Endotoxin Preparations Research Articles

Effect of two different root canal instruments on removal of endotoxins from necrotic root canals (A Randomized clinical trial)

Aim of the study: The aim of the present randomized clinical study was to evaluate the effect of OneShape and Protaper Next rotary system on the removal of endotoxins from teeth with asymptomatic necrotic pulp.Materials and Methods: Thirty patients were randomly allocated into two groups (n=15) according to the type of rotary system used in mechanical preparation; either Group OS (OneShape) or Group PN (Protaper Next). Samples were taken before and after mechanical preparation and quantification of endotoxins was done using Human Elisa kit. Statistical analyses were performed using Mann-Whitney U test to compare between the two groups then Wilcoxon signed- rank test was used to study the changes after treatment within each group. The significance level was set at P ≤ 0.05.Results: Group OS, showed a statistically significant reduction in endotoxins levels after treatment (P-value < 0.001, Effect size = 0.880). In Group PN, there was a statistically significant reduction in endotoxins levels after treatment (P-value <0.001, Effect size = 0.931) and Group OS showed statistically significant lower median % reduction in endotoxins level than Group PN.Conclusion: Protaper Next was more effective than the Oneshape in reducing the endotoxins from teeth with asymptomatic necrotic pulp.

Mass Spectrometry for Profiling LOS and Lipid A Structures from Whole-Cell Lysates: Directly from a Few Bacterial Colonies or from Liquid Broth Cultures.

Lipopolysaccharides (LPSs, endotoxins) are components of the outer cell membrane of most Gram-negative bacteria and can play an important role in a number of diseases of bacteria, including Gram-negative sepsis. The hydrophilic carbohydrate part of LPSs consists of a core oligosaccharide (in the case of an R-type LPS or lipooligosaccharide, LOS) linked to an O-polysaccharide chain (in the case of an S-type LPS), which is responsible for O-specific immunogenicity. The hydrophobic lipid A anchor is composed of a phosphorylated diglucosamine backbone to which varying numbers of ester- and amide-linked fatty acids are attached and this part of the LPSs is associated with endotoxicity. The detailed chemical characterization of endotoxins requires long-lasting large-scale isolation procedures, by which high-purity LPSs can be obtained. However, when a large number of bacterial samples and their LPS content are to be compared prompt, small-scale isolation methods are used for the preparation of endotoxins directly from bacterial cell cultures. The purity of the endotoxins extracted by these methods may not be high, but it is sufficient for analysis.Here, we describe a fast and easy micromethod suitable for extracting small quantities of LOS and a slightly modified micromethod for the detection of the lipid A constituents of the LPSs from bacteria grown in different culture media and evaluate the structures with mass spectrometry. The cellular LOS and lipid A were obtained from crude isolates of heat-killed cells, which were then subjected to matrix-assisted laser desorption/ionization mass spectrometry analysis. The observed ions in the 10-colony samples were similar to those detected for purified samples. The total time for the sample preparation and the MS analysis is less than 3 h.

Nitric oxide production by endotoxin preparations in TLR4-deficient mice

Sepsis and septic shock result from an exacerbated systemic inflammatory reaction to infection. Their incidence is rising, and they have recently become the main cause of death in intensive care units. Septic shock is defined as sepsis accompanied by life-threatening refractory hypotension, for which excessive nitric oxide (NO), produced by inducible NO synthase iNOS, is thought responsible. LPS, a vital outer membrane component of Gram-negative bacteria, mimics most of the septic effects and is widely used as a model for septic shock. TLR4 is the signal-transducing receptor for LPS, evidenced by the resistance of TLR4-deficient C3H/HeJ and C57BL/10ScNJ mice. As expected, we found that TLR4 deficiency precludes LPS-induced cytokine production, independent of the purity of the LPS preparation. However, various conventional LPS preparations induced NO in TLR4-deficient mice to the same level as in control animals, while ultrapure LPS did not, indicating the presence of NO-producing contaminant(s). Nevertheless, despite identical iNOS induction pattern and systemic NO levels, the contaminant does not cause hypotension, hypothermia, or any other sign of morbidity. Using mice deficient for TLR2, TRL3, TLR4, TRL2x4, TLR9, MyD88 or TRIF, we found that the contaminant signals via TLR2 and MyD88. In conclusion, conventional LPS preparations generally used in endotoxic shock research contain TLR2 agonists that induce iNOS and high levels of systemic NO as such, and synergize with LPS towards the production of pro-inflammatory cytokines, morbidity and mortality. Surprisingly, the excessive iNOS-derived systemic NO production induced by impure LPS in TLR4−/− is not accompanied by hypotension or morbidity.

