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Endotoxin tolerance induced by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli: alternations in Toll-like receptor 2 and 4 signaling pathway.  


Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a hyporesponsive state to subsequent challenge, which is termed endotoxin tolerance. In this experiment, we studied the cytokine production in THP-1 cells upon single or repeated Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) or Escherichia coli (E. coli) LPS stimulation by ELISA. In addition, the protein expression profiles of Toll-like receptor 2 (TLR2), TLR4, IL-1 receptor-associated kinase 4 (IRAK4) and IRAK-M and the gene expression changes of Toll-interacting protein (Tollip) and suppressor of cytokine-signaling-1 (SOCS1) were explored to identify possible mechanisms for changes in cytokine secretion. After repeated stimulation with P. gingivalis LPS or E. coli LPS, secretions of TNF-? and IL-1? were decreased significantly compared with those following single challenge, while the levels of IL-10 were increased (p < 0.05). Only comparable levels of IL-8 were confirmed in P. gingivalis LPS-tolerized cells (p > 0.05). In addition, severe downregulation of TLR2 was detected in THP-1 cells retreated with P. gingivalis LPS, and the reduction of TLR4 expression was observed in cells restimulated with E. coli LPS (p < 0.05). Precondition with P. gingivalis LPS or E. coli LPS also led to an enhancement of IRAK-M and SOCS1, while maintaining the expressions of IRAK4 and Tollip. This pattern of cytokine production indicates the different effects of endotoxin tolerance triggered by P. gingivalis LPS and E. coli LPS, which might contribute to limiting inflammatory damage. Moreover, TLR2, TLR4, IRAK-M, and SOCS1 might play important roles in developing tolerance. PMID:24132662

Sun, Ying; Li, Hui; Sun, Meng-Jun; Zheng, Yang-Yu; Gong, Dan-Jun; Xu, Yan



Is Pasteurella tularensis Lipopolysaccharide an Endotoxin.  

National Technical Information Service (NTIS)

Pasteurella tularensis cell wall or lipopolysaccharide (LPS), unlike Salmonella enteritidis LPS, is nontoxic for mice previously sensitized by enterotoxin B. Studies in normal mice challenged with P. tularensis 425, a strain of intermediate virulence, dem...

M. L. Guss



Effects of endotoxin lipopolysaccharide administration on the somatotropic axis  

Microsoft Academic Search

The aim of this work was to study the eVect of chronic activation of the immune system on the somatotropic axis. Accordingly, the changes in growth hormone (GH) secre- tion, circulating insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) in response to endotoxin lipopolysaccharide (LPS) administration were examined in adult male Wistar rats. Acute LPS injection (2·5, 25 or

L Soto; A I Martin; S Millan; E Vara



Interactions of radio-detoxified Escherichia coli endotoxin preparations with the complement system.  

PubMed Central

Escherichia coli O89 lipopolysaccharide (LPS) was treated with different doses of gamma irradiation (5, 10, 15, and 20 Mrad). Various biological activities such as lethal effect, decrease in arterial blood pressure in dogs, and interaction with the complement system were determined for the parent and irradiated preparations. Irradiation of LPS significantly and in a dose-dependent manner decreased its lethal and blood pressure-depressing effects along with its ability to activate the complement system. In contrast, radio-detoxified LPS fixed more strongly the isolated human C1 than did the parent LPS. The possible connection between the toxicity of endotoxin and endotoxin-induced complement activation is discussed. Images

Fust, G; Bertok, L; Juhasz-Nagy, S



Inactivation of Escherichia coli Endotoxin by Soft Hydrothermal Processing?  

PubMed Central

Bacterial endotoxins, also known as lipopolysaccharides, are a fever-producing by-product of gram-negative bacteria commonly known as pyrogens. It is essential to remove endotoxins from parenteral preparations since they have multiple injurious biological activities. Because of their strong heat resistance (e.g., requiring dry-heat sterilization at 250°C for 30 min) and the formation of various supramolecular aggregates, depyrogenation is more difficult than sterilization. We report here that soft hydrothermal processing, which has many advantages in safety and cost efficiency, is sufficient to assure complete depyrogenation by the inactivation of endotoxins. The endotoxin concentration in a sample was measured by using a chromogenic limulus method with an endotoxin-specific limulus reagent. The endotoxin concentration was calculated from a standard curve obtained using a serial dilution of a standard solution. We show that endotoxins were completely inactivated by soft hydrothermal processing at 130°C for 60 min or at 140°C for 30 min in the presence of a high steam saturation ratio or with a flow system. Moreover, it is easy to remove endotoxins from water by soft hydrothermal processing similarly at 130°C for 60 min or at 140°C for 30 min, without any requirement for ultrafiltration, nonselective adsorption with a hydrophobic adsorbent, or an anion exchanger. These findings indicate that soft hydrothermal processing, applied in the presence of a high steam saturation ratio or with a flow system, can inactivate endotoxins and may be useful for the depyrogenation of parenterals, including end products and medical devices that cannot be exposed to the high temperatures of dry heat treatments.

Miyamoto, Toru; Okano, Shinya; Kasai, Noriyuki



Intracellular and extracellular enzymatic deacylation of bacterial endotoxin during localized inflammation induced by Escherichia coli.  

PubMed Central

Acyloxyacyl hydrolase (AOAH), an enzyme that removes the secondary acyl chains of gram-negative bacterial lipid A (endotoxin), has been identified previously in human neutrophils and mouse macrophages. We report here that bovine leukocytes also contain AOAH activity. Although bovine AOAH deacylates bacterial lipopolysaccharide in a manner similar to human AOAH, it is active in vitro over a broader pH range, from 4.0 to 7.0. By using Escherichia coli infection of the bovine mammary gland as a model of localized gram-negative bacterial disease and associated tissue inflammation, AOAH activity per leukocyte increased. In addition, AOAH activity increased in the cell-free portion of infected mammary secretions. These data indicate that AOAH activity increases in leukocytes associated with inflammation induced by gram-negative bacteria and provide additional evidence of its potential involvement in the defense against the effects of bacterial endotoxin. Images

McDermott, C M; Cullor, J S; Fenwick, B W



Isolation and endotoxin activities of lipopolysaccharides from cyanobacterial cultures and complex water blooms and comparison with the effects of heterotrophic bacteria and green alga.  


Massive cyanobacterial water blooms are serious environmental and health problems worldwide. While some cyanobacterial toxins such as peptide microcystins have been investigated extensively, other toxic components of cyanobacteria (e.g. lipopolysaccharides, LPS) are poorly understood. The present study characterized endotoxin activities of LPS isolated from (i) laboratory cyanobacterial cultures, (ii) cyanobacterial water bloom samples dominated by Microcystis sp., Planktothrix sp., Aphanizomenon sp. and Anabaena sp., (iii) heterotrophic Gram-negative bacteria Escherichia coli, Kluyvera intermedia, Pseudomonas putida and Pseudomonas fluorescens and (iv) green alga Pseudokirchneriella subcapitata. Toxicity results derived with Limulus amebocyte lysate assay (LAL-test) showed that endotoxin activities of LPS from both cyanobacteria and heterotrophic bacteria were comparable and the values were within a similar range (1 x 10(3)-1 x 10(6) Endotoxin Units, EU, per mg of isolated LPS). The highest activities among the cyanobacterial samples were observed in the Aphanizomenon sp. dominated water bloom. The results also suggest generally higher endotoxin activities in complex natural samples than in laboratory cyanobacterial cultures. Further, experiments with the eukaryotic green alga P. subcapitata demonstrated a need for careful purification of the LPS extracts prior to testing with the LAL assay as several contaminants may overestimate endotoxin activities. This study shows relatively high pyrogenicity of LPS from various cyanobacteria. Further research should focus on detailed toxicological and ecotoxicological characterization of LPS in massive cyanobacterial water blooms. PMID:17461433

Bernardová, Katerina; Babica, Pavel; Marsálek, Blahoslav; Bláha, Ludek



Action of propolis and medications against Escherichia coli and endotoxin in root canals.  


This study evaluated the action of propolis and intracanal medications against Escherichia coli and endotoxin. Forty-eight dental roots were contaminated with E. coli. The root canals were instrumented with propolis and divided into groups according to the type of intracanal medication: Ca(OH)(2), polymyxin B, or Ca(OH)(2) + 2% chlorhexidine gel. In the control group, saline solution was used without application of intracanal medication. Counts of colony-forming units were carried out and the endotoxin was quantified by the chromogenic Limulus amobocyte lysate assay. The results were evaluated by analysis of variance and the Dunn test (5%). Root canal irrigation with propolis was effective to completely eliminate E. coli and reduce the amount of endotoxins. All intracanal medications contributed to the significant decrease in endotoxins. Only intracanal medications may reduce the amount of endotoxins in the root canals. The greatest efficacy was observed for medications containing Ca(OH)(2). PMID:20868987

Valera, Marcia Carneiro; da Rosa, Jucely Aparecida; Maekawa, Lilian Eiko; de Oliveira, Luciane Dias; Carvalho, Cláudio Antonio Talge; Koga-Ito, Cristiane Yumi; Jorge, Antonio Olavo Cardoso



Antibiotic-induced release of endotoxin from bacteria in vitro  

Microsoft Academic Search

Summary. The ability of cefotaxime, ciprofloxacin, piperacillin and tobramycin to cause release of endotoxin was examined in vitro with cultures of Enterobacter cloacae and Escherichia coli. Endotoxin was measured by a quantitative limulus amoebocyte lysate assay and its presence was confirmed by silver staining of the lipopolysaccharide moiety following SDS-PAGE. The morphology of the bacteria during antibiotic exposure was examined

HEATHER A. CROSBY; J. F. Bion; C. W. PENNS; T. S. J. Elliott



The interaction between Newcastle disease virus and Escherichia coli endotoxin in chickens.  


The interaction between Newcastle disease virus (NDV) and Escherichia coli endotoxin was studied in cell cultures, embryonated chicken eggs, and 8-wk-old chickens. These interactions were evaluated according to the induction of specific or nonspecific resistance in the host system and the virus titer produced in both chicken embryos and chickens. The endotoxin of E. coli induced a decrease in the size of the bursa of Fabricius in live chickens. Escherichia coli endotoxin given intravenously induced plasma antiviral activity in chickens that was interpreted to be interferon, as detected in a vesicular stomatitis virus plaque reduction assay. Endotoxin failed to produced toxic effects in the chicken embryo fibroblasts (CEFs) or to result in any antiviral effect because no change was noted in the number of NDV plaques formed in CEF cultures. When endotoxin was given 3 days before NDV exposure in chickens, the virus titers were significantly (P < 0.05) decreased from a peak of 10(2) to 10(0.18), 10(2.5) to 10(0.18), and 10(2.5) to 0 in the spleens, lungs, and kidneys, respectively, at 72 hr post-NDV inoculation. When endotoxin was given 24 hr after NDV inoculation, the NDV titer significantly (P < 0.05) increased from 10(2.0) to 10(3.5), 10(2.5) to 10(6.5), 10(2.5) to 10(4.5), 0 to 10(2.5) in the spleen, lungs, kidneys, and liver, respectively, at 72 hr after NDV inoculation. In chicken sera, hemagglutination inhibition (HI) titer to NDV was significantly (P < 0.05) enhanced from 1164 to 3127 when endotoxin was given prior to virus inoculation. However, there was a decrease in HI to NDV from 1164 to 727 without a significant difference in chicken sera when NDV was given prior to endotoxin inoculation. PMID:11417810

El Tayeb, A B; Hanson, R P



Tetrahydrobiopterin corrects Escherichia coli endotoxin-induced endothelial dysfunction.  


Acute inflammation causes endothelial dysfunction, which is partly mediated by oxidant stress and inactivation of nitric oxide. The contribution of depletion of tetrahydrobiopterin (BH(4)), the cofactor required for nitric oxide generation, is unclear. In this randomized, double-blind, three-way crossover study, forearm blood flow (FBF) responses to ACh and glyceryltrinitrate (GTN) were measured before and 3.5 h after infusion of Escherichia coli endotoxin (LPS, 20 IU/kg iv) in eight healthy men. The effect of intra-arterial BH(4) (500 microg/min), placebo, or vitamin C (24 mg/min) was studied on separate days 3.5 h after LPS infusion. In addition, human umbilical vein endothelial cells were incubated for 24 h with vitamin C and LPS. ACh and GTN caused dose-dependent forearm vasodilation. The FBF response to ACh, which was decreased by 23 +/- 17% (P < 0.05) by LPS infusion, was restored to baseline reactivity by BH(4) and vitamin C. FBF responses to GTN were not affected by BH(4) or vitamin C. LPS increased leukocyte count, high-sensitivity C-reactive protein, IL-6, IL-1beta, IFN-gamma, monocyte chemoattractant protein-1, pulse rate, and body temperature and decreased platelet count and vitamin C concentration. Vitamin C increased forearm plasma concentration of BH(4) by 32% (P < 0.02). Incubation with LPS and vitamin C, but not LPS alone, increased intracellular BH(4) concentration in human umbilical vein endothelial cells. Impaired endothelial function during acute inflammation can be restored by BH(4) or vitamin C. Vitamin C may exert some of its salutary effects by increasing BH(4) concentration. PMID:15964928

Mittermayer, Friedrich; Pleiner, Johannes; Schaller, Georg; Zorn, Stefan; Namiranian, Khodadad; Kapiotis, Stylianos; Bartel, Gregor; Wolfrum, Mathias; Brügel, Mathias; Thiery, Joachim; Macallister, Raymond J; Wolzt, Michael



A synthetic lipopolysaccharide-binding peptide based on the neutrophil-derived protein CAP37 prevents endotoxin-induced responses in conscious rats.  

PubMed Central

The lipid A component of lipopolysaccharide (LPS) derived from Escherichia coli has been implicated as a significant mediator in the development of circulatory and metabolic dysfunction and lethality associated with sepsis. A synthetic peptide corresponding to amino acid residues 20 through 44 of the neutrophil-derived 37-kDa cationic antimicrobial protein (CAP37 P(20-44)) possesses lipid A binding characteristics which may be useful in attenuating in vivo responses induced during circumstances of endotoxemia, including sepsis. The E. coli LPS to be used in the in vivo study was shown to be attenuated by CAP37 P(20-44) in a dose-dependent manner in the in vitro reaction with Limulus amoebocyte lysate. Intravenous infusion of CAP37 P(20-44) (1.5 or 3.0 mg/kg of body weight) with E. coli LPS (250 microg/kg over 30 min) into conscious, unrestrained rats prevented LPS-induced hyperdynamic and hypodynamic circulatory shock, hyperlactacidemia, and leukopenia in a dose-related fashion. CAP37 P(20-44) (0.2, 1.0, and 5.0 mg/kg) administered intravenously to conscious, actinomycin D-sensitized rats following a lethal dose of LPS neutralized LPS toxicity, resulting in dose-dependent 7-day survival rates of 30, 50, and 80%, respectively. CAP37 P(20-44) (5.0 mg/kg) significantly inhibited the endotoxin-induced increase in circulating tumor necrosis factor alpha in sensitized rats. These data demonstrate that CAP37 P(20-44) has the capacity to abolish in vivo biological responses to LPS that are relevant to human sepsis and to significantly neutralize the toxicity of circulating E. coli LPS.

Brackett, D J; Lerner, M R; Lacquement, M A; He, R; Pereira, H A



Shiga toxin-associated hemolytic uremic syndrome: combined cytotoxic effects of shiga toxin and lipopolysaccharide (endotoxin) on human vascular endothelial cells in vitro.  

PubMed Central

This study explores the in vitro relationship between Shiga toxin-producing Shigella spp. and Escherichia coli and the development of vascular complications in humans following bacillary dysentery. We propose that lipopolysaccharide (LPS; endotoxin) may combine with Shiga toxin to facilitate vascular damage characteristic of hemolytic uremic syndrome. We have examined the direct cytotoxic effects of Shiga toxin and LPS on human umbilical vein endothelial cells (HUVEC) in culture. Shiga toxin alone was cytotoxic to HUVEC, whereas LPS was noncytotoxic at concentrations at or below 10 micrograms/ml. Combinations of LPS with Shiga toxin resulted in a synergistic cytotoxic effect. The synergistic cytotoxic response of HUVEC to Shiga toxin plus LPS was dose dependent for both agents and was maximal at 24 h of exposure. This synergistic response was enhanced by preincubation of HUVEC with LPS. LPS (1 micrograms/ml) alone depressed HUVEC protein synthesis in a transient manner and enhanced the protein synthesis-inhibiting activity of Shiga toxin. The synergistic cytotoxic activity of LPS analogs was as follows, in decreasing order: complete LPS = diphosphoryl lipid A greater than monophosphoryl lipid A greater than deacylated LPS. These results are consistent with a role for Shiga toxin and LPS in the development of hemolytic uremic syndrome at the level of the vascular endothelium in humans.

Louise, C B; Obrig, T G



Effect of E coli endotoxin on the leakage of /sup 14/C-sucrose from phosphatidylcholine liposomes  

SciTech Connect

The effect of E coli endotoxin on the leakage of /sup 14/C-sucrose from phosphatidylcholine liposomes in the absence or presence of Ca/sup 2 +/ was studied. Endotoxin decreased the leakage from liposomes from 27% to 4% in 5 hr when Ca/sup 2 +/ (1 mM) was incorporated into liposomes during sonication. The effect of endotoxin on the leakiness of liposomes was concentration dependent. Ca/sup 2 +/ alone increased the leakage of /sup 14/C-sucrose from liposomes. Mg/sup 2 +/ at concentrations higher than 5 mM exhibited an effect similar to that of Ca/sup 2 +/. These findings suggest that endotoxin increases the molecular packing of phosphatidylcholine bilayers in the presence of Ca/sup 2 +/ or Mg/sup 2 +/. A change in the physical state of membrane lipid bilayers induced by endotoxin may affect the function of biological membranes.

Onji, T.; Liu, M.S.



Induction of reversible shock by Escherichia coli lipopolysaccharide in rats. Changes in serum and cell membrane parameters.  

PubMed Central

Reversible endotoxic shock was induced in adult rats by i.v. injection of Escherichia coli O111:B4 lipopolysaccharide (1.6 mg/100 g). The shock progression was evaluated by measuring serum glucose levels as well as activities of aspartate aminotransferase (GOT) and alkaline phosphatase in serum. A rapid increase of serum glucose levels occurs, after LPS injection, followed by hypoglycaemia (minimum values at 6 h) with progressive reversion to control values. Serum GOT activity increased (twofold) 6 h after endotoxin administration and returned to control values at 72 h. No appreciable changes occurred in serum alkaline phosphatase activity. Endotoxaemia produced a decrease in the cytochrome P-450 levels in all target organs considered: lung, adrenal glands and liver. The progressive decrease in the serum albumin concentration as well as changes of the physical properties of the plasma membranes observed in vivo, can not be explained only by direct interaction of endotoxin with the target organs, underlining the importance of serum mediators in the induction of the shock response.

Bosch, M. A.; Garcia, R.; Pagani, R.; Portoles, M. T.; Diaz-Laviada, I.; Abarca, S.; Ainaga, M. J.; Risco, C.; Municio, A. M.



Underlying mechanisms involved in the decrease of milk secretion during Escherichia coli endotoxin induced mastitis in lactating mice  

PubMed Central

Mastitis, the inflammation of mammary glands resulting from bacterial infection, disrupts milk production in lactating mammary glands. In this study, we injected lipopolysaccharide (LPS), one of the endotoxins from Escherichia coli into mouse mammary glands to disrupt milk production, and we investigated the influence of LPS on nutrient uptake, synthesis, and secretion processes for milk component production in alveolar epithelial cells (AEC). The expression of genes relevant to the three-staged milk component production process (nutrient uptake, synthesis, and secretion of milk components) were down-regulated within 12 h after LPS injection in AEC. The internalization of glucose transporter 1 (GLUT-1) from the basolateral membrane to the cytoplasm occurred in accordance with the down-regulation of gene expression 3 h after LPS injection. The abnormal localization of adipophilin and beta-casein was also observed in the LPS-injected mammary glands. SLC7A1, an amino acid transporter, was up-regulated 3 and 6 h after LPS injection. Furthermore, the inactivation of signal transducer and activator of transcription 5 (STAT5) and the activation of STAT3 and nuclear factor-kappa B (NFkappaB) occurred 3 h after LPS injection. These results indicate that the nutrient uptake, synthesis, and secretion of milk components in AEC are rapidly shut down in the lactating mammary glands after LPS injection.



Role of Lipopolysaccharide and Capsule in the Serum Resistance of Bacteremic Strains of 'Escherichia coli'.  

National Technical Information Service (NTIS)

To define the relative roles of capsule and lipopolysaccharide in the virulence of Escherichia coli obtained from blood, the authors compared the behavior of K1-and K5-encapsulated strains in serum bactericidal and rat virulence assays. Unencapsulated iso...

A. S. Cross K. S. Kim D. C. Wright J. C. Sadoff P. Gemski



Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis  

PubMed Central

Introduction We have previously reported that bacterial toxins, especially endotoxins such as lipopolysaccharides (LPS), might be important causative agents in the pathogenesis of rheumatoid arthritis (RA) in an in vitro model that simulates the potential effects of residing in damp buildings. Since numerous inflammatory processes are linked with the nuclear factor-?B (NF-?B), we investigated in detail the effects of LPS on the NF-?B pathway and the postulated formation of procollagen-endotoxin complexes. Methods An in vitro model of human chondrocytes was used to investigate LPS-mediated inflammatory signaling. Results Immunoelectron microscopy revealed that LPS physically interact with collagen type II in the extracellular matrix (ECM) and anti-collagen type II significantly reduced this interaction. BMS-345541 (a specific inhibitor of I?B kinase (IKK)) or wortmannin (a specific inhibitor of phosphatidylinositol 3-kinase (PI-3K)) inhibited the LPS-induced degradation of the ECM and apoptosis in chondrocytes. This effect was completely inhibited by combining BMS-345541 and wortmannin. Furthermore, BMS-345541 and/or wortmannin suppressed the LPS-induced upregulation of catabolic enzymes that mediate ECM degradation (matrix metalloproteinases-9, -13), cyclooxygenase-2 and apoptosis (activated caspase-3). These proteins are regulated by NF-?B, suggesting that the NF-?B and PI-3K pathways are involved in LPS-induced cartilage degradation. The induction of NF-?B correlated with activation of I?B? kinase, I?B? phosphorylation, I?B? degradation, p65 phosphorylation and p65 nuclear translocation. Further upstream, LPS induced the expression of Toll-like receptor 4 (TLR4) and bound with TLR4, indicating that LPS acts through TLR4. Conclusion These results suggest that molecular associations between LPS/TLR4/collagen type II in chondrocytes upregulate the NF-?B and PI-3K signaling pathways and activate proinflammatory activity.



Endotoxin "priming" potentiates lung vascular abnormalities in response to Escherichia coli hemolysin: an example of synergism between endo- and exotoxin  

PubMed Central

The pore-forming hemolysin of Escherichia coli (HlyA), an important virulence factor in extraintestinal E. coli infections, causes thromboxane generation and related vasoconstriction in perfused rabbit lungs (Seeger, W., H. Walter, N. Suttorp, M. Muhly, and S. Bhakdi. 1989. J. Clin. Invest. 84:220). We investigated the influence of pulmonary vascular "priming" with endotoxin on the responsiveness of the lung to a low-dose HlyA challenge. Rabbit lungs were perfused with Krebs Henseleit buffer containing 0.1-100 ng/ml Salmonella abortus equii lipopolysaccharide (LPS) for 60-180 min. This treatment caused protracted release of tumor necrosis factor into the recirculating medium, but did not induce significant alterations of pulmonary hemodynamics and fluid balance. At a dose of 1 ng/ml, HlyA elicited only moderate thromboxane release (< 200 pg/ml) and pulmonary artery pressure increase (< or = 6 mmHg) in control lungs. Acceleration and potentiation of both the metabolic and vasoconstrictor response occurred in lungs primed with LPS. This priming effect displayed dose (threshold integral of 0.1-1 ng/ml LPS) and time dependencies (threshold integral of 60-90 min LPS incubation). Maximum thromboxane release and pulmonary artery pressure increase surpassed the responses to HlyA in nonprimed lungs by more than 15-fold. Cyclooxygenase inhibition and thromboxane-receptor antagonism blocked these effects. These data demonstrate that LPS priming synergizes with HlyA challenge to provoke vascular abnormalities that are possibly relevant to the pathogenesis of organ failure in severe local and systemic infections.



Escherichia coli lipopolysaccharide impairs the calcium signaling pathway in mesangial cells: role of angiotensin II receptors.  


Sepsis causes impaired vascular reactivity, hypotension and acute renal failure. The ability of the Escherichia coli endotoxin (lipopolysaccharide [LPS]) to impair agonist-induced contractility in mesangial cells, which contributes to LPS-induced renal dysfunction, was evaluated. Agonist-induced intracellular calcium ([Ca(2+)]i) mobilization was analyzed using angiotensin II (AngII). The effect of LPS on the levels of the renin-angiotensin system (RAS) components and the roles of vasodilatation-inducing molecules including AT2 receptor (AT2R) and nitric oxide (NO) in the cell reactivity were also evaluated. Confluent human mesangial cells (HMCs) were stimulated with LPS (0111-B4, 100 microg/mL). AngII-induced [Ca(2+)]i mobilization was measured by fluorometric analysis using Fura-2AM in the absence and presence of an AT2R antagonist (PD123319). The mRNA and protein levels for angiotensinogen, renin, angiotensin-converting enzyme, AT1R and AT2R were analyzed by realtime reverse transcriptase-polymerase chain reaction and Western blot, respectively. NO production was measured by the chemiluminescence method in the culture media after 24, 48 and 72 h of LPS incubation. After 24 h, LPS-stimulated HMCs displayed lower basal [Ca(2+)]i and an impaired response to AngII-induced rise in [Ca(2+)]i. LPS significantly increased AT2R levels, but did not cause significant alterations of RAS components. PD123319 restored both basal and AngII-induced [Ca(2+)]i peak, suggesting an involvement of AT2R in these responses. The expected increase in NO production was significant only after 72 h of LPS incubation and it was unaffected by PD123319. Results showed that LPS reduced the reactivity of HMCs to AngII and suggest that the vasodilatation induced by AT2R is a potential mediator of this response through a pathway independent of NO. PMID:20511680

Maquigussa, Edgar; Arnoni, Carine P; Cristovam, Priscila C; de Oliveira, Andrea S; Higa, Elisa M S; Boim, Mirian A



Role of endotoxin in acute inflammation induced by gram-negative bacteria: specific inhibition of lipopolysaccharide-mediated responses with an amino-terminal fragment of bactericidal/permeability-increasing protein.  

PubMed Central

A recombinant 23-kDa amino-terminal fragment of human bactericidal/permeability-increasing protein (rBPI23), a potent lipopolysaccharide (LPS)-binding/neutralizing protein, was used as a probe to assess the role of endotoxin in the acute inflammatory responses elicited by gram-negative bacteria in rat subcutaneous air pouches. In initial experiments, rBPI23 prevented the Escherichia coli O111:B4 LPS-induced accumulation of polymorphonuclear leukocytes (PMN), tumor necrosis factor alpha (TNF-alpha), and nitrite (a stable end product of nitric oxide formation) in exudate fluids. Significant inhibition of TNF-alpha production was still evident when rBPI23 treatment was delayed for 30 min after LPS instillation. In subsequent experiments, rBPI23 also prevented the nitrite and early (2-h) TNF-alpha accumulation induced by three different strains of formaldehyde-killed gram-negative bacteria (E. coli O7:K1, E. coli O111:B4, and Pseudomonas aeruginosa 12.4.4) but did not inhibit the PMN or late (6-h) TNF-alpha accumulation induced by these bacteria. As with LPS challenge, a significant inhibition of early TNF-alpha production was still evident when rBPI23 treatment was delayed for 30 to 60 min after instillation of killed bacteria. The results indicate that in this experimental model the NO and early TNF-alpha responses to gram-negative bacterial challenge are mediated predominantly by endotoxin, whereas the PMN and late TNF-alpha responses may be mediated by other bacterial components. Moreover, the results indicate that rBPI23 can inhibit the bacterially induced production of certain potentially harmful mediators (TNF-alpha and NO) without entirely blocking the host defense, i.e., PMN response, against the bacteria.

Kohn, F R; Kung, A H



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-1?, IL-6, and TNF-?, 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-1?, IL-6, and TNF-? 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. PMID:24566310

Yang, Li-Ling; Wang, Guo-Quan; Yang, Li-Mei; Huang, Zhi-Bing; Zhang, Wen-Qing; Yu, Lin-Zhong



Low Endotoxin Release from Escherichia coli and Bacteroides fragilis during Exposure to Moxifloxacin  

Microsoft Academic Search

Background: Bacterial endotoxin is known to act as a potent trigger of disseminated coagulation and septic shock. During clinical antibiotic treatment, endotoxin may be released from Gram-negative bacteria. It is known that antibiotic classes differ in their ability to induce endotoxin release. Aim: It was the aim of this study to test the endotoxin-liberating potential of different antibiotics with activity

Matthias Trautmann; Cordula Scheibe; Nele Wellinghausen; Otto Holst; Philipp M. Lepper



Subacute ruminal acidosis induces ruminal lipopolysaccharide endotoxin release and triggers an inflammatory response.  


Subacute ruminal acidosis (SARA) was induced in 3 rumen fistulated Jersey steers by offering them different combinations of wheat-barley pellets and chopped alfalfa hay. Steers were offered 4, 5, and 6 kg/d of pelleted concentrate and 6, 5, and 4 kg/d of chopped alfalfa hay for diets 1, 2, and 3, respectively, during 5-d treatment periods and were fed chopped alfalfa hay between treatment periods. Inducing SARA increased blood concentrations of haptoglobin and serum amyloid-A. Dry matter intake of concentrate and hay decreased from d 1 to 5 in each period. Subacute ruminal acidosis was induced in all steers during d 4 and 5 when concentrate was fed, with ruminal pH remaining below 5.6 for an average of 187 and 174 min/d on these days. Lipopolysaccharide concentration increased significantly during periods of grain feeding compared with times when only hay was fed. Inducing SARA by feeding wheat-barley pellets activated a systemic inflammatory response in the steers. PMID:15778308

Gozho, G N; Plaizier, J C; Krause, D O; Kennedy, A D; Wittenberg, K M



Annexin A5 Binds to Lipopolysaccharide and Reduces Its Endotoxin Activity  

PubMed Central

ABSTRACT Annexin A5 (AnxA5) has a high affinity for phosphatidylserine. The protein is widely used to detect apoptotic cells because phosphatidylserine, a phospholipid that is normally present in the inner leaflets of cytoplasmic membranes, becomes translocated to the outer leaflets during programmed cell death. Here we report the novel observation that AnxA5 binds to Gram-negative bacteria via the lipid A domain of lipopolysaccharide (LPS). Binding of AnxA5 to bacteria was measured quantitatively, confirmed by fluorescence microscopy, and found to be inhibited by antibodies against lipid A. AnxA5 also bound to purified dot-blotted LPS and lipid A. Through ellipsometry, we found that the binding of AnxA5 to purified LPS was calcium dependent and rapid and showed a high affinity—characteristics similar to those of AnxA5 binding to phosphatidylserine. Initial functional studies indicated that AnxA5 can affect LPS activities. AnxA5 inhibited LPS-mediated gelation in the Limulus amebocyte lysate assay. Incubation of LPS with the protein reduced the quantity of tumor necrosis factor alpha (TNF-?) released by cultured monocytes compared to that released upon incubation with LPS alone. Initial in vivo experiments indicated that injection of mice with LPS preincubated with AnxA5 produced serum TNF-? levels lower than those seen after injection of LPS alone. These data demonstrate that AnxA5 binds to LPS and open paths to investigation of the potential biological and therapeutic implications of this interaction.

Rand, Jacob H.; Wu, Xiao-Xuan; Lin, Elaine Y.; Griffel, Alexander; Gialanella, Philip; McKitrick, John C.



Cytoplasmic ATP Hydrolysis Powers Transport of Lipopolysaccharide Across the Periplasm in E. coli*  

PubMed Central

Millions of molecules of lipopolysaccharide (LPS) must be assembled on the E. coli cell surface every division. Biogenesis of LPS requires seven essential lipopolysaccharide transport (Lpt) proteins to move LPS from the inner membrane (IM) through the periplasm to the cell surface. However, no intermediate transport states have been observed. We developed methods to observe intermediate LPS molecules bound to Lpt proteins in the process of being transported in vivo. Movement of individual LPS molecules along these binding sites required multiple rounds of ATP hydrolysis in vitro, which suggests that ATP is used to push a continuous stream of LPS through a transenvelope bridge in discrete steps against a concentration gradient.

Okuda, Suguru; Freinkman, Elizaveta; Kahne, Daniel



Lipopolysaccharide heterogeneity in Escherichia coli J5 variants: analysis by flow cytometry.  


A lipopolysaccharide O-side chain-producing phenotypic variant was isolated from a uridine 5'-diphosphogalactose 4-epimeraseless Escherichia coli J5 rough mutant strain by fluorescence-activated cell sorting with a monoclonal antibody (MAb) specific for the O-side chain of E. coli O111:B4 smooth parent lipopolysaccharide. The variant (J5-2) was recognized by both core- and O-side chain-specific MAbs, while the "original" rough mutant (J5-1) and smooth parent strains stained only with core- and O-side chain-specific MAb, respectively. J5-2 produced complete and incomplete (Rc chemotype) core and O polysaccharide in the presence of galactose. Three other E. coli J5 variants were either phenotypically similar to J5-1 (J5-UK) or distinct from J5-1 and J5-2 (J5-A, -B). The latter phenotype had a lower-molecular-weight core, compared with J5-1 and J5-2, and distinct MAb specificities. Various J5 phenotypes also differed in galactokinase levels, the ability to use galactose, and susceptibility to core-specific MAb binding on solid media. The J5-2 strain showed reciprocal changes in O-side chain and core expression during log-phase growth. E. coli J5 thus undergoes spontaneous alterations in lipopolysaccharide phenotype. PMID:1527415

Evans, M E; Pollack, M; Koles, N L; Hardegen, N J; Panopoulos, D



Neutralization of bacterial endotoxins by frog antimicrobial peptides.  


The ability of skin antimicrobial peptides of the southern bell frog, Litoria raniformis, to neutralize in vitro the endotoxin, proinflammatory lipopolysaccharide (LPS) complex, from two different gram-negative bacterial pathogens, human pathogen Escherichia coli (0111:B4) and frog pathogen Klebsiella pneumoniae, was investigated. The LPS neutralization activity of the natural mixture of skin antimicrobial peptides was measured using chromogenic Limulus amebocyte lysate assays. These skin antimicrobial peptides neutralized the LPSs from both pathogens at physiologically relevant concentrations (IC(50) ?endotoxin agents. PMID:23252916

Schadich, Ermin; Mason, Drusilla; Cole, Anthony L



Inhibition of 3-O-Methyl Glucose Transport in 'Ascaris suum' Midgut by 'Escherichia coli' Endotoxin.  

National Technical Information Service (NTIS)

Movement of 3-0-methyl glucose across the midgut of Ascaris was inhibited by endotoxin. Inhibition was dependent on endotoxin concentration. Despite the fact that the mechanism by which the transport is inhibited is not known, the knowledge that absorptiv...

J. H. Migliaccio



Bacteriophage PhiX174's ecological niche and the flexibility of its Escherichia coli lipopolysaccharide receptor.  


To determine bacteriophage PhiX174's ecological niche, 783 Escherichia coli isolates were screened for susceptibility. Sensitive strains are diverse regarding their phylogenies and core lipopolysaccharides (LPS), but all have rough phenotypes. Further analysis of E. coli K-12 LPS mutants revealed that PhiX174 can use a wide diversity of LPS structures to initiate its infectious process. PMID:20833781

Michel, Alix; Clermont, Olivier; Denamur, Erick; Tenaillon, Olivier



Bacteriophage PhiX174's Ecological Niche and the Flexibility of Its Escherichia coli Lipopolysaccharide Receptor?  

PubMed Central

To determine bacteriophage PhiX174's ecological niche, 783 Escherichia coli isolates were screened for susceptibility. Sensitive strains are diverse regarding their phylogenies and core lipopolysaccharides (LPS), but all have rough phenotypes. Further analysis of E. coli K-12 LPS mutants revealed that PhiX174 can use a wide diversity of LPS structures to initiate its infectious process.

Michel, Alix; Clermont, Olivier; Denamur, Erick; Tenaillon, Olivier



Paeonol suppresses lipopolysaccharide-induced inflammatory cytokines in macrophage cells and protects mice from lethal endotoxin shock.  


Paeonol (2'-hydroxy-4'-methoxyacetophenone) is the main phenolic compound of the radix of Paeonia suffruticosa which has been used as traditional Chinese medicine. In this study, we primarily investigated the anti-inflammatory effects and the underlying mechanisms of paeonol in RAW macrophage cells; and based on these effects, we assessed the protective effects of paeonol on lipopolysaccharide-induced endotoxemia in mice. The in vitro study showed that paeonol regulated the production of TNF-?, IL-1?, IL-6, and IL-10 via inactivation of I?B?, ERK1/2, JNK, and p38 MAPK. In mouse model of lipopolysaccharide-induced endotoxemia, pro- and anti-inflammatory cytokines are significantly regulated, and thus the survival rates of lipolysaccharide-challenged mice are improved by paeonol (150, 200, or 250 mg/kg). Therefore, paeonol has a beneficial activity against lipopolysaccharide-induced inflammation in RAW 264.7 cell and mouse models. PMID:23413967

Chen, Na; Liu, Dianfeng; Soromou, Lanan Wassy; Sun, Jingjing; Zhong, Weiting; Guo, Weixiao; Huo, Meixia; Li, Hongyu; Guan, Shuang; Chen, Zhenwen; Feng, Haihua



Changes in lipopolysaccharide structure induce the sigma(E)-dependent response of Escherichia coli.  


The envelope of Escherichia coli is composed of an asymmetric lipid bilayer containing lipopolysaccharide, phospholipid and outer membrane proteins (OMPs). Physical and chemical stresses impact on the integrity of the outer membrane envelope and trigger the sigma(E)-dependent response, whereby E. coli activates the expression of genes that increase its capacity for folding OMPs and synthesizing lipopolysaccharide (LPS). While it has already been appreciated that misfolded OMPs induce the sigma(E) response, a role for LPS in activating this pathway was hitherto unknown. Here we show that ammonium metavandate (NH4VO3) induces multiple changes in E. coli LPS structure and activates the sigma(E)-dependent response without altering OMP. One such NH4VO3-mediated LPS decoration, the CrcA/PagP-catalysed addition of palmitate to lipid A, appeared to be alone sufficient to activate transcription at sigma(E)-dependent promoters. Furthermore, reduced acylation of LPS, caused by htrB or msbB mutations, also resulted in a constitutive expression of the sigma(E) regulon above wild-type levels. Production of these aberrant outer membrane lipids did not noticeably affect the composition or the amount of OMPs. A model is proposed whereby structural intermediates of the LPS biosynthetic pathway or modified LPS molecules may function as signals that activate the sigma(E) response. PMID:15720549

Tam, Christina; Missiakas, Dominique



Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide  

Microsoft Academic Search

Escherichia coli lipopolysaccharide (LPS)-induced nitrate biosynthesis was studied in LPS-sensitive C3H\\/He and LPS-resistant C3H\\/HeJ mice. Intraperitoneal injection of 15 of LPS led to a temporary 5- to 6-fold increase in blood nitrate concentration in the C3H\\/He strain. Levels of nitrate excreted in the urine were also increased. In contrast, no increase was observed in the C3H\\/HeJ strain with LPS

D. J. Stuehr; M. A. Marletta



Mucosal antibody responses of colonized cattle to Escherichia coli O157-secreted proteins, flagellin, outer membrane proteins and lipopolysaccharide.  


The aim of this work was to characterize adaptive mucosal immune responses to Escherichia coli O157:H7 at the principal site of colonization in the bovine species. Following experimental infection, extracts from terminal rectum mucosal samples were tested for IgA antibodies by immunoblotting against different bacterial antigens including: whole-cell E. coli O157:H7 with and without proteinase treatment, outer membrane and cytoplasmic preparations, secreted protein supernatants and purified E. coli O157 lipopolysaccharide and H7 flagellin. Lipopolysaccharide and H7 flagellin preparations were also used to coat enzyme-linked immunosorbent assay plates to determine mucosal IgG1 and IgA antibody titers. In this work, evidence is presented of strong local IgA immune responses induced following infection at the bovine terminal rectal mucosa directed against multiple antigens including type III secretion-dependent proteins, O157 lipopolysaccharide, H7 flagellin and OmpC. PMID:17995963

Nart, Pablo; Holden, Nicola; McAteer, Sean P; Wang, Dai; Flockhart, Allen F; Naylor, Stuart W; Low, J Christopher; Gally, David L; Huntley, John F



[Effect of pretreatment with radiation-detoxified endotoxin on endotoxin shock in dogs].  


The protective action of the radiation detoxified endotoxin preparation (Tolerin) on the endotoxin shock of dogs was studied. Endotoxin shock was induced by the intravenous administration of 2 mg/kg of Escherichia coli endotoxin. It was found that the intraperitoneal administration of 4 mg of radiation-detoxified endotoxin (Tolerin) 48 hours prior to the inducement of shock prevents the drop in blood pressure and the decrease of cardiac output which promptly appear in the acute phase of endotoxin shock. PMID:7345834

Balogh, A; Bertók, L; Juhász-Nagy, S



Interaction between endotoxin and human monocytes: characteristics of the binding of 3H-labeled lipopolysaccharide and 51Cr-labeled lipid A before and after the induction of endotoxin tolerance.  


Salmonella typhi endotoxin (lipopolysaccharide, LPS) was labeled with tritium and purified by gel filtration. Using this preparation, we found that binding of 3H-labeled LPS (3H-LPS) to isolated human monocytes consisted of a rapid (t1/2 less than 5 min), reversible, temperature-independent phase of surface adsorption that was followed by a slower (t1/2 greater than 20 min) period of binding that was irreversible and temperature-dependent. The interactions between 3H-LPS and monocytes that we measured were dependent both on the concentration of LPS and the cell number. We observed an apparent decrease in 3H-LPS surface binding after initial treatment of the cells with LPS, which was most likely due to an acquired reduction in the number of sites on the monocyte membrane available for the binding of LPS. Estimates of the parameters of the binding of 3H-LPS were calculated from a double-reciprocal plot (1/bound vs. 1/free) of the surface binding data and suggest that the relative binding affinity (Kd) for 3H-LPS was unchanged after pretreatment of the monocytes with LPS; however, the total number of LPS binding sites appeared to be reduced by this manipulation. The results of competition binding experiments also suggest that the binding affinity for 3H-LPS was the same before and after incubation of the cells with LPS. Lipid A, which we extracted from LPS and labeled with chromium-51, exhibited a binding affinity similar to that of 3H-LPS and, like 3H-LPS, could be displaced from the cells by competing concentrations of unfractionated LPS; however, the kinetics of binding of the two labeled ligands differed considerably. Our results suggest that exposure of monocytes to LPS may alter the ability of these cells to interact with, and consequently respond to, LPS. PMID:6587364

Larsen, N E; Sullivan, R



Preliminary Characterization of the Transcriptional Response of the Porcine Intestinal Cell Line IPEC-J2 to Enterotoxigenic Escherichia coli, Escherichia coli, and E. coli Lipopolysaccharide  

PubMed Central

IPEC-J2, a promising in vitro model system, is not well characterized especially on the transcriptional level, in contrast to human counterparts. The aim of this study was to characterize the gene expression in IPEC-J2 cells when coincubated with enterotoxigenic Escherichia coli (ETEC), nonpathogenic E. coli, and E. coli endotoxin. Apical infection of polarized IPEC-J2 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TEER). Microarray analysis showed up-regulation of interleukins when IPEC-J2 were cocultured with E. coli strains this has so far never been measured in this cell line. Highest IL8 expression was found with the ETEC strain possessing the F4 fimbrium, suggesting IPEC-J2 cells to be F4 receptor positive, confirmed in a brush border membrane adhesion assay. It is concluded that the innate immune responses to pathogens and LPS makes the IPEC-J2 cell line a suitable model for research on intestinal host pathogen interaction.

Geens, Marisa M.; Niewold, Theo A.



Monoclonal antibodies specific for Escherichia coli J5 lipopolysaccharide: cross-reaction with other gram-negative bacterial species.  

PubMed Central

Four monoclonal antibodies against Escherichia coli J5 were studied. Each of these monoclonal antibodies reacted with purified lipopolysaccharides from E. coli J5, the deep rough mutant Salmonella minnesota Re595, Agrobacterium tumefaciens, and Pseudomonas aeruginosa PAO1 as well as with the purified lipid A of P. aeruginosa. Enzyme-linked immunosorbent assays using the outer membranes from a variety of gram-negative bacteria demonstrated that these lipid A-specific monoclonal antibodies interacted with between 84 and 97% of the gram-negative bacterial species tested. One of the monoclonal antibodies, 5E4, was shown to interact with 34 of the 35 outer membrane or lipopolysaccharide antigens tested. Immunoenzymatic staining of Western electrophoretic blots of separated P. aeruginosa outer membrane components was used to demonstrate that antibody 5E4 interacted with a similar fast-migrating band, corresponding to rough lipopolysaccharide, from all 17 serotype strains and all 14 clinical isolates of P. aeruginosa. Similarly, iodinated goat anti-mouse immunoglobulin was used to detect the binding of monoclonal antibody 8A1 to a fast-migrating band on Western electrophoretic blots of purified lipopolysaccharides from Klebsiella pneumoniae and both smooth and rough strains of E. coli, Salmonella typhimurium, and S. minnesota. These results suggest considerable conservation of single antigenic sites in the lipid A of gram-negative bacteria. Images

Mutharia, L M; Crockford, G; Bogard, W C; Hancock, R E



Bladder instillation of Escherichia coli lipopolysaccharide alters the muscle contractions in rat urinary bladder via a protein kinase C-related pathway  

SciTech Connect

Uropathogenic Escherichia coli is a common cause of urinary tract infection. We determined the effects of intravesical instillation of E. coli lipopolysaccharide (LPS, endotoxin) on muscle contractions, protein kinase C (PKC) translocation, and inducible nitric oxide synthase (iNOS) expression in rat urinary bladder. The contractions of the isolated rat detrusor muscle evoked by electrical field stimulations were measured short-term (1 h) or long-term (24 h) after intravesical instillation of LPS. One hour after LPS intravesical instillation, bladder PKC-{alpha} translocation from cytosolic fraction to membrane fraction and endothelial (e)NOS protein was elevated, and detrusor muscle contractions were significantly increased. PKC inhibitors chelerythrine and Ro32-0432 inhibited this LPS-enhanced contractile response. Application of PKC activator {beta}-phorbol-12,13-dibutyrate enhanced the muscle contractions. Three hours after intravesical instillation of LPS, iNOS mRNA was detected in the bladder. Immunoblotting study also demonstrated that the induction of iNOS proteins is detected in bladder in which LPS was instilled. 24 h after intravesical instillation of LPS, PKC-{alpha} translocation was impaired in the bladder; LPS did not affect PKC-{delta} translocation. Muscle contractions were also decreased 24 h after LPS intravesical instillation. Aminoguanidine, a selective iNOS inhibitor, blocked the decrease in PKC-{alpha} translocation and detrusor contractions induced by LPS. These results indicate that there are different mechanisms involved in the alteration of urinary bladder contractions after short-term and long-term treatment of LPS; an iNOS-regulated PKC signaling may participate in causing the inhibition of muscle contractions in urinary bladder induced by long-term LPS treatment.

Weng, T.I. [Institute of Toxicology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, 10043, Taiwan (China); Department of Emergency Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Chen, W.J. [Department of Emergency Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Liu, S.H. [Institute of Toxicology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, 10043, Taiwan (China)]. E-mail:



Tumor necrosis factor-? and nitrite\\/nitrate responses during acute mastitis induced by Escherichia coli infection and endotoxin in dairy cows  

Microsoft Academic Search

Concentrations of tumor necrosis factor-? (TNF-?) and of NOx (sum of nitrite and nitrate as indicators of endogenous nitric oxide production) in milk and blood plasma were measured in three mastitis models in dairy cows in early lactation. Escherichia coli P4:037 bacteria or endotoxin 0111:B4 were administered into both left quarters of 12 and 6 cows, respectively. Six of the

J. W Blum; H Dosogne; D Hoeben; F Vangroenweghe; H. M Hammon; R. M Bruckmaier; C Burvenich



Pharmacokinetics of Tulathromycin in Healthy and Neutropenic Mice Challenged Intranasally with Lipopolysaccharide from Escherichia coli  

PubMed Central

Tulathromycin represents the first member of a novel subclass of macrolides, known as triamilides, approved to treat bovine and swine respiratory disease. The objectives of the present study were to assess the concentration-versus-time profile of tulathromycin in the plasma and lung tissue of healthy and neutropenic mice challenged intranasally with lipopolysaccharide (LPS) from Escherichia coli O111:B4. BALB/c mice were randomly allocated into four groups of 40 mice each: groups T-28 (tulathromycin at 28 mg/kg of body weight), T-7, T7-LPS, and T7-LPS-CP (cyclophosphamide). Mice in group T-28 were treated with tulathromycin at 28 mg/kg subcutaneously (s.c.) (time 0 h). The rest of the mice were treated with tulathromycin at 7 mg/kg s.c. (time 0 h). Animals in dose groups T-7-LPS and T7-LPS-CP received a single dose of E. coli LPS intranasally at ?7 h. Mice in group T7-LPS-CP were also rendered neutropenic with cyclophosphamide (150 mg/kg intraperitoneally) prior to the administration of tulathromycin. Blood and lung tissue samples were obtained from 5 mice from each dose group at each sampling time over 144 h after the administration of tulathromycin. There were not statistical differences in lung tissue concentrations among groups T-7, T-7-LPS, and T7-LPS-CP. For all dose groups, the distribution of tulathromycin in the lungs was rapid and persisted at relatively high levels during 6 days postadministration. The concentration-versus-time profile of tulathromycin in lung tissue was not influenced by the intranasal administration of E. coli LPS. The results suggest that in mice, neutrophils may not have a positive influence on tulathromycin accumulation in lung tissue when the drug is administered during either a neutrophilic or a neutropenic state.

Villarino, N.; Brown, S. A.



Differential expression of immunoregulatory genes in monocytes in response to Porphyromonas gingivalis and Escherichia coli lipopolysaccharide  

PubMed Central

Porphyromonas gingivalis lipopolysaccharide (LPS) (strain W50) interacts with Toll-like receptor 2 (TLR-2) leading to cytokine expression and inflammation, and thereby plays a key role in the pathogenesis of periodontal disease. The aims of this study were to investigate gene expression of key regulatory mediators of innate immune responses in a human monocytic cell line (THP-1) to P. gingivalis LPS and to compare these results with those obtained using the TLR-4 ligand, Escherichia coli LPS. Custom-made Taqman low-density arrays were used for expression profiling of 45 different cytokine-related genes. Both types of LPS highly up-regulated interleukin (IL)-1? and IL-1?, IL-18 receptor (IL-18R), IL-18R accessory protein and IL-1 family (IL-1F)9. Expression levels of IL-1F6, IL-1F7 and caspase-1 were unaltered by either LPS. Genes for tumour necrosis factor-?, IL-6, leukaemia inhibitory factor and IL-32 were also highly induced by both LPS. For a subset of genes, including CXC chemokine ligand 5 (CXCL5), expression was induced only by E. coli LPS or was up-regulated more highly by E. coli compared with P. gingivalis LPS in THP-1 monocytes. A similar expression pattern was also observed in dendritic cells. Analysis of signalling pathways which lead to CXCL5 expression indicated that the mechanisms underpinning the differential responses did not involve the recruitment of different adaptor proteins by TLR-2 and TLR-4, and therefore occur downstream of the receptor–adaptor complex. We conclude that differences in signalling pathways activated by TLR-2 and TLR-4 ligands lead to differential innate immune responses which may be important in polymicrobial diseases such as periodontal disease.

Barksby, H E; Nile, C J; Jaedicke, K M; Taylor, J J; Preshaw, P M



Differential expression of immunoregulatory genes in monocytes in response to Porphyromonas gingivalis and Escherichia coli lipopolysaccharide.  


Porphyromonas gingivalis lipopolysaccharide (LPS) (strain W50) interacts with Toll-like receptor 2 (TLR-2) leading to cytokine expression and inflammation, and thereby plays a key role in the pathogenesis of periodontal disease. The aims of this study were to investigate gene expression of key regulatory mediators of innate immune responses in a human monocytic cell line (THP-1) to P. gingivalis LPS and to compare these results with those obtained using the TLR-4 ligand, Escherichia coli LPS. Custom-made Taqman low-density arrays were used for expression profiling of 45 different cytokine-related genes. Both types of LPS highly up-regulated interleukin (IL)-1alpha and IL-1beta, IL-18 receptor (IL-18R), IL-18R accessory protein and IL-1 family (IL-1F)9. Expression levels of IL-1F6, IL-1F7 and caspase-1 were unaltered by either LPS. Genes for tumour necrosis factor-alpha, IL-6, leukaemia inhibitory factor and IL-32 were also highly induced by both LPS. For a subset of genes, including CXC chemokine ligand 5 (CXCL5), expression was induced only by E. coli LPS or was up-regulated more highly by E. coli compared with P. gingivalis LPS in THP-1 monocytes. A similar expression pattern was also observed in dendritic cells. Analysis of signalling pathways which lead to CXCL5 expression indicated that the mechanisms underpinning the differential responses did not involve the recruitment of different adaptor proteins by TLR-2 and TLR-4, and therefore occur downstream of the receptor-adaptor complex. We conclude that differences in signalling pathways activated by TLR-2 and TLR-4 ligands lead to differential innate immune responses which may be important in polymicrobial diseases such as periodontal disease. PMID:19438601

Barksby, H E; Nile, C J; Jaedicke, K M; Taylor, J J; Preshaw, P M



Crystallization of R-form lipopolysaccharides from Salmonella minnesota and Escherichia coli.  

