoresis shift experiments demonstrated a direct interaction of C1INH with LPS from all the strains studied. The binding of both native and reactive center-cleaved, inactive C1INH results in inhibition of LPS-induced proinflammatory cytokine production. Furthermore, we demonstrate the ability of C1INH to bind at the surface of only a restricted number of whole live Gram-negative bacteria as well as mutant bacteria expressing a truncated LPS lacking the O-antigen. These data reveal the interaction of C1INH with a wide range of enteric bacterial LPS and strongly suggest that the interaction between C1INH and the surface of Gram-negative microorganisms is determined by the length of the polysaccharide chain of the endotoxin molecule.

Induction and characterization of endotoxin tolerance in equine peripheral blood mononuclear cells in vitro

Endotoxemia is responsible for severe illness in horses. Individuals can become clinically unresponsive to the endotoxin molecule after an initial exposure; a phenomenon referred to as ‘endotoxin tolerance’ (ET). ET has been observed clinically in horses in vivo; however, cytokine expression associated with ET has not been investigated. The purpose of this study was to develop and validate a method for inducing ET in equine peripheral blood mononuclear cells (PBMC) in vitro, and to describe selected cytokine responses which are associated with ET. ET was induced by culturing cells with three concentrations of endotoxin, and evaluated after a second dose of endotoxin given to challenge the cells. The relative mRNA expression of IL-10 and IL-12 was measured by use of quantitative PCR. ET was induced in all cells exposed to the 2-step endotoxin challenge. The relative mRNA expression of IL-10 in tolerized cells was not different from positive control cells. In contrast, the relative mRNA expression of IL-12 in tolerized cells was decreased by 15-fold after the second endotoxin challenge. This experiment demonstrated a reliable method for the ex vivo induction of ET in equine PBMC. A marked suppression of IL-12 production is associated with ET.

Protective Proteins in Mammalian Milks

Human milk is the ideal nutrient for neonates. Breastfeeding exposes neonates to maternal microflora, provides host protection, and has proteins that mediate immune system development. Lactoferrin (LF) is the major whey protein in mammalian milk, and its multifunctional characteristics have shown importance in preventing infections. Ferric iron binding and natural peptide antibiotic properties of LF likely promote a healthy intestinal microbiome that prevents bacterial translocation and mediates optimal epithelial growth and differentiation. An established asset of LF is stimulation of naïve dendritic cells; this initiates the emergence of neonatal Th1 helper cells, thereby reversing the Th2 bias associated with pregnancy. Moreover, LF promotes development of Peyer patches, which leads to enhanced IgA secretion into the intestinal lumen. The anti-inflammatory properties of LF involve its binding of endotoxin and other proinflammatory molecules released by intestinal pathogens. LF also blocks receptors that microbes use for epithelial invasion, and thus LF mitigates a proinflammatory response by the host during infection. These properties of LF are known from basic science research and preclinical investigations, and they have resulted in the enteral use of bovine and recombinant human LF to prevent neonatal late onset sepsis. Bovine LF has been shown to reduce the incidence of late onset sepsis in extremely preterm infants, but Food and Drug Administration approval of LF for use in the NICU has not taken place. Because LF is currently available only for scientific investigations, the feeding of a mother's milk is encouraged shortly after birth because the concentration of LF is highest in colostrum.

A LONGER DURATION OF POLYMYXIN B-IMMOBILIZED FIBER COLUMN HEMOPERFUSION IMPROVES PULMONARY OXYGENATION IN PATIENTS WITH SEPTIC SHOCK

Endotoxin plays an important role in the pathogenesis of septic shock. Exposure of endothelial cells to endotoxin activates endothelial cells and increases the surface expression of adhesion molecules, markers of endothelial damage in organ dysfunction. Endotoxin adsorption therapy by polymyxin B-immobilized fiber column (PMX) hemoperfusion has been used for the treatment of septic shock patients. In this study, we measured plasma concentrations of endotoxin and soluble adhesion molecules in septic shock patients before and after the PMX treatment then observed on the relationships between actual duration of use and various outcomes. Sixteen patients with septic shock were studied. The 28-day mortality rate was 50%. The elevated plasma concentrations of endotoxin decreased after the PMX treatment in the survivors but not in the nonsurvivors. The norepinephrine dose and plasma concentrations of soluble endothelial leukocyte adhesion molecule 1 and soluble intercellular adhesion molecule 1 significantly (P < 0.05) decreased in the PMX greater-than-2-h (prolonged) group than in the PMX 2-h (conventional) group (-17.8 +/- 14.6 vs. -1.8 +/- 2.7 microg/min, -143.0 +/- 111.0 vs. 0 +/- 2.8 ng/mL, and -126.2 +/- 144.9 vs. 16.5 +/- 108.1 ng/mL, respectively). Changes in the PaO2-FiO2 ratio and the Sequential Organ Failure Assessment score were significantly (P < 0.05) more improved in the PMX greater-than-2-h group than in the PMX 2-h group (75.4 +/- 80.7 vs. 1.2 +/- 49.2 and -0.8 +/- 1.8 vs. 2.2 +/- 1.9 torr, respectively). We thus suggest that a longer duration of PMX treatment may improve the pulmonary oxygenation associated with decreased adhesion molecules in septic shock.