Cellular distribution of lipid A and LPS R595 after in vitro application to isolated human monocytes by freeze-fracture replica immunogold-labelling

We have performed freeze-fracture replica immunogold labelling of endotoxin preparations (lipid A and deep rough mutant LPS Re from Salmonella enterica sv. Minnesota), i.e. adding the endotoxins to human monocytes, labelling with monoclonal Abs recognizing either lipid A or LPS Re (A6 and A20 respectively), and fixing with immunogold secondary Ab. We have found that the endotoxins intercalated into the cell membranes with subsequent internalization by the cells. Surprisingly, membrane uptake took place only in the inner, plasmic leaflet of the plasma membrane, but there was no uptake of the outer leaflet for both compounds. Remarkable labelling could be also found for the two membranes of the nuclear envelope-in the case of lipid A only at the plasmic leaflet, but in the case of LPS Re on both leaflets. Isothermal calorimetric titration of the AB A20 with LPS and phospholipids showed that the Ab may bind not only to LPS but also to negatively charged phosphatidylserine. These results are discussed in the frame of the published concepts of cell activation induced by the endotoxins, i.e. how they are able to cause a conformational change of signalling proteins, such as the TLR4/MD2 complex.

Insecticidal Efficiency of Intergeneric Recombinants between Bacillus thuringiensis and Bacillus subtilis Against Viability of Cotton Leafworm

Bacillus thuringiensis is the most widely applied biological insecticide and is used to manage insects that affect forestry and agriculture. This ubiquitous spore-forming bacterium kills insect larvae largely through the action of insecticidal crystal proteins and is commonly deployed as a direct bacterial spray. Moreover, this study aimed to measuring recombinant efficiency of recombinant bioinsecticides (Bacillus thuringiensis x Bacillus subtilis) against Spodoptera litoralis. For decades, the mechanism of insect killing has been assumed to be toxin-mediated lysis of the gut epithelial cells, which leads to starvation, or B. thuringiensis septicemia. In the present work, two strains of Bacillusstrains belonging to two serotypes and four of their transconjugants were screened with respect to their insecticidal activity against lepidopterous cotton pest. Two strains of Bacillus were screened for their drug resistance to be used as a genetic markers to identify bacterial strains in the conjugation process. B. subtilis was found to be resistant to crystal violet and sensitive to hiconcil. Although, B. thuringiensis was found to be sensitive to crystal violet, except for it was resistant to hiconcil. Bacterial transconjugants isolated from conjugation between both strains were more resistant to both crystal violet and hiconcil. Two groups of crystals and spores have been isolated within Bacillus strains and their transconjugants. The results appeared a highly potent recombinant efficiency in reducing leaves consumption at 72h (crystals), 120h (crystals + endospores) and 144h (crystals + endospors). However, transcongugants B and c was more effective in reducing the rate of consumption at all different times of larval ages. The weight of surviving larvae feeding on leaves sprayed with recombinants of Cry, Cry + End. was markedly reduced especially at 48h of larval age. However, ransconjugants A, B and C appeared recombinant efficiency in reduced survival percentage of larvae at 96h, 120h, 144h and 168h in response to recombinants of both cry, cry + endospores. The same trend was also shown by transconjugant C at 48h and 72h in response to both recombinant bioinsecticides. The results indicated that recombinant endotoxin preparations containing crystals + endospores was more effective against Spodoptera litoralisthan that containing crystals.