PubMed Central

Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) and Escherichia coli K-12 LPS formed three-dimensional crystals, either hexagonal plates (preferential growth along the a axis) or solid columns (preferential growth along the c axis), when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2 at 4 degrees C for 10 days. Analyses of crystals suggested that they consist of hexagonal lattices with the a axis (a side of the lozenge as a unit cell on the basal plane) of 0.462 nm for all these three kinds of LPSs and the c axes (perpendicular to the basal plane) of 5.85, 8.47, and 8.75 nm for S. minnesota Re and Ra LPSs and E. coli K-12 LPS, respectively, and that hydrocarbon chains of the lipid A portion play the leading part in crystallization, whereas the hydrophilic part of the lipid A (the disaccharide backbone) and R core exhibit a disordered structure or are in a random orientation. The phenomenon of doubling of the a axis to 0.924 nm was observed with crystals of S. minnesota Re LPS when they were incubated in 70% ethanol for an additional 180 days, but not with crystals of S. minnesota Ra LPS or E. coli K-12 LPS. S. minnesota S-form LPS possessing the O-antigen-specific polysaccharide and S. minnesota free lipid A obtained by acid hydrolysis of Re LPS did not crystallize under the same experimental conditions. Images FIG. 1 FIG. 2 FIG. 3A-3B FIG. 3C FIG. 4

Kato, N; Ohta, M; Kido, N; Ito, H; Naito, S; Hasegawa, T; Watabe, T; Sasaki, K



Convergent synthesis of the tetrasaccharide repeating unit of the cell wall lipopolysaccharide of Escherichia coli O40  

PubMed Central

Summary A tetrasaccharide repeating unit corresponding to the cell-wall lipopolysaccharide of E. coli O40 was synthesized by using a convergent block glycosylation strategy. A disaccharide donor was coupled to a disaccharide acceptor by a stereoselective glycosylation. A 2-aminoethyl linker was chosen as the anomeric protecting group at the reducing end of the tetrasaccharide. All glycosylation steps are significantly high yielding and stereoselective.

Sau, Abhijit



Plasma alpha 1?acid glycoprotein concentration in broilers: Influence of age, sex and injection of escherichia coli lipopolysaccharide  

Microsoft Academic Search

1. The influence of age, sex and injection of Escherichia coli lipopolysaccharide (LPS) on the plasma concentration of alpha?1 acid glycoprotein (AGP) was determined in broilers using the single radial immunodiffusion method.2. Plasma AGP concentration increased in the 3 d after hatching, and then stabilised at 240 ± 33 ?g\\/ml up to 14 d of age.3. No sex?related differences in

K. Takahashi; N. Kaji; Y. Akiba; K. Tamura



Inhibition of lung phosphodiesterase improves responsiveness to inhaled nitric oxide in isolated-perfused lungs from rats challenged with endotoxin  

Microsoft Academic Search

Objectives: To investigate the ability of phosphodiesterase (PDE) selective inhibitors to improve responsiveness to inhaled nitric oxide (NO) in isolated-perfused lungs of rats pretreated with endotoxin\\/lipopolysaccharide (LPS). Design and setting: Prospective, controlled animal study in the animal research facility of a university hospital. Interventions: Sixteen hours after adult Sprague-Dawley rats were injected intraperitoneally with 0.4 mg\\/kg E. coli 0111:B4 LPS

Alexandra Holzmann; Claire Manktelow; Jörg Weimann; Kenneth D. Bloch; Warren M. Zapol



A reexamination of the O1 lipopolysaccharide antigen group of Escherichia coli.  

PubMed Central

A total of 64 Escherichia coli strains of the O1 serogroup were tested for the migration pattern of their lipopolysaccharides (LPS) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. O1:K1 and O1:K51 strains of the OMP5 outer membrane protein pattern possessed LPS with a doublet pattern (O1A1) or the lowermost band of the O1A1 doublet (O1A2). O1:K1 strains of the OMP9 pattern possessed LPS referred to as O1A, which corresponded to the uppermost band of the O1A1 doublet pattern. A few O1:K? strains possessed LPS of different migration patterns (O1B and O1C). O1A and O1A1 LPS were indistinguishable by chemical techniques, and both reacted with each of 10 different monoclonal antibodies tested. However, O1A1 had an additional epitope within the additional band in each doublet, as demonstrated by adsorption experiments with hyperimmune rabbit sera followed by Western blotting. Furthermore, purified polysaccharide from O1A bacteria was incapable of inhibition in enzyme-linked immunosorbent assays performed with O1A1 LPS as antigen and adsorbed, specific anti-O1A1 antibodies, whereas O1A1 polysaccharide inhibited this reaction. O1B and O1C LPS differed in all respects tested, including chemical composition, from O1A and O1A1 LPS. Images

Moll, A; Kusecek, B; Pluschke, G; Morelli, G; Kamke, M; Jann, B; Jann, K; Achtman, M



Absence of complement fixing antibodies against lipopolysaccharides from Escherichia coli in a subgroup of patients with Crohn's disease.  

PubMed Central

Complement fixing antibodies against different Escherichia coli lipopolysaccharides were determined in patients with Crohn's disease and in healthy individuals and compared with antitetanus toxoid antibodies. All healthy individuals had antilipopolysaccharide antibodies, 10 of 27 patients with Crohn's disease had no antibodies and six had rapidly changing antibody titres. These abnormalities were found in patients with disease in the colon, with arthropathy and fistula. Antilipid A was found at lower titres in Crohn's disease. Neither antitetanus toxoid antibodies, nor immunoglobulin concentrations were different in patients with or without antilipopolysaccharide antibodies. There was no evidence for circulating immune complexes in patients lacking antilipopolysaccharide antibodies. Certain subgroups of patients with Crohn's disease have altered antibody levels to typical enteral antigens which most likely can be explained by local antibody binding to lipopolysaccharides at inflammatory sites, or by changes in immunoregulation in this disease.

Zeitz, M; Hope, U; Wust, B; Galanos, C; Moller, B; Lawley, T J; Riecken, E O



Proteolytic and proteomic changes in milk at quarter level following infusion with Escherichia coli lipopolysaccharide.  


Mastitis is a major disease in dairy cattle, which causes significant economic losses due to decreased milk production, veterinary costs, and discarded milk. Escherichia coli is one of the most prevalent species of gram-negative bacteria that induce clinical mastitis. The objective of the present study was to characterize the proteolytic and proteomic changes in milk in response to infusion with lipopolysaccharide (LPS) at quarter level in a model mastitis system. One quarter of each of 2 cows was infused with 0.1 or 5 ?g of LPS. The somatic cell count of the infused quarters reached a peak 6 h after infusion to a greater extent in the cow infused with 5 ?g of LPS and changes in plasmin activity in milk differed between the 2 animals. Urea-polyacrylamide gel electrophoretograms of milk samples of the cow infused with 5 ?g of LPS obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of ?- and ?(s1)-casein during incubation of milk samples due to indigenous proteolytic activity. Two-dimensional gel electrophoretograms of milk at 0, 6, or 12 h after infusion with LPS showed hydrolysis of ?(s)-casein and ?-casein as well as the appearance of lower molecular weight products. Eleven fragments from proteolysis of the caseins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and, in addition, proteolysis patterns of casein by the indigenous bovine milk proteases plasmin and cathepsin D were studied in model studies using 2-dimensional gel electrophoretograms. Twelve hours after infusion, lower abundance markers of inflammation were identified, including serotransferrin, fibrinogen ? chain, protein S100 A12, and the antimicrobial polypeptide cathelicidin. PMID:22459814

Hinz, K; Larsen, L B; Wellnitz, O; Bruckmaier, R M; Kelly, A L



Replacement of Lipopolysaccharide with Free Lipid A Molecules in Escherichia coli Mutants Lacking All Core Sugars  

PubMed Central

Escherichia coli mutants deficient in 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo) biosynthesis are conditionally lethal, but their phenotypes are bypassed by certain suppressor mutations or by over-expression of MsbA, the inner membrane flippase for core-lipid A. These strains grow on broth with the tetra-acylated precursor lipid IVA replacing lipopolysaccharide (Meredith, T. C. et al. ACS Chem. Biol. 1, 33–42, 2006). Deletion of kdtA, which encodes the Kdo transferase, is possible under these conditions. We now show that lipid IVA reaches the outer surface of the outer membrane in these strains, as judged by its accessibility to the lipase PagL. On the assumption that MsbA is optimized to transport penta- or hexa-acylated lipid A, we over-expressed the lauroyl or the myristoyl transferase of lipid A biosynthesis, encoded by lpxL and lpxM respectively, and demonstrated that kdtA deletion mutants were also viable in this setting. Although E. coli LpxL is stimulated by the presence of the Kdo-disaccharide in its acceptor substrate, LpxL does slowly acylate lipid IVA. Over-expression of LpxL from a plasmid suppressed the lethality of kdtA deletions on nutrient broth at 30 or 37 °C without the need for MsbA over-production. These strains accumulated penta- and hexa-acylated free lipid A containing a secondary laurate chain, or a laurate and a myristate chain, respectively. Deletion of kdtA in strains over-expressing LpxM accumulated penta-acylated lipid A with a secondary myristate moiety. None of the strains lacking kdtA grew in the presence of bile salts at any temperature or on nutrient broth at 42 °C. Our findings show that the main function of Kdo is to provide the right substrates for the acyltransferases LpxL and LpxM, resulting in the synthesis of penta- and hexa-acylated lipid A, which is optimal for the MsbA flippase.

Reynolds, C. Michael; Raetz, Christian R. H.



Modulation of endotoxin-induced monokine release in human monocytes by lipid A partial structures that inhibit binding of 125I-lipopolysaccharide.  

PubMed Central

We have previously shown that the synthetic tetraacyl precursor Ia (compound 406, LA-14-PP, or lipid IVa) was not able to induce the production of tumor necrosis factor, interleukin-1, and interleukin-6 in human monocytes but strongly antagonized lipopolysaccharide (LPS)-induced formation of these monokines. This inhibition was detectable at the level of mRNA production. To achieve a better understanding of molecular basis of this inhibition, we investigated whether lipid A precursor Ia (LA-14-PP), Escherichia coli-type lipid A (LA-15-PP), Chromobacterium violaceum-type lipid A (LA-22-PP), and synthetic lipid A partial structures and analogs (LA-23-PP, LA-24-PP, and PE-4) were able to influence the binding of 125I-LPS to human monocytes and compared this inhibitory activity with the agonistic and antagonistic action in the induction of monokines in human monocytes. 125I-LPS (20 ng per well) was added to human monocytes in the presence or absence of unlabeled rough Re mutant-derived LPS (Re-LPS) or lipid A compounds, and specific LPS binding was determined after 7 h. This binding was found to be dependent on CD14 as shown by the use of an anti-CD14 monoclonal antibody. Compound LA-14-PP was found to inhibit the binding of 125I-LPS to the cells in a similar dose-response to that of unlabeled LPS. This shows that the inhibitory capacity on LPS binding does not correlate with the monokine-inducing capacity because Re-LPS is active in inducing tumor necrosis factor, interleukin-1, and interleukin-6, while LA-14-PP is not. The strong capacity of LA-14-PP to inhibit 125I-LPS binding, however, correlates with the strong inhibitory capacity of this compound on LPS-induced monokine production. Compounds LA-15-PP, LA-23-PP, and LA-24-PP were active in the inhibition of 125I-LPS binding but were 5- to 10-fold weaker than Re-LPS and LA-14-PP. Of all lipid A structures tested, compound LA-22-PP expressed the weakest inhibitory capacity on LPS binding. These compounds showed again that the activity of binding inhibition does not correlate with the monokine-inducing capacity. We assume that the inhibitory effects of lipid A partial structures on LPS-induced monokine production have their origin in a competitive inhibition between LPS and the lipid A partial structures for the same binding site on the cell membrane.

Ulmer, A J; Feist, W; Heine, H; Kirikae, T; Kirikae, F; Kusumoto, S; Kusama, T; Brade, H; Schade, U; Rietschel, E T



Recent advances in biosensor based endotoxin detection.  


Endotoxins also referred to as pyrogens are chemically lipopolysaccharides habitually found in food, environment and clinical products of bacterial origin and are unavoidable ubiquitous microbiological contaminants. Pernicious issues of its contamination result in high mortality and severe morbidities. Standard traditional techniques are slow and cumbersome, highlighting the pressing need for evoking agile endotoxin detection system. The early and prompt detection of endotoxin assumes prime importance in health care, pharmacological and biomedical sectors. The unparalleled recognition abilities of LAL biosensors perched with remarkable sensitivity, high stability and reproducibility have bestowed it with persistent reliability and their possible fabrication for commercial applicability. This review paper entails an overview of various trends in current techniques available and other possible alternatives in biosensor based endotoxin detection together with its classification, epidemiological aspects, thrust areas demanding endotoxin control, commercially available detection sensors and a revolutionary unprecedented approach narrating the influence of omics for endotoxin detection. PMID:23934306

Das, A P; Kumar, P S; Swain, S



Synthesis of the Tetrasaccharide Motif and Its Structural Analog Corresponding to the Lipopolysaccharide of Escherichia coli O75  

PubMed Central

Background Extraintestinal pathogenic E. coli are mostly responsible for a diverse spectrum of invasive human and animal infections leading to the urinary tract infections. Bacterial lipopolysaccharides are responsible for their pathogenicity and their interactions with host immune responses. In spite of several breakthroughs in the development of therapeutics to combat urinary tract infections and related diseases, the emergence of multidrug-resistant bacterial strains is a serious concern. Lipopolysaccharides are attractive targets for the development of long-term therapeutic agents to eradicate the infections. Since the natural sources cannot provide the required amount of oligosaccharides, development of chemical synthetic strategies for their synthesis is relevant to gain access to a reservoir of oligosaccharides and their close analogs. Methodology Two tetrasaccharide derivatives were synthesized from a single disaccharide intermediate. ?-d-mannoside moiety was prepared from ?-d-glucoside moiety following oxidation–reduction methodology. A [2+2] stereoselective block glycosylation strategy has been adopted for the preparation of tetrasaccharide derivative. ?-d-Glucosamine moiety was prepared from ?-d-mannosidic moiety following triflate formation at C-2 and SN2 substitution. A one-pot iterative glycosylation exploiting the orthogonal property of thioglycoside was carried out during the synthesis of tetrasaccharide analog. Results Synthesis of the tetrasaccharide motif (1) and its structural analog (2) corresponding to the lipopolysaccharide of Escherichia coli O75 was successfully achieved in excellent yield. Most of the reactions are clean and high yielding. Both compounds 1 and 2 were synthesized as their 4-methoxyphenyl glycoside, which can act as a temporary anomeric protecting group for further use of these tetrasaccharides in the preparation of glycoconjugates.

Sau, Abhijit; Misra, Anup Kumar



Anti-endotoxin vaccines  

PubMed Central

Gram-negative bacterial (GNB) infections are a leading cause of serious infections both in hospitals and the community. The mortality remains high despite potent antimicrobials and modern supportive care. In the last decade invasive GNB have become increasingly resistant to commonly used antibiotics, and attempts to intervene with novel biological therapies have been unsuccessful. Earlier studies with antibodies directed against a highly conserved core region in the GNB lipopolysaccharide (LPS, or endotoxin) suggested that this approach may have therapeutic benefit, and led to the development of a subunit vaccine that has progressed to phase 1 clinical testing. Since only a few serogroups of GNB cause bacteremia, O-specific vaccines had been developed, but these were not deployed because of the availability of other therapeutic options at the time. Given the likelihood that new antibiotics will not be soon available, the development of vaccines and antibodies directed against endotoxin, both O and core antigens, deserves a “second look”.

Cross, Alan S



Naturally occurring hypothermia is more advantageous than fever in severe forms of lipopolysaccharide- and Escherichia coli-induced systemic inflammation  

PubMed Central

The natural switch from fever to hypothermia observed in the most severe cases of systemic inflammation is a phenomenon that continues to puzzle clinicians and scientists. The present study was the first to evaluate in direct experiments how the development of hypothermia vs. fever during severe forms of systemic inflammation impacts the pathophysiology of this malady and mortality rates in rats. Following administration of bacterial lipopolysaccharide (LPS; 5 or 18 mg/kg) or of a clinical Escherichia coli isolate (5 × 109 or 1 × 1010 CFU/kg), hypothermia developed in rats exposed to a mildly cool environment, but not in rats exposed to a warm environment; only fever was revealed in the warm environment. Development of hypothermia instead of fever suppressed endotoxemia in E. coli-infected rats, but not in LPS-injected rats. The infiltration of the lungs by neutrophils was similarly suppressed in E. coli-infected rats of the hypothermic group. These potentially beneficial effects came with costs, as hypothermia increased bacterial burden in the liver. Furthermore, the hypotensive responses to LPS or E. coli were exaggerated in rats of the hypothermic group. This exaggeration, however, occurred independently of changes in inflammatory cytokines and prostaglandins. Despite possible costs, development of hypothermia lessened abdominal organ dysfunction and reduced overall mortality rates in both the E. coli and LPS models. By demonstrating that naturally occurring hypothermia is more advantageous than fever in severe forms of aseptic (LPS-induced) or septic (E. coli-induced) systemic inflammation, this study provides new grounds for the management of this deadly condition.

Liu, Elaine; Lewis, Kevin; Al-Saffar, Hiba; Krall, Catherine M.; Singh, Anju; Kulchitsky, Vladimir A.; Corrigan, Joshua J.; Simons, Christopher T.; Petersen, Scott R.; Musteata, Florin M.; Bakshi, Chandra S.; Romanovsky, Andrej A.; Sellati, Timothy J.



The presence of endotoxin in powdered infant formula milk and the influence of endotoxin and Enterobacter sakazakii on bacterial translocation in the infant rat  

Microsoft Academic Search

Lipopolysaccharide (LPS) is a heat stable endotoxin that persists during the processing of powdered infant formula milk (IFM). Upon ingestion it may increase the permeability of the neonatal intestinal epithelium and consequently bacterial translocation from the gut. To determine the level of endotoxin present in IFM, 75 samples were collected from seven countries (representing 31 brands) and analysed for endotoxin

Stacy Townsend; Juncal Caubilla Barron; Catherine Loc-Carrillo; Stephen Forsythe



Endotoxin-Induced Renal Failure  

Microsoft Academic Search

Endotoxin-induced hypotension and altered renal microcirculation could lead to tubular injury, particularly at the physiologically hypoxic outer medulla. We explored this hypothesis in isolated perfused kidneys and in vivo in rats subjected to endotoxemia. Rat kidneys were removed 15 min after endotoxin injection in vivo (from Escherichia coli 0127:B8, 1 mg\\/kg i.p.) and perfused with oxygenated medium supplemented with 20

Samuel N. Heyman; Seymour Rosen; David Darmon; Marina Goldfarb; Helena Bitz; Ahuva Shina; Mayer Brezis



Induction of interleukin (IL)-1beta and IL-8 mRNA expression in porcine macrophages by lipopolysaccharide from Serpulina hyodysenteriae.  

PubMed Central

Lipopolysaccharide (LPS) is a classic inducer of inflammatory cytokines and is a key virulence factor for most gram-negative pathogens. The effect of phenol-water (LPS) and butanol-water (endotoxin) extracts from Serpulina hyodysenteriae on inflammatory cytokine mRNA expression from porcine alveolar macrophages was investigated. The LPS and endotoxin extracts from S. hyodysenteriae induced a dose-dependent expression of interleukin 1beta (IL-1beta) and IL-8 which was weak compared with the responses induced by Escherichia coli LPS. In addition, the spirochetal extracts induced no detectable upregulation of mRNA expression for either IL-6 or tumor necrosis factor alpha.

Sacco, R E; Nibbelink, S K; Baarsch, M J; Murtaugh, M P; Wannemuehler, M J



Variable Airway Responsiveness to Inhaled Lipopolysaccharide  

Microsoft Academic Search

Individuals exposed to inhaled endotoxin (lipopolysaccharide (LPS)) can develop airway symptoma- tology and exacerbations of asthma. Moreover, among those occupationally exposed to organic dusts, the progression of airflow obstruction is related to the endotoxin concentration in the bioaero- sol. Not everyone exposed to high concentrations of LPS develops these problems. To determine whether individuals express a differential response to inhaled




The Escherichia coli Lpt Transenvelope Protein Complex for Lipopolysaccharide Export Is Assembled via Conserved Structurally Homologous Domains  

PubMed Central

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components.

Villa, Riccardo; Martorana, Alessandra M.; Okuda, Suguru; Gourlay, Louise J.; Nardini, Marco; Sperandeo, Paola; Deho, Gianni; Bolognesi, Martino; Kahne, Daniel




EPA Science Inventory

Field and laboratory studies were conducted to determine the distribution of algae and bacteria, and investigate sources of endotoxins (lipopolysaccharides) in drinking water. The field survey was performed on five drinking water systems located in Allegheny County, Pennsylvania ...


Obesity Increases Sensitivity to Endotoxin Liver Injury: Implications for the Pathogenesis of Steatohepatitis  

Microsoft Academic Search

Genetically obese fatty\\/fatty rats and obese\\/obese mice exhibit increased sensitivity to endotoxin hepatotoxicity, quickly developing steatohepatitis after exposure to low doses of lipopolysaccharide (LPS). Among obese animals, females are more sensitive to endotoxin liver injury than males. LPS induction of tumor necrosis factor alpha (TNFalpha ), the proven affecter of endotoxin liver injury, is no greater in the livers, white

Shi Qi Yang; Hui Zhi Lin; Mark Clemens; Anna Mae Diehl



Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin  

Microsoft Academic Search

Vertebrates achieve internal homeostasis during infection or injury by balancing the activities of proinflammatory and anti-inflammatory pathways. Endotoxin (lipopolysaccharide), produced by all gram-negative bacteria, activates macrophages to release cytokines that are potentially lethal. The central nervous system regulates systemic inflammatory responses to endotoxin through humoral mechanisms. Activation of afferent vagus nerve fibres by endotoxin or cytokines stimulates hypothalamic-pituitary-adrenal anti-inflammatory responses.

Lyudmila V. Borovikova; Svetlana Ivanova; Minghuang Zhang; Huan Yang; Galina I. Botchkina; Linda R. Watkins; Haichao Wang; Naji Abumrad; John W. Eaton; Kevin J. Tracey



Isolation and Purification of Endotoxin by Hydrolytic Enzymes  

PubMed Central

Various commercial hydrolases were used in an attempt to degrade the endotoxic lipopolysaccharide macromolecule. Some inert components, such as peptides and nucleic acids, could be removed from endotoxin preparations. As a result, endotoxic activity, measured by pyrogenicity, Shwartzman reaction, and mouse lethality, was increased. The remarkable resistance of endotoxin to hydrolases led to the use of such enzymes for the liberation and purification of endotoxin from whole bacterial cells.

Lehrer, Samuel; Nowotny, Alois



Effects of bacterial endotoxins and their detoxified derivatives on serum and liver lipids in mice.  


The influence of different endotoxins (lipopolysaccharides, LPS) obtained from Serratia marcescens 08, Escherichia coli 089, and their derivatives, detoxified either by partial hydrolysis or irradiation, on serum and hepatic lipids and on serum lipase activity in C57Black mice was studied. Endotoxic LPS elevated the serum total lipids and lipoproteins, particularly the very-low-density lipoproteins, and induced a reversible accumulation of triglycerides in the liver. Since nontoxic preparations did not cause such alterations, it is assumed that the toxicity of LPS is an essential factor in causing lipid metabolism disorder. A nearly identical increase in the lipase activity was detected in 5 to 10 hr in the sera of experimental animals treated by both toxic and nontoxic preparations. Results indicated the potential advantage of using detoxified derivatives of bacterial endotoxins in human therapy. PMID:6474473

Gaál, D; Kremmer, T; Bálint, Z; Holczinger, L; Bertók, L; Nowotny, A



Genes for TDP-rhamnose synthesis affect the pattern of lipopolysaccharide heterogeneity in Escherichia coli K-12.  

PubMed Central

The rough lipopolysaccharide (LPS) of commonly used strains of Escherichia coli K-12 has two distinctly different band patterns when analyzed by high-resolution polyacrylamide gel electrophoresis. The LPS of ancestral strains such as W1485F- consists primarily of a single broad gel band. In contrast, the LPS of strains derived from strain Y10 such as AB1133 or C600 gives three sharp gel bands. Complementation studies using DNA fragments from the rfb gene cluster of Shigella dysenteriae 1 indicated that the difference between the two gel patterns is due to a mutation in the gene encoding the TDP-rhamnose synthetase, the final enzyme involved in TDP-rhamnose biosynthesis. This mutation arose during the construction of strain Y10, and not in strain 679-680 as previously thought. The requirement for the rfaS gene for synthesis of the broad major band seen in W1485F- LPS and the shift in gel migration of a component of this band when an rfaQ mutation was introduced indicated that this broad band contained the unique form of rough E. coli LPS which has been termed lipooligosaccharide. This finding indicates that lipooligosaccharide is likely to contain rhamnose and suggests a model in which one of the functions of partial substituents such as rhamnose may be to direct core synthesis into different pathways to produce alternative forms of LPS. Images

Klena, J D; Schnaitman, C A



Proteins required for lipopolysaccharide assembly in Escherichia coli form a trans-envelope complex†  

PubMed Central

The viability of Gram-negative organisms is dependent on the proper placement of lipopolysaccharide (LPS) in the outer leaflet of its outer membrane. LPS is synthesized inside the cell and transported to the surface by seven essential Lpt proteins. How these proteins cooperate to transport LPS is unknown. We show that these Lpt proteins can be found in a membrane fraction that contains inner and outer membranes, and that they co-purify. This constitutes the first evidence that the Lpt proteins form a trans-envelope complex. We suggest that this protein bridge provides a route for LPS transport across the cell envelope.

Chng, Shu-Sin; Gronenberg, Luisa S.; Kahne, Daniel



Endotoxin exposure during late pregnancy alters ovine offspring febrile and hypothalamic-pituitary-adrenal axis responsiveness later in life.  


A growing number of studies indicate that maternal infection during pregnancy is associated with adverse fetal development and neonatal health. In this study, late gestating sheep (day 135) were challenged systemically with saline (0.9%) or Escherichia coli lipopolysaccharide endotoxin (400 ng/kg x 3 consecutive days, or 1.2 microg/kg x 1 day) in order to assess the impact of maternal endotoxemia on the developing fetal neuroendocrine-immune system. During adulthood, cortisol secretion and febrile responses of female offspring and the cortisol response of the male offspring to endotoxin (400 ng/kg), as well as the female cortisol response to adrenocorticotropic hormone (ACTH) challenge, were measured to assess neuroendocrine-immune function. These studies revealed that maternal endotoxin treatment during late gestation altered the female febrile and male and female cortisol response to endotoxin exposure later in life; however, the response was dependent on the endotoxin treatment regime that the pregnant sheep received. The follow-up ACTH challenge suggests that programing of the adrenal gland may be altered in the female fetus during maternal endotoxemia. The long-term health implications of these changes warrant further investigation. PMID:20536335

Fisher, Rebecca E; Karrow, Niel A; Quinton, Margaret; Finegan, Esther J; Miller, Stephan P; Atkinson, Jim L; Boermans, Herman J



Differentiation Between Endotoxin and Non-Endotoxin Pyrogens in Human Albumin Solutions Using an Ex Vivo Whole Blood Culture Assay  

Microsoft Academic Search

Purified E. coli endotoxin, Gram negative bacteria and Gram positive bacteria induce IL-6 secretion by whole blood cultures (WBC's). Polymyxin B at concentrations greater than 2 U\\/ml completely inhibits IL-6 secretion caused by 10 EU\\/ml of endotoxin. Polymyxin B has no effect on IL-6 secretion by WBC's in the absence of endotoxin. the inhibition of endotoxin induced IL-6 secretion is

Edmund J. Pool; Gamieda Johaar; Sandra James; Ignatius Petersen; Patrick Bouic



Mediated effect of endotoxin and lead upon hepatic metabolism  

SciTech Connect

A test was made of the possibility that gram-negative bacterial cell wall lipopolysaccharides acted directly on key glucoregulatory enzymes in rat liver cytosol to cause the characteristic hypoglycemia of severe endotoxemia. Fasted male rats were sensitized to endotoxin by the simultaneous intravenous injection of lead acetate. The minimum systemic dosage of endotoxin necessary to perturb the normal pattern of hepatic glycolytic intermediates was determined by serial testing with diminishing dosages of endotoxin. The hepatocyte concentration of endotoxin was then calculated from this minimum dosage by use of literature data on the fraction of endotoxin delivered to liver cells after a systemic intravenous injection of radiochromium labeled lipopolysaccharides. Accepting a molecular weight of 118,000 daltons for the smallest endotoxin monomer capable of evoking a physiologic response, the molar amount of endotoxin present in 1 gram of hepatocytes was readily calculated. The concentration of glucoregulatory enzymes in parenchymal cells was then estimated from other literature sources. It was found that the amount of endotoxin in the hepatocytes was insufficient to combine directly with even 1 per cent of the quantity of a single key glucoregulatory enzyme in liver parenchyma. Since a one to one stoichiometric reaction between endotoxin and enzyme could not occur in the liver cytosol, a direct interaction mechanism between agonist and biocatalyst can be ruled out. It is concluded that bacterial endotoxin must act on hepatic glucoregulation by an indirect mechanism presumably based upon the release and operation of mediators.

Kuttner, R.E.; Ebata, T.; Schumer, W.



Endotoxin removal by radio frequency gas plasma (glow discharge)  

NASA Astrophysics Data System (ADS)

Contaminants remaining on implantable medical devices, even following sterilization, include dangerous fever-causing residues of the outer lipopolysaccharide-rich membranes of Gram-negative bacteria such as the common gut microorganism E. coli. The conventional method for endotoxin removal is by Food & Drug Administration (FDA)-recommended dry-heat depyrogenation at 250°C for at least 45 minutes, an excessively time-consuming high-temperature technique not suitable for low-melting or heat-distortable biomaterials. This investigation evaluated the mechanism by which E. coli endotoxin contamination can be eliminated from surfaces during ambient temperature single 3-minute to cumulative 15-minute exposures to radio-frequency glow discharge (RFGD)-generated residual room air plasmas activated at 0.1-0.2 torr in a 35MHz electrodeless chamber. The main analytical technique for retained pyrogenic bio-activity was the Kinetic Chromogenic Limulus Amebocyte Lysate (LAL) Assay, sufficiently sensitive to document compliance with FDA-required Endotoxin Unit (EU) titers less than 20 EU per medical device by optical detection of enzymatic color development corresponding to < 0.5 EU/ml in sterile water extracts of each device. The main analytical technique for identification of chemical compositions, amounts, and changes during sequential reference Endotoxin additions and subsequent RFGD-treatment removals from infrared (IR)-transparent germanium (Ge) prisms was Multiple Attenuated Internal Reflection (MAIR) infrared spectroscopy sensitive to even monolayer amounts of retained bio-contaminant. KimaxRTM 60 mm x 15 mm and 50mm x 15mm laboratory glass dishes and germanium internal reflection prisms were inoculated with E. coli bacterial endotoxin water suspensions at increments of 0.005, 0.05, 0.5, and 5 EU, and characterized by MAIR-IR spectroscopy of the dried residues on the Ge prisms and LAL Assay of sterile water extracts from both glass and Ge specimens. The Ge prism MAIR-IR measurements were repeated after employing 3-minute RFGD treatments sequentially for more than 10 cycles to observe removal of deposited matter that correlated with diminished EU titers. The results showed that 5 cycles, for a total exposure time of 15 minutes to low-temperature gas plasma, was sufficient to reduce endotoxin titers to below 0.05 EU/ml, and correlated with concurrent reduction of major endotoxin reference standard absorption bands at 3391 cm-1, 2887 cm-1, 1646 cm -1 1342 cm-1, and 1103 cm-1 to less than 0.05 Absorbance Units. Band depletion varied from 15% to 40% per 3-minute cycle of RFGD exposure, based on peak-to-peak analyses. In some cases, 100% of all applied biomass was removed within 5 sequential 3-minute RFGD cycles. The lipid ester absorption band expected at 1725 cm-1 was not detectable until after the first RFGD cycle, suggesting an unmasking of the actual bacterial endotoxin membrane induced within the gas plasma environment. Future work must determine the applicability of this low-temperature, quick depyrogenation process to medical devices of more complicated geometry than the flat surfaces tested here.

Poon, Angela


The potential of lipopolysaccharide as a real-time biomarker of bacterial contamination in marine bathing water.  


The use of total lipopolysaccharide (LPS) as a rapid biomarker for bacterial pollution was investigated at a bathing and surfing beach during the UK bathing season. The levels of faecal indicator bacteria Escherichia coli (E. coli), the Gram-positive enterococci, and organisms commonly associated with faecal material, such as total coliforms and Bacteroides, were culturally monitored over four months to include a period of heavy rainfall and concomitant pollution. Endotoxin measurement was performed using a kinetic Limulus Amebocyte Lysate (LAL) assay and found to correlate well with all indicators. Levels of LPS in excess of 50 Endotoxin Units (EU) mL(-1) were found to correlate with water that was unsuitable for bathing under the current European regulations. Increases in total LPS, mainly from Gram-negative indicator bacteria, are thus a potential real-time, qualitative method for testing bacterial quality of bathing waters. PMID:24642437

Sattar, Anas A; Jackson, Simon K; Bradley, Graham



Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane.  


Lipopolysaccharide (LPS) is the major glycolipid that is present in the outer membranes (OMs) of most Gram-negative bacteria. LPS molecules are assembled with divalent metal cations in the outer leaflet of the OM to form an impervious layer that prevents toxic compounds from entering the cell. For most Gram-negative bacteria, LPS is essential for growth. In Escherichia coli, eight essential proteins have been identified to function in the proper assembly of LPS following its biosynthesis. This assembly process involves release of LPS from the inner membrane (IM), transport across the periplasm, and insertion into the outer leaflet of the OM. Here, we describe the biochemical characterization of the two-protein complex consisting of LptD and LptE that is responsible for the assembly of LPS at the cell surface. We can overexpress and purify LptD and LptE as a stable complex in a 1:1 stoichiometry. LptD contains a soluble N-terminal domain and a C-terminal transmembrane domain. LptE stabilizes LptD by interacting strongly with the C-terminal domain of LptD. We also demonstrate that LptE binds LPS specifically and may serve as a substrate recognition site at the OM. PMID:20203010

Chng, Shu-Sin; Ruiz, Natividad; Chimalakonda, Gitanjali; Silhavy, Thomas J; Kahne, Daniel



High ambient temperature alleviates the inflammatory response and growth depression in pigs challenged with Escherichia coli lipopolysaccharide.  


Pig production has increased in hot climate countries over recent years, but the effect of exposure to high temperatures on the health status of farm animals has not been investigated thoroughly. It is not clear how the ambient temperature (Ta) might influence responses to inflammatory challenge in pigs. The aim of the present study was to evaluate the effects of high Ta on performance and physiological parameters of growing pigs, subjected to repeated administration of Escherichia coli lipopolysaccharide (LPS). Thirty-seven pigs, each fitted with a jugular catheter, were assigned to one of two Ta conditions: thermo-neutral (TN, 24?°C) or high (HT, 30?°C). After a 14-day adaptation period, and a 7-day measurement period, pigs were administered five repeated injections of LPS at 48?h intervals. Irrespective of Ta, the LPS challenge reduced feed consumption and increased plasma pro-inflammatory cytokines, haptoglobin and cortisol. However, the extent of these responses was greater in pigs at TN than HT. In both groups, plasma thyroxine and triiodothyronine concentrations decreased, following the first LPS injection and thereafter returned to baseline, which occurred faster at HT than at TN. Moreover, the LPS challenge decreased growth and feed efficiency in pigs kept at TN, which was not observed in pigs kept at HT. The results suggest a greater capacity of pigs to limit the physiological and metabolic disturbances caused by inflammatory challenge, when kept at HT, compared to TN. PMID:24792207

Campos, Paulo H R F; Merlot, Elodie; Damon, Marie; Noblet, Jean; Le Floc'h, Nathalie



Protective efficacy of combined administration of lipopolysaccharide of E. coli and chemical radioprotectors under conditions of prolonged irradiation.  


We investigated the protective effectiveness of the lipopolysaccharide of E. coli (LPS) in a combination with a mixture of chemical radioprotectors in female mice of the strain H at various radiation dose rates. LPS in a dose of 0.08 mg per kg of body mass was administered 1, 3, or 24 hours prior to irradiation, the radioprotective mixture (cystamine 90 mg X kg-1 + 5-methoxytryptamine 15 mg X kg-1) was administered 10 minutes before irradiation. Dose rates of 612 mGy X min-1 (irradiation time 10 to 15 minutes), 38 mGy X min-1 (3 to 4 hours), and 8.2 mGy X min-1 (27 to 29 hours) were used. The results showed that isolated administrations of LPS or of the radioprotective mixture increased the resistance of the mice against prolonged irradiation; the combined administration even enhanced the efficacy of the radioprotective action. However, this efficacy depended on the magnitude of the dose rate. At dose rates higher than 38 mGy X min-1 the effectiveness of the chemical protection prevailed, whereas at lower dose rates the biological and especially the combined protection became effective. We demonstrated a slight pyrogenic effect of LPS by measuring oxygen consumption and changes in some parameters of the hematopoiesis. PMID:6380000

Misustová, J; Fjodorov, B A; Hosek, B; Sikulová, J; Kautská, J



Interaction of Antimicrobial Peptide Temporin L with Lipopolysaccharide In Vitro and in Experimental Rat Models of Septic Shock Caused by Gram-Negative Bacteria†  

PubMed Central

Sepsis remains a major cause of morbidity and mortality in hospitalized patients, despite intense efforts to improve survival. The primary lead for septic shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the quest for compounds with antiendotoxin properties is actively pursued. We investigated the efficacy of the amphibian skin antimicrobial peptide temporin L in binding Escherichia coli LPS in vitro and counteracting its effects in vivo. Temporin L strongly bound to purified E. coli LPS and lipid A in vitro, as proven by fluorescent displacement assay, and readily penetrated into E. coli LPS monolayers. Furthermore, the killing activity of temporin L against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Antimicrobial assays showed that temporin L interacted synergistically with the clinically used ?-lactam antibiotics piperacillin and imipenem. Therefore, we characterized the activity of temporin L when combined with imipenem and piperacillin in the prevention of lethality in two rat models of septic shock, measuring bacterial growth in blood and intra-abdominal fluid, endotoxin and tumor necrosis factor alpha (TNF-?) concentrations in plasma, and lethality. With respect to controls and single-drug treatments, the simultaneous administration of temporin L and ?-lactams produced the highest antimicrobial activities and the strongest reduction in plasma endotoxin and TNF-? levels, resulting in the highest survival rates.

Giacometti, Andrea; Cirioni, Oscar; Ghiselli, Roberto; Mocchegiani, Federico; Orlando, Fiorenza; Silvestri, Carmela; Bozzi, Argante; Di Giulio, Antonio; Luzi, Carla; Mangoni, Maria Luisa; Barra, Donatella; Saba, Vittorio; Scalise, Giorgio; Rinaldi, Andrea C.



Structure and Functional Analysis of LptC, a Conserved Membrane Protein Involved in the Lipopolysaccharide Export Pathway in Escherichia coli*  

PubMed Central

LptC is a conserved bitopic inner membrane protein from Escherichia coli involved in the export of lipopolysaccharide from its site of synthesis in the cytoplasmic membrane to the outer membrane. LptC forms a complex with the ATP-binding cassette transporter, LptBFG, which is thought to facilitate the extraction of lipopolysaccharide from the inner membrane and release it into a translocation pathway that includes the putative periplasmic chaperone LptA. Cysteine modification experiments established that the catalytic domain of LptC is oriented toward the periplasm. The structure of the periplasmic domain is described at a resolution of 2.2-? from x-ray crystallographic data. The periplasmic domain of LptC consists of a twisted boat structure with two ?-sheets in apposition to each other. The ?-sheets contain seven and eight antiparallel ?-strands, respectively. This structure bears a high degree of resemblance to the crystal structure of LptA. Like LptA, LptC binds lipopolysaccharide in vitro. In vitro, LptA can displace lipopolysaccharide from LptC (but not vice versa), consistent with their locations and their proposed placement in a unidirectional export pathway.

Tran, An X.; Dong, Changjiang; Whitfield, Chris



Impact of endotoxin challenge in obese pigs.  


Studies exploring the influence of obesity on septic shock remain limited and controversial. Pigs were chosen as a clinically relevant species, resembling to humans in various functions. We hypothesize obesity may impair porcine acute endotoxic shock. Four groups of five "Yucatan" minipigs were studied: lean and obese control groups, lean lipopolysaccharide (LPS) group receiving Escherichia coli endotoxin (LPS) and obese LPS group receiving the same endotoxin dose. We measured hemodynamic and oxygenation parameters, skin microvascular blood flow at rest and during reactive hyperemia, von Willebrand factor, tumor necrosis factor ?, and interleukin 6. All measurements were performed at baseline and at 30, 60, 90, 150, and 300 min. Results were given as median with 25th to 75th interquartile range. Control groups remained stable during the study period. In LPS groups, administration of endotoxin resulted in a typical hypokinetic shock. In obese LPS group at 300 min, we observed a significant impairment of cardiac index (1.2 [1.06-1.45] vs. 1.7 [1.57-1.97] L/min per m, P = 0.008) compared with the lean LPS group; moreover, pulmonary hypertension (mean arterial pressure: 42 [39-47] vs. 32 [28-34] mmHg, P = 0.008), hypoxemia (partial pressure of oxygen: 216 [178-262] vs. 325 [285-414] mmHg, P = 0.02), and lactate levels (5.8 [4.2-6.8] vs. 3.9 [2.2-5.5] mmol/L, P = 0.04) were significantly higher compared with the lean LPS group. Throughout the study, rest flow and peak flow during reactive hyperemia were more decreased in the obese LPS group. Compared with the lean LPS group, tumor necrosis factor ? levels at 60 min (269 [178-428] vs. 126 [105-166] ng/mL, P = 0.03) and interleukin 6 levels at 300 min (101 [61-142] vs. 52 [36-64] ng/mL, P = 0.03) were significantly higher in the obese LPS group. In our model of endotoxic shock, obese pigs developed a more severe hemodynamic failure with pronounced microcirculatory dysfunction and proinflammatory response. PMID:24569508

Duburcq, Thibault; Hubert, Thomas; Saint-Léger, Pierre; Mangalaboyi, Jacques; Favory, Raphael; Gmyr, Valery; Quintane, Laurence; Tailleux, Anne; Staels, Bart; Tournoys, Antoine; Pattou, François; Jourdain, Mercé



Lipid X ameliorates pulmonary hypertension and protects sheep from death due to endotoxin.  

PubMed Central

Lipid X (2,3-diacylglucosamine-1-phosphate) is a novel monosaccharide precursor of lipid A that has some of the physiologic activities of endotoxin but little toxicity. To determine whether lipid X would interfere with the toxic effects of endotoxin, we pretreated sheep with either 100 or 200 micrograms of lipid X per kg of body weight and then challenged them with a potentially fatal dose of Escherichia coli endotoxin (20 micrograms/kg). Twenty-one sheep underwent pulmonary artery catheterization and were monitored for changes in pulmonary artery pressure, temperature, pH, partial O2 pressure, partial CO2 pressure, blood pressure, and cell counts over 7 h. Overall mortality for control animals was 37% versus 5.3% for pretreated animals. None of the 13 animals pretreated with 100 micrograms of lipid X per kg died. These differences in survival were significant (P less than 0.05). Animals pretreated with 100 micrograms of lipid X per kg had significantly lower pulmonary artery pressure during both phases 1 and 2 of endotoxin-induced pulmonary artery hypertension. A higher dose of lipid X, 200 micrograms/kg, produced pulmonary hypertension. Perhaps because lipid X is a subunit of lipid A, lipid X shows a partial pyrogenic effect while also decreasing the pyrogenic activity of complete lipopolysaccharide (LPS). Lipid X did not prevent endotoxin-induced neutropenia or moderate hypotension in response to LPS. Lipid X is a potential prototype compound for a new type of chemotherapy directed at blocking the harmful effects of LPS during bacterial septicemia.

Golenbock, D T; Will, J A; Raetz, C R; Proctor, R A



Surface expression of O-specific lipopolysaccharide in Escherichia coli requires the function of the TolA protein.  


We investigated the involvement of Tol proteins in the surface expression of lipopolysaccharide (LPS). tolQ, -R, -A and -B mutants of Escherichia coli K-12, which do not form a complete LPS-containing O antigen, were transformed with the O7+ cosmid pJHCV32. The tolA and tolQ mutants showed reduced O7 LPS expression compared with the respective isogenic parent strains. No changes in O7 LPS expression were found in the other tol mutants. The O7-deficient phenotype in the tolQ and tolA mutants was complemented with a plasmid encoding the tolQRA operon, but not with a similar plasmid containing a frameshift mutation inactivating tolA. Therefore, the reduction in O7 LPS was attributed to the lack of a functional tolA gene, caused either by a direct mutation of this gene or by a polar effect on tolA gene expression exerted by the tolQ mutation. Reduced surface expression of O7 LPS was not caused by changes in lipid A-core structure or downregulation of the O7 LPS promoter. However, an abnormal accumulation of radiolabelled mannose was detected in the plasma membrane. As mannose is a sugar unique to the O7 subunit, this result suggested the presence of accumulated O7 LPS biosynthesis intermediates. Attempts to construct a tolA mutant in the E. coli O7 wild-type strain VW187 were unsuccessful, suggesting that this mutation is lethal. In contrast, a polar tolQ mutation affecting tolA expression in VW187 caused slow growth rate and serum sensitivity in addition to reduced O7 LPS production. VW187 tolQ cells showed an elongated morphology and became permeable to the membrane-impermeable dye propidium iodide. All these phenotypes were corrected upon complementation with cloned tol genes but were not restored by complementation with the tolQRA operon containing the frameshift mutation in tolA. Our results demonstrate that the TolA protein plays a critical role in the surface expression of O antigen subunits by an as yet uncharacterized involvement in the processing of O antigen. PMID:11069653

Gaspar, J A; Thomas, J A; Marolda, C L; Valvano, M A



Expression of delta-endotoxin cryIA(c) gene of Bacillus thuringiensis in Escherichia coli and Streptomyces lividans.  


The cryIA(c) gene of Bacillus thuringiensis was isolated from plasmid pOS1000. In order to obtain a proper cloning site and open reading frame, some DNA sequences preceding the initiating codon of the gene were replaced by synthetic oligonucleotide sequences. The isolated cryIA(c) was cloned into E. coli expression vector pKK223-3, and production of CryIA(c) protein was detected after induction by IPTG. A recombinant plasmid, pHZ1256, was constructed by insertion of the cryIA(c) gene into Streptomyces vector pHZ1272. pHZ1256 was introduced into Streptomyces lividans, and the production of CryIA(c) protein was confirmed by Western blotting after thiostrepton induction. A bioassy experiment showed that the CryIA(c) protein produced by E. coli and S. lividans caused 93% and 57% mortality to Plutella xylostella, respectively. PMID:10196629

Yang, R; Hu, Z; Deng, Z; Li, J



Differential Regulation of Membrane CD14 Expression and Endotoxin-Tolerance in Alveolar Macrophages  

PubMed Central

SUMMARY CD14 is important in the clearance of bacterial pathogens from lungs. However, the mechanisms that regulate the expression of membrane CD14 (mCD14) on alveolar macrophages (AM) have not been studied in detail. This study examines the regulation of mCD14 on AM exposed to Escherichia coli in vivo and in vitro and explores the consequences of changes in mCD14 expression. The expression of mCD14 was decreased on AM exposed to E. coli in vivo and AM incubated with lipopolysaccharide (LPS) or E. coli in vitro. Polymyxin B abolished LPS effects but only partially blocked the effects of E. coli. Blockade of extracellular signal-regulated kinase pathways attenuated LPS and E. coli-induced decrease in mCD14 expression. Inhibition of proteases abrogated the LPS-induced decrease in mCD14 expression on AM and the release of sCD14 into the supernatants, but did not affect the response to E. coli. The production of TNF-? in response to a second challenge with Staphylococcus aureus or zymosan was decreased in AM following incubation with E. coli but not LPS. These studies show that distinct mechanisms regulate the expression of mCD14 and the induction of endotoxin-tolerance in AM and suggest that AM function is impaired at sites of bacterial infection.

Lin, Shu-Min; Frevert, Charles W.; Kajikawa, Osamu; Wurfel, Mark M.; Ballman, Kimberly; Mongovin, Stephen; Wong, Venus A.; Selk, Amy; Martin, Thomas R.



Both Group 4 Capsule and Lipopolysaccharide O-Antigen Contribute to Enteropathogenic Escherichia coli Resistance to Human ?-Defensin 5  

PubMed Central

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that colonize the small intestine and colon, respectively. To cause disease, these pathogens must overcome the action of different host antimicrobial peptides (AMPs) secreted into these distinct niches. We have shown previously that EHEC expresses high levels of the OmpT protease to inactivate the human cathelicidin LL-37, an AMP present in the colon. In this study, we investigate the mechanisms used by EPEC to resist human ?-defensin 5 (HD-5), the most abundant AMP in the small intestine. Quantitative PCR was used to measure transcript levels of various EPEC surface structures. High transcript levels of gfcA, a gene required for group 4 capsule (G4C) production, were observed in EPEC, but not in EHEC. The unencapsulated EPEC ?gfcA and EHEC wild-type strains were more susceptible to HD-5 than EPEC wild-type. Since the G4C is composed of the same sugar repeats as the lipopolysaccharide O-antigen, an -antigen ligase (waaL) deletion mutant was generated in EPEC to assess its role in HD-5 resistance. The ?waaL EPEC strain was more susceptible to HD-5 than both the wild-type and ?gfcA strains. The ?gfcA?waaL EPEC strain was not significantly more susceptible to HD-5 than the ?waaL strain, suggesting that the absence of -antigen influences G4C formation. To determine whether the G4C and -antigen interact with HD-5, total polysaccharide was purified from wild-type EPEC and added to the ?gfcA?waaL strain in the presence of HD-5. The addition of exogenous polysaccharide protected the susceptible strain against HD-5 killing in a dose-dependent manner, suggesting that HD-5 binds to the polysaccharides present on the surface of EPEC. Altogether, these findings indicate that EPEC relies on both the G4C and the -antigen to resist the bactericidal activity of HD-5.

Thomassin, Jenny-Lee; Lee, Mark J.; Brannon, John R.; Sheppard, Donald C.; Gruenheid, Samantha; Le Moual, Herve



Removal of Endotoxin during Purification of Poly(3-Hydroxybutyrate) from Gram-Negative Bacteria  

Microsoft Academic Search

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ral- stonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of



A Single Nucleotide Exchange in the wzy Gene Is Responsible for the Semirough O6 Lipopolysaccharide Phenotype and Serum Sensitivity of Escherichia coli Strain Nissle 1917  

PubMed Central

Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strain's LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (?) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (?). The wa? and wb? gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa? determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa? gene clusters. The DNA sequence of the wb? gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb?O6 (wb? from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli.

Grozdanov, Lubomir; Zahringer, Ulrich; Blum-Oehler, Gabriele; Brade, Lore; Henne, Anke; Knirel, Yuriy A.; Schombel, Ursula; Schulze, Jurgen; Sonnenborn, Ulrich; Gottschalk, Gerhard; Hacker, Jorg; Rietschel, Ernst T.; Dobrindt, Ulrich



Lipopolysaccharide-responder and nonresponder C3H mouse strains are equally susceptible to an induced Escherichia coli urinary tract infection.  

PubMed Central

Host defense against bacterial urinary tract infections (UTI) includes both inflammatory and immune responses to infecting bacteria. The cellular events leading up to local inflammation are thought to be under genetic control and initiated by lipopolysaccharides (LPS) of gram-negative bacteria such as Escherichia coli. It has been previously reported that mice which lack functional Lps genes are more susceptible to induced E. coli UTI than mice with normal mitogenic responses to LPS. In contrast to these findings, data in this report demonstrate that LPS-responder and nonresponder C3H mouse strains are equally susceptible to E. coli UTI. When C3H/OuJ (Lps(n)/Lps(n)) and C3H/HeJ (Lps(d)/Lps(d)) were intravesically inoculated with equal numbers of uropathogenic E. coli organisms, neither strain was able to effectively resolve the induced UTI. The inability of C3H/OuJ mice to combat the infection was not due to an impaired response to LPS, nor could defect in the local inflammatory response be identified. The results indicate that factors other than LPS responsiveness are also important in determining hose resistance to UTI.

Hopkins, W; Gendron-Fitzpatrick, A; McCarthy, D O; Haine, J E; Uehling, D T



Analysis of lipopolysaccharide biosynthesis in Salmonella typhimurium and Escherichia coli by using agents which specifically block incorporation of 3-deoxy-D-manno-octulosonate.  

PubMed Central

Antibacterial agents which specifically inhibit CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase activity were used to block the incorporation of 3-deoxy-D-manno-octulosonate (KDO) into lipopolysaccharide. Lipopolysaccharide synthesis ceased, molecules similar in structure to lipid A accumulated, and bacterial growth ceased following addition of such agents to cultures of Salmonella typhimurium and Escherichia coli. Although four major species of lipid A accumulated in S. typhimurium, their kinetics of accumulation were different. The least polar of the major species was IVA [O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-a lph a- D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl groups and bearing phosphates at positions 1 and 4'], a molecule previously isolated from bacteria containing a kdsA mutation (C. R. H. Raetz, S. Purcell, M. V. Meyer, N. Qureshi, and K. Takayama, J. Biol. Chem. 260:16080-16088, 1985). Species IVA accumulated first and to the greatest extent following addition of the inhibitor, with other more polar derivatives appearing only after IVA attained half its maximal level. In contrast, only two major species of precursor accumulated in E. coli following addition of the inhibitor. One of these species was identical to IVA from S. typhimurium on the basis of chemical composition, fast atom bombardment mass spectroscopy, and comigration on Silica Gel H, and it also accumulated prior to a more polar species of related structure. We conclude that the addition of KDO to precursor species IVA is the major pathway of lipid A-KDO formation in both S. typhimurium LT2 and E. coli and that accumulation of the more polar species lacking KDO only occurs in response to accumulation of species IVA following inhibition of the normal pathway.

Goldman, R C; Doran, C C; Capobianco, J O



Glucose Metabolism and Role of the Blood in Endotoxin Shock.  

National Technical Information Service (NTIS)

This in vitro study was conducted to explore influences modifying glucose uptake in canine blood administered an estimated LD sub 100 E. coli endotoxin. Particular emphasis was given to assay the role that leukocytes perform in glucose utilization. Result...

L. B. Hinshaw L. T. Archer B. K. Beller G. L. White T. M. Schroeder



Distribution of radiolabeled endotoxin with particular reference to the eye: concise communication  

SciTech Connect

A single systemic injection of endotoxin (lipopolysaccharide or LPS) reproducibly induces a cellular infiltrate in the uveal tract of the rat eye within 24 hr. Other organs are not comparably sensitive to systemic endotoxin. One hypothesis to explain this unique sensitivity is that endotoxin is preferentially bound by ocular tissue. Researchers tested this hypothesis by studying the distribution in the rat of intravenously injected endotoxin that had been radiolabeled with /sup 99m/Tc or /sup 32/P. With either radionuclide the concentration of endotoxin per gram of tissue at a variety of times after injection ranging from 5 min to 3 hr and 45 min, was markedly less in the eye than in liver, kidney, or spleen. A study with radiolabeled albumin indicated that these differences could not be ascribed solely to the organ's blood volume. They demonstrate, therefore, that the eye does not preferentially bind endotoxin, and they are compatible with the hypothesis that endotoxin's ocular effects are indirectly mediated.