Oxidatively Modified Low Density Lipoprotein (LDL) Inhibits TLR2 and TLR4 Cytokine Responses in Human Monocytes but Not in Macrophages

Inflammation characterized by the expression and release of cytokines and chemokines is implicated in the development and progression of atherosclerosis. Oxidatively modified low density lipoproteins, central to the formation of atherosclerotic plaques, have been reported to signal through Toll-like receptors (TLRs), TLR4 and TLR2, in concert with scavenger receptors to regulate the inflammatory microenvironment in atherosclerosis. This study evaluates the role of low density lipoproteins (LDL) and oxidatively modified LDL (oxmLDL) in the expression and release of proinflammatory mediators IκBζ, IL-6, IL-1β, TNFα, and IL-8 in human monocytes and macrophages. Although standard LDL preparations induced IκBζ along with IL-6 and IL-8 production, this inflammatory effect was eliminated when LDL was isolated under endotoxin-restricted conditions. However, when added with TLR4 and TLR2 ligands, this low endotoxin preparation of oxmLDL suppressed the expression and release of IL-1β, IL-6, and TNFα but surprisingly spared IL-8 production. The suppressive effect of oxmLDL was specific to monocytes as it did not inhibit LPS-induced proinflammatory cytokines in human macrophages. Thus, TLR ligand contamination of LDL/oxmLDL preparations can complicate interpretations of inflammatory responses to these modified lipoproteins. In contrast to providing a proinflammatory function, oxmLDL suppresses the expression and release of selected proinflammatory mediators.

Biophysical investigations into the interactions of endotoxins with bile acids

The interaction of selected endotoxin preparations (lipid A from Erwinia carotovora and LPS Re and Ra from Salmonella enterica sv. Minnesota strains R595 and R60, respectively) with selected bile acids was investigated biophysically. Endotoxin aggregates were analyzed for their gel-to-liquid crystalline phase behavior, the type of their aggregates, the conformation of particular functional groups, and their Zeta potential in the absence and presence of the bile acids by applying Fourier-transform infrared spectroscopy, differential scanning calorimetry, measurements of the electrophoretic mobility, and synchrotron radiation X-ray scattering. In addition, the ability of the endotoxins to induce cytokines in human mononuclear cells was tested in the absence and presence of varying concentrations of bile acids. The data show that the endotoxin:bile acid interaction is not governed by Coulomb forces, rather a hydrophobic interaction takes place. This leads to an enhanced formation of the inherent cubic aggregate structures of the endotoxins, concomitant with a slight disaggregation, as evidenced by freeze-fracture electron microscopy. Parallel to this, the addition of bile acids increased the bioactivity of lipid A and, to a lower degree, also that of the tested rough mutant LPS at lower concentrations of the endotoxin preparation, a finding similar as reported for the interaction of other agents such as hemoglobin. These data imply that there are general mechanisms that govern the expression of biological activities of endotoxins.

Lubiprostone, but Not Linaclotide, Protects T84 Cells Against Damage to Barrier Function by Interferon-γ

Purpose: Lubiprostone, a prostone derivative, activates ClC-2 chloride channels and is used for the treatment of chronic idiopathic constipation in adults and irritable bowel syndrome with constipation in adult women. Linaclotide is also being developed for these indications. It is a peptide whose structure is closely related to the bacterial heat-stable endotoxin, STa. Lubiprostone has been shown to promote repair of intestinal barrier properties whereas nothing is known of the effect of linaclotide. The aim of the present study was to compare the effect of lubiprostone and linaclotide on loss of barrier function induced by interferon-γ (IFN-γ), a proinflammatory cytokine which reduces transepithelial resistance (TER) in part through reduction of occludin levels. IFN-γ is thought to be involved in the pathology of celiac sprue enteropathy, inflammatory bowel disease, ulcerative colitis and Crohn's disease. Intestinal barrier breakdown can lead to visceral pain. Methods: T84 cells grown on permeable supports were treated with and without 100 ng/mL IFN-γ and with 100 nM lubiprostone, 200 nM of the 13 amino acid active metabolite of linaclotide (CCEYCCNPACTGC) or 0.1% DMSO (vehicle control) daily for 3 days. TER, flux of FITC-labeled E. coli lipopolysaccharide (LPS, bacterial endotoxin preparation) and occludin levels were measured. Results: Incubation with lubiprostone alone did not affect TER, LPS flux or occludin levels. In contrast, linaclotide alone partially reduced TER, increased LPS flux and decreased occludin levels. IFN-γ reduced TER, increased LPS flux and decreased occludin levels. Lubiprostone, but not linaclotide countered the effects of IFN-γ. Conclusion: In this in vitro study, lubiprostone (but not linaclotide) protected against IFN-γ-induced loss of intestinal barrier function and this may involve preservation of occludin levels. Linaclotide alone caused partial barrier breakdown. These findings suggest that treatment with lubiprostone, but not linaclotide, may lead to reduced visceral damage and reduced pain in inflammatory events in the intestine. Supported by Sucampo Pharmaceuticals, Inc. Disclosure: John Cuppoletti is consultant to, has stock options from and is PI on grant from Sucampo Pharmaceuticals, Inc. All other authors are supported by funds from the research grant. Supported by a grant from Sucampo Pharmaceuticals, Inc.

Morphology, size distribution, and aggregate structure of lipopolysaccharide and lipid A dispersions from enterobacterial origin

Lipopolysaccharides (LPSs) from Gram-negative bacteria are strong elicitors of the human immune systems. There is strong evidence that aggregates and not monomers of LPS play a decisive role at least in the initial stages of cell activation of immune cells such as mononuclear cells. In previous reports, it was shown that the biologically most active part of enterobacterial LPS, hexa-acyl bisphosphorylated lipid A, adopts a particular supramolecular conformation, a cubic aggregate structure. However, little is known about the size and morphology of these aggregates, regarding the fact that LPS may have strong variations in the length of the saccharide chains (various rough mutant and smooth-form LPS). Thus, in the present paper, several techniques for the determination of details of the aggregate morphology such as freeze-fracture and cryo-electron microscopy, analytical ultracentrifugation, laser backscattering analysis, and small-angle X-ray scattering were applied for various endotoxin (lipid A and different LPS) preparations. The data show a variety of different morphologies not only for different endotoxins but also when comparing different applied techniques. The data are interpreted with respect to the suitability of the single techniques, in particular on the basis of available literature data.

The Preparation of a Lipidic Endotoxin Affects Its Biological Activities

Bacterial membrane constituents, such as Ornithine-containing lipid (OL) and the lipid A portion of lipopolysaccharide, trigger various immune responses through recognition by Toll-like receptor (TLR) 4. Usually, these lipids are dissolved in a small amount of aqueous or organic solvent before being added to the culture medium for examination of their biological activities. Macrophages stimulated with OL or lipid A sonically dissolved in saline released both interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). In contrast, macrophages stimulated with OL or lipid A sonically dissolved in ethanol or dimethyl sulfoxide (DMSO) secreted much TNF-alpha, but very little IL-1beta. These results, taken together, indicate that how an endotoxin is prepared affects its biological activities. In addition, electromicroscopic analysis revealed that sonication of air-dried OL or lipid A in DMSO produced larger particles than those produced in saline, suggesting that the process of preparing lipidic TLR4-ligands affects their physical state including particle size, and that the physical state might be an important determinant of biological activity.