Rosenbaum, J.T.; Hendricks, P.A.; Shively, J.E.; McDougall, I.R.



Functional Analysis of the Protein Machinery Required for Transport of Lipopolysaccharide to the Outer Membrane of Escherichia coli  

Microsoft Academic Search

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of trans- port and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of

Paola Sperandeo; Fion K. Lau; Andrea Carpentieri; Cristina De Castro; Antonio Molinaro; Gianni Deho; Thomas J. Silhavy; Alessandra Polissi



Effect of endotoxin on the angiotensin II receptor in cultured vascular smooth muscle cells.  

PubMed Central

1. In some tissues, a decrease in the number of cell surface receptors and alterations of the receptor coupling have been proposed as possible mechanisms mediating the deleterious effects of bacterial endotoxin in septic shock. 2. The effects of bacterial lipopolysaccharide (Escherichia coli 0111-B4; LPS) on vascular angiotensin II and vasopressin receptors have been examined in cultured aortic smooth muscle cells (SMC) of the rat by use of radioligand binding techniques. 3. In vascular SMC exposed to 1 micrograms ml-1 endotoxin for 24 h, a significant increase in angiotensin II binding was found. The change in [125I]-angiotensin II binding corresponded to an increase in the number of receptors whereas the affinity of the receptors was not affected by LPS. In contrast, no change in [3H]-vasopressin binding was observed. 4. The pharmacological characterization of angiotensin II binding sites in control and LPS-exposed cells demonstrated that LPS induced an increase in the AT1 subtype of the angiotensin II receptors. Receptor coupling as evaluated by measuring total inositol phosphates was not impaired by LPS. 5. The effect of LPS on the angiotensin II receptor was dose-, time- and protein-synthesis dependent and was associated with an increased expression of the receptor gene. 6. The ability of LPS to increase angiotensin II binding in cultured vascular SMC was independent of the endotoxin induction of NO-synthase. 7. These results suggest that, besides inducing factors such as cytokines and NO-synthase, endotoxin may enhance the expression of cell surface receptors. The surprising increase in angiotensin II binding in LPS exposed VSM cells may represent an attempt by the cells to compensate for the decreased vascular responsiveness. It may also result from a non-specific LPS-related induction of genes. Images Figure 2

Burnier, M.; Centeno, G.; Waeber, G.; Centeno, C.; Burki, E.



In vivo effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression in striped catfish (Pangasianodon hypophthalmus).  


Lipolysaccharide (LPS), a component of outer membrane protein of gram-negative bacteria, reportedly stimulates fish immune system. However, mechanisms driving this immunomodulatory effect are yet unknown. To determine effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression of striped catfish (Pangasianodon hypophthalmus), juvenile fish (20-25 g) were injected with 3, 15 or 45 mg E.coli LPS/kg and challenged with Edwardsiella ictaluri. Plasma cortisol and glucose were rather low and did not differ (p<0.05) among treatments. All LPS treatments differed regarding blood cell count and immune variables such as plasma and spleen lysozyme, complement activity and antibody titer, 3mg LPS/kg yielding best results; red blood cell count was not affected by LPS treatment. Accumulated mortalities after bacterial challenge were 23.4, 32.8, 37.7 and 52.5% for treatment 3, 15, 45 mg LPS/kg fish and control respectively. Proteomic analysis of peripheral blood mononuclear cells (PBMC) confirmed that LPS induced differentially over-expressed immune proteins such as complement component C3 and lysozyme C2 precursor. Regulation of other proteins such as Wap65, alpha-2 macroglobulin-3 and transferrin precursor was also demonstrated. Striped catfish injected with E.coli LPS enhanced innate immune responses. PMID:23207480

Hang, Bui Thi Bich; Milla, Sylvain; Gillardin, Virginie; Phuong, Nguyen Thanh; Kestemont, Patrick



Characterization of ovine hepatic gene expression profiles in response to Escherichia coli lipopolysaccharide using a bovine cDNA microarray  

Microsoft Academic Search

BACKGROUND: During systemic gram-negative bacterial infections, lipopolysaccharide (LPS) ligation to the hepatic Toll-like receptor-4 complex induces the production of hepatic acute phase proteins that are involved in the host response to infection and limit the associated inflammatory process. Identifying the genes that regulate this hepatic response to LPS in ruminants may provide insight into the pathogenesis of bacterial diseases and

Honghe Cao; Leah C Kabaroff; Qiumei You; Alexander Rodriguez; Herman J Boermans; Niel A Karrow



Dose-Response Relationship to Inhaled Endotoxin in Normal Subjects  

Microsoft Academic Search

Exposure to endotoxin and to its purified derivative lipopolysaccharide (LPS) is related to several oc- cupational pulmonary diseases and to severe domestic asthma. An inhalation of a given dose of pure LPS produces both a systemic and a bronchial inflammatory response. Information on the dose- response relationship to inhaled LPS in normal subjects is a prerequisite to define the safety




FtsH-Mediated Coordination of Lipopolysaccharide Biosynthesis in Escherichia coli Correlates with the Growth Rate and the Alarmone (p)ppGpp  

PubMed Central

The outer membrane is the first line of defense for Gram-negative bacteria and serves as a major barrier for antibiotics and other harmful substances. The biosynthesis of lipopolysaccharides (LPS), the essential component of the outer membrane, must be tightly controlled as both too much and too little LPS are toxic. In Escherichia coli, the cellular level of the key enzyme LpxC, which catalyzes the first committed step in LPS biosynthesis, is adjusted by proteolysis carried out by the essential and membrane-bound protease FtsH. Here, we demonstrate that LpxC is degraded in a growth rate-dependent manner with half-lives between 4 min and >2 h. According to the cellular demand for LPS biosynthesis, LpxC is degraded during slow growth but stabilized when cells grow rapidly. Disturbing the balance between LPS and phospholipid biosynthesis in favor of phospholipid production in an E. coli strain encoding a hyperactive FabZ protein abolishes growth rate dependency of LpxC proteolysis. Lack of the alternative sigma factor RpoS or inorganic polyphosphates, which are known to mediate growth rate-dependent gene regulation in E. coli, did not affect proteolysis of LpxC. In contrast, absence of RelA and SpoT, which synthesize the alarmone (p)ppGpp, deregulated LpxC degradation resulting in rapid proteolysis in fast-growing cells and stabilization during slow growth. Our data provide new insights into the essential control of LPS biosynthesis in E. coli.

Schakermann, Michael; Langklotz, Sina



FtsH-mediated coordination of lipopolysaccharide biosynthesis in Escherichia coli correlates with the growth rate and the alarmone (p)ppGpp.  


The outer membrane is the first line of defense for Gram-negative bacteria and serves as a major barrier for antibiotics and other harmful substances. The biosynthesis of lipopolysaccharides (LPS), the essential component of the outer membrane, must be tightly controlled as both too much and too little LPS are toxic. In Escherichia coli, the cellular level of the key enzyme LpxC, which catalyzes the first committed step in LPS biosynthesis, is adjusted by proteolysis carried out by the essential and membrane-bound protease FtsH. Here, we demonstrate that LpxC is degraded in a growth rate-dependent manner with half-lives between 4 min and >2 h. According to the cellular demand for LPS biosynthesis, LpxC is degraded during slow growth but stabilized when cells grow rapidly. Disturbing the balance between LPS and phospholipid biosynthesis in favor of phospholipid production in an E. coli strain encoding a hyperactive FabZ protein abolishes growth rate dependency of LpxC proteolysis. Lack of the alternative sigma factor RpoS or inorganic polyphosphates, which are known to mediate growth rate-dependent gene regulation in E. coli, did not affect proteolysis of LpxC. In contrast, absence of RelA and SpoT, which synthesize the alarmone (p)ppGpp, deregulated LpxC degradation resulting in rapid proteolysis in fast-growing cells and stabilization during slow growth. Our data provide new insights into the essential control of LPS biosynthesis in E. coli. PMID:23417489

Schäkermann, Michael; Langklotz, Sina; Narberhaus, Franz



[Evaluation of usefulness of the enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to lipopolysaccharides of VTEC strains in patients suspected for Escherichia coli O104:H4 infection in Poland].  


The aim of the study was to investigate the presence of antibodies to lipopolysaccharides obtained by modified Boivin's method from E. coli serotype O104:H4 and O26, O103, O111, O121, O145, O157 in sera of 7 patients with acute diarrhea, suspected in clinical investigation for infection caused by E. coli O104:H4. Additionally, to determine the cut-off levels, the 75 sera from blood donors were tested. The high level of antibodies to LPS E. coli O104 was diagnosed in three patients from family outbreak caused by E. coli serotype O104:H4. In one of those patients, 7-years boy with HUS, we observed also a significant decrease of level of IgA, IgG and IgM antibodies in serum sample obtained in chronic phase of the disease. Furthermore, we showed that two others patients with clinical evidence of VTEC infection, not connected with this family outbreak, had a high level of antibodies to E. coli of other serotypes: one to O157 and one to O103. We did not observe presence of antibodies to LPS from E. coli O26, O111, O121 i O145 in the sera of tested patients. In conclusion, we confirmed that ELISA based on lipopolysaccharides obtained from different serotypes of E. coli may be helpful in laboratory diagnosis of infection caused by VTEC in humans. PMID:22384662

Rokosz, Natalia; Rastawicki, Waldemar; Jagielski, Marek



Influence of Sanitizers on the Lipopolysaccharide Toxicity of Escherichia coli Strains Cultivated in the Presence of Zygosaccharomyces bailii  

PubMed Central

The influence of sublethal concentrations of two sanitizers, liquid iodophor and liquid hypochlorite (LH), on the growth rates and toxicity of food-borne pathogenic Escherichia coli strains grown in the presence of spoilage yeast Zygosaccharomyces bailii was assessed. When grown in combination with Z. bailii both E. coli O113 and E. coli O26 exhibited slower growth rates, except when E. coli O113 was grown in combination with Z. bailii at 0.2% LH. The growth rate of Z. bailii was not impacted by the addition of the sanitizers or by communal growth with E. coli strains. LAL and IL-6 results indicated a decrease in toxicity of pure E. coli cultures with comparable profiles for control and sanitizer exposed samples, although the LAL assay proved to be more sensitive. Interestingly, pure cultures of Z. bailii showed increased toxicity measured by LAL and decreased toxicity measured by IL-6. LAL analysis showed a decrease in toxicity of both E. coli strains grown in combination with Z. bailii, while IL-6 analysis of the mixed cultures showed an increase in toxicity. The use of LAL for toxicity determination in a mixed culture overlooks the contribution made by spoilage yeast, thus demonstrating the importance of using the appropriate method for toxicity testing in mixed microbe environments.

Mogotsi, Lerato; De Smidt, Olga; Venter, Pierre; Groenewald, Willem



Isolation and characterization of murine monoclonal antibodies specific for gram-negative bacterial lipopolysaccharide: association of cross-genus reactivity with lipid A specificity.  

PubMed Central

Somatic cell hybrids secreting monoclonal antibodies against the core-glycolipid portion of enterobacterial endotoxin were derived from mice immunized with Escherichia coli J5 or Salmonella minnesota R595 heat-killed organisms or lipopolysaccharide (LPS). Eight antibodies were selected for their ability to cross-react with several members of a panel of gram-negative bacterial antigens in a radioimmunoassay. This panel represented five genera and two families of organisms: E. coli O111:B4, E. coli O55:B5, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella minnesota, and Serratia marcescens. The binding sites for six of the antibodies were unequivocally localized within the lipid A moiety of the endotoxin molecule by using the radioimmunoassay on LPS and free lipid A. The anti-lipid A antibodies were further characterized for their ability to interact with LPS variants by using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostaining procedure. The monoclonal antibodies bound almost exclusively to the low-molecular-weight species of LPS on the polyacrylamide gel. These components corresponded to LPS isolated from rough strains of organisms (strains which lack O-specific carbohydrate). These results suggested that the cross-reactive component of antisera raised against rough mutants of gram-negative bacteria contain antibodies of lipid A specificity. Moreover, the determinant within the lipid A moiety of LPS may have been accessible to the monoclonal antibodies only in those endotoxin molecules on the outer membrane surface which lack the O-specific carbohydrate. Images

Bogard, W C; Dunn, D L; Abernethy, K; Kilgarriff, C; Kung, P C



Effects of Lipopolysaccharide, Lipid A, Lipid X, and Phorbol Ester on Cultured Bovine Endothelial Cells,  

National Technical Information Service (NTIS)

In pursuing the mechanism of endotoxin action, we examined the effect of lipopolysaccharide (LPS) and its chemically defined components, lipid A and lipid X on cultured bovine endothelial cells. We report that LPS and lipid A caused detachment and altered...

S. L. Gartner D. G. Sieckmann Y. H. Kang L. P. Watson L. D. Homer



Distribution and Role of Lipopolysaccharide in the Pathogenesis of Acute Renal Proximal Tubule Injury.  

National Technical Information Service (NTIS)

Endotoxins (lipopolysaccharides; LPS) are known to cause multiple organ failure, including renal dysfunction. The present report elucidates LPS distribution and effect on renal proximal tubules in an attempt to gain a better understanding of the cellular ...

Y. H. Kang M. C. Falk T. B. Bentley C. H. Lee



Crystallization and preliminary X-ray diffraction studies of the lipopolysaccharide core biosynthetic enzyme ADP-L-glycero-D-mannoheptose 6-epimerase from Escherichia coli K-12.  


ADP-L-glycero-D-mannoheptose 6-epimerase is a 240 kDa NAD-dependent nucleotide diphosphosugar epimerase from Escherichia coli K12 which catalyzes the interconversion of ADP-D-glycero-D-mannoheptose and ADP-L-glycero-D-mannoheptose. ADP-L-glycero-D-mannoheptose is a required intermediate for lipopolysaccharide inner-core and outer-membrane biosynthesis in several genera of pathogenic and non-pathogenic Gram-negative bacteria. ADP-L-glycero-D-mannoheptose 6-epimerase was overexpressed in E. coli and purified to apparent homogeneity by chromatographic methods. Three crystal forms of the epimerase were obtained by a hanging-drop vapor-diffusion method. A native data set for crystal form III was collected in-house on a Rigaku R-AXIS-IIC image plate at 3.0 A resolution. The form III crystals belong to the monoclinic space group P21. The unit-cell parameters are a = 98.94, b = 110.53, c = 180.68 A and beta = 90.94 degrees. Our recent results show that these crystals diffract to 2.0 A resolution at the Cornell High Energy Synchrotron Source. The crystal probably contains six 40 kDa monomers per asymmetric unit, with a corresponding volume per protein mass (Vm) of 4.11 A3 Da-1 and a solvent fraction of 70%. PMID:10089470

Ding, L; Zhang, Y; Deacon, A M; Ealick, S E; Ni, Y; Sun, P; Coleman, W G



The waaL gene is involved in lipopolysaccharide synthesis and plays a role on the bacterial pathogenesis of avian pathogenic Escherichia coli.  


Avian pathogenic Escherichia coli (APEC) is a Gram-negative bacterium that causes avian colibacillosis, resulting in economically devastating to poultry industries worldwide. Lipopolysaccharide (LPS) has been identified as an important virulence factor of E. coli. The waaL gene encodes O-antigen ligase, which is responsible for attaching the O-antigen to lipid A-core oligosaccharide. In this study, a mutant strain ?waaL was constructed from APEC serotype 2 strain DE17. The mutant strain showed a decreased swimming motility and resistance to complement-mediated killing but a similar growth rate in the culture, compared with its parent strain. In addition, the mutant LPS demonstrated different patterns in SDS-PAGE followed by silver staining and western blotting. Besides, the mutant strain significantly decreased its adherence and invasion abilities to DF-1 cells, compared to its parent strain DE17. Deletion of the waaL gene in DE17 reduced the bacterial virulence by 42.2-fold in ducklings, based on measurement of the median lethal dose (LD50). Additional analysis indicated that deletion of the waaL gene increased the biofilm formation ability and reduced the resistance to environmental stress. These results suggest that the waaL gene functions on the APEC LPS synthesis and bacterial pathogenesis. PMID:24970366

Han, Yue; Han, Xiangan; Wang, Shaohui; Meng, Qingmei; Zhang, Yuxi; Ding, Chan; Yu, Shengqing



Effect of an irradiated Escherichia coli endotoxin reparation on the sensitivity to a lymphotropic cytostatic agent in germfree and conventional mice.  


A 18 mg/kg dose of dianhydrodulcitol, a lymphotropic cytostatic agent produced the same death rate among germfree as a 12 mg/kg dose did in conventional mice. Pretreatment with the same dose of an irradiated immunomodulatory endotoxin preparation had increased the sensitivity to these dianhydrodulcitol doses in the same degree in germfree as in conventional mice. A study of the lymphoid organs and the intestinal wall indicate that both in germfree and conventional mice the dianhydrodulcitol sensitivity increasing effect of the endotoxin preparation was due to its stimulation of the lymphoid system. The higher resistance of germfree mice to dianhydrodulcitol is ascribed to their lack of a normal intestinal flora. PMID:6372357

Anderlik, P; Szeri, I; Bános, Z; Wessely, M; Bertók, L; Radnai, B



Gram-Negative Bacterial Lipopolysaccharide Retention by a Positively Charged New-Generation Filter  

Microsoft Academic Search

Endotoxins are lipopolysaccharides (LPS) which constitute the main component of the outer membranes of gram-negative bacteria. Endotoxins at high doses are pathogenic molecules which act as potent activators of the immune system. Mono- cytes and macrophages, following LPS stimulation, release me- diators with powerful biological and pyrogenic activities. More- over, LPS interact with the vascular endothelium and stimulate the complement

Ilaria Bononi; Veronica Balatti; Soccorso Gaeta; Mauro Tognon



A probiotic strain of Escherichia coli, Nissle 1917, given orally exerts local and systemic anti-inflammatory effects in lipopolysaccharide-induced sepsis in mice  

PubMed Central

Background and purpose: Escherichia coli Nissle 1917 is a probiotic strain used in the treatment of intestinal immune diseases, including ulcerative colitis. The aim of the present study was to test if this probiotic bacterium can also show systemic immunomodulatory properties after oral administration. Experimental approach: The probiotic strain was administered to rats or mice for 2 weeks before its assay in two experimental models of altered immune response, the trinitrobenzenesulphonic acid (TNBS) model of rat colitis, localized in the colon, and the lipopolysaccharide (LPS) model of systemic septic shock in mice. Inflammatory status was evaluated both macroscopically and biochemically after 1 week in the TNBS model or after 24 h in the LPS shock model. In addition, splenocytes were obtained from mice and stimulated, ex vivo, with concanavalin A or LPS to activate T or B cells, respectively, and cytokine production (IL-2, IL-5 and IL-10) by T cells and IgG secretion by B cells measured. Key results: E. coli Nissle 1917 was anti-inflammatory in both models of altered immune response. This included a reduction in the pro-inflammatory cytokine tumour necrosis factor-? both in the intestine from colitic rats, and in plasma and lungs in mice treated with LPS. The systemic beneficial effect was associated with inhibited production of the T cell cytokines and by down-regulation of IgG release from splenocyte-derived B cells. Conclusions and implications: The anti-inflammatory effects of E. coli Nissle 1917 given orally were not restricted to the gastrointestinal tract.

Arribas, B; Rodriguez-Cabezas, ME; Camuesco, D; Comalada, M; Bailon, E; Utrilla, P; Nieto, A; Concha, A; Zarzuelo, A; Galvez, J



Endotoxin removal: history of a mission.  


One of the key molecules involved in the pathogenesis of severe sepsis and septic shock is lipopolysaccharide (LPS) or endotoxin, which is a component of the cellular wall of Gram-negative bacteria. Clinical studies have shown that the level of circulating LPS is correlated with illness severity (APACHE II), the onset and amount of organ dysfunction (SOFA) and intensive care unit mortality. Many therapeutic strategies have attempted to neutralize the pathogenic activity of endotoxin in order to interrupt the progression of a septic state towards a worsened clinical framework, i.e. severe sepsis of septic shock. Over the past decades the role of extracorporeal hemoperfusion by means of polymyxin B-based cartridges (PMX-DHP) to bind and neutralize LPS from whole blood has increased in clinical relevance. This is due to an increasing number of studies confirming that a directed therapy of endotoxic shock could significantly influence the course of the septic cascade. This review will outline the meaning of the targeted approach to endotoxin, both highlighting the specific immunologic effect of endotoxin removal by polymyxin B and the evidence of clinical improvements following this kind of therapy in terms of recovery of organ function. PMID:24457488

Ronco, Claudio



The structure of the Escherichia coli O148 lipopolysaccharide core region and its linkage to the O-specific polysaccharide  

PubMed Central

Recently it was demonstrated that Shigella dysenteriae type 1, a cause of severe dysentery epidemics, gained its O-specific polysaccharide (O-SP) from Escherichia coli O148. The O-SPs of these bacteria differ only by a galactose residue in the repeat unit of S. dysenteriae type 1 in place of a glucose residue in E. coli O148. Here we analyzed the core structure and its linkage to the O-SP in E. coli O148 LPS. Both were found to be identical to those of S. dysenteriae type 1 structures, further supporting the relatedness of these two bacteria. The following structure of the core with one repeat unit of the O-SP has been assigned (all have d-configuration except l-Rha):

Kubler-Kielb, Joanna; Lai, Wen-Tzu; Schneerson, Rachel; Vinogradov, Evgeny



Intra-Amniotic Administration of E coli Lipopolysaccharides Causes Sustained Inflammation of the Fetal Skin in Sheep  

PubMed Central

Preterm birth is associated with in utero infection and inflammation. Although the fetal membranes and fetus contribute to the intra-amniotic inflammatory profile, the relationships between a proinflammatory exposure to the fetal compartment and cytokine expression in the fetal skin are unknown. Using an ovine model, we asked whether the fetal skin would generate an extended response to inflammatory stimuli. Relative to control, intra-amniotic lipopolysaccharide (LPS) induced significant increases in cytokine/chemokine (interleukin 1?, IL-8, tumor necrosis factor-?, and monocyte chemoattractant protein 1) expression in skin that lasted for at least 15 days. Histological analysis demonstrated inflammatory cell infiltration in skin between 2 days and 15 days post-LPS exposure. In contrast to the fetal lung, the fetal skin continues to express proinflammatory cytokines for at least 15 days after exposure to LPS. These novel data suggest that the fetal skin may cause prolonged in utero inflammatory response causally associated with preterm birth.

Saito, Masatoshi; Jobe, Alan; Kallapur, Suhas G.; Newnham, John P.; Cox, Thomas; Kramer, Boris; Yang, Huixia; Kemp, Matthew W.



Endotoxin-induced uveitis in rodents.  


Uveitis is a common cause of vision loss, accounting for 10-15 % of all cases of blindness worldwide and affects individuals of all ages, genders, and races. Uveitis represents a broad range of intraocular inflammatory conditions due to complications of autoimmune diseases, bacterial infections, viral infections, and chemical and metabolic injuries. Endotoxin-induced uveitis (EIU) in rodents is an efficient experimental model to investigate the pathological mechanism and pharmacological efficacy of potential drug agents. EIU is characterized by clinically relevant classical signs of inflammation, including inflammatory exudates and cells in the anterior and vitreous chambers. EIU in small animal models such as rats, mice, and rabbits is a short-lived uveal inflammation that can be developed subsequent to administration of bacterial endotoxin, such as lipopolysaccharide. Here, we present a reproducible, reliable, and simplified protocol to induce EIU in mice. This method could be used with similar efficacy for EIU induction in other small animals as well. PMID:23824898

Yadav, Umesh C S; Ramana, Kota V



Monoclonal Antibodies Reactive with K1-Encapsulated Escherichia coli Lipopolysaccharide Are Opsonic and Protect Mice against Lethal Challenge.  

National Technical Information Service (NTIS)

Seven murine monoclonal antibodies (MAbs) directed against 0-side-chain determinants of the K1-encapsulated Bortolussi strain of Escherichia coli (018:K1:H7) were evaluated for their in vitro and in vivo activities. All the MAbs reacted well in Western bl...

B. M. Kaufman A. S. Cross S. L. Futrovsky H. F. Sidberry J. C. Sadoff



Tritiation of endotoxin.  


Tritiated endotoxin was synthesized by three different methods: (1) sodium boro[3H]hydride reduction of native endotoxin; (2) sodium boro[3H]hydride reduction of endotoxin that had been oxidized previously with sodium metaperiodate; and (3) exposure of dry endotoxin to 3H2 gas. Sodium borohydride reduces aldehyde groups and sodium metaperiodate oxidizes vicinal glycol groups to aldehydes. Chromatographic analysis of the three tritiated endotoxins, using agarose, revealed that the biological activity associated with each labeled product appeared at the void volume, and in each case the biological activity coincided with a peak in radioactivity. The labeled product of the first method had a specific radioactivity of 0.18 mCi/g and a biological activity equal to that of native endotoxin. The labeled products of the second and third methods had specific activities of 2.1 mCi/g and 60.0 mCi/g, respectively, while their biological activities were one hundred-fold less than native endotoxin, as determined by the Limulus amebocyte lysate assay. These three labeled endotoxins are potentially ueled endotoxin. PMID:169908

Tomasulo, P A; March, S C; Levin



Class B scavenger receptors SR-BI/BII and CD36 mediate bacterial recognition and pro-inflammatory signaling induced by E. coli, lipopolysaccharide and cytosolic chaperonin 601  

PubMed Central

Class B scavenger receptors (SR-B3) are lipoprotein receptors which also mediate pathogen recognition, phagocytosis and clearance as well as pathogen-induced signaling. In this study we report that three members of the SR-B family namely, CLA-1, CLA-2 and CD36, mediate recognition of bacteria not only through interaction with cell wall lipopolysaccharide (LPS) but also with cytosolic chaperonin 60. HeLa cells stably transfected with any of these SR-Bs demonstrated markedly (3-5-fold) increased binding and endocytosis of E. coli, LPS and chaperonin 60 (GroEL) as revealed by both FACS analysis and confocal microscopy imaging. Increased pathogen (E. coli, LPS and GroEL) binding to SR-Bs was also associated with the dose-dependent stimulation of cytokine secretion in the order of CD36>CLA-2>CLA-1 in HEK293 cells. Pathogen-induced IL-6-secretion was reduced in macrophages from CD36- and SR-BI/II-null mice by 40-50% and 30-40%, respectively. Intravenous GroEL administration increased plasma IL-6 and CXCL1 levels in mice. The cytokine responses were 40-60% lower in CD36?/? relative to WT mice, whereas, increased cytokine responses were found in SR-BI/II?/? mice. While investigating the discrepancy of in vitro vs. in vivo data in SR-BI/II-deficiency, SR-BI/II?/? mice were found to respond to GroEL administration without increases in either plasma corticosterone or aldosterone as normally seen in WT mice. SR-BI/II?/? mice with mineralocorticoid replacement demonstrated a ~40-50% reduction in CXCL1 and IL-6 responses. These results demonstrate that, by recognizing and mediating inflammatory signaling of both bacterial cell wall LPS and cytosolic GroEL, all three SR-B family members play important roles in innate immunity and host defense.

Baranova, Irina N.; Vishnyakova, Tatyana G.; Bocharov, Alexander V.; Leelahavanichkul, Asada; Kurlander, Roger; Chen, Zhigang; Souza, Ana C. P.; Yuen, Peter S. T.; Star, Robert A.; Csako, Gyorgy; Patterson, Amy P.; Eggerman, Thomas L.



Relationships between metabolic changes and clinical signs in pregnant sheep given endotoxin.  

PubMed Central

Groups of four pregnant ewes were allocated to the following feeding and intravenous endotoxin treatments: fed, Escherichia coli endotoxin (50 micrograms/kg X 75), fed, saline, fasted, E. coli endotoxin (50 micrograms/kg X 75) and fasted, saline. Endotoxin administration resulted in depression, fever, hypoglycemia, hypocalcemia and a reduction in nonesterified fatty acid and ketone body concentrations. Depression correlated best with body temperature (r = 0.76), fasted sheep showed smaller increases in body temperature and were less depressed following endotoxin. Three of eight endotoxin treated sheep died, mortality was not related to rectal temperature but was associated with lactic acidosis. Hypoglycemia was not associated with either death or depression. Fed sheep that were unable to stand had lower serum calcium concentrations than standing sheep.

Naylor, J M; Kronfeld, D S



Genetic analysis of lipopolysaccharide core biosynthesis by Escherichia coli K-12: insertion mutagenesis of the rfa locus.  

PubMed Central

Tn10 insertions were selected on the basis of resistance to the lipopolysaccharide (LPS)-specific bacteriophage U3. The majority of these were located in a 2-kilobase region within the rfa locus, a gene cluster of about 18 kb that contains genes for LPS core biosynthesis. The rfa::Tn10 insertions all exhibited a deep rough phenotype that included hypersensitivity to hydrophobic antibiotics, a reduction in major outer membrane proteins, and production of truncated LPS. These mutations were complemented by a Clarke-Carbon plasmid known to complement rfa mutations of Salmonella typhimurium, and analysis of the insert from this plasmid showed that it contained genes for at least six polypeptides which appear to be arranged in the form of a complex operon. Defects in two of these genes were specifically implicated as the cause of the deep rough phenotype. One of these appeared to be rfaG, which encodes a function required for attachment of the first glucose residue to the heptose region of the core. The other gene did not appear to be directly involved in determination of the sugar composition of the core. We speculate that the product of this gene is involved in the attachment of phosphate or phosphorylethanolamine to the core and that it is the lack of one of these substituents which results in the deep rough phenotype. Images

Austin, E A; Graves, J F; Hite, L A; Parker, C T; Schnaitman, C A



Effects of pretreatment with SDZ MRL 953, a novel immunostimulatory lipid A analog, on endotoxin-induced acute lung injury in guinea pigs.  

PubMed Central

SDZ MRL 953 (SDZ), a novel immunostimulatory lipid A analog, has been reported to have immunopharmacological activities similar to those of lipopolysaccharide (LPS) but to have little of the toxicity of LPS. We investigated the effects of pretreatment with SDZ on Escherichia coli endotoxin-induced acute lung injury in guinea pigs. Four experimental groups consisted of saline control (n = 16), SDZ (-12 h) plus LPS (2 mg/kg of SDZ per kg of body weight injected intravenously 12 h before intravenous injection of 2 mg of LPS per kg; n = 15), SDZ (-10 min) plus LPS (SDZ injected 10 min before LPS injection; n = 10), and LPS alone (n = 16). The animals were sacrificed, and lung tissue was sampled 4 h after LPS or saline infusion. Lung injury was assessed by measuring the wet weight-to-dry weight ratio and the level of 125I-labeled albumin accumulation in bronchoalveolar lavage fluid relative to that in plasma. In the SDZ (-12 h) plus LPS group, these two parameters of acute lung injury were decreased compared with those in the LPS alone group. However, they were not decreased in the SDZ (-10 min) plus LPS group. We conclude that SDZ attenuates endotoxin-induced acute lung injury when it is administered 12 h before LPS injection. The attenuating effects of SDZ are speculated to be due to down regulation of the response to endotoxin rather than to receptor blocking.

Nakamura, H; Ishizaka, A; Urano, T; Sayama, K; Sakamaki, F; Terashima, T; Waki, Y; Soejima, K; Tasaka, S; Hasegawa, N



Removal of endotoxin from deionized water using micromachined silicon nanopore membranes  

Microsoft Academic Search

Endotoxins are lipopolysaccharide components of the cell membrane of Gram-negative bacteria that trigger the body's innate immune system and can cause shock and death. Water for medical therapy, including parenteral and dialysate solutions, must be free of endotoxin. This purity is challenging to achieve as many Gram-negative bacteria are endemic in the environment, and can thrive in harsh, nutrient-poor conditions.

Ross A. Smith; Ken Goldman; William H. Fissell; Aaron J. Fleischman; Christian A. Zorman; Shuvo Roy



Functional analysis of the protein machinery required for transport of lipopolysaccharide to the outer membrane of Escherichia coli.  


Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space. PMID:18424520

Sperandeo, Paola; Lau, Fion K; Carpentieri, Andrea; De Castro, Cristina; Molinaro, Antonio; Dehň, Gianni; Silhavy, Thomas J; Polissi, Alessandra



A model for the proteolytic regulation of LpxC in the lipopolysaccharide pathway of Escherichia coli.  


Lipopolysaccharide (LPS) is an essential structural component found in Gram-negative bacteria. The molecule is comprised of a highly conserved lipid A and a variable outer core consisting of various sugars. LPS plays important roles in membrane stability in the bacterial cell and is also a potent activator of the human immune system. Despite its obvious importance, little is understood regarding the regulation of the individual enzymes involved or the pathway as a whole. LpxA and LpxC catalyze the first two steps in the LPS pathway. The reaction catalyzed by LpxA possesses a highly unfavourable equilibrium constant with no evidence of coupling to an energetically favourable reaction. In our model the presence of the second enzyme LpxC was sufficient to abate this unfavourable reaction and confirming previous studies suggesting that this reaction is the first committed step in LPS synthesis. It is believed that the protease FtsH regulates LpxC activity via cleavage. It is also suspected that the activity of FtsH is regulated by a metabolite produced by the LPS pathway; however, it is not known which one. In order to investigate these mechanisms, we obtained kinetic parameters from literature and developed estimates for other simulation parameters. Our simulations suggest that under modest increases in LpxC activity, FtsH is able to regulate the rate of product formation. However, under extreme increases in LpxC activities such as over-expression or asymmetrical cell division then FtsH activation may not be sufficient to regulate this first stage of synthesis. PMID:23831517

Emiola, Akintunde; Falcarin, Paolo; Tocher, Joanne; George, John



Repeated oral administration of lipopolysaccharide from Escherichia coli 0111:B4 modulated humoral immune responses in periparturient dairy cows.  


The objective of this study was to evaluate the effects of repeated oral exposure to LPS on humoral immune responses of periparturient dairy cows. Sixteen Holstein cows were assigned to two treatment groups 2 wk before the expected day of parturition. Cows were administered orally, twice weekly at wk -2, -1 and +1 around parturition, with the following treatments: 3 ml saline; or 3 ml of saline containing LPS from Escherichia coli 0111:B4. The amount of LPS administered during wk -2, -1, and +1 was 0.01, 0.05, or 0.1 µg/kg body weight, respectively. Multiple blood samples were collected by jugular vein and various immune and clinical variables were measured. Results indicated that, on one hand, concentrations of plasma IgG anti-LPS Abs decreased (P??0.05) in the concentrations of serum amyloid A, LPS-binding protein, haptoglobin, cortisol, IgA anti-LPS Abs in the plasma, feed intake, body temperature and rumen contractions rate between the control and treatment groups. To our knowledge, this study is the first to show that repeated oral administration with LPS from E. coli 0111:B4 has the potential to stimulate humoral immune responses in periparturient dairy cows. PMID:22266418

Ametaj, Burim N; Sivaraman, Shanti; Dunn, Suzanna M; Zebeli, Qendrim



Binding of /sup 125/I-labeled endotoxin to bovine, canine, and equine platelets and endotoxin-induced agglutination of canine platelets  

SciTech Connect

Endotoxin from Escherichia coli O127:B8, Salmonella abortus-equi and S minnesota induced clumping of some canine platelets (PLT) at a final endotoxin concentration of 1 microgram/ml. Endotoxin-induced clumping of canine PLT was independent of PLT energy-requiring processes, because clumping was observed with canine PLT incubated with 2-deoxy-D-glucose and antimycin A. The PLT responded to adenosine diphosphate before, but not after, incubation with the metabolic inhibitors. Endotoxin induced a slight and inconsistant clumping of bovine and equine PLT at high (mg/ml) endotoxin concentration. High-affinity binding sites could not be demonstrated on canine, bovine, and equine PLT, using /sup 125/I-labeled E coli O127:B8 endotoxin. Nonspecific binding was observed and appeared to be due primarily to an extraneous coat on the PLT surface that was removed by gel filtration. The endotoxin that was bound to PLT did not appear to modify PLT function. An attempt to identify plasma proteins that bound physiologically relevant amounts of endotoxin was not successful. The significance of the endotoxin-induced clumping or lack of it on the pathophysiology of endotoxemia is discussed.

Meyers, K.M.; Boehme, M.; Inbar, O.



Endotoxin-Binding Affinity of Sevelamer Hydrochloride  

Microsoft Academic Search

Background: Sevelamer hydrochloride has been shown to attenuate circulating biomarkers of inflammation in patients with chronic kidney failure. We hypothesize that sevelamer hydrochloride binds bacterial endotoxin (ET) resulting in a decrease in ET levels and cytokine production. Methods: To assess the ET-binding affinity of sevelamer hydrochloride, purified Escherichia coli ET was incubated with sevelamer hydrochloride (0–50 mg\\/ml). After incubation, ET

Mary C. Perianayagam; Bertrand L. Jaber



Toxic interactions of benzyl alcohol with bacterial endotoxin.  

PubMed Central

Acute toxic interactions of intravenously administered benzyl alcohol and Escherichia coli O55:B5 (Boivin preparation) endotoxin were examined in rodents. Lethality studies in male CD-1 mice demonstrated that these agents were more toxic when administered in combination than when either was administered alone. Prophylactic treatment with diazepam (5 mg/kg intraperitoneally) protected against lethality induced by either the combination or the endotoxin yet offered little, if any, protection against the lethal effects of benzyl alcohol. Similar treatments with naloxone (5 mg/kg intraperitoneally) failed to protect against either endotoxin-induced or benzyl alcohol-induced lethality, but they significantly protected against the lethal effects of the combination. Although hexobarbital-induced sleeping time was prolonged in endotoxin-treated mice (but was normal in benzyl alcohol-treated mice), a more protracted effect on sleeping time was observed in mice treated with both benzyl alcohol and endotoxin. Moreover, male Wistar rats treated with benzyl alcohol (40 mg) showed no evidence of hepatic lesions, but rats treated in combination with sublethal doses of the alcohol (40 mg) and the endotoxin (0.4 mg) developed hepatic lesions which were severe than those observed in rats treated with endotoxin (0.4 mg) alone. A correlation between altered blood chemistry values and severity of hepatic lesions was demonstrated. These data show in vivo toxic interactions between benzyl alcohol and bacterial endotoxin. In addition, our results indicate that the toxic effects induced by the benzyl alcohol-endotoxin combination are due to an enhancement of the lethal properties of bacterial endotoxin. Images

Cebula, T A; El-Hage, A N; Ferrans, V J



Polaprezinc Protects Mice against Endotoxin Shock  

PubMed Central

Polaprezinc (PZ), a chelate compound consisting of zinc and l-carnosine (Car), is an anti-ulcer drug developed in Japan. In the present study, we investigated whether PZ suppresses mortality, pulmonary inflammation, and plasma nitric oxide (NO) and tumor necrosis factor (TNF)-? levels in endotoxin shock mice after peritoneal injection of lipopolysaccharide (LPS), and how PZ protects against LPS-induced endotoxin shock. PZ pretreatment inhibited the decrease in the survival rate of mice after LPS injection. PZ inhibited the increases in plasma NO as well as TNF-? after LPS. Compatibly, PZ suppressed LPS-induced inducible NO synthase mRNA transcription in the mouse lungs. PZ also improved LPS-induced lung injury. However, PZ did not enhance the induction of heat shock protein (HSP) 70 in the mouse lungs after LPS. Pretreatment of RAW264 cells with PZ suppressed the production of NO and TNF-? after LPS addition. This inhibition likely resulted from the inhibitory effect of PZ on LPS-mediated nuclear factor-?B (NF-?B) activation. Zinc sulfate, but not Car, suppressed NO production after LPS. These results indicate that PZ, in particular its zinc subcomponent, inhibits LPS-induced endotoxin shock via the inhibition of NF-?B activation and subsequent induction of proinflammatory products such as NO and TNF-?, but not HSP induction.

Ohata, Shuzo; Moriyama, Chihiro; Yamashita, Atsushi; Nishida, Tadashi; Kusumoto, Chiaki; Mochida, Shinsuke; Minami, Yukari; Nakada, Junya; Shomori, Kohei; Inagaki, Yoshimi; Ohta, Yoshiji; Matsura, Tatsuya



Effect of crude lipopolysaccharide from Escherichia coli O127:B8 on the amebocyte-producing organ of Biomphalaria glabrata (Mollusca).  


Lipopolysaccharide (LPS) is a pathogen associated molecular pattern (PAMP) to which the internal defense system (IDS) of both vertebrates and invertebrates responds. We measured the mitotic response of the hematopoietic tissue of the schistosome-transmitting snail, Biomphalaria glabrata, to crude LPS from Escherichia coli 0127:B8. In a dose-response study, snails were injected with a range of concentrations of crude LPS, and mitotic figures were enumerated in histological sections of amebocyte-producing organ (APO) fixed at 24h post-injection (PI) following a 6h treatment with 0.1% colchicine. In APOs from Salvador strain snails, which are genetically resistant to infection with Schistosoma mansoni, LPS concentrations of 0.01 mg/ml and above triggered a large increase in mitotic activity, whereas in APOs from schistosome-susceptible NIH albino snails, concentrations of 0.1mg/ml elicited a much smaller, but statistically significant increase. A time course study, without colchicine treatment, revealed that in Salvador APOs the mitotic response to 0.1mg/ml occurred by 18 h PI, peaked at 24h, and returned to control levels by 72 h; NIH albino APOs showed no detectible response. When Salvador APOs were exposed to crude LPS in vitro, no increase in mitotic activity occurred, a result suggesting the possible requirement for a peripheral tissue or hemolymph factor. The increased cell proliferation induced by crude LPS represents a novel systemic response of an invertebrate IDS to one or more PAMPs from a Gram-negative bacterium. PMID:21530581

Sullivan, John T; Bulman, Christina A; Salamat, Zahra



CD4+CD25+ regulatory T cells attenuate lipopolysaccharide-induced systemic inflammatory responses and promotes survival in murine Escherichia coli infection.  


It is well established that CD4CD25 regulatory T cells (Tregs) downregulate inflammatory immune responses and help to maintain immune homeostasis. Recent reports have shown that ligation of germline encoded pattern recognition receptors such as Toll-like receptors can stimulate Tregs and therefore implicate Tregs in the pathophysiology of sepsis and other inflammatory diseases. In this report, we show that injection of lipopolysaccharide (LPS) leads to expansion of CD4CD25FoxP3 Tregs, suggesting that these cells may play an important role in immune regulation in LPS-induced acute inflammation. Indeed, genetic or immunological inhibition of Treg function using mice lacking functional Tregs (CD25 KO mice) or anti-CD25 monoclonal antibody (anti-CD25 mAb), respectively, led to acute death in an otherwise nonlethal LPS challenge. This was accompanied by exaggerated production of proinflammatory cytokines. Strikingly, adoptive transfer of CD4CD25 Tregs to CD25 KO mice before LPS challenge rescues mice from death. Unlike LPS, depletion of Tregs followed by concanavalin A (Con A) challenge does not result in mortality, suggesting that Treg depletion does not globally influence all models of acute inflammation. We authenticate our findings by showing that depletion of Tregs leads to mortality in a nonlethal Escherichia coli challenge accompanied by elevated serum levels of proinflammatory cytokines. Collectively, our results indicate that in addition to regulation of LPS-induced acute inflammation, Tregs help to improve bacterial clearance and promote survival in an acute model of bacterial infection. PMID:23635849

Okeke, Emeka B; Okwor, Ifeoma; Mou, Zhirong; Jia, Ping; Uzonna, Jude E



Residues located inside the Escherichia coli FepE protein oligomer are essential for lipopolysaccharide O-antigen modal chain length regulation.  


The Escherichia coli O157?:?H7 FepE protein regulates lipopolysaccharide (LPS) O-antigen (Oag) chain length to confer a very long modal chain length of >80 Oag repeat units (RUs). The mechanism by which FepE regulates Oag modal chain length and the regions within it that are important for its function remain unclear. Studies on the structure of FepE show that the protein oligomerizes. However, the exact size of the oligomer is in dispute, further hampering our understanding of its mechanism. Guided by information previously obtained for regions known to be important for Oag modal chain length determination in the homologous Shigella flexneri WzzBSF protein, a set of FepE mutant constructs with single amino acid substitutions was created. Analysis of the resulting LPS conferred by these mutant His6-FepE proteins showed that amino acid substitutions of leucine 168 (L168) and aspartic acid 268 (D268) resulted in LPS with consistently shortened Oag chain lengths of <80 Oag RUs. Substitution of FepE's transmembrane cysteine residues did not affect function. Chemical cross-linking experiments on mutant FepE proteins showed no consistent correlation between oligomer size and functional activity, and MS analysis of FepE oligomers indicated that the in vivo size of FepE is consistent with a maximum size of a hexamer. Our findings suggest that different FepE residues, mainly located within the internal cavity of the oligomer, contribute to Oag modal chain length determination but not the oligomeric state of the protein. PMID:23393150

Tran, Elizabeth Ngoc Hoa; Morona, Renato



Core-Specific Receptors for Lipopolysaccharide on Hepatocytes,  

National Technical Information Service (NTIS)

The liver has been known to be the principal organ involved in the clearance of endotoxin (lipopolysaccharide, LPS) from the systemic circulation. The uptake of LPS by the liver is rapid, extensive and of apparent high affinity. The identity of the hepati...

J. B. Parent



Endotoxin-binding substances from human leukocytes and platelets.  

PubMed Central

We have found whole human platelets, granulocytes, and mononuclear leukocytes to possess high affinity for the toxic lipopolysaccharide from all gram-negative bacteria tested. We have extracted these cells and platelets with n-butanol-water; all endotoxin-binding activity resided in the organic phase. These endotoxin-binding extracts did not block serologically active groupings on endotoxins or receptors on the erythrocytes. The specificity of these still crude materials was less that that of the highly purified erythrocyte lipopolysaccharide receptor previously described by us, since they bound some bacterial antigens not related to endotoxins. Depending on source, the n-butanol extracts contained 40 to 52% glycerophosphatides (most active), 15 to 22% sphingomyelin, 17% cholesterol, less than 2 to 5% triglycerides, and 7 to 13% inactive peptide. The most active substances in the n-butanol extract were soluble in petroleum ether, whereas the peptide and sphingomyelin were not. Thus, no constituent protein, carbohydrate, or nucleic acid was present in the most highly active material. Polyacrylamide gel electrophoresis of the petroleum ether-soluble material showed for each extract one lipid band only, which was well defined and migrated similarly to phosphatidyllipids. Because of the lipidic nature of the inhibitory substances from leukocytes and platelets we also tested the lipid A component of bacterial endotoxins and some of its derivatives. Lipid A inhibited endotoxin coating of erythrocytes. De-O-acylation of lipid A left amide-linked 3-D-hydroxymyristic acid intact and increased the inhibitory activity of lipid A 20-fold. Complete de-O- and de-N-acylation destroyed its inhibitory effect.

Springer, G F; Adye, J C



Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin  

NASA Astrophysics Data System (ADS)

Vertebrates achieve internal homeostasis during infection or injury by balancing the activities of proinflammatory and anti-inflammatory pathways. Endotoxin (lipopolysaccharide), produced by all gram-negative bacteria, activates macrophages to release cytokines that are potentially lethal. The central nervous system regulates systemic inflammatory responses to endotoxin through humoral mechanisms. Activation of afferent vagus nerve fibres by endotoxin or cytokines stimulates hypothalamic-pituitary-adrenal anti-inflammatory responses. However, comparatively little is known about the role of efferent vagus nerve signalling in modulating inflammation. Here, we describe a previously unrecognized, parasympathetic anti-inflammatory pathway by which the brain modulates systemic inflammatory responses to endotoxin. Acetylcholine, the principle vagal neurotransmitter, significantly attenuated the release of cytokines (tumour necrosis factor (TNF), interleukin (IL)-1?, IL-6 and IL-18), but not the anti-inflammatory cytokine IL-10, in lipopolysaccharide-stimulated human macrophage cultures. Direct electrical stimulation of the peripheral vagus nerve in vivo during lethal endotoxaemia in rats inhibited TNF synthesis in liver, attenuated peak serum TNF amounts, and prevented the development of shock.

Borovikova, Lyudmila V.; Ivanova, Svetlana; Zhang, Minghuang; Yang, Huan; Botchkina, Galina I.; Watkins, Linda R.; Wang, Haichao; Abumrad, Naji; Eaton, John W.; Tracey, Kevin J.



Kinetics of Hydrothermal Inactivation of Endotoxins ?  

PubMed Central

A kinetic model was established for the inactivation of endotoxins in water at temperatures ranging from 210°C to 270°C and a pressure of 6.2 × 106 Pa. Data were generated using a bench scale continuous-flow reactor system to process feed water spiked with endotoxin standard (Escherichia coli O113:H10). Product water samples were collected and quantified by the Limulus amebocyte lysate assay. At 250°C, 5-log endotoxin inactivation was achieved in about 1 s of exposure, followed by a lower inactivation rate. This non-log-linear pattern is similar to reported trends in microbial survival curves. Predictions and parameters of several non-log-linear models are presented. In the fast-reaction zone (3- to 5-log reduction), the Arrhenius rate constant fits well at temperatures ranging from 120°C to 250°C on the basis of data from this work and the literature. Both biphasic and modified Weibull models are comparable to account for both the high and low rates of inactivation in terms of prediction accuracy and the number of parameters used. A unified representation of thermal resistance curves for a 3-log reduction and a 3 D value associated with endotoxin inactivation and microbial survival, respectively, is presented.

Li, Lixiong; Wilbur, Chris L.; Mintz, Kathryn L.



The importance of a lipopolysaccharide-initiated, cytokine-mediated host defense mechanism in mice against extraintestinally invasive Escherichia coli.  

PubMed Central

Extraintestinally invasive Escherichia coli (EC) that possess both a complete LPS and K1 capsule evade both complement-mediated bacteriolysis and neutrophil-mediated killing. Since C3H/HeJ mice that are hyporesponsive to LPS were uniquely susceptible to lethal infection with EC of this phenotype, we speculated there was an LPS-initiated host defense mechanism against this pathogenic phenotype. The LPS-normoresponsive C3H/HeN as well as the C3H/HeJ mice cleared these EC from the circulation within 4 h of intravenous administration. Whereas electron micrographs of the liver demonstrated these EC undergoing degeneration within the phagolysosomes of of both macrophages and Kupffer cells of C3H/HeN mice, these EC replicated within these cells of the C3H/HeJ mice. Restoration of anti-EC activity of C3H/HeJ mice occurred with activation of Kupffer cells and peritoneal macrophages in vivo with BCG and in vitro with IFN-gamma, but not with LPS. Pretreatment of C3H/HeJ mice with a combination of recombinant murine IL-1 and TNF-alpha also restored the killing of K1(+)-EC but did not enhance the killing of a K1(-)-EC mutant. These data are consistent with the hypothesis that (a) there is no intrinsic inability of C3H/HeJ phagocytes to kill EC, but (b) an LPS-initiated, cytokine-mediated host defense mechanism is required for such killing. These studies emphasize the importance of bacterial surface characteristics in the interaction with specific host defenses. Images

Cross, A; Asher, L; Seguin, M; Yuan, L; Kelly, N; Hammack, C; Sadoff, J; Gemski, P



IL18-deficient mice are resistant to endotoxin-induced liver injury but highly susceptible to endotoxin shock  

Microsoft Academic Search

IL-18 is an IL-1-related cytokine which shares biological functions with IL-12. These include the activation of NK cells, induction of IFN-g production and Th1 cell differentiation. In this study we analyzed the effect of IL-18 deficiency on lipopolysaccharide (LPS)-induced liver injury and endotoxin shock in Propionibacterium acnes-primed mice. P. acnes-primed IL-18-deficient (IL-18KO) mice showed resistance to LPS-induced liver injury. Unexpectedly,

Yoshimitsu Sakao; Kiyoshi Takeda; Hiroko Tsutsui; Tsuneyasu Kaisho; Fumiko Nomura; Haruki Okamura; Kenji Nakanishi; Shizuo Akira



Acute Inflammatory Response to Endotoxin in Mice and Humans  

PubMed Central

Endotoxin injection has been widely used to study the acute inflammatory response. In this study, we directly compared the inflammatory responses to endotoxin in mice and humans. Escherichia coli type O113 endotoxin was prepared under identical conditions, verified to be of equal biological potency, and used for both mice and humans. The dose of endotoxin needed to induce an interleukin-6 (IL-6) concentration in plasma of ?1,000 pg/ml 2 h after injection was 2 ng/kg of body weight in humans and 500 ng/kg in mice. Healthy adult volunteers were injected intravenously with endotoxin, and male C57BL/6 mice (n = 4 to 12) were injected intraperitoneally with endotoxin. Physiological, hematological, and cytokine responses were determined. Endotoxin induced a rapid physiological response in humans (fever, tachycardia, and slight hypotension) but not in mice. Both mice and humans exhibited lymphopenia with a nadir at 4 h and recovery by 24 h. The levels of tumor necrosis factor (TNF) and IL-6 in plasma peaked at 2 h and returned to baseline levels by 4 to 6 h. IL-1 receptor antagonist RA and TNF soluble receptor I were upregulated in both mice and humans but were upregulated more strongly in humans. Mice produced greater levels of CXC chemokines, and both mice and humans exhibited peak production at 2 h. These studies demonstrate that although differences exist and a higher endotoxin challenge is necessary in mice, there are several similarities in the inflammatory response to endotoxin in mice and humans.

Copeland, Shannon; Warren, H. Shaw; Lowry, Stephen F.; Calvano, Steve E.; Remick, Daniel



Differentiation between endotoxin and non-endotoxin pyrogens in human albumin solutions using an ex vivo whole blood culture assay.  


Purified E.coli endotoxin, Gram negative bacteria and Gram positive bacteria induce IL-6 secretion by whole blood cultures (WBC's). Polymyxin B at concentrations greater than 2 U/ml completely inhibits IL-6 secretion caused by 10 EU/ml of endotoxin. Polymyxin B has no effect on IL-6 secretion by WBC's in the absence of endotoxin. The inhibition of endotoxin induced IL-6 secretion is Polymyxin B concentration dependent at concentrations less than 1 U/ml. IL-6 induction caused by E.coli is only partially inactivated by 8 U/ml Polymyxin B. Polymyxin B has no effect on IL-6 secretion caused by B.subtilis. Two pyrogenic batches of human serum albumin (HSA), as tested by the rabbit assay for pyrogens, were also investigated. Polymyxin B at 4 U/ml inhibits less than 40 % of IL-6 secretion caused by these pyrogenic HSA batches. All the endotoxin activity in HSA samples spiked with purified endotoxin is inhibited by Polymyxin B indicating that HSA does not protect endotoxin against Polymyxin B inhibition. These results indicate that the pyrogenicity of these HSA batches are caused by Polymyxin B inhibitable and non-inhibitable fractions. This study shows that pyrogenic substances other than endotoxin can contaminate batches of pharmaceutical products and that results obtained using the Limulus Amoebocyte Lysate (LAL) assay does not necessarily indicate the pyrogenic status of pharmaceutical products. The WBC assay for pyrogens, having a broader sensitivity range than the LAL assay, is a better indicator of the pyrogenic status of pharmaceutical products. PMID:10225516

Pool, E J; Johaar, G; James, S; Petersen, I; Bouic, P



Effect of Plasmapheresis on the Immune System in Endotoxin-Induced Sepsis  

Microsoft Academic Search

Background: It has been proposed that plasmapheresis is most effective when applied early in Gram-negative sepsis. We therefore studied the effect of early plasmapheresis on immunity in experimental Escherichia coli endotoxin-induced sepsis. Methods: 20 pigs received 30 ?g\\/kg of E. coli endotoxin. 40 min later, half of the pigs were treated with plasmapheresis which lasted 4 h. The adhesion molecules,

P. Toft; R. Schmidt; A. C. Broechner; B. U. Nielsen; P. Bollen; K. E. Olsen



Paenibacterin, a novel broad-spectrum lipopeptide antibiotic, neutralises endotoxins and promotes survival in a murine model of Pseudomonas aeruginosa-induced sepsis.  