Capacitive biosensor for detection of endotoxin

A capacitive biosensor for the detection of bacterial endotoxin has been developed. Endotoxin-neutralizing protein derived from American horseshoe crab was immobilized to a self-assembled thiol layer on a biosensor transducer (Au). Upon injection of a sample containing endotoxin, a decrease in the observed capacitive signal was registered. Endotoxin could be determined under optimum conditions with a detection limit of 1.0 x 10(-13) M and linearity ranging from 1.0 x 10(-13) to 1.0 x 10(-10) M. Good agreement was achieved when applying endotoxin preparations purified from an Escherichia coli cultivation to the capacitive biosensor system, utilizing the conventional method for quantitative endotoxin determination, the Limulus amebocyte lysate test as a reference. The capacitive biosensor method was statistically tested with the Wilcoxon signed rank test, which proved the system is acceptable for the quantitative analysis of bacterial endotoxin (P<0.05).

Endotoxin Contamination in Recombinant Human Heat Shock Protein 70 (Hsp70) Preparation Is Responsible for the Induction of Tumor Necrosis Factor α Release by Murine Macrophages

Using commercially available recombinant human heat shock protein 70 (rhHsp70), recent studies have shown that rhHsp70 could induce the production of tumor necrosis factor alpha (TNFalpha) by macrophages and monocytes in a manner similar to lipopolysaccharide (LPS) e.g. via CD14 and Toll-like receptor 4-mediated signal transduction pathway. In the current study, we demonstrated that a highly purified rhHsp70 preparation (designated as rhHsp70-1) with a LPS content of 1.4 pg/microg was unable to induce TNFalpha release by RAW264.7 murine macrophages at concentrations up to 5 microg/ml. In contrast, a less purified rhHsp70 preparation (designated as rhHsp70-2) at 1 microg/ml with a LPS content of 0.2 ng/microg was able to induce TNFalpha release to the same extent as that induced by 0.2 ng/ml LPS. Failure of rhHsp70-1 to induce TNFalpha release was not because of defective physical properties since rhHsp70-1 and rhHsp70-2 contained identical hsp70 content as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHsp70 antibody. Both rhHsp70 preparations also had similar enzymatic activities as judged by their ability to remove clathrin from clathrin-coated vesicles. Removal of LPS from rhHsp70-2 by polymyxin B-agarose column or direct addition of polymyxin B to the incubation medium essentially eliminated the TNFalpha-inducing activity of rhHsp70-2. The addition of LPS at the concentration found in rhHsp70-2 to rhHsp70-1 resulted in the same TNFalpha-inducing activity as observed with rhHsp70-2. The TNFalpha-inducing activities of rhHsp-2, LPS alone, and LPS plus rhHsp70-1 were all equally sensitive to heat inactivation. These results suggest that rhHsp-70 does not induce TNFalpha release from murine macrophages and that the observed TNFalpha-inducing activity in the rhHsp70-2 preparation is entirely due to the contaminating LPS.

Endotoxin levels in steam and reservoirs of table-top steam sterilizers.

To document endotoxin levels in "Statim" cassette sterilizer reservoirs and in steam delivered to the cassette in the unwrapped instrument cycle. To document endotoxin levels in sterilizer reservoir water using different management protocols. Endotoxin levels were determined using the Limulus Amebocyte Lysate test. Endotoxin preparations were from Escherichia coli and Ralstonia pickettii. All samples were collected in depyrogenated glassware and stored at -20 degrees C until assayed. The majority of water samples contained < 1.0 Endotoxin Unit (EU)/ml. The highest level found in sterilizers in clinical use was 5.3 EU/ml. Endotoxin was not detected in steam condensate within the limits of the assay. When the endotoxin level in the reservoir water was experimentally enhanced to 200 EU/ml, cassette steam condensate endotoxin levels were from 0.5% to 5% of the reservoir level. Daily and weekly emptying of the cassette reservoir consistently yielded low endotoxin levels as did monthly emptying, but with the latter there was a trend toward higher levels that favors weekly emptying as a precautionary measure. Endotoxin levels in the reservoirs of 23 sterilizers involving 240 samplings were never high enough to yield detectable endotoxin levels in steam in the sterilizer cassette. Regular weekly emptying of sterilizer reservoirs would eliminate the risk of endotoxin transfer during steam sterilization.

Commercial preparations of lipoteichoic acid contain endotoxin that contributes to activation of mouse macrophages in vitro.

Lipoteichoic acids (LTA), cell wall components of gram-positive bacteria, have been reported to induce various inflammatory mediators and to play a key role in gram-positive-microbe-mediated septic shock. In a large number of these studies, investigators used commercially available LTA purified from a variety of gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, and Streptococcus sanguis. We report here that, although these commercially available LTA could be readily shown to stimulate production of nitric oxide (NO) in RAW 264.7 mouse macrophages, the activity was dramatically inhibited by polymyxin B, a relatively specific inhibitor of endotoxin biological activity. One-step purification of the commercially available S. aureus LTA using hydrophobic interaction chromatography resulted in two well-separated peak fractions, one highly enriched for LTA and a second highly enriched for endotoxin. The LTA-enriched fractions did not induce production of NO in RAW 264.7 macrophages, although they caused a dose-dependent induction of NO in the presence of low concentrations of gamma interferon (IFN-gamma) (which by itself induced little NO), regardless of the presence of polymyxin B. In contrast, the endotoxin-enriched fractions by themselves inhibited in high levels of NO in RAW 264.7 macrophages but activity was almost completely inhibited in the presence of polymyxin B. Consistent with these findings, our data also indicate that commercial LTA preparations from S. aureus, B. subtilis, and S. sanguis were not able to induce NO from lipopolysaccharide-hyporesponsive C3H/HeJ mouse peritoneal macrophages, but in the presence of IFN-gamma, these LTA preparations were able to induce relatively high levels of NO from C3H/HeJ macrophages. These results indicate that commercially available LTA can contain contaminating and potentially significant levels of endotoxin that can be expected to contribute to the putative macrophage-stimulating effects of LTA as assessed by NO production. The fact that the purified LTA, by itself, was not able to induce significant levels of NO secretion in RAW 264.7 macrophages supports the conclusion that caution in attributing high-level biological activity to this microbial cell wall constituent should be exercised.

Alternative Forms of Storage Protection: Biological Insecticides for the Control of the Cigarette Beetle (Lasiodermaserricorne) and the Tobacco Moth (Ephestiaelutella)

Abstract A large number of physical and chemical pest control methods have been developed up to now in order to ensure the effective protection of stored tobacco and tobacco products. These methods include the use of fumigants and insecticides, treatment of baled tobacco in cold chambers, monitoring the extent of infestation with pheromone traps and, the most important preventive measure of all, maintaining a strict programme to ensure hygienic storage conditions. To date, very little serious consideration has been given to the practical use of biologically based insecticides in storage pest control. Compared with synthetically-produced pesticides, however, they often have the advantage of being significantly less toxic to users and the environment, while retaining a comparable level of effectiveness on the target organism. One reason for this neglect may certainly be found in the somewhat higher price of bioinsecticides. Another hurdle is presented by the complex mixture of different biological pesticides, which makes it more difficult to obtain official approval for their use in some countries. One possible alternative is, for example, the use of Bacillus thuringiensis endotoxin preparations. The insecticidal activity of certain B.t. kurstaki or B.t. tenebrionis strains against the tobacco moth and cigarette beetle has already been sufficiently documented. Recent research has also looked at the possibility of using natural substances, such as extracts from the neem tree (Azadirachtaindica), as storage protectants. Another very promising development is based on the autodissemination technique, whereby a modified pheromone trap in combination with entomopathogenic viruses or fungi could help to achieve a dramatic increase in mortality rates in stored tobacco pests. The chemical and biological properties of these bioinsecticides will be presented here and their advantages and disadvantages as storage protectants for tobacco discussed.

Modulation of the biological activities of meningococcal endotoxins by association with outer membrane proteins is not inevitably linked to toxicity.