Paenibacterin, produced by Paenibacillus thiaminolyticus OSY-SE, is active both against Gram-negative and Gram-positive pathogens, including antibiotic-resistant strains of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus faecalis. Paenibacterin showed relatively low cytotoxicity against a human kidney cell line (ATCC CRL-2190), with a 50% inhibitory concentration (IC50)?109?g/mL. The cationic paenibacterin molecule binds to the negatively charged Gram-negative endotoxins in vitro, suggesting that paenibacterin can neutralise lipopolysaccharides. In a murine septic shock model, two 500?g doses of paenibacterin significantly increased the survival of mice challenged with a lethal level of P. aeruginosa. Considering that paenibacterin is effective against many strains of antibiotic-resistant pathogens, this study suggests that this antimicrobial agent is a promising candidate as a new drug. PMID:24802906

Huang, En; Yousef, Ahmed E



Influence of various dust sampling and extraction methods on the measurement of airborne endotoxin.  


The influence of various filter types and extraction conditions on the quantitation of airborne endotoxin with the Limulus amebocyte lysate test was studied by using airborne dusts sampled in a potato processing plant. Samples were collected with an apparatus designed to provide parallel samples. Data from the parallel-sampling experiment were statistically evaluated by using analysis of variance. In addition, the influence of storage conditions on the detectable endotoxin concentration was investigated by using commercially available lipopolysaccharides (LPS) and endotoxin-containing house dust extracts. The endotoxin extraction efficiency of 0.05% Tween 20 in pyrogen-free water was seven times higher than that of pyrogen-free water only. Two-times-greater amounts of endotoxin were extracted from glass fiber, Teflon, and polycarbonate filters than from cellulose ester filters. The temperature and shaking intensity during extraction were not related to the extraction efficiency. Repeated freeze (-20 degrees C)-and-thaw cycles with commercial LPS reconstituted in pyrogen-free water had a dramatic effect on the detectable endotoxin level. A 25% loss in endotoxin activity per freeze-thaw cycle was observed. Storage of LPS samples for a period of 1 year at 7 degrees C had no effect on the endotoxin level. House dust extracts showed a decrease of about 20% in the endotoxin level after they had been frozen and thawed for a second time. The use of different container materials (borosilicate glass, "soft" glass, and polypropylene) did not result in different endotoxin levels. This study indicates that the assessment of endotoxin exposure may differ considerably between groups when different sampling, extraction, and storage procedures are employed. PMID:7646014

Douwes, J; Versloot, P; Hollander, A; Heederik, D; Doekes, G



The Core Lipopolysaccharide of Escherichia coli Is a Ligand for the Dendritic-Cell-Specific Intercellular Adhesion Molecule Nonintegrin CD209 Receptor  

Microsoft Academic Search

The dendritic-cell-specific intercellular adhesion molecule nonintegrin (DC-SIGN) CD209 is a receptor for Escherichia coli K-12 that promotes bacterial adherence and phagocytosis. However, the ligand of E. coli for DC-SIGN has not yet been identified. In this study, we found that DC-SIGN did not mediate the phagocytosis of several pathogenic strains of E. coli, including enteropathogenic E. coli, enterohemorrhagic E. coli,

John Klena; Pei Zhang; Olivier Schwartz; Sheila Hull; Tie Chen




EPA Science Inventory

Groups of male CBA/J mice were injected with Salmonella typhimurium lipopolysaccharide (LPS) and irradiated with 2450 MHz (CW) microwaves. High ambient temperature (37C) also potentiated the lethal effect of endotoxin. Microwave irradiation prior to LPS injection, however, did no...


Endotoxin and Cytokines Increase Hepatic Sphingolipid Biosynthesis and Produce Lipoproteins Enriched in Ceramides and Sphingomyelin  

Microsoft Academic Search

Alterations in triglyceride and cholesterol metabolism often accompany inflammatory diseases and infections. We studied the effects of endotoxin (lipopolysaccharide (LPS)) and cytokines on hepatic sphingolipid synthesis, activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid synthesis, and lipoprotein sphingolipid content in Syrian hamsters. Administration of LPS induced a 2-fold increase in hepatic SPT activity. The increase in

Riaz A. Memon; Walter M. Holleran; Arthur H. Moser; Taisuke Seki; Yoshikazu Uchida; John Fuller; Judy K. Shigenaga; Carl Grunfeld; Kenneth R. Feingold


Increased systemic and brain cytokine production and neuroinflammation by endotoxin following ethanol treatment  

Microsoft Academic Search

BACKGROUND: Cytokines and alcohol share a common modulation of inflammation and hormones as well as being implicated in multiple diseases, but the mechanisms are poorly understood. The purpose of this study was to investigate the interaction of liver, serum and brain cytokines as well as whether ethanol would potentiate endotoxin (Lipopolysaccharide, LPS) responses once ethanol had cleared. METHODS: Male C57BL\\/6J

Liya Qin; Jun He; Richard N Hanes; Olivera Pluzarev; Jau-Shyong Hong; Fulton T Crews



Endotoxin Inhibits Intestinal Epithelial Restitution through Activation of Rho-GTPase and Increased Focal Adhesions  

Microsoft Academic Search

Diseases of gut inflammation such as neonatal necro- tizing enterocolitis (NEC) result after an injury to the mucosal lining of the intestine, leading to translocation of bacteria and endotoxin (lipopolysaccharide). Intesti- nal mucosal defects are repaired by the process of intes- tinal restitution, during which enterocytes migrate from healthy areas to sites of injury. In an animal model of NEC,

Selma Cetin; Henri R. Ford; Laura R. Sysko; Charu Agarwal; James Wang; Matthew D. Neal; Catherine Baty; Gerard Apodaca; David J. Hackam



Gram-negative endotoxin: an extraordinary lipid with profound effects on eukaryotic signal transduction1  

Microsoft Academic Search

The lipid A domain of lipopolysaccharide (LPS) is a unique, glucosamine-based phospholipid that makes up the outer monolayer of the outer membrane of most gram-negative bacteria. Because of its profound pharmacological effects on animal cells, especially those of the immune system, lipid A is also known as endo- toxin. Despite decades of earlier work, the precise chemistry of endotoxins and



Recognition of Gram-negative bacteria and endotoxin by the innate immune system  

Microsoft Academic Search

Until about 10 years ago the exact mechanisms controlling cellular responses to the endotoxin – or lipopolysaccharide (LPS) – of Gram-negative bacteria were unknown. Now a considerable body of evidence supports a model where LPS or LPS-containing particles (including intact bacteria) form complexes with a serum protein known as LPS-binding protein; the LPS in this complex is subsequently transferred to

Richard J Ulevitch; Peter S Tobias



Endotoxin and Cancer  

PubMed Central

Objective Exposure to endotoxin, a component of gram-negative bacterial cell walls, is widespread in many industrial settings and in the ambient environment. Heavy-exposure environments include livestock farms, cotton textile facilities, and saw mills. Concentrations are highly variable in non-occupational indoor and outdoor environments. Endotoxin is a potent inflammagen with recognized health effects, including fever, shaking chills, septic shock, toxic pneumonitis, and respiratory symptoms. Somewhat paradoxically, given the putative role of inflammation in carcinogenesis, various lines of evidence suggest that endotoxin may prevent cancer initiation or limit tumor growth. The hypothesis that components of bacteria may retard cancer progression dates back to William B. Coley’s therapeutic experiments (“bacterial vaccine”) in the 1890s. Data sources In this article, we review epidemiologic, clinical trial, and experimental studies pertinent to the hypothesis that endotoxin prevents cancer. Since the 1970s, epidemiologic studies of cotton textile and other endotoxin-exposed occupational groups have consistently demonstrated reduced lung cancer risks. Experimental animal toxicology research and some limited therapeutic trials in cancer patients offer additional support for an anticarcinogenic potential. The underlying biological mechanisms of anticarcinogenesis are not entirely understood but are thought to involve the recruitment and activation of immune cells and proinflammatory mediators (e.g., tumor necrosis factor ? and interleukin-1 and -6). Conclusions In view of the current state of knowledge, it would be premature to recommend endotoxin as a cancer-chemopreventive agent. Nonetheless, further epidemiologic and experimental investigations that can clarify further dose–effect and exposure–timing relations could have substantial public health and basic biomedical benefits.

Lundin, Jessica I.; Checkoway, Harvey



Biosensor of endotoxin and sepsis  

NASA Astrophysics Data System (ADS)

To investigate the relation between biosensor of endotoxin and endotoxin of plasma in sepsis. Method: biosensor of endotoxin was designed with technology of quartz crystal microbalance bioaffinity sensor ligand of endotoxin were immobilized by protein A conjugate. When a sample soliton of plasma containing endotoxin 0.01, 0.03, 0.06, 0.1, 0.5, 1.0Eu, treated with perchloric acid and injected into slot of quartz crystal surface respectively, the ligand was released from the surface of quartz crystal to form a more stable complex with endotoxin in solution. The endotoxin concentration corresponded to the weight change on the crystal surface, and caused change of frequency that occurred when desorbed. The result was biosensor of endotoxin might detect endotoxin of plasma in sepsis, measurements range between 0.05Eu and 0.5Eu in the stop flow mode, measurement range between 0.1Eu and 1Eu in the flow mode. The sensor of endotoxin could detect the endotoxin of plasm rapidly, and use for detection sepsis in clinically.

Shao, Yang; Wang, Xiang; Wu, Xi; Gao, Wei; He, Qing-hua; Cai, Shaoxi



Endotoxin and cytokines alter contractile protein expression in cardiac myocytes in vivo.  


Release of bacterial endotoxin and cytokines induce cardiac failure during sepsis. We investigated the direct effects of E. coli endotoxin (lipopolysaccharide, LPS) and cytokines induced by LPS on the cardiac myocyte gene program. For in vivo-experiments adult Wistar rats were given 600 microg/day LPS i.v. for 24 h or 7 days. In addition, cultured adult rat cardiac myocytes were treated with LPS, interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNFalpha), interferon-gamma (IFNgamma) or IL-6 for 24 h. mRNA expression was evaluated for cardiac-alpha-actin (cAct), skeletal-alpha-actin (skAct), beta- and alpha-myosin heavy chain (MHC). LPS induced betaMHC-mRNA 3.6-fold and repressed alphaMHC 2.7-fold and cAct 2.5-fold after 24 h in vivo. Up-regulation of betaMHC (3-fold) and repression of cAct (2.5-fold) were still observed after 7 days LPS infusion, whereas alphaMHC-mRNA levels had returned to normal. At the protein level, increased expression of betaMHC by LPS treatment occurred already after 24 h and was maintained thereafter. LPS had no influence on skAct-mRNA. Similar changes in contractile protein mRNA expression were observed in LPS-treated cardiomyocytes in culture, whereas the tested cytokines either activated (IL-1beta, IFNgamma) or repressed (TNFalpha, IL-6) both MHC-isoforms and cAct. In conclusion, LPS and proinflammatory cytokines induce changes in contractile protein expression that may contribute to the acute heart failure observed during endotoxaemia. PMID:11680626

Patten, M; Krämer, E; Bünemann, J; Wenck, C; Thoenes, M; Wieland, T; Long, C



Characterization of a lipopolysaccharide mediated neutrophilic hepatitis model in Sprague Dawley rats  

Microsoft Academic Search

Several studies have investigated the role of neutrophils during endotoxin-mediated liver injury, yet the precise mechanism for endotoxin-mediated hepatic neutrophil transmigration is unknown. The primary objective of this study was to establish a reliable lipopolysaccharide (LPS)-mediated necro-hepatitis model to investigate the mecha- nisms of hepatic neutrophil infiltration following LPS administration. Male Sprague Dawley rats were administered a single (5 or

Robert Rose; Atrayee Banerjee; Shashi K. Ramaiah



Heightened endotoxin susceptibility of monocytes and neutrophils during familial Mediterranean fever.  


Familial Mediterranean fever (FMF) is a relapsing autoinflammatory disorder, caused by various mutations in the MEFV gene, which encodes a protein called pyrin, expressed in neutrophils and activated monocytes. Induction of monocyte endotoxin tolerance is observed in FMF patients during attack, whereas monocytes from patients in the attack-free period failed to induce lipopolysaccharide tolerance and exhibited heightened sensitivity to bacterial endotoxin. In this study, we demonstrated that impaired lipopolysaccharide tolerance induction in attack-free FMF patients correlates with both increased lipopolysaccharide-induced proinflammatory cytokine synthesis polarization and a different time-course pattern of lipopolysaccharide-induced changes on monocytic surface expression of CD14 and CD11b coreceptors. We found that this pattern is characterized either by delayed turnover of CD14 or increased surface retention of CD11b receptors on monocytes during stimulation with lipopolysaccharide. In addition, enhancement of lipopolysaccharide-induced apoptosis of neutrophils was observed in FMF patients, and was confirmed based on the fact that neutrophils from FMF patients previously unexposed to Salmonella enteritidis exhibited heightened susceptibility to the lipopolysaccharide of this pathogen similar to that of patients infected with this species. PMID:18294193

Davtyan, Tigran K; Harutyunyan, Vachagan A; Hakobyan, Gagik S; Avetisyan, Samvel A



Comparison of the immune response to sheep erythrocytes, tetanus toxoid and endotoxin in different strains of mice  

Microsoft Academic Search

The primary immune response in 7 inbred and 2 outbred mouse strains to SRBC, tetanus toxin and ‘E.coliendotoxin is compared. Significant variations are found in the different strains concerning their antibody response to these antigens. The differences in antibody production reached with SRBC (direct and indirect PFC) and tetanus toxin are on a quantitative level, those with endotoxin also

J. F. Borel



Measurement of endotoxins in bioaerosols at workplace: a critical review of literature and a standardization issue.  


Endotoxins are lipopolysaccharides found in the outer membrane of most Gram-negative bacteria and cyanobacteria. Worker exposure to endotoxins has been shown in a number of work situations and is associated with both respiratory and systemic pathologies. The lack of an occupational exposure limit is mainly due to the absence of a standard protocol at the international level for sampling and analyzing airborne endotoxins. The bibliographic review in this article takes an exhaustive look at the current knowledge on measuring airborne endotoxins. It shows that, despite several reference documents at the international level, the methods used to measure endotoxin exposure differ considerably from one laboratory to another. Standardization is necessary to reduce interlaboratory variability and, ultimately, to improve the use of interstudy data. The bibliographic review presents the current status of standardization for airborne endotoxin measurement methods in the workplace and summarizes areas for further research. This article is both a reference document for all operators wishing to use such methods and a working document to build international consensus around the measurement of airborne endotoxins. PMID:23002277

Duquenne, Philippe; Marchand, Genevičve; Duchaine, Caroline




EPA Science Inventory

ABSTRACT The endotoxin component of organic dusts causes acute reversible airflow obstruction and airway inflammation. To test the hypothesis that endotoxin alone causes airway remodeling, we have compared the response of two inbred mouse strains to subchronic endotoxin ...


Involvement of cannabinoid receptor-1 activation in mitochondrial depolarizing effect of lipopolysaccharide in human spermatozoa.  


Gram-negative bacteria frequently involved in urogenital tract infections release the endotoxin lipopolysaccharide (LPS); its receptor, toll-like receptor-4 (TLR4), has been recently identified in human spermatozoa, and its direct activation has been suggested in mediating adverse effects of LPS on human spermatozoa. However, the underlying signal transduction remains to be clarified. In other cell types, LPS induces the generation of endocannabinoids, which are involved in mediating endotoxin effects. In human spermatozoa, which exhibit a completely functional endocannabinoid system, the activation of cannabinoid receptor-1 (CB1) inhibited sperm mitochondrial membrane potential (??m). In this study, we tested the hypothesis of a contribution of CB1 activation by sperm-generated endocannabinoids in the adverse effects exerted by LPS on human spermatozoa. The exposure of motile sperm suspensions to E. coli LPS produced a significant decrease in sperm ??m, assessed at flow cytometry with JC-1, similar to that induced by Metanandamide (Met-AEA), a non-hydrolyzable analogue of the endocannabinoid AEA. The LPS-induced inhibition of ??m was prevented by the selective CB1 cannabinoid receptor antagonist, SR141716. However, the inhibition of ??m induced by either LPS or Met-AEA did not affect sperm motility. Consistent with this finding, the CB1-mediated inhibition of ??m was neither associated to mitochondrial generation of reactive oxygen species as evaluated by flow cytometry with MytoSox Red nor to apoptosis pathway activation as evaluated with cytoflorimetric assay for activated caspase-9 and caspase-3. Any oxidative genomic damage was also ruled out with the cytoflorimetric quantification of the oxidized base adduct 8-hydroxy-2'-deoxyguanosine. In conclusion, E. coli LPS inhibited sperm ??m through the activation of CB1, but this effect was not accompanied to the activation of mitochondrial dysfunction-related apoptotic/oxidative mechanisms, which could affect sperm motility and genomic integrity. PMID:24692267

Barbonetti, A; Vassallo, M R C; Costanzo, M; Battista, N; Maccarrone, M; Francavilla, S; Francavilla, F



Feline endotoxin shock: effects on tissue histamine and histidine decarboxylase activity.  

PubMed Central

Tissue histamine levels as well as specific and non-specific histidine decarboxylase were examined before and at various times (5 and 10 min) after the intravenous injection of a lethal dose (2 mg kg-1) of E. coli endotoxin in anaesthetized cats. Histamine levels were increased 5 min after endotoxin, especially in the skin, liver, lung and stomach. There was evidence, in most of the cats, for a rapid and substantial activation of specific histidine decarboxylase especially in the lungs, liver, heart and gastrointestinal tract 5-10 min after endotoxin administration. It is suggested that endotoxin induces the local formation of histamine and that this formation and local release may contribute to the pathophysiology of endotoxin shock in this species.

Parratt, J. R.; Saleh, S.; Waton, N. G.



Endotoxin Structures in the Psychrophiles Psychromonas marina and Psychrobacter cryohalolentis Contain Distinctive Acyl Features  

PubMed Central

Lipid A is the essential component of endotoxin (Gram-negative lipopolysaccharide), a potent immunostimulatory compound. As the outer surface of the outer membrane, the details of lipid A structure are crucial not only to bacterial pathogenesis but also to membrane integrity. This work characterizes the structure of lipid A in two psychrophiles, Psychromonas marina and Psychrobacter cryohalolentis, and also two mesophiles to which they are related using MALDI-TOF MS and fatty acid methyl ester (FAME) GC-MS. P. marina lipid A is strikingly similar to that of Escherichia coli in organization and total acyl size, but incorporates an unusual doubly unsaturated tetradecadienoyl acyl residue. P. cryohalolentis also shows structural organization similar to a closely related mesophile, Acinetobacter baumannii, however it has generally shorter acyl constituents and shows many acyl variants differing by single methylene (-CH2-) units, a characteristic it shares with the one previously reported psychrotolerant lipid A structure. This work is the first detailed structural characterization of lipid A from an obligate psychrophile and the second from a psychrotolerant species. It reveals distinctive structural features of psychrophilic lipid A in comparison to that of related mesophiles which suggest constitutive adaptations to maintain outer membrane fluidity in cold environments.

Sweet, Charles R.; Alpuche, Giancarlo M.; Landis, Corinne A.; Sandman, Benjamin C.



Characterization of Lipopolysaccharides Present in Settled House Dust  

PubMed Central

The 3-hydroxy fatty acids (3-OHFAs) in lipopolysaccharides (LPS) play an important role in determining endotoxin activity, and childhood exposure to endotoxin has recently been associated with reduced risk of atopic diseases. To characterize the 3-OHFAs in house dust (HD), we used gas chromatography-mass spectrometry to assay 190 HD samples. Dust from beds, bedroom floors, family rooms, and kitchen floors was collected as part of a birth cohort study of childhood asthma (study 1) and a longitudinal study of home allergen and endotoxin (study 2). We also measured endotoxin activity with a Limulus assay and computed specific activity (endotoxin activity per nanomole of LPS). Longer-chain (C16:0 and C18:0) 3-OHFAs were predominant in HD compared with short-chain (C10:0, C12:0, and C14:0) acids. Endotoxin activity was positively correlated with short-chain 3-OHFAs in both studies. In study 2, 3-OH C16:0 was negatively correlated and 3-OH C18:0 was not correlated with endotoxin activity, consistent with previous findings that the Limulus assay responds preferentially to LPS containing short-chain 3-OHFAs. Kitchen dust contained the highest concentrations of 3-OH C10:0, the highest endotoxin activities, and the highest specific activities (P < 0.03). Bed dust contained the largest amounts of long-chain 3-OHFAs, the highest concentrations of LPS, and the lowest specific activities. Apartments had significantly different types of LPS (P = 0.03) compared with single-family homes in study 2. These data suggest that the Limulus assay may underestimate exposure to certain types of LPS. Because nontoxic LPS may have immune modulating effects, analysis of 3-OHFAs may be useful in epidemiologic studies.

Park, Ju-Hyeong; Szponar, Bogumila; Larsson, Lennart; Gold, Diane R.; Milton, Donald K.



On the essentiality of lipopolysaccharide to Gram-negative bacteria.  


Lipopolysaccharide is a highly acylated saccharolipid located on the outer leaflet of the outer membrane of Gram-negative bacteria. Lipopolysaccharide is critical to maintaining the barrier function preventing the passive diffusion of hydrophobic solutes such as antibiotics and detergents into the cell. Lipopolysaccharide has been considered an essential component for outer membrane biogenesis and cell viability based on pioneering studies in the model Gram-negative organisms Escherichia coli and Salmonella. With the isolation of lipopolysaccharide-null mutants in Neisseria meningitidis, Moraxella catarrhalis, and most recently in Acinetobacter baumannii, it has become increasingly apparent that lipopolysaccharide is not an essential outer membrane building block in all organisms. We suggest the accumulation of toxic intermediates, misassembly of essential outer membrane porins, and outer membrane stress response pathways that are activated by mislocalized lipopolysaccharide may collectively contribute to the observed strain-dependent essentiality of lipopolysaccharide. PMID:24148302

Zhang, Ge; Meredith, Timothy C; Kahne, Daniel



Uropathogenic Escherichia coli Triggers Oxygen-Dependent Apoptosis in Human Neutrophils through the Cooperative Effect of Type 1 Fimbriae and Lipopolysaccharide  

Microsoft Academic Search

Type 1 fimbriae are the most commonly expressed virulence factor on uropathogenic Escherichia coli .I n addition to promoting avid bacterial adherence to the uroepithelium and enabling colonization, type 1 fimbriae recruit neutrophils to the urinary tract as an early inflammatory response. Using clinical isolates of type 1 fimbriated E. coli and an isogenic type 1 fimbria-negative mutant (CN1016) lacking

Robert Blomgran; Limin Zheng; Olle Stendahl



Effect of radio-detoxified endotoxin on the liver microsomal drug metabolizing enzyme system in rats.  


E. coli endotoxin (LPS) depresses the hepatic microsomal mono-oxygenase activity. Radio-detoxified LPS (TOLERIN: 60 Co irradiated endotoxin preparation) decreases this biotransforming activity to a smaller extent. Phenobarbital, an inducer of this mono-oxygenase system, failed to induce in LPS-treated animals. In radio-detoxified LPS-treated rats, phenobarbital induced the mono-oxygenase and almost fully restored the biotransformation. PMID:6347968

Bertók, L; Szeberényi, S



Maternal supplementation with fishmeal protects against late gestation endotoxin-induced fetal programming of the ovine hypothalamic-pituitary-adrenal axis.  


Adverse uterine environments caused by maternal stress (such as bacterial endotoxin) can alter programming of the fetal hypothalamic-pituitary-adrenal axis (HPAA) rendering offspring susceptible to various adulthood diseases. Thus, protection against this type of stress may be critical for ensuring offspring health. The present study was designed to determine if maternal supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) during pregnancy helps to protect against stress-induced fetal programming. Briefly, 53 ewes were fed a diet supplemented with fishmeal (FM) or soybean meal (SM) from day 100 of gestation (gd100) through lactation. On gd135, half the ewes from each dietary group were challenged with either 1.2 ?g/kg Escherichia coli lipopolysaccharide (LPS) endotoxin, or saline as the control. The offspring's cortisol response to weaning stress was assessed 50 days postpartum by measuring serum cortisol concentrations 0, 6 and 24 h post weaning. Twenty-four hours post-weaning, lambs were subjected to an adrenocorticotropic hormone (ACTH) challenge (0.5 ?g/kg) and serum cortisol concentrations were measured 0, 0.25, 0.5, 1 and 2 h post injection. At 5.5 months of age, offspring were also challenged with 400 ng/kg of LPS, and serum cortisol concentrations were measured 0, 2, 4 and 6 h post challenge. Interestingly, female offspring born to FM+LPS mothers had a greater cortisol response to weaning and endotoxin challenge compared with the other treatments, while female offspring born to SM+LPS mothers had a faster cortisol response to the ACTH stressor. Additionally, males born to FM+LPS mothers had a greater cortisol response to the ACTH challenge than the other treatments. Overall, FM supplementation during gestation combined with LPS challenge alters HPAA responsiveness of the offspring into adulthood. PMID:24901660

Fisher, R E; Or'Rashid, M; Quinton, M; AlZahal, O; Boermans, H J; McBride, B W; Karrow, N A



Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110: identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a.  

PubMed Central

We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity. Images

Yao, Z; Valvano, M A



Unique genome-wide transcriptome profiles of chicken macrophages exposed to Salmonella-derived endotoxin  

PubMed Central

Background Macrophages play essential roles in both innate and adaptive immune responses. Bacteria require endotoxin, a complex lipopolysaccharide, for outer membrane permeability and the host interprets endotoxin as a signal to initiate an innate immune response. The focus of this study is kinetic and global transcriptional analysis of the chicken macrophage response to in vitro stimulation with endotoxin from Salmonella typhimurium-798. Results The 38535-probeset Affymetrix GeneChip Chicken Genome array was used to profile transcriptional response to endotoxin 1, 2, 4, and 8 hours post stimulation (hps). Using a maximum FDR (False Discovery Rate) of 0.05 to declare genes as differentially expressed (DE), we found 13, 33, 1761 and 61 DE genes between endotoxin-stimulated versus non-stimulated cells at 1, 2, 4 and 8 hps, respectively. QPCR demonstrated that endotoxin exposure significantly affected the mRNA expression of IL1B, IL6, IL8, and TLR15, but not IL10 and IFNG in HD 11 cells. Ingenuity Pathway Analysis showed that 10% of the total DE genes were involved in inflammatory response. Three, 9.7, 96.8, and 11.8% of the total DE inflammatory response genes were significantly differentially expressed with endotoxin stimulation at 1, 2, 4 and 8 hps, respectively. The NFKBIA, IL1B, IL8 and CCL4 genes were consistently induced at all times after endotoxin treatment. NLRC5 (CARD domain containing, NOD-like receptor family, RCJMB04_18i2), an intracellular receptor, was induced in HD11 cells treated with endotoxin. Conclusions As above using an in vitro model of chicken response to endotoxin, our data revealed the kinetics of gene networks involved in host response to endotoxin and extend the known complexity of networks in chicken immune response to Gram-negative bacteria such as Salmonella. The induction of NFKBIA, IL1B, IL8, CCL4 genes is a consistent signature of host response to endotoxin over time. We make the first report of induction of a NOD-like receptor family member in response to Salmonella endotoxin in chicken macrophages.



Lipopolysaccharide Is Transferred from High-Density to Low-Density Lipoproteins by Lipopolysaccharide-Binding Protein and Phospholipid Transfer Protein  

PubMed Central

Lipopolysaccharide (LPS), the major outer membrane component of gram-negative bacteria, is a potent endotoxin that triggers cytokine-mediated systemic inflammatory responses in the host. Plasma lipoproteins are capable of LPS sequestration, thereby attenuating the host response to infection, but ensuing dyslipidemia severely compromises this host defense mechanism. We have recently reported that Escherichia coli J5 and Re595 LPS chemotypes that contain relatively short O-antigen polysaccharide side chains are efficiently redistributed from high-density lipoproteins (HDL) to other lipoprotein subclasses in normal human whole blood (ex vivo). In this study, we examined the role of the acute-phase proteins LPS-binding protein (LBP) and phospholipid transfer protein (PLTP) in this process. By the use of isolated HDL containing fluorescent J5 LPS, the redistribution of endotoxin among the major lipoprotein subclasses in a model system was determined by gel permeation chromatography. The kinetics of LPS and lipid particle interactions were determined by using Biacore analysis. LBP and PLTP were found to transfer LPS from HDL predominantly to low-density lipoproteins (LDL), in a time- and dose-dependent manner, to induce remodeling of HDL into two subpopulations as a consequence of the LPS transfer and to enhance the steady-state association of LDL with HDL in a dose-dependent fashion. The presence of LPS on HDL further enhanced LBP-dependent interactions of LDL with HDL and increased the stability of the HDL-LDL complexes. We postulate that HDL remodeling induced by LBP- and PLTP-mediated LPS transfer may contribute to the plasma lipoprotein dyslipidemia characteristic of the acute-phase response to infection.

Levels, J. H. M.; Marquart, J. A.; Abraham, P. R.; van den Ende, A. E.; Molhuizen, H. O. F.; van Deventer, S. J. H.; Meijers, J. C. M.



Additive effect of nitric oxide and prostaglandin-E 2 synthesis inhibitors in endotoxin-induced uveitis in the rabbit  

Microsoft Academic Search

The involvement of nitric oxide (NO) and prostaglandin E2 (PGE2) was investigated in a model of intraocular inflammation induced by intravitreal injection of endotoxin (lipopolysaccharide, LPS, 10 ng) in rabbits. The severity of uveitis, the myeloperoxidase (MPO) activity in iris-ciliary body, and the protein concentration in aqueous humor were determined. Nitric oxide synthase (NOS) and cyclooxygenase (COX) activities were assessed

J. L. Bellot; M. Palmero; C. García-Cabanes; R. Espí; C. Hariton; A. Orst



Endotoxin induction of an inhibitor of plasminogen activator in bovine pulmonary artery endothelial cells  

SciTech Connect

The effects of bacterial lipopolysaccharide (endotoxin) on the fibrinolytic activity of bovine pulmonary artery endothelial cells were examined. Endotoxin suppressed the net fibrinolytic activity of cell extracts and conditioned media in a dose-dependent manner. The effects of endotoxin required at least 6 h for expression. Cell extracts and conditioned media contained a 44-kDa urokinase-like plasminogen activator. Media also contained multiple plasminogen activators with molecular masses of 65-75 and 80-100 kDa. Plasminogen activators in extracts and media were unchanged by treatment of cells with endotoxin. Diisopropyl fluorophosphate (DFP)-abolished fibrinolytic activity of extracts and conditioned media. DFP-treated samples from endotoxin-treated but not untreated cells inhibited urokinase and tissue plasminogen activator, but not plasmin. Inhibitory activity was lost by incubation at pH 3 or heating to 56/sup 0/C for 10 min. These treatments did not affect inhibitory activity of fetal bovine serum. Incubation of /sup 125/I-urokinase with DFP-treated medium from endotoxin-treated cells produced an inactive complex with an apparent molecular mass of 80-85 kDa.

Not Available



Acute Binge Drinking Increases Serum Endotoxin and Bacterial DNA Levels in Healthy Individuals  

PubMed Central

Binge drinking, the most common form of alcohol consumption, is associated with increased mortality and morbidity; yet, its biological consequences are poorly defined. Previous studies demonstrated that chronic alcohol use results in increased gut permeability and increased serum endotoxin levels that contribute to many of the biological effects of chronic alcohol, including alcoholic liver disease. In this study, we evaluated the effects of acute binge drinking in healthy adults on serum endotoxin levels. We found that acute alcohol binge resulted in a rapid increase in serum endotoxin and 16S rDNA, a marker of bacterial translocation from the gut. Compared to men, women had higher blood alcohol and circulating endotoxin levels. In addition, alcohol binge caused a prolonged increase in acute phase protein levels in the systemic circulation. The biological significance of the in vivo endotoxin elevation was underscored by increased levels of inflammatory cytokines, TNF? and IL-6, and chemokine, MCP-1, measured in total blood after in vitro lipopolysaccharide stimulation. Our findings indicate that even a single alcohol binge results in increased serum endotoxin levels likely due to translocation of gut bacterial products and disturbs innate immune responses that can contribute to the deleterious effects of binge drinking.

Gattu, Arijeet; Catalano, Donna; Szabo, Gyongyi



Tumor necrosis factor and interleukin 1 as mediators of endotoxin-induced beneficial effects  

SciTech Connect

Bacterial lipopolysaccharides or endotoxins are known to induce tumor necrosis; enhanced nonspecific resistance to bacterial, viral, and parasitic infections and to radiation sickness; and tolerance to lethal doses of endotoxin. These beneficial effects are achieved by pretreatment with minute amounts of endotoxin. Recombinant tumor necrosis factor (TNF) and interleukin 1 (IL-1) are among the mediators capable of invoking radioprotection or resistance to the consequences of cecal ligation and puncture. Both cytokines are potent inducers of serum colony-stimulating factor (CSF) in C3H/HeJ mice (low responders to endotoxin). The number of splenic granulocyte-macrophage precursors was found to increase 5 days after injection of TNF in these mice. Although with IL-1 no increase in the number of granulocyte-macrophage colonies occurred in culture in the presence of serum CSF, a marked stimulation was observed when TNF was added. This stimulation of myelopoiesis observed in vivo and in vitro may be related to the radioprotective effect of TNF. The data presented suggest that TNF and IL-1 released after injection of endotoxin participate in the mediation of endotoxin-induced enhancement of nonspecific resistance and stimulation of hematopoiesis. 76 references.

Urbaschek, R.; Urbaschek, B.



Removal of endotoxin from deionized water using micromachined silicon nanopore membranes  

NASA Astrophysics Data System (ADS)

Endotoxins are lipopolysaccharide components of the cell membrane of Gram-negative bacteria that trigger the body's innate immune system and can cause shock and death. Water for medical therapy, including parenteral and dialysate solutions, must be free of endotoxin. This purity is challenging to achieve as many Gram-negative bacteria are endemic in the environment, and can thrive in harsh, nutrient-poor conditions. Current methods for removing endotoxin include distillation and reverse osmosis, both of which are resource intensive processes. Membranes that present an absolute barrier to macromolecular passage may be capable of delivering pure water for biomedical applications. In this work, endotoxin has been filtered from aqueous solutions using silicon nanopore membranes (SNMs) with monodisperse pore size distributions. SNMs with critical pore sizes between 26 and 49 nm were challenged with solutions of deionized water spiked with endotoxin and with Pseudomonas cepacia. The filtrate produced by the SNM from Pseudomonas-contaminated water had <1.0 endotoxin unit (EU) ml-1, which meets standards for dialysate purity. This approach suggests a technique for single-step cleanup of heavily contaminated water that may be suitable for field or clinical use.

Smith, Ross A.; Goldman, Ken; Fissell, William H.; Fleischman, Aaron J.; Zorman, Christian A.; Roy, Shuvo



Biological Properties of Lipopolysaccharides from Bordetella Species  

Microsoft Academic Search

Biological activities of lipopolysaccharides (LPS) extracted from Bordetefla perfussis, B. pcrrapertusss and B. bronchisepticu were compared with those of Escbichia coli LPS. The LPS preparations from B. pertussis showed biological activities comparable to those of E. coli LPS in terms of lethal toxicity in galactosamine-sensitized mice, pyrogenicity in rabbits, mitogenicity in C3H\\/He spleen cell cultures, macrophage activation, and induction of




Ultrastructural and Functional Effects of Lipopolysaccharide and Interleukin-2 on Human NK Cells.  

National Technical Information Service (NTIS)

Bacterial endotoxin (lipopolysaccharide, LPS) and interleukin-2 (IL-2) are known to stimulate NK cell mediated cytotoxicity against tumor cells. In the present report we sought to correlate the stimulatory effect of LPS and IL-2 on NK cell activity with u...

Y. H. Kang M. Carl L. P. Watson



Targeting naproxen coupled to human serum albumin to nonparenchymal cells reduces endotoxin-induced mortality in rats with biliary cirrhosis.  


Endotoxin is thought to play a major role in cirrhotic liver disease. Cyclo-oxygenase inhibitors were shown to be partially protective against endotoxin but cannot be used in cirrhotic patients because of renal side-effects. We argued that administration of naproxen (NAP) linked to human serum albumin (HSA), which results in specific delivery of NAP to endothelial cells (EC) and Kupffer cells (KC) and exhibited hepatoprotective effects against lipopolysaccharide (LPS) in vitro, could protect cirrhotic rats from LPS toxicity while preserving renal function. The studies were performed in rats rendered cirrhotic by bile duct ligation (BDL); animals received LPS (Escherichia coli, 800 microg/kg) intravenously. Five groups were studied: LPS alone, rats pretreated with a conventional dose of NAP (50 mg/kg), NAP-HSA (22 mg/kg), NAP equimolar to NAP-HSA (1.5 mg/kg), or the HSA carrier. LPS induced significant mortality (55%); this was not affected by equimolar NAP (57%) but accentuated by conventional NAP (88%). In contrast, NAP-HSA provided significant protection (9%; P < .05). After conventional NAP treatment, significant renal toxicity was observed as evidenced by a marked reduction in sodium excretion (LPS vs. NAP-HSA vs. NAP [50 mg/kg] 33 +/- 22 vs. 50 +/- 39 vs. 4 +/- 3 micromol/h; P < .05). Renal prostaglandin E2 (PGE2) excretion was reduced by NAP in all groups, but most markedly at the conventional dosage (LPS vs. NAP-HSA vs. NAP [50 mg/kg] 132 +/- 115 vs. 39 +/- 19 vs. 9 +/- 8 ng/mL; P < .05). Successful targeting was evidenced by a significant hepatic enrichment of NAP in the NAP-HSA group compared with the equimolar untargeted group (30.16 +/- 9.33 vs. 1.13 +/- 1.95 nmol/g liver). Thus, targeting NAP to EC/KC results in improved survival, higher efficacy, and sparing of renal function in cirrhotic rats. PMID:9397997

Albrecht, C; Meijer, D K; Lebbe, C; Sägesser, H; Melgert, B N; Poelstra, K; Reichen, J



Endotoxin in endodontic infections: a review.  


Gram-negative bacteria play an essential role in primary endodontic infections. They have several virulence factors such as endotoxin, a large molecule that plays a role in the initiation and perpetuation of apical periodontitis. This paper reviews the role of gram-negative bacteria in endodontic infections, structure and mechanisms of action of endotoxin, endotoxin in infected root canals, effects of calcium hydroxide and polymixin B on endotoxin, and applications of endotoxin to measure leakage. PMID:21563594

Mohammadi, Zahed



Allergen specific responses in cord and adult blood are differentially modulated in the presence of endotoxins  

PubMed Central

Background 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. Objectives To examine the effect of endotoxins in allergen preparations on cellular responses in human cord and peripheral blood (PB). Methods The endotoxin content in ? lactoglobulin (BLG), the peanut allergen Ara h 1 and the major birch pollen allergen Bet v 1 was assessed. Proliferation and cytokine response of mononuclear cells towards contaminated and lipopolysaccharide (LPS)-free allergens were evaluated at different time-points. Fractions of contaminated BLG were generated and assayed on their immuno-stimulatory capacity. The involvement of toll-like receptor (TLR) 2 and 4 was investigated by blocking antibodies and TLR-transfected human embryonic kidney cells. Results The proliferative response of cord blood (CB)-derived mononuclear cells towards allergen-preparations at day 3 was related to the level of LPS contamination. At day 7, proliferation was also detected in the absence of endotoxin. Cytokine production in CB was strongly affected by the content of endotoxin, TLR-4 dependent and not related to the allergen content. Allergen- and endotoxin-induced proliferative responses were generally significantly higher in CB than in adult blood. Conclusion Endotoxins in allergen preparations confound allergen-specific cellular responses. The impact of these contaminations varies with the blood source (CB vs. PB), the type of allergen and is time- and dose-dependent. Cite this as: T. Eiwegger, E. Mayer, S. Brix, I. Schabussova, E. Dehlink, B. Bohle, V. Barkholt and Z. Szépfalusi, Clinical and Experimental Allergy, 2008 (38) 1627–1634.

Eiwegger, T; Mayer, E; Brix, S; Schabussova, I; Dehlink, E; Bohle, B; Barkholt, V; Szepfalusi, Z



Grape seed procyanidin extract reduces the endotoxic effects induced by lipopolysaccharide in rats.  


Acute inflammation is a response to injury, infection, tissue damage, or shock. Bacterial lipopolysaccharide (LPS) is an endotoxin implicated in triggering sepsis and septic shock, and LPS promotes the inflammatory response, resulting in the secretion of proinflammatory and anti-inflammatory cytokines such as the interleukins (IL-6, IL-1?, and IL-10) and tumor necrosis factor-? by the immune cells. Furthermore, nitric oxide and reactive oxygen species levels increase rapidly, which is partially due to the activation of inducible nitric oxide synthase in several tissues in response to inflammatory stimuli. Previous studies have shown that procyanidins, polyphenols present in foods such as apples, grapes, cocoa, and berries, have several beneficial properties against inflammation and oxidative stress using several in vitro and in vivo models. In this study, the anti-inflammatory and antioxidant effects of two physiological doses and two pharmaceutical doses of grape seed procyanidin extract (GSPE) were analyzed using a rat model of septic shock by the intraperitoneal injection of LPS derived from Escherichia coli. The high nutritional (75mg/kg/day) and the high pharmacological doses (200mg/kg/day) of GSPE showed anti-inflammatory effects by decreasing the proinflammatory marker NOx in the plasma, red blood cells, spleen, and liver. Moreover, the high pharmacological dose also downregulated the genes Il-6 and iNos; and the high nutritional dose decreased the glutathione ratio (GSSG/total glutathione), further illustrating the antioxidant capability of GSPE. In conclusion, several doses of GSPE can alleviate acute inflammation triggered by LPS in rats at the systemic and local levels when administered for as few as 15 days before the injection of endotoxin. PMID:23439188

Pallarčs, Victor; Fernández-Iglesias, Anabel; Cedó, Lídia; Castell-Auví, Anna; Pinent, Montserrat; Ardévol, Anna; Salvadó, Maria Josepa; Garcia-Vallvé, Santiago; Blay, Mayte



Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides.  


Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS. PMID:21641310

Kaconis, Yani; Kowalski, Ina; Howe, Jörg; Brauser, Annemarie; Richter, Walter; Razquin-Olazarán, Iosu; Ińigo-Pestańa, Melania; Garidel, Patrick; Rössle, Manfred; Martinez de Tejada, Guillermo; Gutsmann, Thomas; Brandenburg, Klaus



DOK3 Negatively Regulates LPS Responses and Endotoxin Tolerance  

PubMed Central

Innate immune activation via Toll-like receptors (TLRs), although critical for host defense against infection, must be regulated to prevent sustained cell activation that can lead to cell death. Cells repeatedly stimulated with lipopolysaccharide (LPS) develop endotoxin tolerance making the cells hypo-responsive to additional TLR stimulation. We show here that DOK3 is a negative regulator of TLR signaling by limiting LPS-induced ERK activation and cytokine responses in macrophages. LPS induces ubiquitin-mediated degradation of DOK3 leading to SOS1 degradation and inhibition of ERK activation. DOK3 mice are hypersensitive to sublethal doses of LPS and have altered cytokine responses in vivo. During endotoxin tolerance, DOK3 expression remains stable, and it negatively regulates the expression of SHIP1, IRAK-M, SOCS1, and SOS1. As such, DOK3-deficient macrophages are more sensitive to LPS-induced tolerance becoming tolerant at lower levels of LPS than wild type cells. Taken together, the absence of DOK3 increases LPS signaling, contributing to LPS-induced tolerance. Thus, DOK3 plays a role in TLR signaling during both naďve and endotoxin-induced tolerant conditions.

Peng, Qisheng; O'Loughlin, Jason L.; Humphrey, Mary Beth



The Role of Cyclooxygenases in Endotoxin and Interleukin1Induced Hypophagia  

Microsoft Academic Search

Endotoxin (lipopolysaccharide, LPS) and interleukin-1 (IL-1) reduce food intake in rodents. Cyclooxygenase (COX) inhibitors have long been known to attenuate these responses, but recent work has revealed the existence of two distinct isoforms of the enzyme, COX1 and COX2, with different characteristics and functions. Therefore, we reassessed the COX involvement using inhibitors with different selectivities for COX1 and COX2. Feeding

Adrian J. Dunn; Artur H. Swiergiel



Pirfenidone prevents endotoxin-induced liver injury after partial hepatectomy in rats  

Microsoft Academic Search

Background\\/Aims: Massive liver resection causes a variety of complications including endotoxemia. Pirfenidone (PFD) is a new experimental drug used as antifibrotic agent. Studies were performed to investigate whether PFD influences the survival rate of animals with endotoxin-induced liver injury after partial hepatectomy, and the mechanisms involved.Methods: Rats were treated with lipopolysaccharide (LPS) 48 h after 70% hepatectomy. PFD was administered

Hideto Tsuchiya; Masaki Kaibori; Hidesuke Yanagida; Norio Yokoigawa; A-Hon Kwon; Tadayoshi Okumura; Yasuo Kamiyama



Surface Expression of Neutrophil CXCR4 is Down-Modulated by Bacterial Endotoxin  

Microsoft Academic Search

The chemokine receptor CXCR4 and its unique ligand, stromal-derived factor 1 (SDF-1), play critical roles in the retention\\u000a of hematopoietic cells within bone marrow and in their mobilization into the circulation. Surface CXCR4 down-regulation in\\u000a hematopoietic cells is associated with a loss of retention of the cells in bone marrow. Lipopolysaccharide (LPS), commonly\\u000a referred to as endotoxin, induces neutrophilia in

Hyun Kyung Kim; Ji-Eun Kim; Junho Chung; Kyou-Sup Han; Han-Ik Cho



Interactions of surfactant protein D with bacterial lipopolysaccharides. Surfactant protein D is an Escherichia coli-binding protein in bronchoalveolar lavage.  

PubMed Central

Surfactant protein D (SP-D) is a collagenous glycoprotein that is secreted into the pulmonary airspaces by alveolar type II and nonciliated bronchiolar cells. SP-D exhibits Ca(++)-dependent carbohydrate binding in vitro and is structurally related to the collagenous C-type lectins, including serum conglutinin, serum mannose-binding proteins, and surfactant protein A. Preliminary studies showed calcium- and saccharide-dependent binding of fluorescein-conjugated or radioiodinated SP-D to a variety of microorganisms, including Gram-negative bacteria and fungi. A laboratory strain of Escherichia coli (Y1088) was chosen to further examine the mechanism(s) of binding. Binding of SP-D to Y1088 was time dependent, saturable, and inhibited by cold SP-D or competing saccharides; Scatchard analysis gave a Kd of 2 x 10(-11) M. At higher concentrations, SP-D also caused Ca(++)-dependent agglutination of Y1088 that was inhibited by alpha-glucosyl-containing saccharides, antisera to the carbohydrate-binding domain of SP-D, or Y1088 LPS. Lectin blots showed specific binding of 125I-SP-D to Y1088 LPS, as well as LPS from other several strains of enteric Gram-negative bacteria. Immunogold studies demonstrated strong and uniform surface labeling of the bacteria. Rat and human bronchoalveolar lavage (BAL) caused Ca(++)-dependent agglutination of E. coli that was dose dependent and inhibited by competing saccharides or anti-SP-D. SP-D was selectively and efficiently adsorbed from rat BAL by incubation with E. coli, and incubation of E. coli with radiolabeled rat type II cell medium revealed that SP-D is the major E. coli-binding protein secreted by freshly isolated cells in culture. We suggest that SP-D plays important roles in the lung's defense against Gram-negative bacteria. Images

Kuan, S F; Rust, K; Crouch, E



Upregulation of ICAM-1 expression on J774.2 macrophages by endotoxin involves activation of NF-kappaB but not protein tyrosine kinase: comparison to induction of iNOS.  

PubMed Central

This study compares the signal transduction pathway which leads to the upregulation of intercellular adhesion molecule-1 (ICAM-1) expression with that of the increase in the expression of inducible nitric oxide synthase (iNOS) protein and activity caused by endotoxin in cultured J774.2 macrophages. Treatment of J774.2 cells with lipopolysaccharide E. coli (LPS) induced a concentration-dependent increase in the expression of ICAM-1 on the cell surface within 4 h and an increase in iNOS protein and activity at 24 h. The upregulation of ICAM-1 expression on J774.2 macrophages caused by LPS was significantly inhibited by pretreatment of the cells with inhibitors of the activation of the nuclear transcription factor NF-kappaB, such as L-1-tosylamido-2-phenylethylchloromethyl ketone (TPCK), pyrrolidine dithiocarbamate (PDTC), rotenone or calpain inhibitor I, but not by the tyrosine kinase inhibitors, tyrphostin AG126 or genistein. In contrast, genistein or tyrphostin AG126 also prevented the induction of iNOS protein and activity in J774.2 macrophages elicited by LPS. Thus, the increase in the expression of ICAM-1 on J774.2 macrophages by endotoxin involves the activation of NFkappaB, but not of protein tyrosine kinase.

Ruetten, H; Thiemermann, C; Perretti, M



Expression of genes kdsA and kdsB involved in 3-deoxy-D-manno-octulosonic acid metabolism and biosynthesis of enterobacterial lipopolysaccharide is growth phase regulated primarily at the transcriptional level in Escherichia coli K-12.  


We have cloned and sequenced a cluster of six open reading frames containing gene kdsA from Escherichia coli K-12. The gene encodes 3-deoxy-D-manno-octulosonate 8-phosphate synthetase (KDO-8-phosphate synthetase), which catalyzes formation of 3-deoxy-D-manno-octulosonic acid (KDO), an essential component of enterobacterial lipopolysaccharide. We have also identified two other genes, hemA and prfA, at the beginning of the cluster. Deletion analysis shows that kdsA, the terminal gene of this putative operon, is transcribed from its own promoter located within the cluster rather than from two promoters preceding this group of six open reading frames. Northern (RNA) blot analysis as well as lacZ operon fusion experiments reveal that the expression of gene kdsA occurs maximally in the early log phase and falls to a low level in the late log and stationary phases. Hence, this gene is subjected to growth phase-dependent regulation at the transcriptional level. Similarly, we show that expression of gene kdsB, which codes for the CTP:CMP-3-deoxy-D-manno-octulosonate cytidyltransferase (CMP-KDO-synthetase), is also growth regulated. This enzyme catalyzes the activation of KDO via formation of CMP-KDO, which is necessary for the incorporation of KDO into lipid A. We have identified the promoter of gene kdsB, whose expression is growth regulated in the same way as that of kdsA. Despite the fact that transcription of genes kdsA and kdsB is shut off as cells enter stationary phase, KDO-8-phosphate synthetase as well as CMP-KDO-synthetase activities are still present at various levels during stationary-phase growth of an E. coli K-12 culture. PMID:7543480

Strohmaier, H; Remler, P; Renner, W; Högenauer, G



Expression of genes kdsA and kdsB involved in 3-deoxy-D-manno-octulosonic acid metabolism and biosynthesis of enterobacterial lipopolysaccharide is growth phase regulated primarily at the transcriptional level in Escherichia coli K-12.  

PubMed Central

We have cloned and sequenced a cluster of six open reading frames containing gene kdsA from Escherichia coli K-12. The gene encodes 3-deoxy-D-manno-octulosonate 8-phosphate synthetase (KDO-8-phosphate synthetase), which catalyzes formation of 3-deoxy-D-manno-octulosonic acid (KDO), an essential component of enterobacterial lipopolysaccharide. We have also identified two other genes, hemA and prfA, at the beginning of the cluster. Deletion analysis shows that kdsA, the terminal gene of this putative operon, is transcribed from its own promoter located within the cluster rather than from two promoters preceding this group of six open reading frames. Northern (RNA) blot analysis as well as lacZ operon fusion experiments reveal that the expression of gene kdsA occurs maximally in the early log phase and falls to a low level in the late log and stationary phases. Hence, this gene is subjected to growth phase-dependent regulation at the transcriptional level. Similarly, we show that expression of gene kdsB, which codes for the CTP:CMP-3-deoxy-D-manno-octulosonate cytidyltransferase (CMP-KDO-synthetase), is also growth regulated. This enzyme catalyzes the activation of KDO via formation of CMP-KDO, which is necessary for the incorporation of KDO into lipid A. We have identified the promoter of gene kdsB, whose expression is growth regulated in the same way as that of kdsA. Despite the fact that transcription of genes kdsA and kdsB is shut off as cells enter stationary phase, KDO-8-phosphate synthetase as well as CMP-KDO-synthetase activities are still present at various levels during stationary-phase growth of an E. coli K-12 culture.

Strohmaier, H; Remler, P; Renner, W; Hogenauer, G



Prevention of experimental endotoxin shock by a monocyte activator.  

PubMed Central

In patients with polytrauma or major surgery, severe bacterial infections leading to septic shock and multiorgan failure are still a major cause of death. Prevention of septic shock in patients at risk would be an alternative to treatment of patients with overt septic shock. We therefore conducted a trial with the monocyte activator muramyl tripeptide phosphatidylethanolamine (MTP-PE) in an experimental pig model. Liposome encapsulated MTP-PE (50 micrograms/kg of body weight) or liposomes alone were given intravenously at 72 or 24 h before endotoxemia was induced by lipopolysaccharide (LPS), simultaneously with the induction of endotoxin shock, or 1 h thereafter. Pretreatment with MTP-PE at 72 and 24 h before endotoxemia was induced resulted in a reduction of endotoxin shock-induced mortality from 81.8% (9 of 11 animals) in the control group to 8.3% (1 of 12 animals) of the MTP-PE-pretreated animals (P < 0.001). The administration of MTP-PE 24 h before the induction of endotoxin shock was more effective (P < 0.01) than administration of MTP-PE 72 h before endotoxemia was induced (P = 0.05). The pretreated animals did not develop fever or cardiovascular complications, and pulmonary function was significantly improved. Furthermore, the alpha-form of the soluble CD14 LPS receptor in pig serum showed a marked decrease in LPS-treated animals, and this decrease was reduced by MTP-PE pretreatment at 24 h before endotoxemia was induced. When MTP-PE was given simultaneously with the induction of septic shock or 1 h thereafter, it did not influence either mortality or morbidity. In conclusion, pretreatment of pigs with MTP-PE improves several parameters of endotoxin shock and it reduces mortality. Patients with high risk of developing septic complications might benefit from a pretreatment with this monocyte-activating substance.

Passlick, B; Labeta, M O; Izbicki, J R; Ostertag, P; Loffler, T; Siebeck, M; Pichlmeier, U; Schweiberer, L; Ziegler-Heitbrock, H W



Effect of the lipopolysaccharide antagonist Planktothrix sp. FP1 cyanobacterial extract on human polymorphonuclear leukocytes.  


CyP is a lipopolysaccharide (LPS)-like molecule extracted from the freshwater cyanobacterium Oscillatoria planktothrix FP1, which has been reported to be a potent competitive inhibitor of bacterial LPS. In the present study the ability of CyP to affect human polymorphonuclear leukocyte (PMN) function was investigated. PMNs were isolated from venous blood by standard density-gradient centrifugation. Cell migration was measured by use of the Boyden chamber assay. Interleukin (IL)-8 and tumor necrosis factor (TNF)-? production was measured using a sandwich-type enzyme-linked immunosorbent assay. PMN intracellular reactive oxygen species (ROS) levels were assessed by the use of a fluorescent probe coupled to spectrophotometry. CyP 10-100 ?g/ml was chemotactic for PMNs without affecting the chemotactic response to either E. coli LPS or N-formyl-Met-Leu-Phe (fMLP). CyP per se did not affect PMN production of either IL-8 or TNF-?, but concentration-dependently reduced LPS-induced production of both cytokines. On the contrary, CyP had no effect either on fMLP-induced production of IL-8 or on PMN oxidative burst (at rest and after stimulation with fMLP), a response which is known to be independent from LPS-operated pathways. In human PMNs CyP behaves as a selective and effective LPS antagonist. These findings support the therapeutic potential of CyP in endotoxin-dependent disease. PMID:21115122

Maio, Ramňna Consučlo; Cosentino, Marco; Rossetti, Carlo; Molteni, Monica; Lecchini, Sergio; Marino, Franca



Interactions of lipopolysaccharide with lipid membranes, raft models - a solid state NMR study.  


Lipopolysaccharide (LPS) is a major component of the external leaflet of bacterial outer membranes, key pro-inflammatory factor and an important mediator of host-pathogen interactions. In host cells it activates the complement along with a pro-inflammatory response via a TLR4-mediated signalling cascade and shows preference for cholesterol-containing membranes. Here, we use solid state (13)C and (31)P MAS NMR to investigate the interactions of LPS from three bacterial species, Brucella melitensis, Klebsiella pneumoniae and Escherichia coli, with mixed lipid membranes, raft models. All endotoxin types are found to be pyrophosphorylated and Klebsiellar LPS is phosphonylated, as well. Carbon-13 MAS NMR indicates an increase in lipid order in the presence of LPS. Longitudinal (31)P relaxation, providing a direct probe of LPS molecular and segmental mobility, reveals a significant reduction in (31)P T1 times and lower molecular mobility in the presence of ternary lipid mixtures. Along with the ordering effect on membrane lipid, this suggests a preferential partitioning of LPS into ordered bilayer sphingomyelin/cholesterol-rich domains. We hypothesise that this is an important evolutionary drive for the selection of GPI-anchored raft-associated LPS-binding proteins as a first line of response to membrane-associated LPS. PMID:23567915

Ciesielski, Filip; Griffin, David C; Rittig, Michael; Moriyón, Ignacio; Bonev, Boyan B



Bacterial lipopolysaccharide (LPS) and interleukin 1 (IL1) exert multiple physiological effects in the tilapia Oreochromis mossambicus (Teleostei)  

Microsoft Academic Search

To gain insight in immuno-endocrine communication in teleosts the physiological effects of interleukin 1 and bacterial lipopolysaccharide in teleosts were investigated. Tilapia (Oreochromis mossambicus) were treated with murine interleukin 1 and E. coli lipopolysaccharide in vivo, and lipopolysaccharide was administered to pituitary lobes and head kidneys in vitro. The integument of the fish appeared to be a sensitive target for

P. H. M. Balm; E. van Lieshout; J. Lokate; S. E. Wendelaar Bonga



Enhancement of endotoxin activity by muramyldipeptide.  


Synthetic muramyldipeptide (MDP), the minimum structural moiety of bacterial peptidoglycan for adjuvant and related activities, sensitized mice for two types of lethal shock induced by lipopolysaccharide (LPS): an early anaphylactoid shock and late endotoxin shock. In relation to the late reaction in MDP-primed mice, enhanced production of inflammatory cytokines was induced in response to various bacterial components. MDP showed a priming effect in mice not only when administered parentally but also via the oral route. MDP activated human monocytic THP-1 cells in a CD14-, Toll-like receptor 2 (TLR2)- and TLR4-independent manner to increase expression of MyD88, a common adaptor and signaling molecule for TLRs, and exhibited synergistic cytokine inducing effects with TLR4 agonists (LPS, synthetic lipid A), TLR2 agonist (synthetic lipopeptide), and TLR9 agonist (bacterial CpGDNA) in THP-1 cells in culture. Consistent with these findings, MDP primed TLR2 knockout mice as well as wild-type controls, but not TLR4-mutated C3H/HeJ mice, to enhance production of tumor necrosis factor-alpha upon stimulation with synthetic lipid A. In contrast to the BCG- and Propionibacterium acnes-priming system, MDP primed mice in an interferon-gamma-independent manner. Further studies are required to elucidate the mechanisms of the synthetic and priming activities of MDP for various bacterial components. PMID:12537692

Takada, Haruhiko; Yokoyama, Shigekuni; Yang, Shuhua



Regulation of serum tumor necrosis factor in glucocorticoid-sensitive and -resistant rodent endotoxin shock models.  

PubMed Central

Bolus injection of lethal or sublethal doses of endotoxin or lipopolysaccharide (LPS) results in the rapid and transient rise in tumor necrosis factor (TNF) levels in serum in mammals. TNF levels peak between 1 and 2 h after LPS injection in mice and guinea pigs and approach basal levels by 6 h. Although the kinetics of TNF in serum appear similar between these two species, guinea pigs respond to a lethal dose of LPS of 20 mg/kg by producing approximately 10-fold more TNF than mice do. These two endotoxin shock models also differ in their sensitivity to glucocorticoids. TNF levels in serum are not reduced in the lethal endotoxin shock model in guinea pigs after treatment with dexamethasone at 25 mg/kg. In contrast, TNF levels in mouse serum are inhibited by more than 90% after treatment with dexamethasone at 3 mg/kg. Coincident with the TNF peak in serum is a leukopenia which approaches control levels by 6 h in dexamethasone-treated mice, while remaining depressed in dexamethasone-treated guinea pigs. Treatment with dexamethasone at 25 mg/kg did not save guinea pigs from endotoxin lethality, whereas long-term survival of mice under identical conditions was apparent. These results suggest that the relative glucocorticoid resistance observed in guinea pigs is also apparent in a lethal endotoxin shock model in which dexamethasone does not modulate TNF levels or result in increased survival as occurs in mice. The lack of clear efficacy for steroid therapy in human clinical septic shock trials would suggest that the guinea pig endotoxin model may be a more predictive system than the mouse model for the identification of novel agents useful in the treatment of endotoxin shock.

Zuckerman, S H; Bendele, A M




EPA Science Inventory

SUBCHRONIC ENDOTOXIN INHALATION CAUSES CHRONIC AIRWAY DISEASE IN ENDOTOXIN-SENSITIVE BUT NOT ENDOTOXIN-RESISTANT MICE. D. M. Brass, J. D. Savov, *S. H. Gavett, ?C. George, D. A. Schwartz. Duke Univ Medical Center Durham, NC, *U.S. E.P.A. Research Triangle Park, NC, ?Univ of Iowa,...


The Limulus Amoebocyte Lysate (LAL) assay for the detection of endotoxin in fat emulsions for total parenteral nutrition (TPN).  


Fat emulsions spiked with Escherichia coli endotoxin were tested with Limulus Amoebocyte Lysate (LAL) reagent, using both the micro method and the tube method. The effect of vortexing, ultrasonication and addition of Pyrosperse on the dispersion of endotoxin in these emulsions was compared. The advantage of using the tube method, rather than the micro method, is shown. Ultrasonication may substitute vortex mixing. Pyrosperse had no convincing effect. PMID:6435392

Paulssen, J; Michaelsen, P



Equine platelets inhibit E. coli growth and can be activated by bacterial lipopolysaccharide and lipoteichoic acid although superoxide anion production does not occur and platelet activation is not associated with enhanced production by neutrophils.  


Activated platelets can contribute to host defense through release of products with bactericidal actions such as antimicrobial peptides and reactive oxygen species (ROS), as well as by forming heterotypic aggregates with neutrophils and enhancing their antimicrobial properties. Whilst release of vasoactive mediators from equine platelets in response to stimuli including bacterial lipopolysaccharide (LPS) has been documented, neither ROS production, nor the effects of activated platelets on equine neutrophil ROS production, have been reported. This study first sought evidence that activated equine platelets inhibit bacterial growth. Platelet superoxide production in response to stimuli including Escherichia coli-derived LPS and lipoteichoic acid (LTA) from Staphylococcus aureus was then determined. The ability of LPS and LTA to up-regulate platelet P-selectin expression and induce platelet-neutrophil aggregate formation was investigated and the effect of co-incubating activated platelets with neutrophils on superoxide production measured. Growth of E. coli was inhibited in a time-dependent manner, and to a similar extent, by addition of platelet rich plasma (PRP) or platelet poor plasma (PPP) obtained by centrifugation of PRP. Activation of platelets in PRP by addition of thrombin led to a significant increase in the inhibitory action between 0.5 and 2h. Although phorbol myristate acetate (PMA) caused superoxide production by equine platelets in a protein kinase C-dependent manner, thrombin, platelet activating factor (PAF), LPS, LTA and formyl-methionyl-leucyl phenylalanine (FMLP) were without effect. LPS and LTA did induce platelet activation, measured as an increase in P-selectin expression (% positive cells: 17±3 (un-stimulated); 63±6 (1?g/ml LPS); 64±6 (1?g/ml LTA); n=5) but not platelet superoxide production or heterotypic aggregate formation. Co-incubation of activated platelets with neutrophils did not increase neutrophil superoxide production. This study has demonstrated for the first time that when activated, equine platelets, like those of other species, are capable of releasing ROS that could assist in bacterial killing. However, the findings suggest that neither superoxide production by platelets nor enhancement of production by neutrophils is likely to play a significant role. Nevertheless, as has been reported in man, equine PPP and PRP did inhibit E. coli growth in vitro, and addition of thrombin significantly increased the inhibitory effect of PRP. This suggests that products released from activated platelets could contribute to antimicrobial activity in the horse. The factors in equine plasma and released by activated platelets that are responsible for inhibiting bacterial growth have yet to be determined. PMID:23332730

Aktan, I; Dunkel, B; Cunningham, F M



Long-lasting protection in brain trauma by endotoxin preconditioning.  


We investigated the occurrence of endotoxin (lipopolysaccharide, LPS) preconditioning in traumatic brain injury (TBI), evaluating the time window of LPS-induced protection, its persistence, and the associated molecular mechanisms. Mice received 0.1?mg/kg LPS or saline intraperitoneally and subsequently TBI (by controlled cortical impact brain injury) at various time intervals. Mice receiving LPS 3, 5, or 7 days before TBI showed attenuated motor deficits at 1 week after injury compared with mice receiving saline. Those receiving LPS 5 days before injury had also a reduced contusion volume (7.9±1.3 versus 12±2.3?mm(3)) and decreased cell death. One month after injury, the protective effect of LPS on contusion volume (14.5±1.2 versus 18.2±1.2?mm(3)) and neurologic function was still present. Traumatic brain injury increased glial fibrillary acidic protein, CD11b, CD68, tumor necrosis factor-?, interleukin (IL)-10, and IL-6 mRNA expression 24?hours after injury. Lipopolysaccharide administered 5 (but not 9) days before injury increased the expression of CD11b (233%) and of interferon ? (500%) in uninjured mice, while it reduced the expression of CD68 (by 46%) and increased that of IL-6 (by 52%) in injured mice. Lipopolysaccharide preconditioning conferred a long-lasting neuroprotection after TBI, which was associated with a modulation of microglia/macrophages activity and cytokine production. PMID:21468087

Longhi, Luca; Gesuete, Raffaella; Perego, Carlo; Ortolano, Fabrizio; Sacchi, Noemi; Villa, Pia; Stocchetti, Nino; De Simoni, Maria-Grazia



Relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in postpartum cows with dystocia or retained placenta.  


A study was conducted to investigate the relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in cows that experienced a dystocia or retained their placenta. Fifteen healthy cows, 31 cows with retained placenta (RP) and 13 cows that had dystocia were clinically examined 1 or 2 days after parturition when a uterine swab for bacteriological examination was taken. In addition, plasma and uterine lochia samples were collected to determine lipopolysaccharide (LPS) and the plasma IgG anti-LPS concentrations. Subsequently, 15 RP and 6 dystocia cows were initially left untreated and another uterine swab was collected at 2 and 4 wk postpartum. Immediately after calving, RP cows had significantly higher LPS levels in uterine lochia (average of 2.24 x 10(4) Endotoxin Units (EU)/mL) as compared to dystocia and healthy postpartum cows (average of 0.10 and 0.26 EU/mL, respectively). However, plasma LPS levels were below the detection limit (<0.036 EU/mL platelet-rich plasma) in all groups of cows. IgG anti-LPS levels in plasma were not significantly different between the 3 groups immediately postpartum (average of 26, 16 and 44 Median Units (MU)/mL) for healthy, dystocia and RP cows, respectively), but they were significantly lower when compared to plasma IgG anti-LPS levels of healthy cows at more than 2 months postpartum (mean 83 MU/mL). High LPS levels in lochia at 1 or 2 days postpartum were significantly related to abnormal cervical discharge, the presence of Escherichia coli, black pigmented gram-negative anaerobes and Clostridium spp. shortly after calving, and Arcanobacterium pyogenes and gram-negative anaerobes in the uterus at 14 days postpartum. These results suggest that the presence of E. coli and LPS (endotoxins) in lochia early postpartum favor the development of uterine infections by A. pyogenes and gram-negative anaerobes later postpartum. LPS were not observed in plasma, suggesting that either they are not absorbed into the blood, or they are efficiently detoxified by IgG anti-LPS or other detoxification mechanisms. PMID:11131320

Dohmen, M J; Joop, K; Sturk, A; Bols, P E; Lohuis, J A



Deferoxamine reduces tissue damage during endotoxin-induced mastitis in dairy cows.  


The protective effects of 3 antioxidants on polymorphonuclear neutrophil-induced damage to mammary cells were evaluated in vivo using an endotoxin-induced mastitis model. Fifteen healthy, midlactation cows with no history of clinical Escherichia coli mastitis were randomly assigned to 1 of the 3 treatment groups corresponding to each modulator to be evaluated, that is, deferoxamine, catechin, and glutathione ethyl ester. Each cow had 1 quarter infused with saline and 1 quarter infused with the selected modulator; a third quarter was infused with lipopolysaccharides (LPS), whereas the fourth quarter received a combination of LPS and the modulator. Infusion of LPS caused acute mastitis as determined by visual observations and by large increases in milk somatic cell count, BSA, and proteolytic activity. These parameters were not affected by antioxidant administration. The extent of cell damage was evaluated by measuring milk levels of lactate dehydrogenase and N-acetyl-beta-D-glucosaminidase activity. Levels of these parameters were several times higher after LPS administration. Intramammary infusions of catechin or glutathione ethyl ester did not exert any protective effect, whereas infusion of deferoxamine, a chelator of iron, decreased milk lactate dehydrogenase and NA-Gase activity, suggesting a protective effect against neutrophil-induced damage. The protective effect of deferoxamine was also evidenced by a lower milk level of haptoglobin. The proteolytic activity of mastitic milk was not influenced by the presence of deferoxamine. Overall, our results suggest that local infusion of deferoxamine may be an effective tool to protect mammary tissue against neutrophil-induced oxidative stress during bovine mastitis. PMID:16960060

Lauzon, K; Zhao, X; Lacasse, P



Effect of ciprofloxacin on lethal and sublethal challenge with endotoxin and on early cytokine responses in a murine in vivo model  

Microsoft Academic Search

The influence of ciprofloxacin on immune responses has been suggested by results of in vitro and in vivo studies. This effect was studied using a murine model that measured mortality and early cytokine responses after challenge with endotoxin. C57\\/BL6mice weighing between 18 and 21 g were given a single intraperitoneal dose of lipopolysaccharide (LPS), ranging from 200 to 1000 µ

Murli U. Purswani; Susan J. Eckert; Harman K. Arora; Gary J. Noel


Recombinant Interleukin-1 Alpha and Recombinant Tumor Necrosis Factor Alpha Synergize In vivo to Induce Early Endotoxin Tolerance and Associated Hematopoietic Changes,  

National Technical Information Service (NTIS)

Endotoxin, the lipopolysaccharide (LPS) derived from gram-negative bacteria, invokes a wide range of responses in susceptible hosts. It is known that virtually all responses to LPS are mediated by the action of macrophage-derived cytokines (such as interl...

E. N. Kaufman M. D. Tate R. Neta S. N. Vogel



Enhanced Resistance of Gastric Mucosa to Damaging Agents in the Rat Stomach Adapted to Helicobacter pylori Lipopolysaccharide  

Microsoft Academic Search

Background and Aim: Lipopolysaccharide (LPS) has been proposed to act as one of numerous virulence factors in the Helicobacter pylori (HP)-infected stomach. However, little is known as to whether the gastric mucosa can withstand the repeated LPS insult, and how the possible adaptation to this endotoxin influences the damage induced by strong irritants. We determined the effect of a single

Tomasz Brzozowski; Peter C. Konturek; Anthony P. Moran; Slawomir Kwiecien; Robert Pajdo; Stanislaw J. Konturek; Danuta Drozdowicz; Agata Ptak; Wieslaw Pawlik; Eckhart G. Hahn



Performance, serum biochemical responses, and gene expression of intestinal folate transporters of young and older laying hens in response to dietary folic acid supplementation and challenge with Escherichia coli lipopolysaccharide.  


The present study was conducted to investigate the effects of dietary folic acid (FA) supplementation on performance, serum biochemical indices, and mRNA abundance of intestinal folate transporters in young and older laying hens after acute lipopolysaccharide (LPS) challenge. Two experiments were conducted separately involving 48 Shaver White young laying hens (24 wk of age) in experiment 1 and 48 Shaver White older laying hens (58 wk of age) in experiment 2. Birds were fed 2 diets in a complete randomized design. The diets were wheat-soybean meal based, with or without supplemental 4 mg of FA/kg of diet. Birds were fed for 8 wk, during which time feed consumption and egg production were monitored. At the end of each feeding experiment, 6 hens from each dietary treatment were injected intravenously with 8 mg/kg of BW of either Escherichia coli LPS or sterile saline. Four hours after injection, blood and intestinal samples were collected for further analysis. Compared with the control, dietary FA supplementation increased egg weight and egg mass and decreased serum glucose levels in the young laying hens, and reduced serum uric acid in the older laying hens (P < 0.05). Relative to saline injection, plasma homocysteine, serum calcium, and phosphorus levels were found to be lower in both young and older laying hens after LPS challenge (P < 0.05). Other serum biochemical variables and the mRNA expression of 2 folate transport genes in the small and large intestine were differentially affected by LPS challenge, and some of those responses varied with the age of the birds. Additionally, interactions between diet and LPS challenge were specifically found in the older laying hens. In summary, in addition to improving production performance, there were effects of dietary FA supplementation and its interaction with LPS challenge on biochemical constituents, and age played a role in the development of responses to diet and bacterial LPS infections. PMID:24570431

Jing, M; Munyaka, P M; Tactacan, G B; Rodriguez-Lecompte, J C; O, K; House, J D



Domain Organization of the Polymerizing Mannosyltransferases Involved in Synthesis of the Escherichia coli O8 and O9a Lipopolysaccharide O-antigens*  

PubMed Central

The Escherichia coli O9a and O8 polymannose O-polysaccharides (O-PSs) serve as model systems for the biosynthesis of bacterial polysaccharides by ATP-binding cassette transporter-dependent pathways. Both O-PSs contain a conserved primer-adaptor domain at the reducing terminus and a serotype-specific repeat unit domain. The repeat unit domain is polymerized by the serotype-specific WbdA mannosyltransferase. In serotype O9a, WbdA is a bifunctional ?-(1?2)-, ?-(1?3)-mannosyltransferase, and its counterpart in serotype O8 is trifunctional (?-(1?2), ?-(1?3), and ?-(1?2)). Little is known about the detailed structures or mechanisms of action of the WbdA polymerases, and here we establish that they are multidomain enzymes. WbdAO9a contains two separable and functionally active domains, whereas WbdAO8 possesses three. In WbdCO9a and WbdBO9a, substitution of the first Glu of the EX7E motif had detrimental effects on the enzyme activity, whereas substitution of the second had no significant effect on activity in vivo. Mutation of the Glu residues in the EX7E motif of the N-terminal WbdAO9a domain resulted in WbdA variants unable to synthesize O-PS. In contrast, mutation of the Glu residues in the motif of the C-terminal WbdAO9a domain generated an enzyme capable of synthesizing an altered O-PS repeat unit consisting of only ?-(1?2) linkages. In vitro assays with synthetic acceptors unequivocally confirmed that the N-terminal domain of WbdAO9a possesses ?-(1?2)-mannosyltransferase activity. Together, these studies form a framework for detailed structure-function studies on individual domains and a strategy applicable for dissection and analysis of other multidomain glycosyltransferases.

Greenfield, Laura K.; Richards, Michele R.; Vinogradov, Evgeny; Wakarchuk, Warren W.; Lowary, Todd L.; Whitfield, Chris



Lipopolysaccharide O antigen size distribution is determined by a chain extension complex of variable stoichiometry in Escherichia coli O9a.  


The lengths of bacterial polysaccharides can be critical for their biological function. Unlike DNA or protein synthesis, where polymer length is implicit in the nucleic acid template, the molecular mechanisms for regulating polysaccharide length are poorly understood. Two models are commonly cited: a "molecular clock" regulates length by controlling the duration of the polymer extension process, whereas a "molecular ruler" determines length by measurement against a physical structure in the biosynthetic complex. Escherichia coli O9a is a prototype for the biosynthesis of O polysaccharides by ATP-binding cassette transporter-dependent processes. The length of the O9a polysaccharide is determined by two proteins: an extension enzyme, WbdA, and a termination enzyme, WbdD. WbdD is known to self-oligomerize and also to interact with WbdA. Changing either enzyme's concentration can alter the polysaccharide length. We quantified the O9a polysaccharide length distribution and the enzyme concentration dependence in vivo, then made mathematical models to predict the polymer length distributions resulting from hypothetical length-regulation mechanisms. Our data show qualitative features that cannot be explained by either a molecular clock or a molecular ruler model. Therefore, we propose a "variable geometry" model, in which a postulated biosynthetic WbdA-WbdD complex assembles with variable stoichiometry dependent on relative enzyme concentration. Each stoichiometry produces polymers with a distinct, geometrically determined, modal length. This model reproduces the enzyme concentration dependence and modality of the observed polysaccharide length distributions. Our work highlights limitations of previous models and provides new insight into the mechanisms of length control in polysaccharide biosynthesis. PMID:24733938

King, Jerry D; Berry, Scott; Clarke, Bradley R; Morris, Richard J; Whitfield, Chris



Determination of endotoxin levels and their impact on interleukin-1 generation in continuous ambulatory peritoneal dialysis and hemodialysis.  


Endotoxins represent a family of ubiquitous bacterial lipopolysaccharides found in water and raw materials. These substances have the ability to generate interleukin-1 (IL-1) and induce fever, as well as other acute phase phenomena. A study was undertaken to determine levels of background endotoxin in (1) continuous ambulatory peritoneal dialysis solution, (2) spent dialysate subsequent to overnight dwell, (3) hemodialysis solution, and (4) Limulus amebocyte lysate-reactive material (LAL-RM) in hemodialyzers and patient plasma. Levels of endotoxin in all of the above cases were less than thought to be required to induce biological activity, such as pyrogenicity, through IL-1 generation. Although nanogram amounts of LAL-RM are associated with some hollow-fiber membranes as well as the plasma of patients on those membranes, this material per se does not appear to produce IL-1 in vitro. PMID:3293624

Pearson, F C; Dubczak, J; Weary, M; Anderson, J



Immobilization of ?-polylysine onto the probe surface for molecular adsorption type endotoxin detection system  

NASA Astrophysics Data System (ADS)

Patients with renal failure become not able to expel the waste product, and they accumulate the toxic products for themselves. They therefore must use the hemodialysis to weed out the metabolic decomposition product. Hemodialysis for chronic renal failure is the most popular treatment method with artificial organs. However, hemodialysis patients must continue the treatment throughout their life, the results of long term extracorporeal dialysis, those patients develop the various complications and diseases, for example, dialysis amyloidosis etc. Dialysis amyloidosis is one of the refractory complications, and the prevention of this complication is important. Recently, endotoxin is thought to be the most likely cause of the complication. Endotoxin is one of the major cell wall components of gram-negative bacteria, and it forms the complex with proteins and lipopolysaccharide (LPS). It has various biological activities, e.g. attack of fever, when it gets mixed into human blood. In addition, it is known that large amount of endotoxin exists in living environment, and medicine is often contaminated with endotoxin. When contaminated dialyzing fluids are used to hemodialysis, above-mentioned dialysis amyloidosis is developed. Therefore, it is important that the detection and removal of endotoxin from dialyzing fluids. Until now, the measurement methods using Limulus Amebosyte Lysate (LAL) reagent were carried out as the tests for the presence of endotoxin. However, these methods include several different varieties of measurement techniques. The following are examples of them, gelatinization method, turbidimetric assay method, colorimetric assay method and fluoroscopic method. However, these techniques needed 30-60 minutes for the measurement. From these facts, they are not able to use as a "real-time endotoxin detector". The detection of endotoxin has needed to carry out immediately, for that reason, a new "real-time" detection method is desired. We focused attention to adsorption reaction between ?-polylysine and endotoxin. ?-polylysine has the structure of straight chain molecule composed by 25-30 residues made by lysine, and it is used as an antimicrobial agent, moreover, cellulose beads with immobilized ?-polylysine is used as the barrier filter for endotoxin removal. Therefore, it is expected that the endotoxin be adsorbed to the immobilized ?-polylysine onto the probe. As the result of this reaction, the mass of the probe is increased, and endotoxin can be detected by using of Quartz Crystal Microbalance (QCM). In our previous research, we have already acquired the proteins immobilization technique onto Au and Si surface. In this report, the proposal of molecular adsorption type endotoxin detection system, and the immobilization of ?-polylysine onto the probe are described. We use X-ray Photoelectron Spectroscopy (XPS) to confirm the ?-polylysine immobilization, and the adsorptive activity of immobilized ?-polylysine is measured by XPS and AFM. The purpose of this study is to bring about the realization of "Real-time endotoxin detection system".

Ooe, Katsutoshi; Tsuji, Akihito; Nishishita, Naoki; Hirano, Yoshiaki



Domain organization of the polymerizing mannosyltransferases involved in synthesis of the Escherichia coli O8 and O9a lipopolysaccharide O-antigens.  


The Escherichia coli O9a and O8 polymannose O-polysaccharides (O-PSs) serve as model systems for the biosynthesis of bacterial polysaccharides by ATP-binding cassette transporter-dependent pathways. Both O-PSs contain a conserved primer-adaptor domain at the reducing terminus and a serotype-specific repeat unit domain. The repeat unit domain is polymerized by the serotype-specific WbdA mannosyltransferase. In serotype O9a, WbdA is a bifunctional ?-(1?2)-, ?-(1?3)-mannosyltransferase, and its counterpart in serotype O8 is trifunctional (?-(1?2), ?-(1?3), and ?-(1?2)). Little is known about the detailed structures or mechanisms of action of the WbdA polymerases, and here we establish that they are multidomain enzymes. WbdA(O9a) contains two separable and functionally active domains, whereas WbdA(O8) possesses three. In WbdC(O9a) and WbdB(O9a), substitution of the first Glu of the EX(7)E motif had detrimental effects on the enzyme activity, whereas substitution of the second had no significant effect on activity in vivo. Mutation of the Glu residues in the EX(7)E motif of the N-terminal WbdA(O9a) domain resulted in WbdA variants unable to synthesize O-PS. In contrast, mutation of the Glu residues in the motif of the C-terminal WbdA(O9a) domain generated an enzyme capable of synthesizing an altered O-PS repeat unit consisting of only ?-(1?2) linkages. In vitro assays with synthetic acceptors unequivocally confirmed that the N-terminal domain of WbdA(O9a) possesses ?-(1?2)-mannosyltransferase activity. Together, these studies form a framework for detailed structure-function studies on individual domains and a strategy applicable for dissection and analysis of other multidomain glycosyltransferases. PMID:22989876

Greenfield, Laura K; Richards, Michele R; Vinogradov, Evgeny; Wakarchuk, Warren W; Lowary, Todd L; Whitfield, Chris



Comparative Analysis of Hepatic CD14 Expression between Two Different Endotoxin Shock Model Mice: Relation between Hepatic Injury and CD14 Expression  

PubMed Central

CD14 is a glycoprotein that recognizes gram-negative bacterial lipopolysaccharide (LPS) and exists in both membrane-bound and soluble forms. Infectious and/or inflammatory diseases induce CD14 expression, which may be involved in the pathology of endotoxin shock. We previously found that the expression of CD14 protein differs among the endotoxin shock models used, although the reasons for these differences are unclear. We hypothesized that the differences in CD14 expression might be due to liver injury, because the hepatic tissue produces CD14 protein. We investigated CD14 expression in the plasma and liver in the carrageenan (CAR)-primed and D-galN-primed mouse models of endotoxin shock. Our results showed that severe liver injury was not induced in CAR-primed endotoxin shock model mice. In this CAR-primed model, the higher mRNA and protein expression of CD14 was observed in the liver, especially in the interlobular bile duct in contrast to D-galN-primed-endotoxin shock model mice. Our findings indicated that the molecular mechanism(s) underlying septic shock in CAR-primed and D-galN-primed endotoxin shock models are quite different. Because CD14 expression is correlated with clinical observations, the CAR-primed endotoxin shock model might be useful for studying the functions of CD14 during septic shock in vivo.

Hozumi, Hiroyasu; Tada, Rui; Murakami, Taisuke; Adachi, Yoshiyuki; Ohno, Naohito



Enhanced Phagocytosis and Bactericidal Activity of Hepatic Reticuloendothelial System During Endotoxin Tolerance  

PubMed Central

The effects of tolerance to Escherichia coli endotoxin on the phagocytic and bactericidal activity of the hepatic reticuloendothelial system against viable E. coli were examined using ex vivo perfused rat livers. Livers were isolated from control and endotoxin-tolerant rats and perfused with a medium containing 5% homologous serum from either control or tolerant rats. After the addition of the E. coli (2 × 107 cells per ml) to the perfusate, the hepatic clearance of the bacteria was followed for 30 min. The highest activation of the hepatic reticuloendothelial system was observed when serum from tolerant animals was added to the perfusate. Under these conditions phagocytosis was 47% (12% in controls), and 37 to 38% of the bacteria were killed (5% in controls). This activation was less when livers obtained from tolerant rats were perfused with serum from controls or with saline only. The data suggests that, during endotoxin tolerance, humoral factors play an important role in the activation of the hepatic reticulendothelial system, although a direct stimulation of Kupffer cells also occurs. The enhancement of phagocytosis by tolerant serum did not require the presence of homologous antibodies and involved the activation of the alternative complement pathway, since it was lost after removal of factor B activity. On the other hand, stimulation of intracellular killing required both complement and specific antibodies. The data suggest a role of endotoxin in the activation of humoral and cellular mechanisms involved in the host resistance to gram-negative bacterial infection.

Ruggiero, Giuseppe; Andreana, Augusto; Utili, Riccardo; Galante, Domenico



Deletion of the Ron receptor tyrosine kinase domain in mice provides protection from endotoxin-induced acute liver failure  

Microsoft Academic Search

The targeted deletion of the cytoplasmic domain of the Ron receptor tyrosine kinase (TK) in mice leads to exaggerated responses to injury in several murine models of inflammation as well as increased lethality in response to endotoxin (lipopolysaccharide [LPS]). Using a well-characterized model of LPS-induced acute liver failure (ALF) in galactosamine (GalN)-sensitized mice, we show that Ron TK?\\/? mice display

Mike A. Leonis; Sandra J. F. Degen; Susan E. Waltz



Cellular Localisation and Dynamics of Nitric Oxide Synthase Expression in the Rat Anterior Segment during Endotoxin-Induced Uveitis  

Microsoft Academic Search

The present study examined the temporal pattern and cellular localisation of nitric oxide synthase in Endotoxin-Induced Uveitis (EIU). Lewis rats (n=40) received a single footpad injection of 200 ?g of bacterial lipopolysaccharide. Animals were killed at 0, 2, 4, 6, 12, 24, 48 and 72 hr after injection and ocular tissues prepared as iris-ciliary body wholemounts or frozen sections of




Contribution of CD14 to Endotoxin-Induced Liver Injury May Depend on Types of Macrophage Activation in Rats  

Microsoft Academic Search

Activated Kupffer cells and hepatic macrophages can produce massive liver necrosis through microcirculatory disturbance due to sinusoidal fibrin deposition. This mechanism is involved in the development of liver injury after endotoxin administration in rats pretreated with heat-killedPropionibacterium acnes(P.acnes) or undergoing 70% liver resection. The significance of CD14, a receptor for lipopolysaccharide and its binding protein, was evaluated in both models

Keiko Toshima; Satoshi Mochida; Keiko Ishikawa; Atsushi Matsui; Masahiro Arai; Itsuro Ogata; Kenji Fujiwara



DNA microarray analysis of gene expression in iris and ciliary body of rat eyes with endotoxin-induced uveitis  

Microsoft Academic Search

The purposes of this study are to determine the genes that are up- or down-regulated in eyes with endotoxin-induced uveitis (EIU) by an oligonucleotide microarray system, and to determine the temporal and spatial changes in expression of selected genes that show strong up-regulation. EIU was induced by a footpad injection of lipopolysaccharide (LPS) in male Lewis rats. The expression of

Kouichi Ohta; Takanobu Kikuchi; Teruyoshi Miyahara; Nagahisa Yoshimura



Biophysical Mechanisms of the Neutralization of Endotoxins by Lipopolyamines  

PubMed Central

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.

Sil, Diptesh; Heinbockel, Lena; Kaconis, Yani; Rossle, Manfred; Garidel, Patrick; Gutsmann, Thomas; David, Sunil A; Brandenburg, Klaus



Arginase Activity Mediates Retinal Inflammation in Endotoxin-Induced Uveitis  

PubMed Central

Arginase has been reported to reduce nitric oxide bioavailability in cardiovascular disease. However, its specific role in retinopathy has not been studied. In this study, we assessed the role of arginase in a mouse model of endotoxin-induced uveitis induced by lipopolysaccharide (LPS) treatment. Measurement of arginase expression and activity in the retina revealed a significant increase in arginase activity that was associated with increases in both mRNA and protein levels of arginase (Arg)1 but not Arg2. Immunofluorescence and flow cytometry confirmed this increase in Arg1, which was localized to glia and microglia. Arg1 expression and activity were also increased in cultured Muller cells and microglia treated with LPS. To test whether arginase has a role in the development of retinal inflammation, experiments were performed in mice deficient in one copy of the Arg1 gene and both copies of the Arg2 gene or in mice treated with a selective arginase inhibitor. These studies showed that LPS-induced increases in inflammatory protein production, leukostasis, retinal damage, signs of anterior uveitis, and uncoupling of nitric oxide synthase were blocked by either knockdown or inhibition of arginase. Furthermore, the LPS-induced increase in Arg1 expression was abrogated by blocking NADPH oxidase. In conclusion, these studies suggest that LPS-induced retinal inflammation in endotoxin-induced uveitis is mediated by NADPH oxidase-dependent increases in arginase activity.

Zhang, Wenbo; Baban, Babak; Rojas, Modesto; Tofigh, Sohrab; Virmani, Suvika K.; Patel, Chintan; Behzadian, M. Ali; Romero, Maritza J.; Caldwell, Robert W.; Caldwell, Ruth B.



Piceatannol suppresses endotoxin-induced ocular inflammation in rats.  


Anti-inflammatory effect of piceatannol, a naturally occurring polyphenol and a potent free radical scavenger, on ocular inflammation is not known. We examined the anti-inflammatory role of piceatannol in ocular inflammatory response due to endotoxin-induced uveitis (EIU) in rats. EIU was induced in Lewis rats by subcutaneous injection of lipopolysaccharide (LPS; 150 ug/rat). Piceatannol (30mg/kg body wt, i.p) was injected either 2h prior to or 1h post LPS induction. A significant increase in the number of infiltrating cells, total protein, and various cytokines and chemokines in AqH were observed in the EIU rat eyes as compared to control groups. However, pre- or post-treatment of piceatannol significantly blocked the LPS-induced changes. Further, piceatannol also suppressed the expression of cyclooxygenase-2 (Cox-2), inducible nitric oxide synthase (iNOS) and activation of NF-?B in the ciliary bodies as well as retina. Further, piceatannol also inhibited the expression of Cox-2, iNOS, and phosphorylation of NF-?B in primary human non-pigmented ciliary epithelial cells (HNPECs) treated with LPS. Similarly, piceatannol also diminished LPS-induced level of NO and prostaglandin E2 in HNPECs. Thus our results demonstrate an anti-inflammatory role of piceatannol in suppressing ocular inflammation induced by endotoxin in rats. PMID:23892029

Kalariya, Nilesh M; Shoeb, Mohammad; Reddy, Aramati B M; Sawhney, Rahul; Ramana, Kota V



[Blood oxygen transport, prooxidant -- antioxidant status, and vasoactive characteristics of vascular endothelium in rats treated with endotoxin and taurine].  


Experiments on a group of 74 pregnant rats upon intramuscular introduction of E. coli lipopolysaccharides during pregnancy revealed the correction effect of taurine on the blood oxygen transport function, prooxidant - antioxidant status, and vasoactive characteristics of vascular endothelium. PMID:24800521

Milosh, T S; Maksimovich, N E



Mifepristone (RU486) restores humoral and T cell-mediated immune response in endotoxin immunosuppressed mice  

PubMed Central

Sepsis and septic shock can be caused by Gram-positive and -negative bacteria and other microorganisms. In the case of Gram-negative bacteria, endotoxin, a normal constituent of the bacterial wall, also known as lipopolysaccharide (LPS), has been considered as one of the principal agents causing the undesirable effects in this critical illness. The response to LPS involves a rapid secretion of proinflammatory cytokines such as tumour necrosis factor (TNF)-?, interleukin (IL)-1, IL-6, interferon (IFN)-? and the concomitant induction of anti-inflammatory mediators such as IL-10, transforming growth factor (TGF)-? or glucocorticoids, which render the host temporarily refractory to subsequent lethal doses of LPS challenge in a process known as LPS or endotoxin tolerance. Although protective from the development of sepsis or systemic inflammation, endotoxin tolerance has also been pointed out as the main cause of the non-specific humoral and cellular immunosuppression described in these patients. In this report we demonstrate, using a mouse model, that mifepristone (RU486), a known glucocorticoid receptor antagonist, could play an important role in the restoration of both adaptive humoral and cellular immune response in LPS immunosuppressed mice, suggesting the involvement of endogenous glucocorticoids in this phenomenon. On the other hand, using cyclophosphamide and gemcitabine, we demonstrated that regulatory/suppressor CD4+CD25+forkhead boxP3+ and GR-1+CD11b+ cells do not play a major role in the establishment or the maintenance of endotoxin tolerance, a central mechanism for inducing an immunosuppression state.

Rearte, B; Maglioco, A; Balboa, L; Bruzzo, J; Landoni, V I; Laborde, E A; Chiarella, P; Ruggiero, R A; Fernandez, G C; Isturiz, M A



Network Topologies and Dynamics Leading to Endotoxin Tolerance and Priming in Innate Immune Cells  

PubMed Central

The innate immune system, acting as the first line of host defense, senses and adapts to foreign challenges through complex intracellular and intercellular signaling networks. Endotoxin tolerance and priming elicited by macrophages are classic examples of the complex adaptation of innate immune cells. Upon repetitive exposures to different doses of bacterial endotoxin (lipopolysaccharide) or other stimulants, macrophages show either suppressed or augmented inflammatory responses compared to a single exposure to the stimulant. Endotoxin tolerance and priming are critically involved in both immune homeostasis and the pathogenesis of diverse inflammatory diseases. However, the underlying molecular mechanisms are not well understood. By means of a computational search through the parameter space of a coarse-grained three-node network with a two-stage Metropolis sampling approach, we enumerated all the network topologies that can generate priming or tolerance. We discovered three major mechanisms for priming (pathway synergy, suppressor deactivation, activator induction) and one for tolerance (inhibitor persistence). These results not only explain existing experimental observations, but also reveal intriguing test scenarios for future experimental studies to clarify mechanisms of endotoxin priming and tolerance.

Fu, Yan; Glaros, Trevor; Zhu, Meng; Wang, Ping; Wu, Zhanghan; Tyson, John J.; Li, Liwu; Xing, Jianhua



Preparation, characterization, and immunological properties in mice of Escherichia coli O157 O-specific polysaccharide-protein conjugate vaccines.  

PubMed Central

Escherichia coli O157 causes severe enteritis and the extraintestinal complication of hemolytic-uremic syndrome, with their highest incidence occurring in children. We postulated that serum immunoglobulin G (IgG) antibodies to the O-specific polysaccharide of lipopolysaccharide (LPS) may confer protective immunity to enteric pathogens by inducing bactericidal reactions against the ingested organisms in the jejunum (J. B. Robbins, C. Chu, and R. Schneerson, Clin. Infect. Dis. 15:346-361, 1992; S. C. Szu, R. Gupta, and J. B. Robbins, p. 381-394, in I. K. Wachsmuth, P. A. Blake, and O. Olsvik, ed., Vibrio cholerae, 1994). Because polysaccharide-protein conjugates induce serum IgG antibodies in infants, we bound the O-specific polysaccharide of E. coli O157 to proteins. E. coli O157 LPS, treated with acetic acid or hydrazine, was derivatized with adipic acid dihydrazide and bound to proteins by carbodiimide-mediated condensation. Conjugates of these adipic hydrazide derivative were prepared with bovine serum albumin, formalin-treated exotoxin C of Clostridium welchii (Pig Bel toxoid), or Pseudomonas aeruginosa recombinant exoprotein A. The conjugates had low levels of endotoxin and elicited serum antibodies with bactericidal activity to the O157 LPS. The largest increase in LPS antibodies was of the IgG class. Clinical evaluation of E. coli O157-toxoid conjugates is planned. Images

Konadu, E; Robbins, J B; Shiloach, J; Bryla, D A; Szu, S C



Preparation, characterization, and immunological properties in mice of Escherichia coli O157 O-specific polysaccharide-protein conjugate vaccines.  


Escherichia coli O157 causes severe enteritis and the extraintestinal complication of hemolytic-uremic syndrome, with their highest incidence occurring in children. We postulated that serum immunoglobulin G (IgG) antibodies to the O-specific polysaccharide of lipopolysaccharide (LPS) may confer protective immunity to enteric pathogens by inducing bactericidal reactions against the ingested organisms in the jejunum (J. B. Robbins, C. Chu, and R. Schneerson, Clin. Infect. Dis. 15:346-361, 1992; S. C. Szu, R. Gupta, and J. B. Robbins, p. 381-394, in I. K. Wachsmuth, P. A. Blake, and O. Olsvik, ed., Vibrio cholerae, 1994). Because polysaccharide-protein conjugates induce serum IgG antibodies in infants, we bound the O-specific polysaccharide of E. coli O157 to proteins. E. coli O157 LPS, treated with acetic acid or hydrazine, was derivatized with adipic acid dihydrazide and bound to proteins by carbodiimide-mediated condensation. Conjugates of these adipic hydrazide derivative were prepared with bovine serum albumin, formalin-treated exotoxin C of Clostridium welchii (Pig Bel toxoid), or Pseudomonas aeruginosa recombinant exoprotein A. The conjugates had low levels of endotoxin and elicited serum antibodies with bactericidal activity to the O157 LPS. The largest increase in LPS antibodies was of the IgG class. Clinical evaluation of E. coli O157-toxoid conjugates is planned. PMID:7927787

Konadu, E; Robbins, J B; Shiloach, J; Bryla, D A; Szu, S C



Cytokine Inducing Activities of Rhizobial and Mesorhizobial Lipopolysaccharides of Different Lethal Toxicity  

Microsoft Academic Search

The lethality and cytokines-inducing activity of lipopolysaccharides (LPS) obtained from nodulating bacteria, Rhizobium leguminosarum and Mesorhizobium loti, were compared to those of Salmonella typhimurium LPSThe activity of Rleguminosarum LPS was almost comparable to Salmonella endotoxin in terms of lethality, Limulus lysate gelating activity and in vivo tumor necrosis factor-? (TNF-?), interleukin-1ß (IL-lß), interleukin-6 (IL-6) and interferon-? (IFN-?) induction capacityIn contrast

T. Urbanik-Sypniewska; A. Choma; J. Kutkowska; T. KamiŃska; M. Kandefer-SzerszeŃ; R. Russa; J. Dolecka



Effects of lipopolysaccharide treatment on feeding of goldfish: role of appetite-regulating peptides  

Microsoft Academic Search

The gram-negative bacteria-derived endotoxin lipopolysaccharide (LPS) is known to play an important role in immune and neurological manifestations during bacterial infections. In mammals, peripheral or brain administration of LPS induces anorexia and is thought to exert its effects through activation of pro-inflammatory cytokines. In this study, we investigated the effects of peripheral (intraperitoneal, IP) and central (intracerebroventricular, ICV) injections of

Helene Volkoff; Richard Ector Peter



Lipopolysaccharide-binding protein: localization in secretory granules of Paneth cells in the mouse small intestine  

Microsoft Academic Search

Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase protein involved in the host’s response to endotoxin and\\u000a mainly synthesized and secreted to the blood by the liver. But in addition, LBP is also made by extrahepatic cells, including\\u000a the enterocyte-like cell line Caco-2. To study in closer detail the synthesis and storage of LBP in the intestinal mucosal\\u000a epithelium, we performed

Gert H. Hansen; Karina Rasmussen; Lise-Lotte Niels-Christiansen; E. Michael Danielsen



Kinetics of lipopolysaccharide clearance by Kupffer and parenchyma cells in perfused rat liver  

Microsoft Academic Search

We studied the kinetics of [3H]lipopolysaccharide ([3H]LPS) (endotoxin) binding to Kupffer cells and hepatocytes at the level of the microtubular system after treatment with gadolinium chloride (GdCl3) and colchicine. Liver perfusion in Sprague–Dawley rats involves both portal vein and thoracic inferior vena cava cannulations as inlet and outlet, respectively. The subhepatic inferior vena cava is ligated to prevent perfusate leakage.

Anwar B Bikhazi; Abdo R Jurjus; Maud T Kamal; Ali M Al-Housseini; Rola N Saab; Wael A Jaroudi; Khalil M Bitar



Effects of urethane, ambient temperature and injection route on rat body temperature and metabolism due to endotoxins.  

PubMed Central

1. We have investigated the influence of environmental temperature, anaesthesia and route of administration on rectal temperature and other metabolic responses to two preparations of bacterial endotoxin in male adult Wistar rats. 2. Urethane anaesthesia, environmental temperatures of 20 and 28 degrees C, subcutaneous (S.C.) and intraperitoneal (I.P.) routes of administration and butanol and trichloroacetic acid (TCA) extracts of E. coli endotoxin (1.2 mg/kg) were used. 3. In addition to rectal temperature, serum zinc, albumin and urea concentrations and liver protein, RNA and zinc contents were measured. 4. Fevers were produced by injections of both endotoxins, by either route at 28 degrees C. Butanol-extracted endotoxin produced a more rapid response than the TCA extract via the I.P. route whereas the TCA extract produced a higher temperature than the butanol extract when the S.C. route was used. 5. Fevers were inhibited at an environmental temperature of 20 degrees C and by anaesthesia, while the former had no effect on compositional changes the latter inhibited the fall in serum zinc in response to subcutaneous doses of either endotoxin and the increase in liver zinc content in response to the butanol extract of endotoxin. 6. At 20 degrees C a marked fall in rectal temperature occurred in conscious rats 2 h after receiving the TCA but not the butanol extract of endotoxin. Temperature depression was more severe when endotoxin was administered by the I.P. route. 7. Serum urea was elevated in conscious rats by the TCA extract of endotoxin via both routes but only by the I.P. route for the butanol extract of endotoxin. In anaesthetized animals only the TCA extract of endotoxin raised serum urea concentration when given intraperitoneally. 8. Serum albumin and liver protein and RNA were unaffected by endotoxin injections over the 7 h time course of the study. 9. Rectal temperature responses to endotoxins were influenced in direction and magnitude by all variables employed in the study, while compositional changes were unaffected by environmental temperature but influenced to varying degrees by urethane anaesthesia and the route of administration employed.

Bibby, D C; Grimble, R F



Endotoxin and nanobacteria in polycystic kidney disease  

Microsoft Academic Search

Endotoxin and nanobacteria in polycystic kidney disease.BackgroundMicrobes have been suspected as provocateurs of polycystic kidney disease (PKD), but attempts to isolate viable organisms have failed. Bacterial endotoxin is the most often reported microbial product found in PKD fluids. We assessed potential microbial origins of endotoxin in cyst fluids from 13 PKD patients and urines of PKD and control individuals.MethodsFluids were

J. Thomas Hjelle; Marcia A. Miller-Hjelle; Ian R. Poxton; E. Olavi Kajander; Neva Ciftcioglu; Monica L. Jones; Robert C. Caughey; Robert Brown; Paul D. Millikin; Frank S. Darras



The adsorption of endotoxin molecule in a microporous polyethylene hollow fibre membrane.  

PubMed Central

The microporous polyethylene hollow fibre membrane is capable of adsorbing small-sized lipopolysaccharides (LPS) prepared by sonication dispersion, column chromatography on Sephadex G-75 and filtration through a filter membrane with a nominal pore size of 0.025 micron. Small-sized LPS had a one-thousandth of endotoxin activity as compared to intact LPS, when determined by the Synthetic Chromogenic Substrate method of LAL with a specific endotoxin activity of 73.7 ng/micrograms LPS. Fluorescent microscopy of fluorescein conjugated LPS on a microporous polyethylene hollow fibre showed that fluorescein-LPS was adsorbed through the entire depth of the membrane texture. Accordingly the adsorption capacity of the filter for small-sized LPS was determined as 1.65 mg LPS/3.68 m2 surface/116 mg fibre/module. Images Fig. 5

Sawada, Y.; Fujii, R.; Igami, I.; Kawai, A.; Kamiki, T.; Niwa, M.



Inhibition of biological effects of endotoxins by phenothiazines.  


The importance of change transfer reaction involving endotoxins of bacteria and interactions between endotoxin-induced tumor necrosis factor (TNF) were investigated both in vitro and in vivo in the presence of several phenothiazines. Complex formation between endotoxins and ring-substituted phenothiazines, benzodiazepines, amantadine and promethazine was measured using spectrophotometric methods. The endotoxin-induced hypotensive effect was prevented by phenothiazine pretreatment of the dogs; however, the endotoxin-phenothiazine complex had the same effect as the endotoxin itself. The tumor necrosis factor-inducing ability of endotoxin in human monocytes was prevented by promethazine. The TNF induction by endotoxin was inhibited by promethazine in vivo. PMID:1525340

Molnár, J; Mandi, Y; Regely, K; Tarnoky, K; Nakamura, M J



Mosquitocidal activity of the CryIC delta-endotoxin from Bacillus thuringiensis subsp. aizawai.  

PubMed Central

The cloned 135-kDa CryIC delta-endotoxin from Bacillus thuringiensis is a lepidopteran-active toxin, displaying high activity in vivo against Spodoptera litoralis and Spodoptera frugiperda larvae and in vitro against the S. frugiperda Sf9 cell line. Here, we report that the CryIC delta-endotoxin cloned from B. thuringienesis subsp. aizawai HD-229 and expressed in an acrystalliferous B. thuringiensis strain is also toxic to Aedes aegypti, Anophles gambiae, and Culex quinquefasciatus mosquito larvae. Furthermore, when solubilized and proteolytically activated by insect gut extracts, CryIC is cytotoxic to cell lines derived from the first two of these dipteran insects. This activity was not observed for two other lepidopteran-active delta-endotoxins, CryIA(a) and CryIA(c). However, in contrast to the case with a lepidopteran and dipteran delta-endotoxin cloned from B. thuringiensis subsp. aizawai IC1 (M.Z. Haider, B. H. Knowles, and D. J. Ellar, Eur. J. Biochem. 156:531-540, 1986), no differences in the in vitro specificity or processing of CryIC were found when it was activated by lepidopteran or dipteran gut extract. The recombinant CryIC delta-endotoxin expressed in Escherichia coli was also toxic to A. aegypti larvae. By contrast, a second cryIC gene cloned from B. thuringiensis subsp. aizawai 7.29 (V. Sanchis, D. Lereclus, G. Menou, J. Chaufaux, S. Guo, and M. M. Lecadet, Mol. Microbiol. 3:229-238, 1989) was nontoxic. DNA sequencing showed that the two genes were identical. However, CryIC from B. thuringiensis subsp. aizawai 7.29 had been cloned with a truncated C terminus, and when it was compared with the full-length CryIC delta-endotoxin, it was found to be insoluble under alkaline reducing conditions. These results show that CryIC from B. thuringiensis subsp. aizawai is a dually active delta-endotoxin.

Smith, G P; Merrick, J D; Bone, E J; Ellar, D J



Bacterial endotoxins and nonspecific resistance.  


The stress situations, including medical intervention (e.g. operations, antitumor drugs, irradiation, etc.) decrease the nonspecific resistance of the body. In these situations patients people have greater chance to get an opportunistic infection than healthy ones. The restoration or elevation of the activity of immune system in injured patients is a very important task of medicine. Minute amounts of bacterial endotoxin (LPS)--given parenterally--can elevate the nonspecific resistance. Unfortunately this beneficial influence is associated with noxious properties. Irradiation (60 Co-gamma; 150 kGy) is a good technique for the detoxification of LPS. The radiodetoxified endotoxin (RD-LPS) preparation (so-called TOLERIN) is less toxic but its beneficial properties is preserved. On the basis of animal experiments and clinical trials TOLERIN could be a suitable preparation for regeneration of the lymphoreticular-immune system and elevation of nonspecific resistance. PMID:9554170

Bertók, L



Endotoxin-induced selective dysfunction of rabbit polymorphonuclear leukocytes in response to endogenous chemotactic factors.  