Meningococcal sepsis results partly from overproduction of host cytokines after macrophages interact with endotoxin. To obtain less toxic and highly immunomodulatory meningococcal endotoxins for prophylactic purposes, we investigated the relationship between endotoxicity and immunomodulatory activity of several endotoxin preparations from Neisseria meningitidis group B. Using the D-galactosamine-sensitized mouse model to determine endotoxin lethality, we found that the toxicity of purified lipooligosaccharide (LOS) from M986, a group B disease strain, was three to four times higher than those of purified LOSs from the noncapsulated strains M986-NCV-1 and OP-, the truncated-LOS mutant. The LOSs of outer membrane vesicles (OMVs) and detergent-treated OMVs (D-OMVs) from the three strains were 2 to 3 and over 300 times less toxic than the purified LOSs, respectively. Intraperitoneal administration of these preparations induced production of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in serum 2 h after injections. However, repeated doses of low- and high-toxicity preparations induced lower amounts of TNF-alpha and IL-6, i.e., LOS tolerance. Injection of mice with low doses of LOS was as effective as injection with high doses in inducing tolerance. Peritoneal macrophages from tolerant mice pretreated with either high- or low-toxicity LOS preparations produced only a fraction of the amounts of TNF-alpha and IL-6 produced by control groups in response to LOS ex vivo. Despite tolerance to LOS induced by pretreatment with reduced-toxicity preparations, killing of N. meningitidis M986 by macrophages from these animals was enhanced. Protection was achieved when mice treated with LOS, and especially that of D-OMVs, were challenged with live N. meningitidis. The least toxic LOS, that in D-OMVs, was most effective in inducing hyporesponsiveness to endotoxin in mice but protected them against challenge with N. meningitidis. No inevitable link between toxicity and host immune modulation and responses was shown. Our results show that LOS is responsible for both toxicity and immunomodulation. When LOS is tightly associated with outer membrane proteins in D-OMV, it reduces toxicity but enhances beneficial effects compared to results with its purified form. Thus, systematic and critical evaluation of D-OMVs as adjuvants or as portions of group B meningococcal vaccines may help improve survival and outcome in meningococcal sepsis.

Bacterial-induced release of inflammatory mediators by bronchial epithelial cells.

This review focuses on bacterial induction and release of inflammatory cytokines and adhesion molecules by human bronchial epithelial cells, with special reference to Haemophilus influenzae, a pathogen commonly associated with chronic bronchitis. Studies investigating the mechanisms underlying bacterial colonization of the airways and bacterial-induced chronic airway inflammation have suggested that these are likely to involve localization of bacteria to the site(s) of infection in the respiratory tract and induction of a local airway inflammation resulting in the initiation of epithelial damage. We have hypothesized that the gross airway epithelial damage observed in chronic infective lung disease is an indirect consequence of proteolytic enzymes and toxic oxygen radicals generated by large numbers of neutrophils infiltrating the airways. Furthermore, the infiltration and activation of the neutrophils is a consequence of increased release of proinflammatory mediators from the host respiratory epithelium, induced by bacterial products, such as endotoxin. This hypothesis is based on studies which have demonstrated that the concentrations of circulating cytokines, such as interleukin (IL)-8 and tumour necrosis factor-alpha (TNF-alpha), which have profound effects on neutrophil activity, are increased in endotoxaemia and that airway epithelial cells are a rich source of these cytokines. Support for this hypothesis is provided by studies of cultured human bronchial epithelial cells incubated either in the absence or presence of purified endotoxin preparations from nontypable and type b H. influenzae strains which have demonstrated that these endotoxins lead to significantly increased expression and/or release of proinflammatory mediators, including IL-6, IL-8, TNF-alpha and intercellular adhesion molecule-1 (ICAM-1). Treatment of the cells with steroids can downregulate the expression and/or release of these inflammatory mediators. Additionally, these studies have demonstrated that culture medium collected from endotoxin-treated cultures, 24 h after treatment, significantly increases neutrophil chemotaxis and adhesion to human endothelial cells in vitro.