PubMed Central

To assess the mechanism and specificity of polymorphonuclear leukocyte (PMN) dysfunction induced by endotoxin, rabbits were injected intravenously with 100 micrograms of Escherichia coli endotoxin, and PMN function was studied 18 to 24 h later. Compared to PMN from normal rabbits, peripheral blood PMN from rabbits injected with endotoxin showed diminished chemotactic responsiveness to two endogenous peptides, C5a (complement) and platelet-derived growth factor, and to two endogenous lipids, leukotriene B4 and platelet-activating factor. The chemotactic response to the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), was unimpaired. In contrast to migration, endotoxin injection resulted in inhibition of the secretory response to the two endogenous peptides but not to the lipids or to FMLP. At a 1:4 (vol/vol) dilution, the plasma either 1 or 24 h after the endotoxin injection inhibited normal PMN chemotactic responses to C5a but not to FMLP. Similarly, at a 1:10 dilution, this plasma inhibited normal PMN chemotactic responses to leukotriene B4. The factor responsible for inhibiting responses to leukotriene B4 was anionic, specific for leukotriene B4 responses, and greater than 12,000 daltons. These data may be relevant to understanding PMN dysfunction during gram-negative sepsis.

Hartiala, K T; Langlois, L; Goldstein, I M; Rosenbaum, J T



The lipid A of Burkholderia multivorans C1576 smooth-type lipopolysaccharide and its pro-inflammatory activity in a cystic fibrosis airways model  

Microsoft Academic Search

Cystic fibrosis is an autosomal recessive disorder and it is characterised by chronic bacterial airway infection which leads to progressive lung deterioration, sometimes with fatal outcome. Burkholderia multivorans and Burkholderia cenocepacia are the species responsible for most of the infections of cystic fibrosis patients. Lipopolysaccharide endotoxins (LPSs) are among the foremost factors of pathogenesis of Gram-negative infection and, in particular,

Teresa Ieranň; Paola Cescutti; Maria Rosaria Leone; Alessandro Luciani; Roberto Rizzo; Valeria Raia; Rosa Lanzetta; Michelangelo Parrilli; Luigi Maiuri; Alba Silipo; Antonio Molinaro



Interaction of Antimicrobial Peptide Temporin L with Lipopolysaccharide In Vitro and in Experimental Rat Models of Septic Shock Caused by Gram-Negative Bacteria  

Microsoft Academic Search

Sepsis remains a major cause of morbidity and mortality in hospitalized patients, despite intense efforts to improve survival. The primary lead for septic shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the quest for compounds with antiendotoxin properties is actively pursued. We investigated the efficacy

Andrea Giacometti; Oscar Cirioni; Roberto Ghiselli; Federico Mocchegiani; Fiorenza Orlando; Carmela Silvestri; Argante Bozzi; A. Di Giulio; C. Luzi; M. L. Mangoni; D. Barra; V. Saba; G. Scalise; A. C. Rinaldi



In Vitro Evaluation of the Effects of Low-Intensity Nd:YAG Laser Irradiation on the Inflammatory Reaction Elicited by Bacterial Lipopolysaccharide Adherent to Titanium Dental Implants  

Microsoft Academic Search

Background: The bacterial endotoxin lipopolysaccharide (LPS) represents a prime pathogenic factor of peri-implantitis because of its ability to adhere tenaciously to dental titanium implants. Despite this, the current therapeutic approach to this disease remains based mainly on bacterial decontamina- tion, paying little attention to the neutralization of bioactive bacterial products. The purpose of the present study was to evaluate whether

Marco Giannelli; Daniele Bani; Alessia Tani; Alessandro Pini; Martina Margheri; Sandra Zecchi-Orlandini; Paolo Tonelli; Lucia Formigli



Increased responsiveness to intravenous lipopolysaccharide challenge in steers grazing endophyte-infected tall fescue compared with steers grazing endophyte-free tall fescue  

Microsoft Academic Search

Fescue toxicosis in cattle occurs as a result of consump- tion of ergot alkaloids in endophyte-infected (E+, Neotyphodium coenophialum) tall fescue (Festuca arundinacea). The condition is characterized by pyrexia, decreased weight gains, rough hair coats, and decreased calving rates. The objective of this experiment was to investigate whether steers grazing E+ fescue have altered host response to lipopolysaccharide (endotoxin, LPS)

N M Filipov; F N Thompson; J A Stuedemann; T H Elsasser; S Kahl; R P Sharma; L H Stanker; C K Smith



Inhibition of IRAK-4 activity for rescuing endotoxin LPS-induced septic mortality in mice by lonicerae flos extract  

SciTech Connect

Highlights: •Lonicerae flos extract (HS-23) is a clinical candidate, Phase I for sepsis treatment. •Here, HS-23 or its major constituents rescued LPS-induced septic mortality in mice. •As a mechanism, they directly inhibited IRAK-4-catalyzed kinase activity. •Thus, they suppressed LPS-induced expression of NF-?B/AP-1-target inflammatory genes. -- Abstract: Lonicerae flos extract (HS-23) is a clinical candidate currently undergoing Phase I trial in lipopolysaccharide (LPS)-injected healthy human volunteers, but its molecular basis remains to be defined. Here, we investigated protective effects of HS-23 or its major constituents on Escherichia coli LPS-induced septic mortality in mice. Intravenous treatment with HS-23 rescued LPS-intoxicated C57BL/6J mice under septic conditions, and decreased the levels of cytokines such as tumor necrosis factor ? (TNF-?), interleukin (IL)-1? and high-mobility group box-1 (HMGB-1) in the blood. Chlorogenic acid (CGA) and its isomers were assigned as major constituents of HS-23 in the protection against endotoxemia. As a molecular mechanism, HS-23 or CGA isomers inhibited endotoxin LPS-induced autophosphorylation of the IL-1 receptor-associated kinase 4 (IRAK-4) in mouse peritoneal macrophages as well as the kinase activity of IRAK-4 in cell-free reactions. HS-23 consequently suppressed downstream pathways critical for LPS-induced activation of nuclear factor (NF)-?B or activating protein 1 (AP-1) in the peritoneal macrophages. HS-23 also inhibited various toll-like receptor agonists-induced nitric oxide (NO) production, and down-regulated LPS-induced expression of NF-?B/AP-1-target inflammatory genes in the cells. Taken together, HS-23 or CGA isomers exhibited anti-inflammatory therapy against LPS-induced septic mortality in mice, at least in part, mediated through the inhibition of IRAK-4.

Park, Sun Hong; Roh, Eunmiri [College of Pharmacy, Chungbuk National University, Cheongju 361-763 (Korea, Republic of)] [College of Pharmacy, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Kim, Hyun Soo [Pharmaceutical R and D Center, Huons Co., Ltd., Anyang (Korea, Republic of)] [Pharmaceutical R and D Center, Huons Co., Ltd., Anyang (Korea, Republic of); Baek, Seung-Il [College of Pharmacy, Chungbuk National University, Cheongju 361-763 (Korea, Republic of)] [College of Pharmacy, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Choi, Nam Song [Pharmaceutical R and D Center, Huons Co., Ltd., Anyang (Korea, Republic of)] [Pharmaceutical R and D Center, Huons Co., Ltd., Anyang (Korea, Republic of); Kim, Narae; Hwang, Bang Yeon; Han, Sang-Bae [College of Pharmacy, Chungbuk National University, Cheongju 361-763 (Korea, Republic of)] [College of Pharmacy, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Kim, Youngsoo, E-mail: [College of Pharmacy, Chungbuk National University, Cheongju 361-763 (Korea, Republic of)] [College of Pharmacy, Chungbuk National University, Cheongju 361-763 (Korea, Republic of)



Inhibition of IRAK-4 activity for rescuing endotoxin LPS-induced septic mortality in mice by lonicerae flos extract.  


Lonicerae flos extract (HS-23) is a clinical candidate currently undergoing Phase I trial in lipopolysaccharide (LPS)-injected healthy human volunteers, but its molecular basis remains to be defined. Here, we investigated protective effects of HS-23 or its major constituents on Escherichia coli LPS-induced septic mortality in mice. Intravenous treatment with HS-23 rescued LPS-intoxicated C57BL/6J mice under septic conditions, and decreased the levels of cytokines such as tumor necrosis factor ? (TNF-?), interleukin (IL)-1? and high-mobility group box-1 (HMGB-1) in the blood. Chlorogenic acid (CGA) and its isomers were assigned as major constituents of HS-23 in the protection against endotoxemia. As a molecular mechanism, HS-23 or CGA isomers inhibited endotoxin LPS-induced autophosphorylation of the IL-1 receptor-associated kinase 4 (IRAK-4) in mouse peritoneal macrophages as well as the kinase activity of IRAK-4 in cell-free reactions. HS-23 consequently suppressed downstream pathways critical for LPS-induced activation of nuclear factor (NF)-?B or activating protein 1 (AP-1) in the peritoneal macrophages. HS-23 also inhibited various toll-like receptor agonists-induced nitric oxide (NO) production, and down-regulated LPS-induced expression of NF-?B/AP-1-target inflammatory genes in the cells. Taken together, HS-23 or CGA isomers exhibited anti-inflammatory therapy against LPS-induced septic mortality in mice, at least in part, mediated through the inhibition of IRAK-4. PMID:24269819

Park, Sun Hong; Roh, Eunmiri; Kim, Hyun Soo; Baek, Seung-Il; Choi, Nam Song; Kim, Narae; Hwang, Bang Yeon; Han, Sang-Bae; Kim, Youngsoo



Lipopolysaccharide Sequestrants: Structural Correlates of Activity and Toxicity in Novel Acylhomospermines  

PubMed Central

Lipopolysaccharides (LPS), otherwise termed ‘endotoxins’, are outer-membrane constituents of Gram-negative bacteria. Lipopolysaccharides play a key role in the pathogenesis of ‘Septic Shock’, a major cause of mortality in the critically ill patient. Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time. We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS and, using animal models of sepsis, have shown that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy. In this paper, the interactions of a series of acylated homologated spermine compounds with lipopolysaccharide (LPS) have been characterized. The optimal acyl chain length for effective sequestration of LPS was identified to be C16 for the mono-acyl compounds. The most promising of these compounds, 4e, binds LPS with an ED50 of 1.37 ?M. Nitric oxide production in murine J774A.1 cells, as well as TNF-? in human blood, are inhibited in a dose-dependent manner by 4e at concentrations orders of magnitude lower than toxic doses. Administration of 4e to d-galactosamine-sensitized mice challenged with supralethal doses of LPS provided significant protection against lethality. Potent anti-endotoxic activity, low toxicity, and ease of synthesis render this class of compounds candidate endotoxin-sequestering agents of potential significant therapeutic value.

Miller, Kelly A.; Kumar, E.V.K. Suresh; Wood, Stewart J.; Cromer, Jens R.; Datta, Apurba; David*, Sunil A.



Dry-heat destruction of lipopolysaccharide: dry-heat destruction kinetics.  

PubMed Central

Dry-heat destruction kinetics of lipopolysaccharides from Escherichia coli, Serratia marcescens, and Salmonella typhosa at 170 to 250 degrees C are described. The destruction rate seems to follow the second order and can be linearized by the equation, log y = a + b . -10cx. Because c is the slope, 1/c = D3. Both a and b are constant at a given temperature and are linear functions of temperature. The D(3)170, D(3)190, D(3)210, D(3)230, and D(3)250 values for E. coli lipopolysaccharide are 251, 99.4, 33.3, 12.3, and 4.99 min, respectively, with a z value of 46.4 min. The D values for lipopolysaccharides from S. marcescens and S. typhosa are not significantly different from those from E. coli lipopolysaccharide.

Tsuji, K; Harrison, S J



Human exposure to endotoxins and fecal indicators originating from water features.  


Exposure to contaminated aerosols and water originating from water features may pose public health risks. Endotoxins in air and water and fecal bacteria in water of water features were measured as markers for exposure to microbial cell debris and enteric pathogens, respectively. Information was collected about wind direction, wind force, distance to the water feature, the height of the water feature and the tangibility of water spray. The mean concentration of endotoxins in air nearby and in water of 31 water features was 10 endotoxin units (EU)/m(3) (Geometric Mean (GM), range 0-85.5 EU/m(3) air) and 773 EU/mL (GM, range 9-18,170 EU/mL water), respectively. Such mean concentrations may be associated with respiratory health effects. The water quality of 26 of 88 water features was poor when compared to requirements for recreational water in the Bathing Water Directive 2006/7/EC. Concentrations greater than 1000 colony forming units (cfu) Escherichia coli per 100 mL and greater than 400 cfu intestinal enterococci per 100 mL increase the probability of acquiring gastrointestinal health complaints. Regression analyses showed that the endotoxin concentration in air was significantly influenced by the concentration of endotoxin in water, the distance to the water feature and the tangibility of water spray. Exposure to air and water near water features was shown to lead to exposure to endotoxins and fecal bacteria. The potential health risks resulting from such exposure to water features may be estimated by a quantitative microbial risk assessment (QMRA), however, such QMRA would require quantitative data on pathogen concentrations, exposure volumes and dose-response relationships. The present study provides estimates for aerosolisation ratios that can be used as input for QMRA to quantify exposure and to determine infection risks from exposure to water features. PMID:24231029

de Man, H; Heederik, D D J; Leenen, E J T M; de Roda Husman, A M; Spithoven, J J G; van Knapen, F



Endotoxin contamination in the dental surgery.  


Dental waterlines contain large numbers of Gram-negative bacteria. Endotoxin, a component of such organisms, has significant health implications. Paired samples of dental unit water and the aerosols generated during dental procedures were collected, and assayed for bacteria and endotoxin levels, using heterotrophic plate counts and the Limulus amoebocyte lysate test. Consistent with published studies, the extent of bacterial contamination in the dental waters sampled for this investigation surpassed the levels associated with potable water, with counts in excess of 2.0x10(6) c.f.u. ml(-1) in some samples. Correspondingly high concentrations of endotoxin [up to 15 000 endotoxin units (EU) ml(-1)] were present in the water. A statistically significant Spearman correlation coefficient of rho=0.94 between endotoxin (EU ml(-1)) and bacterial load (c.f.u. ml(-1)) was demonstrated. All of the aerosol samples contained detectable endotoxin. Further studies of the consequences of dental endotoxin exposure, and evaluation of means to prevent exposure, are warranted. PMID:17761488

Huntington, M K; Williams, J F; Mackenzie, C D



Novel Bacillus thuringiensis ?-Endotoxin Active against Locusta migratoria manilensis ?  

PubMed Central

A novel ?-endotoxin gene was cloned from a Bacillus thuringiensis strain with activity against Locusta migratoria manilensis by PCR-based genome walking. The sequence of the cry gene was 3,432 bp long, and it encoded a Cry protein of 1,144 amino acid residues with a molecular mass of 129,196.5 kDa, which exhibited 62% homology with Cry7Ba1 in the amino acid sequence. The ?-endotoxin with five conserved sequence blocks in the amino-terminal region was designated Cry7Ca1 (GenBank accession no. EF486523). Protein structure analysis suggested that the activated toxin of Cry7Ca1 has three domains: 227 residues forming 7 ?-helices (domain I); 213 residues forming three antiparallel ?-sheets (domain II); and 134 residues forming a ?-sandwich (domain III). The three domains, respectively, exhibited 47, 44, and 34% sequence identity with corresponding domains of known Cry toxins. SDS-PAGE and Western blot analysis showed that Cry7Ca1, encoded by the full-length open reading frame of the cry gene, the activated toxin 1, which included three domains but without the N-terminal 54 amino acid residues and the C terminus, and the activated toxin 2, which included three domains and N-terminal 54 amino acid residues but without the C terminus, could be expressed in Escherichia coli. Bioassay results indicated that the expressed proteins of Cry7Ca1 and the activated toxins (toxins 1 and 2) showed significant activity against 2nd instar locusts, and after 7 days of infection, the estimated 50% lethal concentrations (LC50s) were 8.98 ?g/ml for the expressed Cry7Ca1, 0.87 ?g/ml for the activated toxin 1, and 4.43 ?g/ml for the activated toxin 2. The ?-endotoxin also induced histopathological changes in midgut epithelial cells of adult L. migratoria manilensis.

Wu, Yan; Lei, Cheng-Feng; Yi, Dan; Liu, Peng-Ming; Gao, Mei-Ying




PubMed Central

Cortisone acetate administered to mice at the same time as either the LD50 or 2 x LD50 of endotoxin significantly protected against lethality. Delaying the injection of cortisone to 1, 2, or 4 hours after that of endotoxin resulted in loss of protection with the possible exception of a 1 hour delay with the LD50 of endotoxin. Associated with this loss of protection was the failure of the hormone to induce liver tryptophan pyrrolase. Normal mice given only cortisone showed an increase in enzyme activity nearly three times that of control values when assays were carried out either 4 or 17 hours after the hormone was given. Endotoxin-poisoned mice showed normal levels of enzyme activity with concurrent injection of cortisone but depressed levels of enzyme when the cortisone injection was delayed for only 1 hour or more. Apparently, therefore, enzyme induction (or maintenance) is related to survival in endotoxin poisoning. In line with this hypothesis was the observation that inhibitors of enzyme (protein) synthesis were found to potentiate the lethal action of endotoxin and to prevent the protective effect of cortisone. The inhibitors employed were actinomycin D, ethionine, 2-thiouracil, and 8-azaguanine. Activity of liver tryptophan pyrrolase was lowered by endotoxin and elevated by cortisone. When the two were given concurrently, normal enzyme activity was maintained. Chloramphenicol, an active inhibitor of protein synthesis in microorganisms but with limited effect in mammals, was without observable influence in these respects. Mice 18 hours postinfection with Salmonella typhimurium, strain SR-11, given at a level that caused first deaths on the 3rd day, had a lower than normal activity of liver tryptophan pyrrolase and responded to cortisone induction with a smaller increase in enzyme level than that found in control mice. Each is characteristic of endotoxin poisoning. Animals 42 hours postinfection were free of these signs of endointoxication, an observation in agreement with earlier experiments where other measures of endotoxin were employed.

Berry, L. Joe; Smythe, Dorothy S.



Heterogeneity of Rhizobium lipopolysaccharides.  

PubMed Central

The lipopolysaccharides ( LPSs ) from strains of Rhizobium leguminosarum, Rhizobium trifolii, and Rhizobium phaseoli were isolated and partially characterized by mild acid hydrolysis and by polyacrylamide gel electrophoresis. Mild acid hydrolysis results in a precipitate which can be removed by centrifugation or extraction with chloroform. The supernatant contains polysaccharides which, in general, are separated into two fractions ( LPS1 and LPS2 ) by Sephadex G-50 gel filtration chromatography. The higher-molecular-weight LPS1 fractions among the various Rhizobium strains are highly variable in composition and reflect the variability reported in the intact LPSs (R. W. Carlson and R. Lee, Plant Physiol. 71:223-228, 1983; Carlson et al., Plant Physiol. 62:912-917, 1978; Zevenhuizen et al., Arch. Microbiol. 125:1-8, 1980). The LPS1 fraction of R. leguminosarum 128C53 has a higher molecular weight than all other LPS1 fractions examined. All LPS2 fractions examined are oligosaccharides with a molecular weight of ca. 600. The major sugar component of all LPS2 oligosaccharides is uronic acid. The LPS2 compositions are similar for strains of R. leguminosarum and R. trifolii, but the LPS2 from R. phaseoli was different in that it contained glucose, a sugar not found in the other LPS2 fractions or found only in trace amounts. Polyacrylamide gel electrophoretic analysis shows that each LPS contains two banding regions, a higher-molecular-weight heterogeneous region often containing many bands and a lower-molecular-weight band. The lower-molecular-weight bands of all LPSs have the same electrophoretic mobility, which is greater than that of lysozyme. The banding pattern of the heterogeneous regions varies among the different Rhizobium strains. In the case of R. leguminosarum 128C53 LPS, the heterogeneous region of a higher molecular weight than is this region from all other Rhizobium strains examined and consists of many bands separated from one another by a small and apparently constant molecular weight interval. When the heterogeneous region of R. Leguminosarum 128C53 LPS was cut from the gel and analyzed, its composition was found to be that of the intact LPS, whereas the lower-molecular-weight band contains only sugars found in the LPS2 oligosaccharide. In the case of R. leguminosarum 128C63 and R. trifolii 0403 LPSs, the heterogeneous regions are similar and consist of several band s separated by a large-molecular-weight interval with a the major band of these heterogeneous regions having the lowest molecular weight with an electrophoretic mobility near that of beta-lactoglobulin. The heterogeneous region from R. phaseoli 127K14 consists of several bands with electrophoretic mobilities near that of beta-lactoglobulin, whereas this region from R. trifolii 162S7 shows a continuous staining region, indicating a great deal of heterogeneity. The results described in this paper are discussed with regard to the reported properties of Escherichia coli and Salmonella LPSs. Images

Carlson, R W



If blocking potency of ivabradine is preserved under elevated endotoxin levels in human atrial myocytes  

PubMed Central

Lower heart rate is associated with better survival in patients with multiple organ dysfunction syndrome (MODS), a disease mostly caused by sepsis. The benefits of heart rate reduction by ivabradine during MODS are currently being investigated in the MODIfY clinical trial. Ivabradine is a selective inhibitor of the pacemaker current If and since If is impaired by lipopolysaccharide (LPS, endotoxin), a trigger of sepsis, we aimed to explore If blocking potency of ivabradine under elevated endotoxin levels in human atrial cardiomyocytes. Treatment of myocytes with S-LPS (containing the lipid A moiety, a core oligosaccharide and an O-polysaccharide chain) but not R595 (an O-chain lacking LPS-form) caused If inhibition under acute and chronic septic conditions. The specific interaction of S-LPS but not R595 to pacemaker channels HCN2 and HCN4 proves the necessity of O-chain for S-LPS–HCN interaction. The efficacy of ivabradine to block If was reduced under septic conditions, an observation that correlated with lower intracellular ivabradine concentrations in S-LPS- but not R595-treated cardiomyocytes. Computational analysis using a sinoatrial pacemaker cell model revealed that despite a reduction of If under septic conditions, ivabradine further decelerated pacemaking activity. This novel finding, i.e. If inhibition by ivabradine under elevated endotoxin levels in vitro, may provide a molecular understanding for the efficacy of this drug on heart rate reduction under septic conditions in vivo, e.g. the MODIfY clinical trial.

Scheruebel, Susanne; Koyani, Chintan N.; Hallstrom, Seth; Lang, Petra; Platzer, Dieter; Machler, Heinrich; Lohner, Karl; Malle, Ernst; Zorn-Pauly, Klaus; Pelzmann, Brigitte



Lipopolysaccharide priming enhances expression of effectors of immune defence while decreasing expression of pro-inflammatory cytokines in mammary epithelia cells from cows  

PubMed Central

Background Udder infections with environmental pathogens like Escherichia coli are a serious problem for the dairy industry. Reduction of incidence and severity of mastitis is desirable and mild priming of the immune system either through vaccination or with low doses of immune stimulants such as lipopolysaccharide LPS was previously found to dampen detrimental effects of a subsequent infection. Monocytes/macrophages are known to develop tolerance towards the endotoxin LPS (endotoxin tolerance, ET) as adaptation strategy to prevent exuberant inflammation. We have recently observed that infusion of 1 ?g of LPS into the quarter of an udder effectively protected for several days against an experimentally elicited mastitis. We have modelled this process in primary cultures of mammary epithelial cells (MEC) from the cow. MEC are by far the most abundant cells in the healthy udder coming into contact with invading pathogens and little is known about their role in establishing ET. Results We primed primary MEC cultures for 12 h with LPS (100 ng/ml) and stimulated three cultures either 12 h or 42 h later with 107/ml particles of heat inactivated E. coli bacteria for six hours. Priming-related alterations in the global transcriptome of those cells were quantified with Affymetrix microarrays. LPS priming alone caused differential expression of 40 genes and mediated significantly different response to a subsequent E. coli challenge of 226 genes. Expression of 38 genes was enhanced while that of 188 was decreased. Higher expressed were anti-microbial factors (?-defensin LAP, SLPI), cell and tissue protecting factors (DAF, MUC1, TGM1, TGM3) as well as mediators of the sentinel function of MEC (CCL5, CXCL8). Dampened was the expression of potentially harmful pro-inflammatory master cytokines (IL1B, IL6, TNF-?) and immune effectors (NOS2, matrix metalloproteases). Functional network analysis highlighted the reduced expression of IL1B and of IRF7 as key to this modulation. Conclusion LPS-primed MEC are fitter to repel pathogens and better protected against misguided attacks of the immune response. Attenuated is the exuberant expression of factors potentially promoting immunopathological processes. MEC therefore recapitulate many aspects of ET known so far from professional immune cells.



The suppression by lipopolysaccharide of cytochrome P450-dependent renal vasodilation in the rat is mediated by nitric oxide  

Microsoft Academic Search

The isolated perfused kidney of the rat was used to examine the hypothesis that lipopolysaccharide-induced nitric oxide (NO) production inhibits cytochrome P450-dependent vasodilation. The vasodilator responses to arachidonic acid and bradykinin were examined as the response to arachidonic acid is wholly dependent, and that to bradykinin partly dependent on cytochrome P450 metabolism. In endotoxin-treated rats, the vasodilator response to arachidonic

Adebayo O. Oyekan



The Role of Glucose in Endotoxin Shock.  

National Technical Information Service (NTIS)

The primary purpose of the review is to consider the present state of knowledge pretaining to the role of glucose in endotoxin shock. The report covers changing blood levels of glucose, relationship of glucose and insulin, mechanisms of hypoglycemia, and ...

L. B. Hinshaw



Detection of endotoxin in the plasma of patients with gram-negative bacterial sepsis by the Limulus amoebocyte lysate assay.  

PubMed Central

A total of 120 Limulus amoebocyte lysate (LAL) determinations were made on plasma obtained from normal, healthy human blood donors. Results demonstrated a mean endotoxin level in blood of 0.02 to 1.57 pg/ml. The amount of Escherichia coli endotoxin added to human plasma samples can be quantitated by both nephelometry and turbidimetry. Endotoxin-spiked samples were shown to be significantly different from unspiked samples. When plasma samples were collected from 45 patients hospitalized at three centers, a strong association was demonstrated between a positive Limulus amoebocyte lysate assay and a septic condition. Sensitivity, specificity, and false-positive and false-negative rates for the Limulus amoebocyte lysate assay as a diagnostic test for gram-negative bacteremia were estimated.

Pearson, F C; Dubczak, J; Weary, M; Bruszer, G; Donohue, G



Isoforskolin pretreatment attenuates lipopolysaccharide-induced acute lung injury in animal models.  


Isoforskolin was isolated from Coleus forskohlii native to Yunnan in China. We hypothesize that isoforskolin pretreatment attenuates acute lung injury induced by lipopolysaccharide (endotoxin). Three acute lung injury models were used: situ perfused rat lung, rat and mouse models of endotoxic shock. Additionally, lipopolysaccharide stimulated proinflammatory cytokine production was evaluated in human mononuclear leukocyte. In situ perfused rat lungs, pre-perfusion with isoforskolin (100, and 200 ?M) and dexamethasone (65 ?M, positive control) inhibited lipopolysaccharide (10 mg/L) induced increases in lung neutrophil adhesion rate, myeloperoxidase activity, lung weight Wet/Dry ratio, permeability-surface area product value, and tumor necrosis factor (TNF)-? levels. In rats, pretreatments with isoforskolin (5, 10, and 20 mg/kg, i.p.) and dexamethasone (5mg/kg, i.p.) markedly reduced lipopolysaccharide (6 mg/kg i.v.) induced increases of karyocyte, neutrophil counts and protein content in bronchoalveolar lavage fluid, and plasma myeloperoxidase activity. Lung histopathology showed that morphologic changes induced by lipopolysaccharide were less pronounced in the isoforskolin and dexamethasone pretreated rats. In mice, 5 mg/kg isoforskolin and dexamethasone caused 100% and 80% survival, respectively, after administration of lipopolysaccharide (62.5mg/kg, i.v., 40% survival if untreated). In human mononuclear leukocyte, isoforskolin (50, 100, and 200 ?M) and dexamethasone (10 ?M) pre-incubation lowered lipopolysaccharide (2 ?g/mL) induced secretion of the cytokine TNF-?, and interleukins (IL)-1?, IL-6, and IL-8. In conclusion, pretreatment with isoforskolin attenuates lipopolysaccharide-induced acute lung injury in several models, and it is involved in down-regulation of inflammatory responses and proinflammatory cytokines TNF-?, IL-1?, IL-6, and IL-8. PMID:21272678

Yang, Weimin; Qiang, Dongjin; Zhang, Min; Ma, Limei; Zhang, Yonghui; Qing, Chen; Xu, Yunlong; Zhen, Chunlan; Liu, Jikai; Chen, Yan-Hua



Administration to mouse of endotoxin from gram-negative bacteria leads to activation and apoptosis of T lymphocytes.  


Lipopolysaccharide (LPS) from gramnegative bacteria is a well-known T cell-independent B lymphocyte mitogen and macrophage/monocyte activator. While the conventional view holds that LPS is ignored by T cells, we report here that administration of LPS to mice activates all B cells, but also engages most CD4 and CD8 T cells, as measured by the expression of the activation markers CD69 and CD25 and by size increase. T cells recruited in endotoxin-treated mice showed, following in vitro stimulation by concanavalin A, altered patterns of cytokine production. In vivo, massive T cell apoptosis was evidenced in the days following LPS exposure. The present observation may contribute novel insights into the mechanisms of endotoxin shock and of the immunological consequences of gram-negative infections. PMID:9521057

Castro, A; Bemer, V; Nóbrega, A; Coutinho, A; Truffa-Bachi, P



Binding interactions of bacterial lipopolysaccharide and the cationic amphiphilic peptides polymyxin B and WLBU2.  


Passage of blood through a sorbent device for removal of bacteria and endotoxin by specific binding with immobilized, membrane-active, bactericidal peptides holds promise for treating severe blood infections. Peptide insertion in the target membrane and rapid/strong binding is desirable, while membrane disruption and release of degradation products to the circulating blood is not. Here we describe interactions between bacterial endotoxin (lipopolysaccharide, LPS) and the membrane-active, bactericidal peptides WLBU2 and polymyxin B (PmB). Analysis of the interfacial behavior of mixtures of LPS and peptide using air-water interfacial tensiometry and optical waveguide lightmode spectroscopy strongly suggests insertion of intact LPS vesicles by the peptide WLBU2 without vesicle destabilization. In contrast, dynamic light scattering (DLS) studies show that LPS vesicles appear to undergo peptide-induced destabilization in the presence of PmB. Circular dichroism spectra further confirm that WLBU2, which shows disordered structure in aqueous solution and substantially helical structure in membrane-mimetic environments, is stably located within the LPS membrane in peptide-vesicle mixtures. We therefore expect that presentation of WLBU2 at an interface, if tethered in a fashion which preserves its mobility and solvent accessibility, will enable the capture of bacteria and endotoxin without promoting reintroduction of endotoxin to the circulating blood, thus minimizing adverse clinical outcomes. On the other hand, our results suggest no such favorable outcome of LPS interactions with polymyxin B. PMID:24905681

Ryder, Matthew P; Wu, Xiangming; McKelvey, Greg R; McGuire, Joseph; Schilke, Karl F



Intestinal integrity, endotoxin transport and detoxification in pigs divergently selected for residual feed intake.  


Microbes and microbial components potentially impact the performance of pigs through immune stimulation and altered metabolism. These immune modulating factors can include endotoxin from gram negative bacterial outer membrane component, commonly referred to as lipopolysaccharide (LPS). In this study, our objective was to examine the relationship between intestinal barrier integrity, endotoxin and inflammation with feed efficiency (FE), using pig lines divergently selected for residual feed intake (RFI) as a model. Twelve gilts (62 ± 3 kg BW) from the low RFI (LRFI, more efficient) and 12 from the high RFI (HRFI, less efficient) were used. Individual performance data was recorded for 5 wk. At the end of the experimental period, ADFI of LRFI pigs was less (P < 0.001), ADG not different between the 2 lines (P = 0.72) but the G:F of LRFI pigs was greater than for HRFI pigs (P = 0.019). Serum endotoxin concentration (P < 0.01) and the acute phase protein haptoglobin (P < 0.05) were greater in HRFI pigs. Transepithelial resistance of the ileum, transport of fluorescein isothiocyanate labeled-Dextran and-LPS in ileum and colon, as well as tight junction protein mRNA expression in ileum, did not differ between the lines, indicating the 2 lines did not differ in transport characteristics at the intestinal level. Ileum inflammatory markers, myeloperoxidase (P < 0.05) and IL-8 (P < 0.10), were found to be greater in HRFI pigs. Alkaline phosphatase (ALP) activity was significantly increased in the LRFI pigs in ileum and liver tissues and negatively correlated with blood endotoxin (P < 0.05). Lysozyme activity in the liver was not different between the lines; however, the LRFI pigs had a twofold greater lysozyme activity in ileum (P < 0.05). Despite the difference in their activity, ALP or lysozyme mRNA expression was not different between the lines in either tissue. Decreased endotoxin and inflammatory markers and the enhanced activities of antimicrobial enzymes in the LRFI line may not fully explain the difference in the FE between the lines, but they have the potential to prevent the growth potential in HRFI pigs. Further studies are needed to identify the other mechanisms that may contribute to the greater endotoxin and acute phase proteins in the HRFI pigs and the greater FE in the LRFI pigs. PMID:23463550

Mani, V; Harris, A J; Keating, A F; Weber, T E; Dekkers, J C M; Gabler, N K



Homogeneity of lipopolysaccharides from Acholeplasma.  

PubMed Central

Five methods were employed to determine the heterogeneity or homogeneity of lipopolysaccharides from four acholeplasmal species, Acholeplasma axanthum, A. granularum, A. laidlawii, and A. modicum. A axanthum lipopolysaccharide behaved as a single component in all tests. A. granularum exhibited two components of identical composition and antigenic specificity. A. modicum lipopolysaccharide behaved as three components in two tests, but all three were similar in composition and identical serologically. The separable components of lipopolysaccharides from A. granularum and A. modicum probably represent size differences only. A. laidlwii lipopolysaccharide contained two distinct components by all methods. One was identified as the previously reported amino sugar polymer, whereas the other was a lipopolysaccharide containing both neutral and amino sugars. Images

Smith, P F



Mepacrine-Induced Sensitization of Rats to Bacterial Endotoxin.  

National Technical Information Service (NTIS)

Investigations in the past several years have implicated prostaglandin release in the pathogenesis of endotoxin shock. These studies have focused on the observation that prostaglandin levels are increased following endotoxin administration. Furthermore, i...

S. L. Gartner K. M. Shakir



Use of Isoproterenol and Phenoxybenzamine in Treatment of Endotoxin Shock.  

National Technical Information Service (NTIS)

This report evaluates five approaches to the treatment of endotoxin shock in the dog. The effect of endotoxin on physiological parameters and survival was noted. Dogs treated with hydrocortisone, isoproterenol, or a combination of the two, showed temporar...

J. A. Vick H. P. Ciuchta J. H. Manthei



Effect of bacterial endotoxin on placentation of rats.  


The effect of bacterial endotoxin on placentation in rats was studied on 160 CFY pregnant rats. Based on this experiment, it was concluded that (i) the endotoxin (1 mg/animal i.p.) inhibited placentation (in 90% of animal). (ii) The endotoxin-induced fetopathy almost exclusively resulted in abortion. (iii) The fetuses reacted to endotoxin with relatively the same degrees of susceptibility. (iv) The growth of surviving fetuses seemed to be undisturbed. (v) Endotoxin-induced damages in mothers first of all depend on the individual susceptibility of these pregnant animals and (vi) the endotoxin tolerance induced by radio-detoxified endotoxin (TOLERIN) significantly protects both the mothers and the fetuses against endotoxin challenge. PMID:2082637

Szöcs, G; Csordás, T; Bertók, L



Altered Toll-like Receptor 2-mediated Endotoxin Tolerance Is Related to Diminished Interferon ? Production  

PubMed Central

Induction of endotoxin tolerance leads to a reduced inflammatory response after repeated challenge by LPS and is important for resolution of inflammation and prevention of tissue damage. Enterobacterial LPS is recognized by the TLR4 signaling complex, whereas LPS of some non-enterobacterial organisms is capable of signaling independently of TLR4 utilizing TLR2-mediated signal transduction instead. In this study we report that Porphyromonas gingivalis LPS, a TLR2 agonist, fails to induce a fully endotoxin tolerant state in a human monocytic cell line (THP-1) and mouse bone marrow-derived macrophages. In contrast to significantly decreased production of human IL-8 and TNF-? and, in mice, keratinocyte-derived cytokine (KC), macrophage inflammatory protein-2 (MIP-2), and TNF-? after repeated challenge with Escherichia coli LPS, cells repeatedly exposed to P. gingivalis LPS responded by producing less TNF-? but sustained elevated secretion of IL-8, KC, and MIP-2. Furthermore, in endotoxin-tolerant cells, production of IL-8 is controlled at the signaling level and correlates well with NF-?B activation, whereas TNF-? expression is blocked at the gene transcription level. Interferon ? plays an important role in attenuation of chemokine expression in endotoxin-tolerized cells as shown in interferon regulatory factor-3 knock-out mice. In addition, human gingival fibroblasts, commonly known not to display LPS tolerance, were found to be tolerant to repeated challenge by LPS if pretreated with interferon ?. The data suggest that the inability of the LPS-TLR2 complex to induce full endotoxin tolerance in monocytes/macrophages is related to diminished production of interferon ? and may partly explain the involvement of these LPS isoforms in the pathogenesis of chronic inflammatory diseases.

Zaric, Svetislav S.; Coulter, Wilson A.; Shelburne, Charles E.; Fulton, Catherine R.; Zaric, Marija S.; Scott, Aaron; Lappin, Mark J.; Fitzgerald, Denise C.; Irwin, Christopher R.; Taggart, Clifford C.



Thromboxane A2 mediates lung vasoconstriction but not permeability after endotoxin.  

PubMed Central

The effect of dazoxiben, a selective thromboxane (Tx) synthetase inhibitor, on systemic and pulmonary hemodynamics, eicosanoids, and lung permeability was assessed in awake goats with lung lymph fistulae following infusion of Escherichia coli endotoxin (1 microgram/kg). Animals received endotoxin either with no treatment or pretreatment with a bolus (25 mg/kg) followed by a maintenance infusion (10 mg/kg per h) of dazoxiben. In untreated animals, the peak rise of 26.8 cm H2O in pulmonary artery (Ppa) and of 13.5 cm H2O in wedge (Pw) pressures occurred at the same time as the peak elevations in plasma thromboxane B2 (T X B2). Maximum reduction in cardiac output (Qt) also occurred at the same time. Lung lymph flow (QL) increased during this period and remained elevated for at least 6 h after endotoxin. T X B2 levels had returned from a peak of 13.1 to 0.7 ng/ml by 2 h. In dazoxiben-treated animals, plasma concentrations of T X B2 were never significantly elevated. Increases in Ppa and Pw were markedly reduced and decreased Qt was transient. QL in treated animals began to increase by 30 min after endotoxin and reached a peak by 2 h. Increased QL in treated animals was not as great as in the untreated animals. Moreover, lymph-plasma protein ratios increased significantly in treated animals. Plasma prostaglandin (PG)F2 alpha and 6-keto-PGF1 alpha concentrations were elevated in both groups after endotoxin with values significantly greater in treated animals. We conclude that selective inhibition of Tx ameliorates many adverse hemodynamic consequences of endotoxemia but does not prevent lung permeability changes.

Winn, R; Harlan, J; Nadir, B; Harker, L; Hildebrandt, J



Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 2. Lipid A, its structural variability, the linkage to the core oligosaccharide, and the biological activity of the lipopolysaccharide.  


Plesiomonas shigelloides is a Gram-negative bacterium associated with waterborne infections, which is common in tropical and subtropical habitats. Contrary to the unified antigenic classification of P. shigelloides, data concerning the structure and activity of their lipopolysaccharides (LPS and endotoxin) are limited. This study completes the structural investigation of phenol- and water-soluble fractions of P. shigelloides O74 (strain CNCTC 144/92) LPS with the emphasis on lipid A heterogeneity, describing the entire molecule and some of its biological in vitro activities. Structures of the lipid A and the affinity-purified decasaccharide obtained by de-N,O-acylation of P. shigelloides O74 LPS were elucidated by chemical analysis combined with electrospray ionization multiple-stage mass spectrometry (ESI-MS(n)), MALDI-TOF MS, and NMR spectroscopy. Lipid A of P. shigelloides O74 is heterogeneous, and three major forms have been identified. They all were asymmetric, phosphorylated, and hexaacylated, showing different acylation patterns. The beta-GlcpN4P-(1-->6)-alpha-GlcpN1P disaccharide was substituted with the primary fatty acids: (R)-3-hydroxytetradecanoic acid [14:0(3-OH)] at N-2 and N-2' and (R)-3-hydroxydodecanoic acid [12:0(3-OH)] at O-3 and O-3'. The heterogeneity among the three forms (I-III) of P. shigelloides O74 lipid A was attributed to the substitution of the acyl residues at N-2' and O-3' with the secondary acyls: (I) cis-9-hexadecenoic acid (9c-16:1) at N-2' and 12:0 at O-3', (II) 14:0 at N-2' and 12:0 at O-3', and (III) 12:0 at N-2' and 12:0 at O-3'. The pro-inflammatory cytokine-inducing activities of P. shigelloides O74 LPS were similar to those of Escherichia coli O55 LPS. PMID:16939196

Lukasiewicz, Jolanta; Dzieciatkowska, Monika; Niedziela, Tomasz; Jachymek, Wojciech; Augustyniuk, Anna; Kenne, Lennart; Lugowski, Czeslaw



Endotoxin exposure in allergy and asthma: Reconciling a paradox  

Microsoft Academic Search

Well-established evidence links endotoxin exposure, especially in the workplace, to airways disease. Endotoxin can increase disease severity by acting as a natural adjuvant to augment asthma and atopic inflammation. Recent studies suggest that it can even act on its own, causing a distinct endotoxic form of asthma. Other studies, however, contradict the paradigm that endotoxin's influence is solely a negative

Andrew H. Liu



Levels of bacterial endotoxin in air of animal houses determined with the use of gas chromatography-mass spectrometry and Limulus test.  


Air samples were collected on glass fibre filters in 22 animal houses and 3 hay storage barns and examined for the presence of bacterial endotoxin with the Limulus (LAL) test and the gas chromatography-tandem mass spectrometry (GC-MSMS) technique, based on detection of 3-hydroxy fatty acids (3-OH-FAs) as chemical markers of the endotoxin lipopolysaccharide. The median concentrations of airborne endotoxin determined with LAL test in poultry houses, sheep sheds, piggeries, cow barns, and horse stables were respectively 62.49 microg/m3, 26.2 microg/m3, 3.8 microg/m3, 1.65 microg/m3, and 1.14 microg/m3, while those determined with the GC-MSMS technique were respectively 1.06 microg/m3, 7.91 microg/m3, 0.2 microg/m3, 0.31 microg/m3, and 1.42 microg/m3. The median concentrations of airborne endotoxin determined with LAL test and GC-MSMS technique in hay storage barns were much smaller, 0.09 microg/m3 and 0.03 microg/m3, respectively. The concentrations of airborne endotoxin (LPS) detected with GC-MSMS method in the air of sheep sheds were significantly greater than in all other examined facilities, while those detected in hay storage barns were significantly smaller than in all other examined facilities (p<0.05). The concentrations of airborne endotoxin determined with LAL test and GC-MSMS analysis exceeded in most of animal houses examined (91% by each method) the threshold limit value for airborne endotoxin of 0.1 microg/m3 proposed by various authors. A significant correlation (p<0.05) between the concentrations of endotoxin determined with the LAL and GC-MSMS techniques was found in the air samples collected in poultry houses and sheep sheds, but not in other examined facilities. 3-OH FAs with C14-C18 chains were predominant in the air of the facilities under study. A significant correlation (p<0.05) was found between the concentrations of endotoxin determined with LAL test and the amounts of 3-OH FAs with C14-C16 chains. In conclusion, endotoxin in the concentrations detected in this study may present a respiratory hazard to both humans and livestock animals. PMID:18247467

Pomorska, Dorota; Larsson, Lennart; Skórska, Czes?awa; Sitkowska, Jolanta; Dutkiewicz, Jacek



Lipopolysaccharides of Salmonella T Mutants  

PubMed Central

The composition of lipopolysaccharides derived from various Salmonella T forms was studied. All T1-form lipopolysaccharides examined contained 14 to 22% each of both d-galactose and pentose in addition to 4 to 9% each of ketodeoxyoctonic acid, heptose, d-glucosamine, and d-glucose. The pentose was identified as d-ribose. The T2-form lipopolysaccharide examined did not contain a significant amount of pentose, nor more than the usual amounts of d-galactose. Periodate oxidation of T1 (lipo) polysaccharides followed by NaBH4 reduction revealed that ribose was almost quantitatively protected, galactose was destroyed, and threitol and mannose were newly formed. The latter two products probably originated from 4-linked galactose and heptose, respectively. Ribose and galactose were found in specific precipitates of T1 lipopolysaccharide with anti-T1 antiserum but were not found in specific precipitates of alkali-treated T1 lipopolysaccharide and of Freeman degraded polysaccharide with anti-T1 serum Ribose and galactose are present in these degraded preparations in the form of nondialyzable polymers. The T1-form mutant lipopolysaccharides lacked the O-specific sugars constituting the side-chains in the wild-type antigens. They did not produce the soluble O-specific haptenic polysaccharide known to be accumulated in RI strains. With these properties, T1 lipopolysaccharides resemble RII lipopolysaccharides. Like RII degraded polysaccharides, T1-degraded polysaccharides also contained glucosamine. Furthermore, strong cross-reactions were found to exist between T1 and RII lipopolysaccharides in both hemagglutination inhibition assays and in precipitation tests. It is proposed that T1 lipopolysaccharides represent RII lipopolysaccharides to which polymers consisting of ribose and galactose are attached.

Wheat, R. W.; Berst, M.; Ruschmann, E.; Luderitz, O.; Westphal, O.



Endotoxin-induced renal failure. II. A role for tubular hypoxic damage.  


Endotoxin-induced hypotension and altered renal microcirculation could lead to tubular injury, particularly at the physiologically hypoxic outer medulla. We explored this hypothesis in isolated perfused kidneys and in vivo in rats subjected to endotoxemia. Rat kidneys were removed 15 min after endotoxin injection in vivo (from Escherichia coli 0127:B8, 1 mg/kg i.p.) and perfused with oxygenated medium supplemented with 20 amino acids and endotoxin. Glomerular filtration rate and filtration fraction markedly declined (0.4 +/- 0. 1 ml/min and 1.1 +/- 0.1, respectively) as compared with control kidneys (0.7 +/- 0.1 ml/min and 1.8 +/- 0.1, n = 8-12 per group; p < 0.05). Hypoxic injury to medullary thick ascending limbs in the innermost outer medulla increased (47 +/- 9% of tubules vs. 16 +/- 8% in controls, p < 0.05). When rats were preconditioned with an additional endotoxin injection 16 h earlier (a manipulation that markedly reduces cortical and medullary blood flow), glomerular filtration rate and filtration fraction further declined to 0.1 +/- 0.0 ml/min and 0.4 +/- 0.1, respectively (p < 0.01), and tubular sodium reabsorption fell to 81 +/- 12 vs 98 +/- 0% in controls (p < 0.05). Tubular damage, however, did not increase (20 +/- 7%), probably reflecting a decline in reabsorptive workload and oxygen requirement. In rats subjected to a single or two repeated daily doses of endotoxin (1 mg/kg i.p.) plasma creatinine comparably rose 41% on the average over 24 h, creatinine clearance fell by 27% (p < 0.0001), but tubular damage was absent. By contrast, in rats preconditioned with indomethacin and the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (10 mg/kg), the addition of endotoxin markedly augmented outer medullary hypoxic tubular damage both in S(3) segments (27 +/- 10 vs 1 +/- 1%) and in medullary thick ascending limbs (38 +/- 11 vs. 10 +/- 5%, n = 7-8; p < 0.05). It is concluded that under special conditions, such as altered medullary oxygen balance or defective nitric oxide or prostaglandin synthesis, endotoxin may predispose to hypoxic outer medullary tubular damage. PMID:10940727

Heyman, S N; Rosen, S; Darmon, D; Goldfarb, M; Bitz, H; Shina, A; Brezis, M



Evidence that an L-arginine/nitric oxide dependent elevation of tissue cyclic GMP content is involved in depression of vascular reactivity by endotoxin.  

PubMed Central

1. The aim of this investigation was to study the relationship between contractile responsiveness, activation of the L-arginine pathway and tissue levels of guanosine 3':5'cyclic monophosphate (cylic GMP) in aortic rings removed from rats 4 h after intraperitoneal administration of bacterial endotoxin (E. coli. lipopolysaccharide, LPS, 20 mg kg-1). 2. LPS-treatment resulted in a reduction of the sensitivity and maximal contractile response to noradrenaline (NA). 3. Depression of the maximal contractile response was restored to control by 6-anilo-5,8-quinolinedione (LY 83583, 10 microM), which prevents activation of soluble guanylate cyclase. 4. Cyclic GMP levels in tissue from LPS-treated rats were 2 fold greater than cyclic GMP levels detected in tissue from control (saline-treated) rats. The LPS-induced increase in cyclic GMP content was observed both in the presence and absence of functional endothelium. 5. Addition of L-arginine 1 mM) to maximally contracted aortic rings produced significantly relaxation of rings from LPS-treated rats but not rings from control animals. In the LPS-treated group, addition of L-arginine was also associated with a significant increase in cyclic GMP content. L-Arginine had no effect on the cyclic GMP content of control rings. D-Arginine (1 mM) was without effect. 6. In rings from LPS-treated rats, NG-nitro-L-arginine methyl ester (L-NAME, 300 microM), an inhibitor of nitric oxide (NO) production, increased the contractile response to NA and prevented the LPS-induced increase in cyclic GMP content.(ABSTRACT TRUNCATED AT 250 WORDS)

Fleming, I.; Julou-Schaeffer, G.; Gray, G. A.; Parratt, J. R.; Stoclet, J. C.



Deletion mutants of the gene encoding delta-endotoxin specific to scarabaeid beetles: minimum region of the gene required to express the activity.  


Various deletion genes for the delta-endotoxin protein of Bacillus thuringiensis strain Buibui were made. The truncated proteins were produced in E. coli, and their larvicidal activity against the cupreous chafer was checked. The fifth block of the five blocks described as "conserved" was shown to be necessary for the activity. PMID:7670204

Minami, M; Hori, H; Ogiwara, K; Sato, R; Ohba, M; Iwahana, H



Application of quartz tuning forks for detection of endotoxins and Gram-negative bacterial cells by monitoring of Limulus Amebocyte Lysate coagulation.  


Endotoxins, pyrogens of bacterial origin, are a significant threat in many areas of life. Currently, the test most commonly used for endotoxin level determination is LAL (Limulus Amebocyte Lysate) assay. This paper presents application of commercially available low-frequency piezoelectric tuning forks (QTFs) for endotoxin detection. Measurement of the decrease in the QTF oscillation amplitude provides information about the viscosity changes, occurring in the tested sample upon addition of LAL. That method was used to determine the concentrations of endotoxins and bacterial cells (E. coli O157:H19). The relevance of the obtained results was confirmed using a commercially available colorimetric LAL assay. The constructed system can detect bacterial endotoxins in the range of 0.001-5EU/ml and bacterial cells in the range of 10(2)-10(7)CFU/ml. The presented technique requires very simple sample preparation and the sensor response is obtained using compact, portable readout electronics. The single test cost is low compared to commercial endotoxin assays and other novel systems based on micromechanical sensors. PMID:24632139

Cha?upniak, Andrzej; Waszczuk, Karol; Ha?ubek-G?uchowska, Katarzyna; Piasecki, Tomasz; Gotszalk, Teodor; Rybka, Jacek



Mutations in firA, encoding the second acyltransferase in lipopolysaccharide biosynthesis, affect multiple steps in lipopolysaccharide biosynthesis.  

PubMed Central

The product of the firA (ssc) gene is essential for growth and for the integrity of the outer membrane of Escherichia coli and Salmonella typhimurium. Recently, Kelly and coworkers (T. M. Kelly, S. A. Stachula, C. R. H. Raetz, and M. S. Anderson, J. Biol. Chem., 268:19866-19874, 1993) identified firA as the gene encoding UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase, the third step in lipid A biosynthesis. We studied the effects of six different mutations in firA on lipopolysaccharide synthesis. All of the firA mutants of both E. coli and S. typhimurium examined had a decreased lipopolysaccharide synthesis rate. E. coli and S. typhimurium strains defective in firA produced a lipid A that contains a seventh fatty acid, a hexadecanoic acid, when grown at the nonpermissive temperature. Analysis of the enzymatic activity of other enzymes involved in lipid A biosynthesis revealed that the firA mutations pleiotropically affect lipopolysaccharide biosynthesis. In addition to that of UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase, the enzymatic activity of the lipid A 4' kinase (the sixth step of lipid A biosynthesis) was decreased in strains with each of the firA mutations examined. However, overproduction of FirA was not accompanied by overexpression of the lipid A 4' kinase. Images

Roy, A M; Coleman, J



Complexing of bacterial lipopolysaccharide with lung surfactant.  

PubMed Central

Lipopolysaccharides (LPS) from Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Serratia marcescens, or Pseudomonas aeruginosa were mixed with pulmonary surfactant to investigate their in vitro interaction. After 6 h of incubation at 37 degrees C, LPS-surfactant mixtures were examined by sucrose density gradient centrifugation. The E. coli LPS-surfactant mixture was examined by immunoelectron microscopy with protein A-colloidal gold. The binding that occurred between LPS and the surfactant vesicles resulted in a complex with a density higher than the density of the surfactant alone. The protein A-colloidal gold identified LPS in the LPS-surfactant complexes. The toxicity of E. coli LPS was enhanced by complexing with the surfactant when compared with the intraperitoneal injection into CF1 mice, even at a 64:1 ratio of surfactant to LPS. The complexing of LPS and surfactant in the lung may alter the physiologic properties of surfactant that contribute to the physiopathological changes observed with some types of pneumonia. Images

Brogden, K A; Cutlip, R C; Lehmkuhl, H D



Low endotoxic activity of lipopolysaccharides isolated from Bradyrhizobium, Mesorhizobium, and Azospirillum strains.  


The endotoxic activities of lipopolysaccharides (LPS) isolated from different strains of rhizobia and rhizobacteria (Bradyrhizobium, Mesorhizobium, and Azospirillum) were compared to those of Salmonella enterica sv. Typhimurium LPS. The biological activity of all the examined preparations, measured as Limulus lysate gelation, production of tumor necrosis factor (TNF), interleukin-1? (IL-1?), and interleukin-6 (IL-6), and nitrogen oxide (NO) induction in human myelomonocytic cells (line THP-1), was considerably lower than that of the reference enterobacterial endotoxin. Among the rhizobial lipopolysaccharides, the activities of Mesorhizobium huakuii and Azospirillum lipoferum LPSs were higher than those of the LPS preparations from five strains of Bradyrhizobium. The weak endotoxic activity of the examined preparations was correlated with differences in lipid A structure compared to Salmonella. PMID:21091983

Komaniecka, Iwona; Zdzisinska, Barbara; Kandefer-Szerszen, Martyna; Choma, Adam



Ambient endotoxin concentrations in PM10 from Southern California.  

PubMed Central

Concentrations of endotoxin in urban air pollution have not previously been extensively characterized. We measured 24-hr levels of PM10 (particulate matter < 10 microm in aerodynamic diameter) and the associated endotoxin component once every 6 weeks for 1 year in 13 communities in Southern California. All the samples collected had detectable PM10 and endotoxin levels. The geometric mean PM10 was 34.6 microg/m3 [geometric SD (GSD), 2.1; range, 3.0-135]. By volume, the endotoxin geometric mean was 0.44 endotoxin units (EU)/m3 (GSD, 3.1; range, 0.03-5.44). Per unit material collected, the geometric mean of endotoxin collected was 13.6 EU/mg (GSD, 3.2; range, 0.7-96.8). No correlation was found between endotoxin concentrations and other ambient pollutants concurrently measured [ozone, nitrogen dioxide, total acids, or PM2.5 (particulate matter < 2.5 micro m in aerodynamic diameter]. PM10 and endotoxin concentrations were significantly correlated, most strongly in summer. Samples collected in more rural and agricultural areas had lower PM10 and mid-range endotoxin levels. The high desert and mountain communities had lower PM10 levels but endotoxin levels comparable with or higher than the rural agricultural sites. By volume, endotoxin levels were highest at sites downwind of Los Angeles, California, which were also the locations of highest PM10. Endotoxin concentrations measured in this study were all < 5.5 EU/m3, which is lower than recognized thresholds for acute adverse health effects for occupational exposures but in the same range as indoor household concentrations. This study provides the first extensive characterization of endotoxin concentration across a large metropolitan area in relation to PM10 and other pollutant monitoring, and supports the need for studies of the role of endotoxin in childhood asthma in urban settings.

Mueller-Anneling, Linda; Avol, Ed; Peters, John M; Thorne, Peter S



Lipopolysaccharide inhibition of rat hepatic microsomal epoxide hydrolase and glutathione S-transferase gene expression irrespective of nuclear factor-?B activation  

Microsoft Academic Search

Lipopolysaccharide (LPS) is an endotoxin involved in septic shock syndrome and potentiates toxicant-induced liver injury. The effects of LPS on the constitutive and inducible expression of hepatic microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST) genes were studied in rats. Northern blot analysis showed that treatment of rats with LPS caused suppression in mEH and GST gene expression. The mEH

Sung Hee Choi; Sang Geon Kim



Effect of pH on Porphyromonas gingivalis endotoxin affinity for resins.  


This study evaluated the effect of pH on Porphyromonas gingivalis lipopolysaccharide (LPS) affinity for polymethyl methacrylate, polyethyl methacrylate, and polyethyl and polyisobutyl methacrylate resins. Specimens were exposed to 1,010 endotoxin units LPS in potassium phosphate buffer at pH 6, pH 7, or pH 8. Control specimens were incubated in LPS-free water. Sequence I evaluated LPS uptake and release from resin when exposure and elution pH were identical, whereas Sequence II evaluated LPS release from resin when elution pH differed from exposure pH. A slightly acidic pH decreased LPS affinity for all resins compared to pH 7. A slightly alkaline pH increased LPS affinity for the polyethyl methacrylate resin but decreased LPS affinity for the others compared to pH 7. The pH may affect resin-LPS affinity by altering LPS molecular charge. PMID:8957858

Knoernschild, K L; Tompkins, G R; Lefebvre, C A; Griffiths, L L; Schuster, G S



Sirt1 Deletion Leads to Enhanced Inflammation and Aggravates Endotoxin-Induced Acute Kidney Injury  

PubMed Central

Bacterial endotoxin has been known to induce excessive inflammatory responses and acute kidney injury. In the present study, we used a mouse model of endotoxemia to investigate the role of Sirt1 in inflammatory kidney injury. We examined molecular and cellular responses in inducible Sirt1 knockout (Sirt1?/?) mice and wild type littermates (Sirt1+/+) in lipopolysaccharide (LPS)-induced kidney injury. Our studies demonstrated that Sirt1 deletion caused aggravated kidney injury, which was associated with increased inflammatory responses including elevated pro-inflammatory cytokine production, and increased ICAM-1 and VCAM-1 expression. Inflammatory signaling such as STAT3/ERK phosphorylation and NF-?B activation was markedly elevated in kidney tissues of Sirt1 knockout mice after LPS challenge. The results indicate that Sirt1 is protective against LPS-induced acute kidney injury by suppressing kidney inflammation and down-regulating inflammatory signaling.

Gao, Rong; Chen, Jiao; Hu, Yuxin; Li, Zhenyu; Wang, Shuxia; Shetty, Sreerama; Fu, Jian



Carbohydrates inhibit the potentiating effect of bacteria, endotoxin and virus on basophil histamine release.  


Histamine release caused by calcium ionophore A23187 and anti-IgE was examined in leukocyte suspensions from 8 healthy individuals. Staphylococcus aureus, lipopolysaccharide (LPS) from Salmonella typhimurium and influenza A virus were found to enhance the histamine release but did not release histamine per se. The potentiation of mediator release depends on a non-transient signal since the potentiating effect was also obtained by preincubation of the cells with LPS followed by wash-out and stimulation of the cells with anti-IgE. The potentiation was abolished or reduced by galactose, N-acetyl-glucosamine, alpha-methyl-D-glucoside, alpha-methyl-D-mannoside, N-acetylneuraminic acid and lactose, but not by glucose. These findings indicate that the enhancement of mediator release by bacteria, endotoxin, and virus depends on a sugar-mediated reaction. PMID:1695460

Norn, S; Clementsen, P; Kristensen, K S; Hannoun, C; Jarlřv, J O



Sensitivity of mice to lipopolysaccharide is increased by a high saturated fat and cholesterol diet  

Microsoft Academic Search

BACKGROUND: It was hypothesized that a pro-atherogenic, high saturated fat and cholesterol diet (HCD) would increase the inflammatory response to E. coli endotoxin (LPS) and increase its concentration in plasma after administration to mice. METHODS: C57Bl\\/6 mice were fed a HCD or a control diet (CD) for 4 weeks, and then treated with saline, 0.5, 1 or 2 mg LPS\\/kg,

Hong Huang; Tongzheng Liu; Jane L Rose; Rachel L Stevens; Dale G Hoyt



Reactivity of retinal blood flow to 100% oxygen breathing after lipopolysaccharide administration in healthy subjects  

Microsoft Academic Search

Administration of low doses of Escherichia coli endotoxin (LPS) to humans enables the study of inflammatory mechanisms. The purpose of the present study was to investigate the retinal vascular reactivity after LPS infusion. In a randomized placebo-controlled cross-over study, 18 healthy male volunteers received 20IU\\/kg LPS or placebo as an intravenous bolus infusion. Outcome parameters were measured at baseline and

Julia Kolodjaschna; Fatmire Berisha; Michael Lasta; Elzbieta Polska; Gabriele Fuchsjäger-Mayrl; Leopold Schmetterer



Effect of inhibition of inducible nitric oxide synthase and guanylyl cyclase on endotoxin-induced delay in gastric emptying and intestinal transit in mice.  


Nitric oxide (NO) is postulated to play a role in endotoxin-induced ileus. We investigated the effect of selective blockade of inducible NO synthase (iNOS) and guanylyl cyclase on endotoxin-induced ileus in mice. Thirty minutes before injection of lipopolysaccharides (LPS), mice were pretreated with L-NAME (N omega-nitro-L-arginine methyl ester, non-selective NOS inhibitor), 1400W (N-(3-(aminomethyl)benzyl)acetamide, selective iNOS inhibitor), ODQ (1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, guanylyl cyclase inhibitor), dimethyl sulfoxide (DMSO, vehicle), or dexamethasone. After 18 h, general well being deteriorated and the mice developed hypothermia and a significant delay in gastric emptying and intestinal transit as measured by Evans blue. 1400W completely reversed the endotoxin-induced delay in gastric emptying, while L-NAME did not have these beneficial effects. On the contrary, even in control mice, L-NAME delayed gastric emptying. Dexamethasone, DMSO, and ODQ mimicked the effect of 1400W on endotoxin-induced delay in gastric emptying. The endotoxin-induced delay in transit was significantly improved only by 1400W. None of the drugs reversed the hypothermia. In LPS mice treated with L-NAME, the behavior scale increased even further, while it decreased after treatment with 1400W. In conclusion, selective inhibition of iNOS reverses the endotoxin-induced delay in gastric emptying and transit and improves general well being. The pathway used by NO, derived from iNOS, may involve inhibition of guanylyl cyclase or radical scavenging. PMID:12166774

De Winter, Benedicte Y; Bredenoord, Albert J; De Man, Joris G; Moreels, Tom G; Herman, Arnold G; Pelckmans, Paul A



Comparison of the rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay for endotoxin in hepatitis B vaccines and the effect of aluminum hydroxide.  


The rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay have been used to detect endotoxins in vaccines, but interactions between the endotoxins and proteins or aluminum hydroxide can interfere with the results. Currently, the rabbit pyrogen test is used to detect endotoxin in hepatitis B (HB) vaccines even though the HB surface protein, the active ingredient, is over-expressed in and purified from eukaryotic cells which lack endotoxin. Therefore, we examined the possibility of replacing the animal tests with the more efficient LAL test. To this end, we determined whether the aluminum hydroxide in the HB vaccines affects the rabbit pyrogen test and the LAL assay. HB vaccines and HB protein solutions spiked with lipopolysaccharide (LPS) produced almost the same dose-dependent temperature rise in rabbits, indicating that the aluminum hydroxide in the HB vaccine does not interfere with the pyrogenic response in rabbit. In contrast, a spike recovery study showed that aluminum hydroxide interfered with the LAL clot and kinetic assays; however, the LAL clot assay was effective at detecting endotoxin without loss of LAL activity after serial dilution of the samples. Furthermore, there was good correlation in the LAL clot assay between the amount of LPS added and the amount recovered. However, both turbidimetric and chromogenic kinetic assays displayed no correlation between the LPS amount added and recovered. Our results suggest that the LAL clot assay is sensitive and reliable when samples are properly prepared, and can be used to replace the rabbit pyrogen test for the detection of endotoxin in HB vaccines. PMID:16055344

Park, Chul-Yong; Jung, Seung-Ha; Bak, Jong-Phil; Lee, Sun-Suk; Rhee, Dong-Kwon



TLR4 activation of TRPC6-dependent calcium signaling mediates endotoxin-induced lung vascular permeability and inflammation  

PubMed Central

Lung vascular endothelial barrier disruption and the accompanying inflammation are primary pathogenic features of acute lung injury (ALI); however, the basis for the development of both remains unclear. Studies have shown that activation of transient receptor potential canonical (TRPC) channels induces Ca2+ entry, which is essential for increased endothelial permeability. Here, we addressed the role of Toll-like receptor 4 (TLR4) intersection with TRPC6-dependent Ca2+ signaling in endothelial cells (ECs) in mediating lung vascular leakage and inflammation. We find that the endotoxin (lipopolysaccharide; LPS) induces Ca2+ entry in ECs in a TLR4-dependent manner. Moreover, deletion of TRPC6 renders mice resistant to endotoxin-induced barrier dysfunction and inflammation, and protects against sepsis-induced lethality. TRPC6 induces Ca2+ entry in ECs, which is secondary to the generation of diacylglycerol (DAG) induced by LPS. Ca2+ entry mediated by TRPC6, in turn, activates the nonmuscle myosin light chain kinase (MYLK), which not only increases lung vascular permeability but also serves as a scaffold to promote the interaction of myeloid differentiation factor 88 and IL-1R–associated kinase 4, which are required for NF-?B activation and lung inflammation. Our findings suggest that TRPC6-dependent Ca2+ entry into ECs, secondary to TLR4-induced DAG generation, participates in mediating both lung vascular barrier disruption and inflammation induced by endotoxin.

Tauseef, Mohammad; Knezevic, Nebojsa; Chava, Koteswara R.; Smith, Monica; Sukriti, Sukriti; Gianaris, Nicholas; Obukhov, Alexander G.; Vogel, Stephen M.; Schraufnagel, Dean E.; Dietrich, Alexander; Birnbaumer, Lutz; Malik, Asrar B.



Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14  

SciTech Connect

CD14 functions as a key pattern recognition receptor for a diverse array of Gram-negative and Gram-positive cell-wall components in the host innate immune response by binding to pathogen-associated molecular patterns (PAMPs) at partially overlapping binding site(s). To determine the potential contribution of CD14 residues in this pattern recognition, we have examined using solution NMR spectroscopy, the binding of three different endotoxin ligands, lipopolysaccharide, lipoteichoic acid, and a PGN-derived compound, muramyl dipeptide to a {sup 15}N isotopically labeled 152-residue N-terminal fragment of sCD14 expressed in Pichia pastoris. Mapping of NMR spectral changes upon addition of ligands revealed that the pattern of residues affected by binding of each ligand is partially similar and partially different. This first direct structural observation of the ability of specific residue combinations of CD14 to differentially affect endotoxin binding may help explain the broad specificity of CD14 in ligand recognition and provide a structural basis for pattern recognition. Another interesting finding from the observed spectral changes is that the mode of binding may be dynamically modulated and could provide a mechanism for binding endotoxins with structural diversity through a common binding site.

Albright, Seth; Chen Bin; Holbrook, Kristen [Biochemistry, Cellular and Molecular Biology Department, University of Tennessee, M407 Walters Life Sciences, 1410 Cumberland Avenue, Knoxville, TN 37996-0840 (United States); Jain, Nitin U. [Biochemistry, Cellular and Molecular Biology Department, University of Tennessee, M407 Walters Life Sciences, 1410 Cumberland Avenue, Knoxville, TN 37996-0840 (United States)], E-mail:



Proteomic analysis of differentially expressed proteins in caprine milk during experimentally induced endotoxin mastitis.  


The goal of the current study was to identify proteins in goat milk before and at 18 h following intramammary challenge with lipopolysaccharide (LPS). Initial evaluation of protein profiles generated using 2-dimensional gel electrophoresis on skim milk samples from a group of 6 goats collected before challenge and at 18, 24, and 48 h after LPS challenge revealed little change in the abundance of casein proteins, and minimal changes in the presence or abundance of the plasma protein serum albumin, which is known to leak into milk during coliform mastitis in dairy cattle. Proteins in baseline milk samples and in milk from the same goats 18 h post-LPS challenge were excised from the gels, and peptides were sequenced using nano-flow liquid chromatography coupled with tandem mass spectrometry. Despite the overwhelming presence of casein proteins and ?-lactoglobulin, the lower abundance proteins ?-2-microglobulin, fatty acid-binding protein, serum albumin, and retinol-binding protein were detected in skim milk samples from healthy goats. Skim milk samples 18 h postchallenge were characterized by the sustained presence and abundance of the casein proteins, and by the presence of haptoglobin, serum amyloid A, lactoferrin, cathelicidin-1, and cathelicidin-3. No marked differences in the intensity of the spot corresponding to serum albumin were observed in gels of skim milk samples 18 h postchallenge, which could indicate that the breakdown of the blood-milk barrier during endotoxin mastitis may not be as profound in goats as has been observed in dairy cattle. Nonetheless, the occurrence of an inflammatory response was supported by elevated somatic cell counts in the goat milk following inoculation with endotoxin, as well as by the presence of both antimicrobial and acute phase proteins. The results provide information about the composition of proteins in goat milk as well as added knowledge of the host response during endotoxin mastitis in goats. PMID:23498005

Olumee-Shabon, Z; Swain, T; Smith, E A; Tall, E; Boehmer, J L



If blocking potency of ivabradine is preserved under elevated endotoxin levels in human atrial myocytes.  


Lower heart rate is associated with better survival in patients with multiple organ dysfunction syndrome (MODS), a disease mostly caused by sepsis. The benefits of heart rate reduction by ivabradine during MODS are currently being investigated in the MODIfY clinical trial. Ivabradine is a selective inhibitor of the pacemaker current If and since If is impaired by lipopolysaccharide (LPS, endotoxin), a trigger of sepsis, we aimed to explore If blocking potency of ivabradine under elevated endotoxin levels in human atrial cardiomyocytes. Treatment of myocytes with S-LPS (containing the lipid A moiety, a core oligosaccharide and an O-polysaccharide chain) but not R595 (an O-chain lacking LPS-form) caused If inhibition under acute and chronic septic conditions. The specific interaction of S-LPS but not R595 to pacemaker channels HCN2 and HCN4 proves the necessity of O-chain for S-LPS-HCN interaction. The efficacy of ivabradine to block If was reduced under septic conditions, an observation that correlated with lower intracellular ivabradine concentrations in S-LPS- but not R595-treated cardiomyocytes. Computational analysis using a sinoatrial pacemaker cell model revealed that despite a reduction of If under septic conditions, ivabradine further decelerated pacemaking activity. This novel finding, i.e. If inhibition by ivabradine under elevated endotoxin levels in vitro, may provide a molecular understanding for the efficacy of this drug on heart rate reduction under septic conditions in vivo, e.g. the MODIfY clinical trial. PMID:24583250

Scheruebel, Susanne; Koyani, Chintan N; Hallström, Seth; Lang, Petra; Platzer, Dieter; Mächler, Heinrich; Lohner, Karl; Malle, Ernst; Zorn-Pauly, Klaus; Pelzmann, Brigitte



Protective effects of inhaled carbon monoxide in endotoxin-induced cholestasis is dependent on its kinetics.  


Carbon monoxide (CO), a product of heme oxygenase (HMOX), has many beneficial biological functions and is a promising therapeutic agent for many pathological conditions. However, the kinetics of inhaled CO and its protective role in endotoxin-induced cholestasis is not fully known. Thus, our objective was to characterize the kinetics of inhaled CO and then investigate its use in early phase experimental endotoxin-induced cholestasis. Female Wistar rats were randomly divided into 4 groups: CON (control), LPS (lipopolysaccharide, 6 mg/kg), CO (250 ppm COx1h), and CO + LPS. Rats were sacrificed at 0-12 h after LPS administration. Tissues and blood were collected for liver injury markers and tissue CO distribution measurements. Livers were harvested for measurements of Hmox activity, Hmox1 mRNA expression, cytokines (IL10, IL6, TNF), and bile lipid and pigment transporters. Half-lives of CO in spleen, blood, heart, brain, kidney, liver, and lungs were 2.4 ± 1.5, 2.3 ± 0.8, 1.8 ± 1.6, 1.5 ± 1.2, 1.1 ± 1.1, 0.6 ± 0.3, 0.6 ± 0.2 h, respectively. CO treatment increased liver IL10 mRNA and decreased TNF expression 1 h after LPS treatment and prevented the down-regulation of bile acid and bilirubin hepatic transporters (Slc10a1, Abcb11, and Abcc2, p < 0.05), an effect closely related to the kinetics. The protective effect of CO against cholestatic liver injury persisted even 12 h after CO exposure, as shown by attenuation of serum cholestatic markers in CO-treated animals. CO exposure substantially attenuated endotoxin-induced cholestatic liver injury and was directly related to the kinetics of inhaled CO. This data underscores the importance of the kinetics of inhaled CO for the proper design of experimental and clinical studies of using CO as a treatment strategy. PMID:24148277

Vanova, K; Suk, J; Petr, T; Cerny, D; Slanar, O; Vreman, H J; Wong, R J; Zima, T; Vitek, L; Muchova, L



Analusis by 252Cf plasma desorption mass spectrometry of Bordetella pertussis endotoxin after nitrous deamination  

NASA Astrophysics Data System (ADS)

Endotoxic lipopolysaccharides (LPSs) are the major components of Gram-negative bacterial outer membrane. Like many amphipathic molecules, they pose problems of heterogeneity, purity, solubility, and aggregation. Nevertheless, PDMS has recently have been applied to unmodified endotoxins composed of LPS having uip to five sugar units in their saccharide chain. The B. Pertussis LPSs, most of which have a dodecasaccharide domain, ahve been analysed by classical methods and the masses of the separate lipid and saccharide domains determined after rupture of the bond linking them. However, the acid treatment employed for these and most chemical analyses can also modify structures in the vicinity of the bond. In order to investigate this biologically-important region, the endotoxin was treated to nitrous deamination, which shortens the saccharide chain to five sugars, but preserves the acid-labile region of the LPS. The PDM spectrum of this derivative, which required new conditions for its desorption, confirmed the structure analysis and demonstrated the presence of at least four molecular species.

Deprun, C.; Karibian, D.; Caroff, M.



Effect of UHMWPE Particle Size, Dose, and Endotoxin on in vitro Macrophage Response.  


Ultra-high molecular weight polyethylene wear debris generated by a prosthetic hip or knee has been linked to osteolysis and the limited lifespan of the implant. However, research results are conflicting with regard to which characteristics of the polyethylene wear debris are most inflammatory. The goal of this study was to determine whether particle size, number, and the presence of endotoxin significantly contribute to increased secretion of pro-inflammatory mediators by macrophages in vitro in response to polyethylene wear debris generated by a hip simulator. The results show that the prevailing inflammatory factor is endotoxin. The macrophages released only minimal levels of TNF-? and IL-6 in response to cleaned polyethylene particles, but these cytokines were released in significantly higher amounts in response to particles spiked with lipopolysaccharide (LPS). The number (up to 500 particles per cell) and size of the particles tested in this study had no significant influence on any of the measured outputs (macrophage viability, TNF-?, IL-6, or PGE2) unless associated with LPS. PMID:24941405

Alley, Carie; Haggard, Warren; Smith, Richard



Organ Dysfunction among Piglets Treated with Inhaled Nitric Oxide and Intravenous Hydrocortisone during Prolonged Endotoxin Infusion  

PubMed Central

Objective It has previously been shown that a combination of inhaled nitric oxide (iNO) and intravenous (IV) steroid attenuates endotoxin-induced organ damage in a 6-hour porcine endotoxemia model. We aimed to further explore these effects in a 30-hour model with attention to clinically important variables. Design Randomized controlled trial. Setting University animal laboratory. Subjects Domestic piglets (n?=?30). Interventions Animals were randomized into 5 groups (n?=?6 each): 1) Controls, 2) LPS-only (endotoxin/lipopolysaccharide (LPS) infusion), 3) LPS + iNO, 4) LPS + IV steroid, 5) LPS + iNO + IV steroid. Measurements and Main Results Exposure to LPS temporarily increased pulmonary artery mean pressure and impeded renal function with elevated serum creatinine and acidosis compared to a control group over the 30-hour study period. Double treatment with both iNO and IV steroid tended to blunt the deterioration in renal function, although the only significant effect was on Base Excess (p?=?0.045). None of the LPS + iNO + IV steroid treated animals died during the study period, whereas one animal died in each of the other LPS-infused groups. Conclusions This study suggests that combined early therapy with iNO and IV steroid is associated with partial protection of kidney function after 30 hours of experimental LPS infusion.

Goranson, Sofie Paues; Gozdzik, Waldemar; Harbut, Piotr; Ryniak, Stanislaw; Zielinski, Stanislaw; Haegerstrand, Caroline Gillis; Kubler, Andrzej; Hedenstierna, Goran; Frostell, Claes; Albert, Johanna



Adsorption of inflammatory cytokines and endotoxin by mesoporous polymers and activated carbons  

Microsoft Academic Search

Adsorption of E. coli lipopolysaccharide (LPS) and an inflammatory cytokine TNF-? from model solutions on uncoated ‘hyper-crosslinked’ polystyrene polymers MN200 and MN500 and activated carbons Carboxen 1003 and Carboxen 1010 has been studied. It has been shown that TNF? can be efficiently removed by both non-functionalised MN200 and MN500 functionalised with cation exchange functional groups. On the contrary, surface chemistry

M. C. Murphy; S. Patel; G. J. Phillips; J. G. Davies; A. W. Lloyd; V. M. Gun'ko; S. V. Mikhalovsky



Isolation, characterization, and biological properties of an endotoxin-like material from the gram-positive organism Listeria monocytogenes.  

PubMed Central

The bacterial component responsible for the induction of transient cold agglutinin syndrome in rabbits after intravenous injection of heat-killed Listeria monocytogenes type 4B has been purified and biologically and chemically characterized. A purified immunoglobulin M cold agglutinin was prepared from high-titer sera resulting from the immunization of rabbits with heat-killed L. monocytogenes type 4B and was subsequently used to monitor the purification of the bacterial component responsible for its induction. The bacterial component was isolated from a hot phenol-water extract of lyophilized L. monocytogenes type 4B by multiple molecular sieve chromatography. Upon chemical analysis the purified material was found to be strikingly similar in chemical composition to gram-negative lipopolysaccharide endotoxins. The material contained 15% total fatty acid (of which 50% was beta-hydroxymyristic acid), 40 to 45% neutral sugar (glucose, galactose, and rhamnose), 11.5% amino sugar, 12% uronic acid, 2.5% 2-keto-3-deoxyoctonic acid, 2% heptose, 0.87% phosphorus, and 1.6% amino acid, thereby accounting for 85 to 90% of the weight of the component. Electron micrographs of the purified material were similar to those of lipopolysaccharide preparations from gram-negative organisms. The purified material exist in aqueous solutions as large aggregates, but can be dissociated into a single smaller subunit (3.1S) by dialysis against sodium dodecyl sulfate buffer. The listerial component was toxic and pyrogenic to rabbits, producing symptoms typical of gram-negative endotoxins. Activity in the limulus lysate gelation assay and in the carbocyanine dye assay provides a further link of this material with classical gram-negative endotoxins. Images

Wexler, H; Oppenheim, J D



Endotoxin contamination of Agaricus blazei Murrill extract enhances murine immunologic responses and inhibits the growth of sarcoma 180 implants in vivo.  


Agaricus blazei Murrill, a native mushroom of Brazil, has been reported to be an immunoreactant with anti-tumor effect. There are many reports on the anti-tumor effect of Agaricus blazei Murrill; however, the precise mechanism of its effect is not fully understood. In this study, we tried to confirm the anti-tumor effect of Agaricus blazei Murrill against Sarcoma 180 cells in a mouse model and found that an inhibitory effect on tumor growth was induced by peritoneal injection of a freeze-dried, hot water extract of Agaricus blazei Murrill (FAG). We noted that there were differences among each sample in terms of anti-tumor activity. We hypothesized that this was because some contaminants of FAG were affecting the anti-tumor activity. We evaluated cytokine secretion from mouse peritoneal cells incubated with FAG. While high interleukin-6 and tumor necrosis factor-? secretions were observed in response to crude FAG, they were dramatically decreased by the removal of endotoxin from the FAG using an endotoxin-specific polymyxin B-conjugated affinity column. The reductions were synergistically recovered by adding an amount of lipopolysaccharide equivalent to the amount of contaminated endotoxin. Thus, these data suggest that the contaminated endotoxin of Agaricus blazei Murrill may act as an immunomodulator of anti-tumor activity. PMID:20932249

Kobayashi, Hitoshi; Masumoto, Junya



mRNA and protein stability regulate the differential expression of pro- and anti-inflammatory genes in endotoxin-tolerant THP-1 cells.  


The products of proinflammatory genes such as interleukin-1beta (IL-1beta) and cyclooxygenase-2 (COX-2) initiate many of the events associated with sepsis. Transcription of these genes is subsequently down-regulated, whereas expression of anti-inflammatory genes such as secretory interleukin-1 receptor antagonist (sIL-1 RA) is maintained. Differential expression is associated with endotoxin tolerance, a cellular phenomenon common to sepsis and characterized by reduced proinflammatory gene expression after repeated exposure to lipopolysaccharide. As a model for endotoxin tolerance, we examined the expression of COX-2 and sIL-1 RA in a human promonocyte cell line, THP-1. We observed a 5-fold decrease in COX-2 protein in endotoxin-tolerant cells relative to control cells. In contrast, sIL-1 RA protein increased 5-fold in control and tolerant cells and remained elevated. Decreased COX-2 production is due to repressed transcription and not enhanced mRNA degradation. In addition, COX-2 protein is turned over rapidly. Transcription of sIL-1 RA is also repressed during tolerance. However, sIL-1 RA mRNA is degraded more slowly than COX-2 mRNA, allowing continued synthesis of sIL-1 RA protein that is very stable. These results indicate that differential expression during endotoxin tolerance occurs by transcriptional repression of COX-2 and by protein and mRNA stabilization of sIL-1 RA. PMID:10766854

Learn, C A; Mizel, S B; McCall, C E



Interception of the endotoxin-induced arterial hyporeactivity to vasoconstrictors.  


Septic shock is a severe pathophysiologic condition characterized by vasodilation, hypotension, hypoperfusion, tissue hypoxia, multiple organ failure and death. It is unclear what causes the septic vasodilation that may result from general dysfunction of vascular smooth muscles (VSMs) or selective disruption of vasomotor balances in VSMs. The latter could be due to enhanced vasorelaxation and/or depressed vasoconstriction. Understanding these may lead to pharmacological interventions to septic vasodilation. Therefore, we performed studies in isolated and perfused mesenteric arterial rings. A 20-h exposure of the rings to lipopolysaccharide (LPS, 1?g/ml) led to hyporeactivity to phenylephrine (PE). However, the responses of the LPS-treated rings to high concentrations of KCl (60mM) and ATP remained comparable to control rings, suggesting that contractility of VSMs is retained. The hyporeactivity was marginally affected by atropine, indomethacin and L-NAME, suggesting that endothelium-dependent vasorelaxation does not play a major role. In addition to PE, the LPS-treated rings were hyporeactive to dopamine, histamine and angiotensin II. They showed intermediate hyporeactivity to the thromboxane-A2 receptor agonist U46619. Little hyporeactivity to endothelin-1 (ET-1), serotonin (5-HT) and vasopressin was found. ET-1-induced vasoconstriction occurred without endothelium, whereas the effect of 5-HT was endothelium dependent. Although rings were hyporeactive to some of the vasopressors, their vasoconstriction effects were significantly potentiated by PE co-application. Taken together, these data suggest that the endotoxin-induced vasodilation may not result from general dysfunction of VSMs, neither from the endothelium-dependent vasorelaxation. The promising vascular response to various vasoconstrictors found in this study warrants further investigations of therapeutic potentials of these agents. PMID:24792896

Zhang, Shuang; Cui, Ningren; Li, Shanshan; Guo, Lei; Wu, Yang; Zhu, Daling; Jiang, Chun



Antidiabetic Drug Metformin Suppresses Endotoxin-Induced Uveitis in Rats  

PubMed Central

Purpose. To investigate the therapeutic effects of metformin, a commonly used antidiabetic drug, in preventing endotoxin-induced uveitis (EIU) in rats. Methods. EIU in Lewis rats was developed by subcutaneous injection of lipopolysaccharide (LPS; 150 ?g). Metformin (300 mg/kg body weight, intraperitoneally) or its carrier was injected either 12 hours before or 2 hours after LPS induction. Three and 24 hours after EIU, eyes were enucleated and aqueous humor (AqH) was collected. The MILLIPLEX-MAG Rat cytokine-chemokine magnetic bead array was used to determine inflammatory cytokines. The expression of Cox-2, phosphorylation of AMPK, and NF-?B (p65) were determined immunohistochemically. Primary human nonpigmented ciliary epithelial cells (HNPECs) were used to determine the in vitro efficacy of metformin. Results. Compared with controls, the EIU rat AqH had significantly increased number of infiltrating cells and increased levels of various cytokines and chemokines (TNF-?, MCP-1, IL-1?, MIP-1?, IL-6, Leptin, and IL-18) and metformin significantly prevented the increase. Metformin also prevented the expression of Cox-2 and phosphorylation of p65, and increased the activation of AMPK in the ciliary bodies and retinal tissues. Moreover, metformin prevented the expression of Cox-2, iNOS, and activation of NF-kB in the HNPECs and decreased the levels of NO and PGE2 in cell culture media. Conclusions. Our results for the first time demonstrate a novel role of the antidiabetic drug, metformin, in suppressing uveitis in rats and suggest that this drug could be developed to prevent uveitis complications.

Kalariya, Nilesh M.; Shoeb, Mohammad; Ansari, Naseem H.; Srivastava, Satish K.; Ramana, Kota V.



Endotoxin-induced effects on nucleotide catabolism in mouse kidney.  


Extracellular adenosine 5'-triphosphate (ATP) acts as a proinflammatory mediator. Adenosine, the final product of ATP breakdown, is an anti-inflammatory compound, acting mainly on adenosine A(2A) receptors. Considering that the kidney is an organ strongly affected during systemic inflammatory responses and that ectonucleotidases are responsible for the control of extracellular nucleotide and nucleoside levels, we examined the endotoxin-induced effects on ectonucleotidases in kidney membranes of mice, and whether CGS-21680 hydrochloride (3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid), a selective adenosine A(2A) receptor agonist, antagonizes the lipopolysaccharide (LPS)-induced effects on nucleotide catabolism in kidney. Animals were injected intraperitoneally with 12 mg/kg LPS and/or 0.5mg/kg CGS-21680 or saline. Nucleotidase activities were determined in kidney membrane preparations and ATP metabolism was measured by high performance liquid chromatography (HPLC) assay. Analysis of ectonucleotidase expression was carried out by semi-quantitative semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Exposure to endotoxemia promoted an increase in ATP and p-Nitrophenyl thymidine 5'-monophosphate (p-Nph-5'-TMP) hydrolysis, and a decrease in adenosine 5'-monophosphate (AMP) hydrolysis. CGS-21680 treatment failed to reverse these changes. HPLC analysis indicated a decrease in extracellular ATP and adenosine levels in groups treated with LPS and LPS plus CGS-21680. The expression pattern of ectonucleotidases revealed an increase in Entpd3, Enpp2, and Enpp3 mRNA levels after LPS injection. These findings indicate that nucleotide and nucleoside availability in mouse kidney is altered at different stages of endotoxemia, in order to protect the integrity of this organ when exposed to systemic inflammation. PMID:22108548

Vuaden, Fernanda C; Savio, Luiz Eduardo B; Ramos, Denise B; Casali, Emerson A; Bogo, Maurício R; Bonan, Carla D




PubMed Central

Paraffin oil containing oil red O and emulsified with lipopolysaccharide obtained from Escherichia coli was ingested rapidly by guinea pig polymorphonuclear leukocytes or human peripheral blood granulocytes and monocytes after opsonization by fresh homologous serum. The initial rate of engulfment of the particles was spectrophotometrically assayed by determination of cell-associated oil red O and reflected the opsonic activity of the serum. This activity was resistant to dialysis but labile to heat, hydrazine, and zymosan, required divalent cations, and was maximal in the presence of Ca++ and Mg++. It was associated with the fixation of [125I]C3 to the lipopolysaccharide particles. Genetically C3-deficient serum had no opsonic activity, and this activity was restored by the addition of purified C3. Normal and C4-deficient guinea pig serum and normal, C2-, and C4-deficient human sera were equally effective in opsonizing lipopolysaccharide particles and lipopolysaccharide particles sensitized with heat-inactivated lipopolysaccharide immune serum. Cord serum deficient in glycine-rich beta-glycoprotein (GBG) (properdin factor B) had diminished opsonic activity which was improved by addition of purified GBG. Thus, C3 fixation to lipopolysaccharide particles occurs by means of the properdin system, and the opsonization and ingestion of lipopolysaccharide particles constitutes a quantitative functional assay of this pathway.

Stossel, Thomas P.; Alper, Chester A.; Rosen, Fred S.



Applicability of bacterial endotoxins test to various blood products by the use of endotoxin-specific lysates.  


Endotoxin contamination is a serious threat to the safety of parenteral drugs, and the rabbit pyrogen test has played a crucial role in controlling this contamination. Although the highly sensitive endotoxin test has replaced the pyrogen test for various pharmaceuticals, the pyrogen test is still implemented as the control test for most blood products in Japan. We examined the applicability of the endotoxin test to blood products for reliable detection and quantification of endotoxin. Nineteen types of blood products were tested for interfering factors based on spike/recovery of endotoxin by using 2 types of endotoxin-specific lysate reagents for photometric techniques. Interfering effects on the endotoxin test by the products could be eliminated by diluting from 1/2 to 1/16, with the exception of antithrombin III. However, conventional lysate reagents that also react with non-pyrogenic substances, such as (1-3)-?-D-glucan, produced results that were not relevant to endotoxin content or pyrogenicity. Our results showed that the endotoxin test would be applicable to most blood products if used with appropriate endotoxin-specific lysate reagents. PMID:20702107

Ochiai, Masaki; Yamamoto, Akihiko; Naito, Seishiro; Maeyama, Jun-Ichi; Masumi, Atsuko; Hamaguchi, Isao; Horiuchi, Yoshinobu; Yamaguchi, Kazunari



A static magnetic field attenuates lipopolysaccharide-induced neuro-inflammatory response via IL-6-mediated pathway.  


Abstract An effective method for controlling brain damage and neurodegeneration caused by inflammation remains elusive. Down-expression of the lipopolysaccharide (LPS)-induced inflammatory cytokines resulting in endotoxin tolerance is reported as an alternative anti-infection treatment. Nonetheless, because the dosage and action site are hard to control, endotoxin tolerance caused by low-dose LPS injection in brain tissue may induce side effects. The aim of this study was to test the hypothesis that static magnetic fields (SMF) stimulate endotoxin tolerance in brain tissue. In this study, survival rate and pathological changes in brain tissues of LPS-challenged mice were examined with and without SMF treatment. In addition, the effects of SMF exposure on growth rate and cytokine expression of LPS-challenged BV-2 microglia cells were monitored. Our results showed that SMF pre-exposure had positive effects on the survival rate and histological outcomes of LPS-treated mice. Furthermore, SMF exposure significantly decreased IL-6 expression in BV-2 cells (p?endotoxin tolerance. We suggest that SMF has potential as an alternative simulation source for controlling LPS-induced excess neuro-inflammatory response. PMID:23781996

Shen, Li-Kuo; Huang, Haw-Ming; Yang, Po-Chieh; Huang, Yung-Kai; Wang, Peter Da-Yun; Leung, Ting-Kai; Chen, Chi-Jen; Chang, Wei-Jen



Effect of vasopressors on organ blood flow during endotoxin shock in pigs  

SciTech Connect

A volume-resuscitated porcine endotoxin shock model was used to evaluate the effect on organ blood flow of increasing systemic arterial blood pressure with vasopressors. Administration of 0.05-0.2 mg/kg of Escherichia coli endotoxin (E) reduced mean arterial blood pressure (MAP) to 50 mmHg, decreased systemic vascular resistance to 50% of control, and did not change cardiac output or heart rate. Blood flow measured with radiolabeled microspheres to brain, kidney, spleen, and skeletal muscle was reduced during endotoxin shock, but blood flow to left ventricle, small and large intestine, and stomach remained at pre-endotoxin levels throughout the study period. Four groups of animals were used to evaluate the effect of vasopressor therapy. Vasopressors were administered starting 60 min after E exposure, and the dose of each was titrated to increase MAP to 75 mmHg. Despite the increase in MAP, brain blood flow did not increase in any group. Norepinephrine alone increased blood flow to the left ventricle. The dose of norepinephrine required to increase MAP by 20-25 mmHg during E shock was 30 times the does required for a similar increase in MAP in animals not receiving E. The authors conclude 1) that hypotension in the fluid resuscitated porcine E shock model is primarily the result of peripheral vasodilatation, 2) that the vascular response to vasoconstrictors in this model is markedly attenuated following E administration, 3) that blood pressure elevation with norepinephrine, dopamine, and phenylephrine neither decreases blood flow to any organs nor increases blood flow to organs with reduced flow, and 4) that norepinephrine, dopamine, and phenylephrine affect regional blood flow similarly in this model.

Breslow, M.J.; Miller, C.F.; Parker, S.D.; Walman, A.T.; Traystman, R.J.



Preparation and Preclinical Evaluation of a Novel Liposomal Complete-Core Lipopolysaccharide Vaccine  

Microsoft Academic Search

Our objective is to develop a prophylactic vaccine strategy that can be evaluated for surgical and other high-risk hospitalized patients. In this paper, we describe the preparation and preclinical evaluation of a liposomal complete-core lipopolysaccharide (LPS) vaccine that is nontoxic and broadly antigenic. Complete- core (Ra-chemotype) LPSs were isolated from four gram-negative bacterial strains (Escherichia coli K-12, E. coli R1,




Citric acid effects on brain and liver oxidative stress in lipopolysaccharide-treated mice.  


Citric acid is a weak organic acid found in the greatest amounts in citrus fruits. This study examined the effect of citric acid on endotoxin-induced oxidative stress of the brain and liver. Mice were challenged with a single intraperitoneal dose of lipopolysaccharide (LPS; 200 ?g/kg). Citric acid was given orally at 1, 2, or 4?g/kg at time of endotoxin injection and mice were euthanized 4?h later. LPS induced oxidative stress in the brain and liver tissue, resulting in marked increase in lipid peroxidation (malondialdehyde [MDA]) and nitrite, while significantly decreasing reduced glutathione, glutathione peroxidase (GPx), and paraoxonase 1 (PON1) activity. Tumor necrosis factor-alpha (TNF-?) showed a pronounced increase in brain tissue after endotoxin injection. The administration of citric acid (1-2?g/kg) attenuated LPS-induced elevations in brain MDA, nitrite, TNF-?, GPx, and PON1 activity. In the liver, nitrite was decreased by 1?g/kg citric acid. GPx activity was increased, while PON1 activity was decreased by citric acid. The LPS-induced liver injury, DNA fragmentation, serum transaminase elevations, caspase-3, and inducible nitric oxide synthase expression were attenuated by 1-2?g/kg citric acid. DNA fragmentation, however, increased after 4?g/kg citric acid. Thus in this model of systemic inflammation, citric acid (1-2?g/kg) decreased brain lipid peroxidation and inflammation, liver damage, and DNA fragmentation. PMID:24433072

Abdel-Salam, Omar M E; Youness, Eman R; Mohammed, Nadia A; Morsy, Safaa M Youssef; Omara, Enayat A; Sleem, Amany A



Endotoxin Inhalation Alters Lung Development in Neonatal Mice  

PubMed Central

Background Childhood asthma is a significant public health problem. Epidemiologic evidence suggests an association between childhood asthma exacerbations and early life exposure to environmental endotoxin. Although the pathogenesis of endotoxin-induced adult asthma is well studied, questions remain about the impact of environmental endotoxin on pulmonary responsiveness in early life. Methods We developed a murine model of neonatal/juvenile endotoxin exposures approximating those in young children and evaluated the lungs inflammatory and remodeling responses. Results Persistent lung inflammation induced by the inhalation of endotoxin in early life was demonstrated by the influx of inflammatory cells and pro-inflammatory mediators to the airways and resulted in abnormal alveolarization. Conclusions Results of this study advance the understanding of the impact early life endotoxin inhalation has on the lower airways, and demonstrates the importance of an experimental design that approximates environmental exposures as they occur in young children.

Kulhankova, Katarina; George, Caroline L.S.; Kline, Joel N.; Darling, Melissa; Thorne, Peter S.



Survey of Innate Immune Responses to Burkholderia pseudomallei in Human Blood Identifies a Central Role for Lipopolysaccharide  

PubMed Central

B. pseudomallei is a gram-negative bacterium that causes the tropical infection melioidosis. In northeast Thailand, mortality from melioidosis approaches 40%. As exemplified by the lipopolysaccharide-Toll-like receptor 4 interaction, innate immune responses to invading bacteria are precipitated by activation of host pathogen recognition receptors by pathogen associated molecular patterns. Human melioidosis is characterized by up-regulation of pathogen recognition receptors and pro-inflammatory cytokine release. In contrast to many gram-negative pathogens, however, the lipopolysaccharide of B. pseudomallei is considered only weakly inflammatory. We conducted a study in 300 healthy Thai subjects to investigate the ex vivo human blood response to various bacterial pathogen associated molecular patterns, including lipopolysaccharide from several bacteria, and to two heat-killed B. pseudomallei isolates. We measured cytokine levels after stimulation of fresh whole blood with a panel of stimuli. We found that age, sex, and white blood cell count modulate the innate immune response to B. pseudomallei. We further observed that, in comparison to other stimuli, the innate immune response to B. pseudomallei is most highly correlated with the response to lipopolysaccharide. The magnitude of cytokine responses induced by B. pseudomallei lipopolysaccharide was significantly greater than those induced by lipopolysaccharide from Escherichia coli and comparable to many responses induced by lipopolysaccharide from Salmonella minnesota despite lower amounts of lipid A in the B. pseudomallei lipopolysaccharide preparation. In human monocytes stimulated with B. pseudomallei, addition of polymyxin B or a TLR4/MD-2 neutralizing antibody inhibited the majority of TNF-? production. Challenging existing views, our data indicate that the innate immune response to B. pseudomallei in human blood is largely driven by lipopolysaccharide, and that the response to B. pseudomallei lipopolysaccharide in blood is greater than the response to other lipopolysaccharide expressing isolates. Our findings suggest that B. pseudomallei lipopolysaccharide may play a central role in stimulating the host response in melioidosis.

Chantratita, Narisara; Tandhavanant, Sarunporn; Myers, Nicolle D.; Seal, Sudeshna; Arayawichanont, Arkhom; Kliangsa-ad, Aroonsri; Hittle, Lauren E.; Ernst, Robert K.; Emond, Mary J.; Wurfel, Mark M.; Day, Nicholas P. J.; Peacock, Sharon J.; West, T. Eoin



Adipokinetic hormone enhances nodule formation and phenoloxidase activation in adult locusts injected with bacterial lipopolysaccharide  

Microsoft Academic Search

Interactions between the locust endocrine and immune systems have been studied in vivo in relation to nodule formation and activation of the prophenoloxidase cascade in the haemolymph. Injection of bacterial lipopolysaccharide (LPS) extracted from Escherichia coli induces nodule formation in larval and adult locusts but does not increase phenoloxidase activity in the haemolymph. Nodule formation starts rapidly after injection of

Graham Goldsworthy; Shashi Chandrakant; Kwaku Opoku-Ware



Genome-Wide Expression Analysis of Lipopolysaccharide-Induced Mastitis in a Mouse Model  

Microsoft Academic Search

To better understand the acute host response to Escherichia coli mastitis, we analyzed gene expression patterns of approximately 23,000 transcripts 4 h after an intramammary infusion of lipopolysaccharide (LPS) in a mouse model. A total of 489 genes were significantly affected, of which 391 were induced and 98 were repressed. Gene ontology analysis demonstrated that most of the induced genes

Jiamao Zheng; Anjanette D. Watson; David E. Kerr



Predictors of Endotoxin Levels in U.S. Housing  

PubMed Central

Background The relationship of domestic endotoxin exposure to allergy and asthma has been widely investigated. However, few studies have evaluated predictors of household endotoxin, and none have done so for multiple locations within homes and on a national scale. Objectives We assayed 2,552 house dust samples in a nationwide study to understand the predictors of household endotoxin in bedroom floors, family room floors, beds, kitchen floors, and family room sofas. Methods Reservoir house dust from five locations within homes was assayed for endotoxin and demographic and housing information was assessed through questionnaire and onsite evaluation of 2,456 residents of 831 homes selected to represent national demographics. We performed repeated-measures analysis of variance (rANOVA) for 37 candidate variables to identify independent predictors of endotoxin. Meteorologic data were obtained for each primary sampling unit and tested as predictors of indoor endotoxin to determine if wetter or warmer microclimates were associated with higher endotoxin levels. Results Weighted geometric mean endotoxin concentration ranged from 18.7 to 80.5 endotoxin units (EU)/mg for the five sampling locations, and endotoxin load ranged from 4,160 to 19,500 EU/m2. Bivariate analyses and rANOVA demonstrated that major predictors of endotoxin concentration were sampling location in the home, census division, educational attainment, presence of children, current dog ownership, resident-described problems with cockroaches, food debris, cockroach stains, and evidence of smoking observed by field staff. Low household income entered the model if educational attainment was removed. Conclusion Increased endotoxin in household reservoir dust is principally associated with poverty, people, pets, household cleanliness, and geography.

Thorne, Peter S.; Cohn, Richard D.; Mav, Deepak; Arbes, Samuel J.; Zeldin, Darryl C.



Disulfide rearrangement triggered by translocon assembly controls lipopolysaccharide export.  


The presence of lipopolysaccharide (LPS) on the cell surface of Gram-negative bacteria is critical for viability. A conserved ?-barrel membrane protein LptD (lipopolysaccharide transport protein D) translocates LPS from the periplasm across the outer membrane (OM). In Escherichia coli, this protein contains two disulfide bonds and forms the OM LPS translocon with the lipoprotein LptE. Here, we identified seven in vivo states on the oxidative-folding pathway of LptD. Proper assembly involved a nonfunctional intermediate containing non-native disulfides. Intermediate formation required the oxidase DsbA, and subsequent maturation to the active form with native disulfides was triggered by LptE. Thus, disulfide bond-dependent protein folding of LptD requires the proper assembly of a two-protein complex to promote disulfide bond rearrangement. PMID:22936569

Chng, Shu-Sin; Xue, Mingyu; Garner, Ronald A; Kadokura, Hiroshi; Boyd, Dana; Beckwith, Jonathan; Kahne, Daniel



Smooth muscle F-actin disassembly and RhoA/Rho-kinase signaling during endotoxin-induced alterations in pulmonary arterial compliance.  


Endotoxemia is associated with changed pulmonary vascular function with respect to vasoreactivity, endothelial permeability, and activation of inducible nitric oxide synthase II (NOSII). However, whether altered passive arterial wall mechanics contribute to this endotoxin-induced pulmonary vascular dysfunction is still unknown. Therefore, we investigated whether endotoxin affects the passive arterial mechanics and compliance of isolated rat pulmonary arteries. Pulmonary arteries of pentobarbital-anesthetized Wistar rats (n = 55) were isolated and exposed to Escherichia coli endotoxin (50 microg/ml) for 20 h. Endotoxin increased pulmonary artery diameter and compliance (transmural pressure = 13 mmHg) in an endothelium-, Ca2+-, or NOSII-induced NO release-independent manner. Interestingly, the endotoxin-induced alterations in the passive arterial mechanics were accompanied by disassembly of the smooth muscle cell (SMC) F-actin cytoskeleton. Disassembly of F-actin by incubation of control arteries with the cytoskeleton-disrupting agent cytochalasin B or the Rho-kinase inhibitor Y-27632 induced a similar increase in passive arterial diameter and compliance. In contrast, RhoA activation by lysophosphatidic acid prevented the endotoxin-induced alterations in the pulmonary SMC F-actin cytoskeleton and passive mechanics. In conclusion, these findings indicate that disassembly of the SMC F-actin cytoskeleton and RhoA/Rho-kinase signaling act as mediators of endotoxin-induced changes in the pulmonary arterial mechanics. They imply the involvement of F-actin rearrangement and RhoA/Rho-kinase signaling in endotoxemia-induced vascular lung injury. PMID:14514519

Boer, Christa; van Nieuw Amerongen, Geerten P; Groeneveld, A B Johan; Scheffer, Gert Jan; de Lange, Jaap J; Westerhof, Nico; van Hinsbergh, Victor W M; Sipkema, Pieter



Enhanced Toxicity for Mice of Pactamycin with Bacterial Endotoxin  

PubMed Central

Combinations of pactamycin and Salmonella typhosa 0901 endotoxin, administered simultaneously, killed more BALB/c mice than comparable doses of either agent alone The slopes of the dose-response curves for combinations of endotoxin and pactamycin were parallel to both that for endotoxin alone and the antitumor drug alone; therefore, no new mechanism of toxicity has been evoked by the combination. The synergistic toxicity of endotoxin and pactamycin was due to an in vivo interaction rather than a direct reaction between the two agents. Phenobarbital pretreatment protected the mice from toxicity of the antitumor agent alone but not from the lethal action of the combination. Pretreatment with endotoxin increased the resistance of the mice to both endotoxin alone and the combination. Third agents unable to protect mice from the synergistic toxicity of endotoxin and pactamycin were ?[p-(fluoren-9-ylidenemethyl)-phenyl]-2-piperidine-ethanol, neomycin, phenylbutazone, polymyxin B, and tybamate. Prednisolone pretreatment alleviated the toxicity of the combination. For the restricted series of killed bacteria and bacterial products tested for capability to enhance the toxicity of pactamycin, only gram-negative bacterial cells were potent. These results indicated that pactamycin rendered the mice more susceptible to endotoxin and that endotoxin was the causal lethal agent.

Bradley, S. G.; Rose, W. C.



Crystal structure of LpxC, a zinc-dependent deacetylase essential for endotoxin biosynthesis  

PubMed Central

The outer leaflet of the outer membrane of the Gram-negative bacterium serves as a permeability barrier and is composed of lipopolysaccharide, also known as endotoxin. The membrane anchor of lipopolysaccharide is lipid A, the biosynthesis of which is essential for cell viability. The first committed step in lipid A biosynthesis is catalyzed by UDP-(3-O-(R-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase (LpxC), a zinc-dependent deacetylase. Here we report the crystal structure of LpxC from Aquifex aeolicus, which reveals a new ?+? fold reflecting primordial gene duplication and fusion, as well as a new zinc-binding motif. The catalytic zinc ion resides at the base of an active-site cleft and adjacent to a hydrophobic tunnel occupied by a fatty acid. This tunnel accounts for the specificity of LpxC toward substrates and inhibitors bearing appropriately positioned 3-O-fatty acid substituents. Notably, simple inhibitors designed to target interactions in the hydrophobic tunnel bind with micromolar affinity, thereby representing a step toward the structure-based design of a potent, broad-spectrum antibacterial drug.

Whittington, Douglas A.; Rusche, Kristin M.; Shin, Hyunshun; Fierke, Carol A.; Christianson, David W.



Infusion of freshly isolated autologous bone marrow derived mononuclear cells prevents endotoxin-induced lung injury in an ex-vivo perfused swine model.  