Biological characterization of Pseudomonas aeruginosa endotoxin released by antibiotic treatment in vitro

The supernatants taken from Pseudomonas aeruginosa cultures in human sera or chemically defined M9 medium in the presence of ceftazidime (CAZ) contained high levels of endotoxin, while those of imipenem (IPM) yielded relatively lower levels of endotoxin. The membrane-filtered supernatants were used as a source of endotoxin and the biological activities of the endotoxin were examined in comparison with those of hot-phenol water-extracted LPS. The CAZ-released endotoxin preparation contained large amounts of protein. The protein, however, appeared to lack significant endotoxic activity through the endotoxin-protein component, since the endotoxin did not show any toxic effect in D(+)-galactosamine (GaIN)-sensitized C3H/HeJ mice in vivo or macrophage activation in vitro. The activities of CAZ- and IPM-released endotoxins as assessed by chromogenic Limulus amebocyte coagulation test were fundamentally identical to those of purified P. aeruginosa LPS and Escherichia coli LPS, since their regression lines were parallel. The biological effects of CAZ-released endotoxin, such as lethal toxicity in GaIN-sensitized mice, in vitro induction of TNF and NO production by peritoneal macrophages, and MAP-kinase activation in macrophages of LPS-responsive C3H/He and LPS-low responsive C3H/HeJ mice, were similar to those of the LPS. Macrophage activation by CAZ-released endotoxin as well as LPS was mainly dependent on serum factors and CD14 antigen. Polymyxin B blocked the activity. These findings indicate that the endotoxic activity of CAZ-released endotoxin is due primarily to LPS (lipid A), although such preparations contain a high level of protein released from or produced by the organisms. Finally, the possibility that the treatment of P. aeruginosa infection with some kind of antibiotics may induce endotoxic shock was suggested in a mouse model.

Effects of Escherichia coli and Porphyromonas gingivalis lipopolysaccharide on pregnancy outcome in the golden hamster

This report describes the effects of two gram-negative bacterial endotoxin (lipopolysaccharide [LPS]) preparations on hamster pregnancy outcome variables. Single intravenous challenges with Escherichia coli and Porphyromonas gingivalis LPS on day 8 of pregnancy produced dose-dependent effects on fetal weight malformation and fetal resorption with E. coli LPS having potent embryolethal effects. Premating maternal exposure to P. gingivalis produced embryolethal effects similar to those of E. coli. These data suggest that maternal exposure to P. gingivalis LPS prior to and during pregnancy can induce deleterious effects on the developing fetus.

Differential blocking of coagulation-activating pathways of Limulus amebocyte lysate.

The coagulation of Limulus amebocyte lysate (LAL) can be activated through two pathways, one initiated by endotoxin and the other by beta-glucans. The two pathways join at the step of activation of the proclotting enzyme. We report here that the endotoxin-activated pathway can be differentially inhibited by two methods in a Limulus enzyme-linked immunosorbent assay (ELISA), either by the combined use of dimethyl sulfoxide and polymyxin B or by a monoclonal antibody against Limulus factor C. LAL reactivities to 10 different endotoxin preparations could be inhibited by the former method by a factor of 10(4) to 10(6) and could be blocked almost totally by the latter method, irrespective of the source of endotoxin. The sensitivity of the assay was approximately 50 pg/ml both for curdlan from Alcaligenes faecalis and for laminarin from Laminaria digitata. We also found that the beta-glucan-activated pathway could be totally blocked by laminarin (> 1 microgram/ml) without affecting the endotoxin-activated pathway, allowing endotoxin to be quantitated specifically by the Limulus ELISA with a detection limit of 0.005 endotoxin unit per ml. The use of uninhibited and differentially inhibited ELISAs demonstrated that different LAL preparations showed much greater variation in assaying beta-glucans than in assaying endotoxins. The LAL reactivity of normal human plasma was found to be due to the activation of the beta-glucan pathway, but not the endotoxin pathway, of LAL.