INTRODUCTION: The acute respiratory distress syndrome (ARDS), affects up to 150,000 patients per year in the United States. We and other groups have demonstrated that bone marrow derived mesenchymal stromal stem cells prevent ARDS induced by systemic and local administration of endotoxin (lipopolysaccharide (LPS)) in mice. METHODS: A study was undertaken to determine the effects of the diverse populations of bone marrow derived cells on the pathophysiology of ARDS, using a unique ex-vivo swine preparation, in which only the ventilated lung and the liver are perfused with autologous blood. Six experimental groups were designated as: 1) endotoxin alone, 2) endotoxin + total fresh whole bone marrow nuclear cells (BMC), 3) endotoxin + non-hematopoietic bone marrow cells (CD45 neg), 4) endotoxin + hematopoietic bone marrow cells (CD45 positive), 5) endotoxin + buffy coat and 6) endotoxin + in vitro expanded swine CD45 negative adherent allogeneic bone marrow cells (cultured CD45neg). We measured at different levels the biological consequences of the infusion of the different subsets of cells. The measured parameters were: pulmonary vascular resistance (PVR), gas exchange (PO2), lung edema (lung wet/dry weight), gene expression and serum concentrations of the pro-inflammatory cytokines IL-1?, TNF-? and IL-6. RESULTS: Infusion of freshly purified autologous total BMCs, as well as non-hematopoietic CD45(-) bone marrow cells significantly reduced endotoxin-induced pulmonary hypertension and hypoxemia and reduced the lung edema. Also, in the groups that received BMCs and cultured CD45neg we observed a decrease in the levels of IL-1? and TNF-? in plasma. Infusion of hematopoietic CD45(+) bone marrow cells or peripheral blood buffy coat cells did not protect against LPS-induced lung injury. CONCLUSIONS: We conclude that infusion of freshly isolated autologous whole bone marrow cells and the subset of non-hematopoietic cells can suppress the acute humoral and physiologic responses induced by endotoxemia by modulating the inflammatory response, mechanisms that do not involve engraftment or trans-differentiation of the cells. These observations may have important implications for the design of future cell therapies for ARDS. PMID:23497755

Rojas, Mauricio; Parker, Richard E; Thorn, Natalie; Corredor, Claudia; Iyer, Smita S; Bueno, Marta; Mroz, Lyle; Cardenes, Nayra; Mora, Ana L; Stecenko, Arlene A; Brigham, Kenneth L



Endotoxins, arachidonic acid, and superoxide formation.  


The pathophysiologic relevance of altered eicosanoid metabolism and free-radical production during endotoxemia is reviewed. A direct or complement-mediated release of eicosanoids--depending on the species, the tissue, and the kind of endotoxin--has been found to be associated with endotoxemic shock. Prostacyclin appears to be partially responsible for the early decrease in blood pressure and clearly induces the sustained hypotension of endotoxemia but is irrelevant to its fatal outcome. In contrast, simultaneous administration of vasodilating prostanoids such as prostaglandin E1 and metabolically stable analogues of prostacyclin prevents endotoxin lethality. Thromboxane A2 may contribute to platelet aggregation and vasoconstriction, but its quantitative importance often is obscured by other mediators. The peptidoleukotrienes, in particular leukotriene D4, contribute to lung damage and--in galactosamine-sensitized rats, at least--to fatal liver destruction. Little is known about the role of leukotriene B4 in endotoxemia. Endotoxin suppresses rather than stimulates superoxide release from phagocytes in vitro but leads to massive free-radical formation mediated by complement activation in vivo and, thereby, contributes to lung injury. PMID:2825324

Flohé, L; Giertz, H



Sequence context induced antimicrobial activity: insight into lipopolysaccharide permeabilization.  


Lactoferrampin (WR17, Trp 268-Arg 284), an antimicrobial peptide, is known to have significant antibacterial and candidacidal activities. However, there are no previous studies explaining how WR17 permeabilizes the outer membrane of Gram negative bacteria and neutralizes endotoxins. In this study we used a series of assays like antimicrobial activity, calcein leakage, NPN dye uptake and endotoxin neutralization assay to show that the sequence context of WR17 modulates its multi-faceted activities. We determined the high resolution NMR structure of WR17 in LPS and found that the N-ter region forms a helix (Trp1-Phe11) and orients itself at an angle of 45° into the lipopolysaccharide (LPS) micelle, whereas the C-ter region (Lys13-Arg17) remains as a flexible extended random coil. We also verified this result through in silico molecular modeling simulation. Isothermal titration calorimetry showed that the interaction of WR17 and its analogues with LPS was primarily endothermic in nature. Using several fluorescence techniques such as anisotropy and red edge excitation shift assay we revealed motional restriction for Trp1 of WR17 in LPS. The distance between the indole ring of Trp1 of WR17 and the polar head group of LPS is around 7 Ĺ, as obtained from the depth of insertion assay. Additionally, MD simulation demonstrated that the incorporation of the peptide in LPS is achieved with the help of the K(13)xK(15)xR(17) motif at the C-terminus. This novel anchoring "K(13)NKSR(17)" motif is currently being utilized in our ongoing research to design novel anti-endotoxic molecules. PMID:24714742

Ghosh, Anirban; Datta, Aritreyee; Jana, Jagannath; Kar, Rajiv Kumar; Chatterjee, Chiradip; Chatterjee, Subhrangsu; Bhunia, Anirban



Expression of low endotoxin 3-O-sulfotransferase in Bacillus subtilis and Bacillus megaterium.  


A key enzyme for the biosynthesis and bioengineering of heparin, 3-O-sulfotransferase-1 (3-OST-1), was expressed and purified in Gram-positive Bacillus subtilis and Bacillus megaterium. Western blotting, protein sequence analysis, and enzyme activity measurement confirmed the expression. The enzymatic activity of 3-OST-1 expressed in Bacillus species were found to be similar to those found when expressed in Escherichia coli. The endotoxin level in 3-OST-1 from B. subtilis and B. megaterium were 10(4)-10(5)-fold lower than that of the E. coli-expressed 3-OST-1, which makes the Bacillus expression system of particular interest for the generation of pharmaceutical grade raw heparin from nonanimal sources. PMID:23912211

Wang, Wenya; Englaender, Jacob A; Xu, Peng; Mehta, Krunal K; Suwan, Jiraporn; Dordick, Jonathan S; Zhang, Fuming; Yuan, Qipeng; Linhardt, Robert J; Koffas, Mattheos



The Origin of 8-Amino-3,8-dideoxy-d-manno-octulosonic Acid (Kdo8N) in the Lipopolysaccharide of Shewanella oneidensis*  

PubMed Central

Lipopolysaccharide (LPS; endotoxin) is an essential component of the outer monolayer of nearly all Gram-negative bacteria. LPS is composed of a hydrophobic anchor, known as lipid A, an inner core oligosaccharide, and a repeating O-antigen polysaccharide. In nearly all species, the first sugar bridging the hydrophobic lipid A and the polysaccharide domain is 3-deoxy-d-manno-octulosonic acid (Kdo), and thus it is critically important for LPS biosynthesis. Modifications to lipid A have been shown to be important for resistance to antimicrobial peptides as well as modulating recognition by the mammalian innate immune system. Therefore, lipid A derivatives have been used for development of vaccine strains and vaccine adjuvants. One derivative that has yet to be studied is 8-amino-3,8-dideoxy-d-manno-octulosonic acid (Kdo8N), which is found exclusively in marine bacteria of the genus Shewanella. Using bioinformatics, a candidate gene cluster for Kdo8N biosynthesis was identified in Shewanella oneidensis. Expression of these genes recombinantly in Escherichia coli resulted in lipid A containing Kdo8N, and in vitro assays confirmed their proposed enzymatic function. Both the in vivo and in vitro data were consistent with direct conversion of Kdo to Kdo8N prior to its incorporation into the Kdo8N-lipid A domain of LPS by a metal-dependent oxidase followed by a glutamate-dependent aminotransferase. To our knowledge, this oxidase is the first enzyme shown to oxidize an alcohol using a metal and molecular oxygen, not NAD(P)+. Creation of an S. oneidensis in-frame deletion strain showed increased sensitivity to the cationic antimicrobial peptide polymyxin as well as bile salts, suggesting a role in outer membrane integrity.

Gattis, Samuel G.; Chung, Hak Suk; Trent, M. Stephen; Raetz, Christian R. H.



Acute Hypoxia Decreases E. coli LPS-Induced Cytokine Production and NF-?B Activation in Alveolar Macrophages*  

PubMed Central

Reductions in alveolar oxygenation during lung hypoxia/reoxygenation (H/R) injury are common after gram-negative endotoxemia. However, the effects of H/R on endotoxin-stimulated cytokine production by alveolar macrophages are unclear and may depend upon thresholds for hypoxic oxyradical generation in situ. Here TNF-? and IL-? production were determined in rat alveolar macrophages stimulated with E. coli lipopolysaccharide (LPS, serotype O55:B5) while exposed to either normoxia for up to 24 h, to brief normocarbic hypoxia (1.5 h at an atmospheric PO2 = 10 ± 2 mm Hg), or to combined H/R. LPS-induced TNF-? and IL-? were reduced at the peak of hypoxia and by reoxygenation in LPS + H/R cells (P < 0.01) compared with normoxic controls despite no changes in reduced glutathione (GSH) or in PGE2 production. Both TNF-? mRNA and NF-?B activation were reduced by hypoxia that suppressed superoxide anion generation. Thus, dynamic reductions in the ambient PO2 of alveolar macrophages that do not deplete GSH suppress LPS-induced TNF-? expression, IL-? production, and NF-?B activation even as oxyradical production is decreased.

Matuschak, George M.; Nayak, Ravi; Doyle, Timothy M.; Lechner, Andrew J.



In vitro toxicity and interactions of environmental contaminants (Arochlor 1254 and mercury) and immunomodulatory agents (lipopolysaccharide and cortisol) on thymocytes from lake trout (Salvelinus namaycush)  

USGS Publications Warehouse

The immunotoxicity of chemical combinations commonly encountered by the lake trout (Salvelinus namaycush) immune system was the focus of this study. It was hypothesised that combinations of an environmental contaminant (mercuric chloride or Aroclor 1254) and an immunomodulatory agent (bacterial endotoxin or cortisol) might interact to produce a greater toxicity than that of the environmental contaminant alone at concentrations typically encountered in piscine blood and other tissues. Thus lake trout thymocytes were isolated and treated with mercuric chloride or Aroclor 1254 in the presence and absence of cortisol or lipopolysaccharide. Incubations were performed for 6 or 20h at 4A?C or 10A?C. Lipopolysaccharide did not affect the toxicity of either contaminant. In contrast, cortisol enhanced the toxicity of both environmental contaminants. Hence, stressors that lead to increased cortisol production, but not lipopolysaccharide directly, may increase the toxicity of mercury and Aroclor 1254 to lake trout thymocytes.

Miller, Gregory G.; Sweet, Leonard I.; Adams, Jean V.; Omann, Geneva M.; Passino-Reader, Dora R.; Meier, Peter G.



Effect of radiodetoxified endotoxin and trace elements on reticulo-endothelial system damaged by ethanol in rats.  


For the demonstration of RES activity 99-mTc labelled nano-albumon micro-colloid solution (prepared by OSSKI, Budapest, Hungary) was injected into the prepared jugular vein of narcotized rats and the clearance rate was investigated. The effects of various materials have been observed on RES in vivo. Continuous consumption of 20% ethanol solution for 15 weeks decreased the phagocytic activity. One hundred microgram/100 g body weight (i.v.) of the radio-detoxified endotoxin preparation (RD-LPS, TOLERIN) prepared from Escherichia coli endotoxin by 60Co-gamma irradiation (150 KGy) increased the granulopectic activity of RES of intoxicated animals. The preparation containing trace elements also increased the former activity and the treatment consisting of the use of both trace elements and RD-LPD was as effective as the RD-LPD per sc. PMID:7866731

Gál, K; Bertók, L



Expression of a Bacillus thuringiensis ?-endotoxin gene by Bacillus pumilus  

Microsoft Academic Search

The ?-endotoxin genes from Bacillus thuringiensis were introduced into a rhizosphere-inhabiting Bacillus pumilus isolate to create a ?-endotoxin expression and delivery system for subterranean feeding insects such as the larvae of pale western cutworm (Agrotis orthogonia Morrison (Lepidoptera: Noctuidae)). Preliminary experiments indicated that Bacillus thuringiensis subsp. kurstaki cultures were toxic to pale western cutworm larvae. Three different cry genes from

L. B. Selinger; G. G. Khachatourians; J. R. Byers; M. F. Hynes



Endotoxin contamination in wound dressings made of natural biomaterials.  


Contamination by endotoxin of nine kinds of wound dressings made of natural biomaterials (calcium alginate, collagen, chitin, and poly-L-leucine) was examined with the use of water extracts. By applying the Limulus amoebocyte lysate (LAL) test, high concentrations of endotoxin were detected in extracts from three kinds of products made of calcium alginate. These extracts evoked fever in rabbits and induced the release of a proinflammatory (pyrogenic) cytokine, interleukin-6 (IL-6), from human monocytic cells (MM6-CA8). The effects disappeared when the extracts were treated with endotoxin-removing gel column chromatography or with an endotoxin antagonist, B464, confirming that the contaminating pyrogen was endotoxin. A noteworthy finding was that one of the endotoxin-containing extracts showed very weak IL-6-inducibility in human monocytic cells in contrast to its high pyrogenicity to rabbits. The discrepancy could be explained based on differences between humans and rabbits in sensitivity to the endotoxin, because the extract showed higher proinflammatory-cytokine (TNF-alpha)-inducibility in rabbit whole-blood cells (WBCs) than human WBCs. The results suggest that the LAL test is a useful method of detecting endotoxin contamination in wound dressings and the MM6-CA8 assay is a good supplement to the LAL test for evaluating pyrogenicity in humans accurately. PMID:12808594

Nakagawa, Y; Murai, T; Hasegawa, Chie; Hirata, M; Tsuchiya, T; Yagami, T; Haishima, Y



Endotoxin exacerbates immunologically induced liver injury in cooperation with interferon- ?  

Microsoft Academic Search

Objective and Design: To investigate the role of endotoxin in the development of immunologically induced liver injury. Materials and Methods: A new model of liver injury induced in BALB\\/c mice by delayed-type hypersensitivity to picryl chloride and its in vitro assay for the interaction between liver nonparenchymal and parenchymal cells were used. Results: Plasma endotoxin in the injured liver correlated

X. Chen; J. Cao; Q. Xu



Effect of irradiated haemophilus influenzae endotoxin preparations in mice.  


A detoxified substance (rdLPS) was produced from Haemophilus influenzae endotoxin by ionizing radiation and its capacity to prevent attacks of dyspnoea elicited by endotoxin inhalation in mice has been studied. The rdLPS proved to be an effective stimulant of aspecific immune resistance of mice but it could only partly prevent attacks of dyspnoea. PMID:6609516

Csukás, Z; Wessely, M; Slowik, F; Bertók, L




EPA Science Inventory

Lime addition is a common practice for treating biosolids in order to meet EPA 503 requirements for land application. Since this treatment kills the majority of microorganisms, will it increase the level of endotoxins present in biosolids? And, if endotoxin levels are increased, ...


Pathogenesis of pulmonary edema associated with intracisternal endotoxin in dogs.  


To examine the role of central nervous system injury in the pathogenesis of pulmonary edema, we injected Escherichia coli endotoxin (5 mg/kg) into the cisterna magna of six dogs (group E) and compared, over 4 h, both the pulmonary edema and cerebrospinal fluid (CSF) abnormalities with those in six control dogs (group C). In group E, intracisternal endotoxin raised intracranial pressure from 21 +/- 6 to 38 +/- 8 cmH2O (P less than 0.001), CSF total protein from 18 +/- 6 to 54 +/- 19 mg/dl (P less than 0.001), and CSF malondialdehyde from 0.12 +/- 0.11 to 0.61 +/- 0.35 nmol/ml (P less than 0.05); all were unchanged in group C. When the pulmonary wedge pressure was maintained at 10 mmHg by fluid infusion, extravascular thermal volume in group E increased from 7.2 +/- 1.2 to 12.0 +/- 2.7 ml/kg (P less than 0.005) at 4 h when the excised lungs weighed 13.6 +/- 1.5 g/kg; in group C, extravascular thermal volume did not increase, and the excised lungs weighed less (10.8 +/- 1.3 g/kg, P less than 0.05) than those in group E. The dry weights of the lungs were not different between groups, and the alveolar lining fluid-to-plasma albumin ratio in both groups remained low, 0.1-0.2. Fluid infusion in group E (9.2 +/- 2.9 liters) caused colloid oncotic pressure to decrease 4.5 +/- 2.8 mmHg; colloid oncotic pressure fell less (0.8 +/- 1.9 mmHg, P less than 0.001) in group C as less fluid (2.2 +/- 1.5 liters, P less than 0.001) was required to maintain pulmonary wedge pressure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2189863

Nahum, A; Wood, L D; Crawford, G; Ripper, R; Segil, L; Sznajder, J I



Prevention of endotoxin-induced leukocytic infiltration of lung with radio-detoxified endotoxin (TOLERIN) pretreatment.  


Five mg of bacterial endotoxin (LPS) can provoke a serious leukocytic pulmonary infiltration in rats. The same dose of radiation-detoxified LPS (RD-LPS; 150 kGy 60Co-gamma irradiated) can produce only slight pulmonary infiltration. However, 100 micrograms RD-LPS--as pretreatment--can prevent the LPS-induced leukocytic infiltration of the lung. PMID:3434087

Szabó, L G; Tóth, T; Benedek, G; Bertók, L



Production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus.  

PubMed Central

Rat astrocytes, immunologically competent glial cells of the central nervous system (CNS), released a variety of cytokines after activation. Lipopolysaccharide-stimulated astrocytes produced tumor necrosis factor (TNF) as demonstrated by Northern blot analysis using a mouse TNF probe and by functional assay. Biological activity of rat astrocyte-derived TNF was neutralized by rabbit antiserum against recombinant murine TNF. Stimulation of astrocytes by lipopolysaccharide also activated the interleukin 1 and interleukin 6 genes. We have also investigated whether a neurotropic paramyxovirus, Newcastle disease virus, triggers cytokine production by astrocytes. This virus induced astrocytes to produce TNF, lymphotoxin, interleukin 6, and alpha- and beta-interferons. Thus, stimulation by endotoxin and virus activated distinct, yet overlapping, sets of cytokine genes. We propose that astrocytes and the cytokines they produce may play a significant role in the pathogenesis of immunologically and/or virally mediated CNS disease, in CNS intercellular communication, and in the interactions between the nervous and immune systems. Images

Lieberman, A P; Pitha, P M; Shin, H S; Shin, M L



Biosynthesis of the Polymannose Lipopolysaccharide O-antigens from Escherichia coli Serotypes O8 and O9a Requires a Unique Combination of Single- and Multiple-active Site Mannosyltransferases*  

PubMed Central

The Escherichia coli O9a and O8 O-antigen serotypes represent model systems for the ABC transporter-dependent synthesis of bacterial polysaccharides. The O9a and O8 antigens are linear mannose homopolymers containing conserved reducing termini (the primer-adaptor), a serotype-specific repeat unit domain, and a terminator. Synthesis of these glycans occurs on the polyisoprenoid lipid-linked primer, undecaprenol pyrophosphoryl-GlcpNAc, by two conserved mannosyltransferases, WbdC and WbdB, and a serotype-specific mannosyltransferase, WbdA. The glycan structure and pattern of conservation in the O9a and O8 mannosyltransferases are not consistent with the existing model of O9a biosynthesis. Here we establish a revised pathway using a combination of in vivo (mutant complementation) experiments and in vitro strategies with purified enzymes and synthetic acceptors. WbdC and WbdB synthesize the adaptor region, where they transfer one and two ?-(1?3)-linked mannose residues, respectively. The WbdA enzymes are solely responsible for forming the repeat unit domains of these O-antigens. WbdAO9a has two predicted active sites and polymerizes a tetrasaccharide repeat unit containing two ?-(1?3)- and two ?-(1?2)-linked mannopyranose residues. In contrast, WbdAO8 polymerizes trisaccharide repeat units containing single ?-(1?3)-, ?-(1?2)-, and ?-(1?2)-mannopyranoses. These studies illustrate assembly systems exploiting several mannosyltransferases with flexible active sites, arranged in single- and multiple-domain formats.

Greenfield, Laura K.; Richards, Michele R.; Li, Jianjun; Wakarchuk, Warren W.; Lowary, Todd L.; Whitfield, Chris



Biosynthesis of the polymannose lipopolysaccharide O-antigens from Escherichia coli serotypes O8 and O9a requires a unique combination of single- and multiple-active site mannosyltransferases.  


The Escherichia coli O9a and O8 O-antigen serotypes represent model systems for the ABC transporter-dependent synthesis of bacterial polysaccharides. The O9a and O8 antigens are linear mannose homopolymers containing conserved reducing termini (the primer-adaptor), a serotype-specific repeat unit domain, and a terminator. Synthesis of these glycans occurs on the polyisoprenoid lipid-linked primer, undecaprenol pyrophosphoryl-GlcpNAc, by two conserved mannosyltransferases, WbdC and WbdB, and a serotype-specific mannosyltransferase, WbdA. The glycan structure and pattern of conservation in the O9a and O8 mannosyltransferases are not consistent with the existing model of O9a biosynthesis. Here we establish a revised pathway using a combination of in vivo (mutant complementation) experiments and in vitro strategies with purified enzymes and synthetic acceptors. WbdC and WbdB synthesize the adaptor region, where they transfer one and two ?-(1?3)-linked mannose residues, respectively. The WbdA enzymes are solely responsible for forming the repeat unit domains of these O-antigens. WbdA(O9a) has two predicted active sites and polymerizes a tetrasaccharide repeat unit containing two ?-(1?3)- and two ?-(1?2)-linked mannopyranose residues. In contrast, WbdA(O8) polymerizes trisaccharide repeat units containing single ?-(1?3)-, ?-(1?2)-, and ?-(1?2)-mannopyranoses. These studies illustrate assembly systems exploiting several mannosyltransferases with flexible active sites, arranged in single- and multiple-domain formats. PMID:22875852

Greenfield, Laura K; Richards, Michele R; Li, Jianjun; Wakarchuk, Warren W; Lowary, Todd L; Whitfield, Chris



Applicability of bacterial endotoxins test to various blood products by the use of endotoxin-specific lysates  

Microsoft Academic Search

Endotoxin contamination is a serious threat to the safety of parenteral drugs, and the rabbit pyrogen test has played a crucial role in controlling this contamination. Although the highly sensitive endotoxin test has replaced the pyrogen test for various pharmaceuticals, the pyrogen test is still implemented as the control test for most blood products in Japan. We examined the applicability

Masaki Ochiai; Akihiko Yamamoto; Seishiro Naito; Jun-ichi Maeyama; Atsuko Masumi; Isao Hamaguchi; Yoshinobu Horiuchi; Kazunari Yamaguchi



Bacterial lipopolysaccharides and innate immunity  

Microsoft Academic Search

Bacterial lipopolysaccharides (LPS) are the major outer surface membrane components present in almost all Gram-negative bacteria and act as extremely strong stimulators of innate or natural immunity in diverse eukaryotic species ranging from insects to humans. LPS consist of a poly- or oligosaccharide region that is anchored in the outer bacterial membrane by a specific carbohydrate lipid moiety termed lipid

Christian Alexander; E. T. Rietschel



The Lipopolysaccharide-Binding Protein Is a Secretory Class 1 Acute-Phase Protein Whose Gene Is Transcriptionally Activated by APRF\\/STAT3 and Other Cytokine-Inducible Nuclear Proteins  

Microsoft Academic Search

Acute-phase reactants (APRs) are proteins synthesized in the liver following induction by interleukin-1 (IL-1),IL-6,andglucocorticoids,involvingtranscriptionalgeneactivation.Lipopolysaccharide-bindingprotein (LBP) is a recently identified hepatic secretory protein potentially involved in the pathogenesis of sepsis, capable of binding the bacterial cell wall product endotoxin and directing it to its cellular receptor, CD14. In order to examine the transcriptional induction mechanisms by which the LBP gene is




Lipoteichoic Acids from Lactobacillus johnsonii Strain La1 and Lactobacillus acidophilus Strain La10 Antagonize the Responsiveness of Human Intestinal Epithelial HT29 Cells to Lipopolysaccharide and Gram-Negative Bacteria  

Microsoft Academic Search

Intestinal epithelial cells (IECs) respond to lipopolysaccharide (LPS) from gram-negative bacteria in the presence of the soluble form of CD14 (sCD14), a major endotoxin receptor. Since sCD14 is also known to interact with gram-positive bacteria and their components, we looked at whether sCD14 could mediate their effects on human IECs. To this end, we examined the production of proinflammatory cytokines

Karine Vidal; Anne Donnet-Hughes; Dominique Granato



Differential sensitivity of Staphylo coccus epidermidis and Escherichici coli to lysozyme  

Microsoft Academic Search

Lysozyme is a natural host defence mechanism in the human body that destroys the cell walls of bacteria, and interferes with the toxic and destructive activity of endotoxins found in the lipopolysaccharide layer of the cell wall of gram-negative bacteria.Disc sensitivity methods currently in use in microbiology manuals, to measure lysozyme sensitivity of gram-positive bacteria, show small zones of inhibition

Isaiah A. Benathen; La Shon McKenzie



Visualization and analysis of lipopolysaccharide distribution in binary phospholipid bilayers  

SciTech Connect

Lipopolysaccharide (LPS) is an endotoxin released from the outer membrane of Gram-negative bacteria during infections. It have been reported that LPS may play a role in the outer membrane of bacteria similar to that of cholesterol in eukaryotic plasma membranes. In this article we compare the effect of introducing LPS or cholesterol in liposomes made of dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine on the solubilization process by Triton X-100. The results show that liposomes containing LPS or cholesterol are more resistant to solubilization by Triton X-100 than the binary phospholipid mixtures at 4 {sup o}C. The LPS distribution was analyzed on GUVs of DPPC:DOPC using FITC-LPS. Solid and liquid-crystalline domains were visualized labeling the GUVs with LAURDAN and GP images were acquired using a two-photon microscope. The images show a selective distribution of LPS in gel domains. Our results support the hypothesis that LPS could aggregate and concentrate selectively in biological membranes providing a mechanism to bring together several components of the LPS-sensing machinery.

Henning, Maria Florencia [Instituto de Investigaciones Bioquimicas La Plata (INIBIOLP), CCT-La Plata, CONICET, Facultad de Ciencias Medicas, UNLP, Calles 60 y 120, 1900 La Plata (Argentina)] [Instituto de Investigaciones Bioquimicas La Plata (INIBIOLP), CCT-La Plata, CONICET, Facultad de Ciencias Medicas, UNLP, Calles 60 y 120, 1900 La Plata (Argentina); Sanchez, Susana [Laboratory for Fluorescence Dynamics, University of California-Irvine, Irvine, CA (United States)] [Laboratory for Fluorescence Dynamics, University of California-Irvine, Irvine, CA (United States); Bakas, Laura, E-mail: [Instituto de Investigaciones Bioquimicas La Plata (INIBIOLP), CCT-La Plata, CONICET, Facultad de Ciencias Medicas, UNLP, Calles 60 y 120, 1900 La Plata (Argentina) [Instituto de Investigaciones Bioquimicas La Plata (INIBIOLP), CCT-La Plata, CONICET, Facultad de Ciencias Medicas, UNLP, Calles 60 y 120, 1900 La Plata (Argentina); Departamento de Ciencias Biologicas, Facultad de Ciencias Exactas, UNLP, Calles 47 y 115, 1900 La Plata (Argentina)



Home Endotoxin Exposure and Wheeze in Infants: Correction for Bias Due to Exposure Measurement Error  

PubMed Central

Exposure to elevated levels of endotoxin in family-room dust was previously observed to be significantly associated with increased wheeze in the first year of life among a cohort of 404 children in the Boston, Massachusetts, metropolitan area. However, it is likely that family-room dust endotoxin was a surrogate for airborne endotoxin exposure. Therefore, a related substudy characterized the relationship between levels of airborne household endotoxin and the level of endotoxin present in house dust, in addition to identifying other significant predictors of airborne endotoxin in the home. We now reexamine the relationship between endotoxin exposure and wheeze under the assumption that the level of airborne endotoxin in the home is the exposure of interest and that the amount of endotoxin in household dust is a surrogate for this exposure. We applied a measurement error correction technique, using all available data to estimate the effect of endotoxin exposure in terms of airborne concentration and accounting for the measurement error induced by using house-dust endotoxin as a surrogate measure in the portion of the data in which airborne endotoxin could not be directly measured. After adjusting for confounding by lower respiratory infection status and race/ethnicity, endotoxin exposure was found to be significantly associated with a nearly 6-fold increase in prevalence of wheeze for a one interquartile range increase in airborne endotoxin (95% confidence interval, 1.2–26) among the 360 children in households with dust endotoxin levels between the 5th and 95th percentiles.

Horick, Nora; Weller, Edie; Milton, Donald K.; Gold, Diane R.; Li, Ruifeng; Spiegelman, Donna



Key structures of bacterial peptidoglycan and lipopolysaccharide triggering the innate immune system of higher animals: Chemical synthesis and functional studies  

PubMed Central

Chemistry-based investigation is reviewed which led to identification of the active entities responsible for the immunostimulating potencies of peptidoglycan and lipopolysaccharide. Though these glycoconjugates which ubiquitously occur in wide range of bacteria as the essential components of their cell envelopes have long been known to enhance the immunological responses of higher animals, neither the precise chemical structures required nor the mechanism of their action remained to be elucidated until early 1970s. Chemical synthesis of partial structures of peptidoglycan proved N-acetylmuramyl-L-alanyl-D-isoglutamine to be the minimum structure responsible for the activity and led to later identification of its receptor protein Nod2 present in animal cells. Another active partial structure of peptidoglycan, ?-D-glutamyl-meso-diaminopimelic acid, and its receptor Nod1 were also identified as well. With regard to lipopolysaccharide, its glycolipid part named lipid A was purified and the structure studied. Chemically synthesized lipid A according to the newly elucidated structure exhibited full activity described for lipopolysaccharide known as endotoxin. Synthetic homogeneous lipid A and its structural analogues and labeled derivatives enabled precise studies of their interaction with receptor proteins and the mechanism of their action. Chemical synthesis of homogeneous partial structures of peptidoglycan and lipopolysaccharide gave unequivocal evidences for the concept that definite small molecular parts of these complex macromolecular bacterial glycoconjugates are specifically recognized by their respective receptors and trigger our defense system now widely recognized as innate immunity.

Kusumoto, Shoichi; Fukase, Koichi; Shiba, Tetsuo



Endotoxin-triggered haematological interactions in Fusobacterium necrophorum infections.  


The haematological mechanisms in the course of liver abscess formation were evaluated. They were examined by employing viable cells of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme in comparison with their endotoxins. Whole cell infection with F.n. necrophorum led to neutrophilia and to a concomitant monocytosis in parallel with those responses induced by the in vivo injection of its endotoxin. Viable infection with F.n. funduliforme was characterized by a sustained endotoxin-related monocytosis against neutropenia. The stimulatory impact of endotoxin on monocytes when released from a viable F.n. funduliforme infection suggested an inherently peculiar mechanism which differed from the induction of both neutrophilia and monocytosis when F.n. funduliforme endotoxin was administered alone. The neutrophilic inducing capacity of the F.n. necrophorum endotoxin was equally illustrated by its positive chemotactic effect on polymorphonuclear neutrophils in vitro. The data presented here emphasize the virulence of F.n. necrophorum viewed in reference to changes in leucocyte trafficking and as complemented by a relatively high endotoxin content. PMID:10817519

Garcia, G G; Goto, Y; Shinjo, T



Streptomycetes in house dust: associations with housing characteristics and endotoxin  

PubMed Central

In addition to mold, indoor bioaerosols also contain bacterial components that may have implications for human health. Endotoxin is a cell wall component in Gram-negative bacteria present at varying levels indoors that has been found to have respiratory health implications. Streptomyces is a large genus of Gram-positive bacteria, and some species have been shown to produce inflammatory reactions in vitro and in vivo. The aim of this study was to determine predictors of streptomycetes levels in house dust, and to compare the variation in streptomycetes levels with that in endotoxin levels. Dust was collected by floor vacuuming from 178 homes in the Cincinnati metropolitan area. streptomycetes levels were measured by quantitative PCR and endotoxin was assayed by the Limulus Amebocyte Lysate method. Associations between home characteristics and bacterial contaminants, expressed as concentration and load, were investigated through multiple regression analyses. The presence of two or more dogs was a strong predictor of both streptomycetes and endotoxin levels. Season of dust collection and levels of outdoor molds were predictors of streptomycetes but not endotoxin levels. In contrast, number of inhabitants was a significant predictor of endotoxin load only. Neither streptomycetes nor endotoxin levels were associated with metrics of moisture damage.

Johansson, Elisabet; Vesper, Stephen; Levin, Linda; LeMasters, Grace; Grinshpun, Sergey; Reponen, Tiina



MitoQ administration prevents endotoxin-induced cardiac dysfunction.  


Sepsis elicits severe alterations in cardiac function, impairing cardiac mitochondrial and pressure-generating capacity. Currently, there are no therapies to prevent sepsis-induced cardiac dysfunction. We tested the hypothesis that administration of a mitochondrially targeted antioxidant, 10-(6'-ubiquinonyl)-decyltriphenylphosphonium (MitoQ), would prevent endotoxin-induced reductions in cardiac mitochondrial and contractile function. Studies were performed on adult rodents (n = 52) given either saline, endotoxin (8 mg x kg(-1) x day(-1)), saline + MitoQ (500 microM), or both endotoxin and MitoQ. At 48 h animals were killed and hearts were removed for determination of either cardiac mitochondrial function (using polarography) or cardiac pressure generation (using the Langendorf technique). We found that endotoxin induced reductions in mitochondrial state 3 respiration rates, the respiratory control ratio, and ATP generation. Moreover, MitoQ administration prevented each of these endotoxin-induced abnormalities, P < 0.001. We also found that endotoxin produced reductions in cardiac pressure-generating capacity, reducing the systolic pressure-diastolic relationship. MitoQ also prevented endotoxin-induced reductions in cardiac pressure generation, P < 0.01. One potential link between mitochondrial and contractile dysfunction is caspase activation; we found that endotoxin increased cardiac levels of active caspases 9 and 3 (P < 0.001), while MitoQ prevented this increase (P < 0.01). These data demonstrate that MitoQ is a potent inhibitor of endotoxin-induced mitochondrial and cardiac abnormalities. We speculate that this agent may prove a novel therapy for sepsis-induced cardiac dysfunction. PMID:19657095

Supinski, G S; Murphy, M P; Callahan, L A



Dust sampling methods for endotoxin - an essential, but underestimated issue.  


Exposure to farming environment in early life has been associated with lower risk for allergic diseases possibly caused by increased exposure to endotoxin. The aims of this study were to compare the reproducibility of different sampling methods for endotoxin, and to determine whether environmental characteristics have different effect on endotoxin levels of different sample types. The reproducibility of sampling methods (bed dust, floor dust, vacuum cleaner dust bag dust, settled dust and air samples) was studied with repeated sampling (five visits during 1 year) in five farming and five urban homes. To examine determinants of endotoxin for different types of dust sample, sampling was conducted once in 12 farming and 17 urban homes. Endotoxin was analyzed using Limulus Amebocyte Lysate assay. Bed dust samples had the best reproducibility (intraclass correlation, ICC=66%), but the difference between farming and non-farming homes was not clear with this sample type. The reproducibility of floor (ICC=52%) and settled dust (ICC=51%) was moderate. With these sample types the difference between farming and non-farming homes was clear. Settled dust had some seasonal variation. Based on this study, the best compromise for sampling for endotoxin appears to be floor dust sample followed by bed and settled dust samples. Practical Implications Endotoxins have been widely measured, even though the validity of different sample types to reflect the endotoxin exposure level of an indoor environment is poorly known. This study shows that bed dust samples have the best reproducibility, but they do not reflect the differences in exposure due to environmental factors such as farming. Floor dust samples with moderate reproducibility may be the best choice for sampling of endotoxin in large field studies. PMID:16420494

Hyvärinen, A; Roponen, M; Tiittanen, P; Laitinen, S; Nevalainen, A; Pekkanen, J



Eicosapentaenoic acid preserves diaphragm force generation following endotoxin administration  

PubMed Central

Introduction Infections produce severe respiratory muscle weakness, which contributes to the development of respiratory failure. An effective, safe therapy to prevent respiratory muscle dysfunction in infected patients has not been defined. This study examined the effect of eicosapentaenoic acid (EPA), an immunomodulator that can be safely administered to patients, on diaphragm force generation following endotoxin administration. Methods Rats were administered the following (n = 5/group): (a) saline, (b) endotoxin, 12 mg/kg IP, (c) endotoxin + EPA (1.0 g/kg/d), and (d) EPA alone. Diaphragms were removed and measurements made of the diaphragm force-frequency curve, calpain activation, caspase activation, and protein carbonyl levels. Results Endotoxin elicited large reductions in diaphragm specific force generation (P < 0.001), and increased diaphragm caspase activation (P < 0.01), calpain activation (P < 0.001) and protein carbonyl levels (P < 0.01). EPA administration attenuated endotoxin-induced reductions in diaphragm specific force, with maximum specific force levels of 27 ± 1, 14 ± 1, 23 ± 1, and 24 ± 1 N/cm2, respectively, for control, endotoxin, endotoxin + EPA, and EPA treated groups (P < 0.001). EPA did not prevent endotoxin induced caspase activation or protein carbonyl formation but significantly reduced calpain activation (P < 0.02). Conclusions These data indicate that endotoxin-induced reductions in diaphragm specific force generation can be partially prevented by administration of EPA, a nontoxic biopharmaceutical that can be safely given to patients. We speculate that it may be possible to reduce infection-induced skeletal muscle weakness in critically ill patients by administration of EPA.



Effective Immunomodulatory Treatment of Escherichia coli Experimental Sepsis with Thalidomide  

PubMed Central

Thalidomide, an agent which inhibits biosynthesis of tumor necrosis factor alpha (TNF-?) and which is used to treat a variety of chronic inflammatory conditions, was investigated as therapy for experimental sepsis. Sepsis was induced by intraperitoneal injection of 107 CFU of Escherichia coli per kg of body weight to 80 Wistar rats divided into four groups. Group A consisted of 24 control animals that did not receive any pretreatment, group B consisted of 18 vehicle-treated control animals pretreated with seed oil, group C consisted of 30 rats administered thalidomide diluted in seed oil at a dose of 50 mg/kg 30 min before bacterial challenge, and group D consisted of eight animals that were not challenged with E. coli but that were used for white blood cell count determination. Sepsis was determined by measurement of vital signs before and 6 h after bacterial challenge. After 6 h the animals were killed and blood was sampled for culture; white blood cell count determination; and determination of endotoxin (lipopolysaccharide), TNF-?, interleukin-1? (IL-1?), and IL-6 levels. The levels of these cytokines were also estimated in the supernatants of human monocytes pretreated with thalidomide after exposure to the isolate. Sepsis developed in all vehicle-treated control animals and controls by 6 h after bacterial challenge but in only 10 animals (33.3%) pretreated with thalidomide (P < 0.0001). Six hours after bacterial challenge all animals had similar levels of endotoxemia, IL-1?, and IL-6. The mean white blood cell count for groups A, B, and C were 5,631.1, 2,638.9, and 8,169.3 cells/?l, respectively (P value between groups, <0.0001); the TNF-? levels were 77.3, 107.2, and 26.1 pg/ml, respectively (P values between groups, <0.0001). Pretreatment of human monocytes with thalidomide prevented the secretion of TNF-? and IL-1? but not that of IL-6. It is concluded that thalidomide exerts a considerable anti-inflammatory effect by preventing evolution to sepsis and by decreasing the level of production of TNF-? and therefore deserves to be further evaluated in research for the therapy of sepsis.

Giamarellos-Bourboulis, Evangelos J.; Poulaki, Helen; Kostomitsopoulos, Nikolaos; Dontas, Ismene; Perrea, Despina; Karayannacos, Panayotis E.; Giamarellou, Helen



Selective endothelin-A receptor blockade attenuates endotoxin-induced pulmonary hypertension and pulmonary vascular dysfunction  

PubMed Central

Abstract Endothelin-1 is a potent mediator of sepsis-induced pulmonary hypertension (PH). The pulmonary vascular effects of selective blockade of endothelin receptor subtype A (ETAR) during endotoxemia remain unknown. We hypothesized that selective ETAR antagonism attenuates endotoxin-induced PH and improves pulmonary artery (PA) vasoreactivity. Adult male Sprague-Dawley rats (250–450 g) received lipopolysaccharide (LPS; Salmonella typhimurium; 20 mg/kg intraperitoneally) or vehicle 6 hours before hemodynamic assessment and tissue harvest. The selective ETAR antagonist sitaxsentan (10 or 20 mg/kg) or vehicle was injected intravenously 3 hours after receipt of LPS. Right ventricular systolic pressure, mean arterial pressure (MAP), cardiac output (CO), oxygenation (P/F ratio), and serum bicarbonate were measured. Bronchoalveolar lavage (BAL) cell differential and lung wet-to-dry ratios were obtained. Endothelium-dependent and endothelium-independent vasorelaxations were determined in isolated PA rings. PA interleukin (IL)-1?, IL-6, tumor necrosis factor ? (TNF-?), and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) were measured. LPS caused PH, decreased MAP, CO, and serum bicarbonate, and increased PA IL-1?, IL-6, TNF-?, and iNOS mRNA. Sitaxsentan attenuated sepsis-induced PH and increased MAP. The P/F ratio, CO, serum bicarbonate, and BAL neutrophilia were not affected by sitaxsentan. In isolated PA rings, while not affecting phenylephrine-induced vasocontraction or endothelium-dependent relaxation, sitaxsentan dose-dependently attenuated LPS-induced alterations in endothelium-independent relaxation. PA cytokine mRNA levels were not significantly attenuated by ETAR blockade. We conclude that ETAR blockade attenuates endotoxin-induced alterations in systemic and PA pressures without negatively affecting oxygenation. This protective effect appears to be mediated not by attenuation of sepsis-induced cardiac dysfunction, acidosis, or alveolar inflammation but rather by improved endothelium-independent vasorelaxation.



The role of phosphodiesterase 3 in endotoxin-induced acute kidney injury  

PubMed Central

Background Acute kidney injury frequently accompanies sepsis. Endotoxin is known to reduce tissue levels of cAMP and low levels of cAMP have been associated with renal injury. We, therefore, hypothesized that endotoxin induced renal injury by activating phosphodiesterase 3 (PDE3) which metabolizes cAMP and that amrinone an inhibitor of PDE3 would prevent the renal injury. Methods Animals were divided into three groups (n = 7/group): 1) Control (0.9% NaCl infusion without LPS); 2) LPS (0.9% NaCl infusion with LPS); 3) Amrinone+LPS (Amrinone infusion with LPS). Either lipopolysaccharide (LPS) or vehicle was injected via the jugular vein and the rats followed for 3 hours. We explored the expression of PDE3 isoenzymes and the concentrations of cAMP in the tissue. Results The PDE3B gene but not PDE3A was upregulated in the kidney of LPS group. Immunohistochemistry also showed that PDE3B was expressed in the distal tubule in the controls and LPS caused PDE3B expression in the proximal as well. However, PDE3A was not expressed in the kidney either in the control or LPS treated groups. Tissue level of cAMP was decreased after LPS and was associated with an increase in blood urea nitrogen, creatinine, ultrastructural proximal tubular changes, and expression of inducible nitric oxide synthase (iNOS) in the endotoxemic kidney. In septic animals the phosphodiesterase 3 inhibitor, amrinone, preserved the tissue cAMP level, renal structural changes, and attenuated the increased blood urea nitrogen, creatinine, and iNOS expression in the kidney. Conclusion These findings suggest a significant role for PDE3B as an important mediator of LPS-induced acute kidney injury.



Intervention in endotoxin shock by sulfatide (I3SO3-GalCer) with a concomitant reduction in tumor necrosis factor alpha production.  

PubMed Central

Accumulating evidence indicates that tumor necrosis factor alpha (TNF-alpha) is a principal mediator of endotoxin shock. We previously reported that the action as well as the production of TNF requires the adhesion of leukocytes to the endothelium through integrin beta2 and intercellular adhesion molecule 1. In order to elucidate the roles of the initial interaction of the leukocytes with the endothelium through the selectins, we have examined the effects of a ligand for L- and P-selectins, sulfatide, on endotoxin shock in mice. Consistent with previous reports, a single injection of a high dose of endotoxin caused acute lethality, marked hypotension, leukopenia, and elevation in serum TNF-alpha levels. Pretreatment with sulfatide prevented acute lethality and hypotension, but not leukopenia, with a concomitant reduction in the increase in serum TNF-alpha levels. Moreover, pretreatment with sulfatide inhibited lipopolysaccharide (LPS)-induced TNF-alpha production by a human monocytic cell line, THP-1, in a dose-dependent manner. These results suggest either that selectin is critically involved in conferring the responsiveness of leukocytes to LPS or that sulfatide interferes with the intracellular signaling pathway which leads to TNF-alpha gene activation.

Higashi, H; Suzuki, Y; Mukaida, N; Takahashi, N; Miyamoto, D; Matsushima, K



Additive effect of nitric oxide and prostaglandin-E2 synthesis inhibitors in endotoxin-induced uveitis in the rabbit.  


The involvement of nitric oxide (NO) and prostaglandin E2 (PGE2) was investigated in a model of intraocular inflammation induced by intravitreal injection of endotoxin (lipopolysaccharide, LPS, 10 ng) in rabbits. The severity of uveitis, the myeloperoxidase (MPO) activity in iris-ciliary body, and the protein concentration in aqueous humor were determined. Nitric oxide synthase (NOS) and cyclooxygenase (COX) activities were assessed respectively by nitrite and PGE2 levels in aqueous humor. Treatment with inhibitors of NOS (NG-nitro-L-arginine methyl ester, L-NAME, 50 mg/kp i.p.) or COX (diclofenac, 30 micrograms, topically), alone or in combination, were compared to a saline-treated group. Diclofenac or L-NAME alone reduced or delayed the intensity of uveitis, and partially decreased the protein concentration in aqueous humor; diclofenac, but not L-NAME, partially reduced the polymorphonuclear leukocyte infiltration in the iris ciliary body as indicated by the MPO activity. Treatment with both inhibitors in combination diminished the clinical uveitis, the disruption of the blood-aqueous barrier and the MPO activity in the iris-ciliary body. We conclude that NO and PGE2 have additive effects in endotoxin-induced uveitis in rabbits, and that the inhibition of both pathways would improve the therapeutical management of uveitis. PMID:8741011

Bellot, J L; Palmero, M; García-Cabanes, C; Espí, R; Hariton, C; Orts, A



Aspirin-triggered resolvin D1 down-regulates inflammatory responses and protects against endotoxin-induced acute kidney injury.  


The presence of endotoxin in blood can lead to acute kidney injury (AKI) and septic shock. Resolvins, the endogenous lipid mediators derived from docosahexaenoic acid, have been reported to exhibit potent anti-inflammatory action. Using a mouse model of lipopolysaccharide (LPS)-induced AKI, we investigated the effects of aspirin-triggered resolvin D1 (AT-RvD1) on inflammatory kidney injury. Administration of AT-RvD1 1h after LPS challenge protected the mice from kidney injury as indicated by the measurements of blood urea nitrogen, serum creatinine, and morphological alterations associated with tubular damage. The protective effects were evidenced by decreased neutrophil infiltration in the kidney indicating reduction in inflammation. AT-RvD1 treatment restored kidney cell junction protein claudin-4 expression, which was otherwise reduced after LPS challenge. AT-RvD1 treatment inhibited endotoxin-induced NF-?B activation and suppressed LPS-induced ICAM-1 and VCAM-1 expression in the kidney. Moreover, AT-RvD1 treatment markedly decreased LPS-induced IL-6 level in the kidney and blocked IL-6-mediated signaling including STAT3 and ERK phosphorylation. Our findings demonstrate that AT-RvD1 is a potent anti-inflammatory mediator in LPS-induced kidney injury, and AT-RvD1 has therapeutic potential against AKI during endotoxemia. PMID:24709673

Chen, Jiao; Shetty, Sreerama; Zhang, Ping; Gao, Rong; Hu, Yuxin; Wang, Shuxia; Li, Zhenyu; Fu, Jian



Interleukin-10 mediates the protective effect of Linomide by reducing CXC chemokine production in endotoxin-induced liver injury  

PubMed Central

The immunomodulator Linomide has been shown to protect against septic liver injury by reducing hepatic accumulation of leukocytes although the detailed anti-inflammatory mechanisms remain elusive. This study examined the effect of Linomide on the production of CXC chemokines, including macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC) and interleukin-10 (IL-10) in lipopolysaccharide (LPS)/D-galactosamine (Gal)-induced liver injury in mice. It was found that pretreatment with 300 mg kg?1 of Linomide markedly suppressed leukocyte recruitment, perfusion failure, and hepatocellular damage and apoptosis in the liver of endotoxemic mice. Administration of Linomide inhibited endotoxin-induced gene expression of MIP-2 and KC and significantly reduced the hepatic production of MIP-2 and KC by 63 and 80%, respectively. Moreover, it was found that Linomide increased the liver content of IL-10 by more than three-fold in endotoxemic mice. The protective effect of Linomide against endotoxin-induced inflammation and liver injury was abolished in IL-10-deficient mice, suggesting that the beneficial effect of Linomide is dependent on the function of IL-10. Taken together, these novel findings suggest that the protective effect of Linomide is mediated via local upregulation of IL-10, which in turn decreases the generation of CXC chemokines and pathological recruitment of leukocytes in the liver of endotoxemic mice.

Li, Xiang; Klintman, Daniel; Sato, Tohru; Hedlund, Gunnar; Schramm, Rene; Jeppsson, Bengt; Thorlacius, Henrik



Experimental application of a synthetic luminescent substrate assay using endotoxin-specific limulus amebocyte lysate to human blood.  


A synthetic luminescent substrate method, using a mutant-type luciferase whose luminescence intensity is more than ten times as intense as the wild type, was developed recently. We conducted the first basic studies on clinical application of the novel endotoxin measurement method. We assessed and established measurement conditions, including reagent concentrations and reaction time, so that it would be possible to apply the luminescent synthetic substrate method proposed by Noda et al. to measurements in human blood. When we added lipopolysaccharide (LPS) to water, it was possible to measure LPS at a concentration of 0.1 pg/ml, whereas it was possible to measure LPS in tenfold diluted and heated plasma at a concentration of 1 pg/ml. When plasma was further diluted, inhibiting activity decreased considerably. Thus, it will be necessary to completely eliminate the inhibitor present in plasma. However, the shortest time after collecting the specimen in which it was possible to make measurements was 30-40 min, suggesting that if an assay is established, it will be possible to use the method as a novel blood endotoxin assay. PMID:22569792

Onodera, Chiaki; Takahashi, Gaku; Kan, Shigenori; Shozushima, Tatsuyori; Matsumoto, Naoya; Inada, Katsuya; Endo, Shigeatsu



Cardiopulmonary Effects of Volume Loading of Primates in Endotoxin Shock.  

National Technical Information Service (NTIS)

Myocardial performance was evaluated in rhesus monkeys after endotoxin shock, and the responses to fluid loading with colloid measured in both anesthetized control and experimental groups. Minute work and cardiac output (CO) were decreased in five monkeys...

L. J. Greenfield R. H. Jackson R. C. Elkins J. J. Coalson L. B. Hinshaw



Vasoactive Mediators as the Trigger Mechanism of Endotoxin Shock.  

National Technical Information Service (NTIS)

The role of histamine, catecholamines, and serotonin was investigated in the early phase of endotoxin shock. Histamine induced hemodynamic alterations similar to indotoxin; however, these changes could also be induced by larger amounts of acetylcholine. P...

E. D. Jacobson B. Mehlman J. P. Kalas



Activation of the Classic and Alternate Complement Pathways by Endotoxin.  

National Technical Information Service (NTIS)

The ability of bacterial endotoxin (LPS) to activate the complement system was studied in guinea pig serum (GPS). In serum chelated with ethyleneglycol tetraacetic acid (EGTA) 10 mM, which permits alternate complement pathway activation but inhibits class...

D. P. Fine



Occupational exposure to airborne endotoxins during poultry processing  

Microsoft Academic Search

Airborne gram?negative bacterial endotoxin levels were quantified in a live chicken hanging (shackling) room of a poultry processing plant. The mean respirable dust levels at the entrance and exit of the shackling line were 1.13 ± 0.72 and 0.72 ± 0.06 mg\\/m, respectively, or approximately 6% of the total dust. Endotoxins constituted 43.3 ±2.8 ?g per gram of respirable dust.

Stephen A. Olenchock; Steven W. Lenhart; Judith C. Mull



Lipopolysaccharide induces endoplasmic store Ca2+-dependent inflammatory responses in lung microvessels.  


The pulmonary microvasculature plays a critical role in endotoxin-induced acute lung injury. However, the relevant signaling remain unclear. Specifically the role of endothelial Ca(2+) in the induction of endotoxin-mediated responses in lung microvessels remains undefined. Toward elucidating this, we used the isolated blood-perfused rat lung preparation. We loaded microvessels with the Ca(2+) indicator, Fura 2 AM and then determined Ca(2+) responses to infusions of lipopolysaccharide (LPS) into the microvessels. LPS induced a more than two-fold increase in the amplitude of cytosolic Ca(2+) oscillations. Inhibiting inositol 1,4,5 trisphosphate receptors on endoplasmic reticulum (ER) Ca(2+) stores with Xestospongin C (XeC), blocked the LPS-induced increase in the Ca(2+) oscillation amplitude. However, XeC did not affect entry of external Ca(2+) via plasma membrane Ca(2+) channels in lung microvascular endothelial cells. This suggested that LPS augmented the oscillations via release of Ca(2+) from ER stores. In addition, XeC also blocked LPS-mediated activation and nuclear translocation of nuclear factor-kappa B in lung microvessels. Further, inhibiting ER Ca(2+) release blunted increases in intercellular adhesion molecule-1 expression and retention of naďve leukocytes in LPS-treated microvessels. Taken together, the data suggest that LPS-mediated Ca(2+) release from ER stores underlies nuclear factor-kappa B activation and downstream inflammatory signaling in lung microvessels. Thus, we show for the first time a role for inositol 1,4,5 trisphosphate-mediated ER Ca(2+) release in the induction of LPS responses in pulmonary microvascular endothelium. Mechanisms that blunt this signaling may mitigate endotoxin-induced morbidity. PMID:23675486

Kandasamy, Kathirvel; Bezavada, Lavanya; Escue, Rachel B; Parthasarathi, Kaushik



A critical role for human caspase-4 in endotoxin sensitivity.  


Response to endotoxins is an important part of the organismal reaction to Gram-negative bacteria and plays a critical role in sepsis and septic shock, as well as other conditions such as metabolic endotoxemia. Humans are generally more sensitive to endotoxins when compared with experimental animals such as mice. Inflammatory caspases mediate endotoxin-induced IL-1? secretion and lethality in mice, and caspase-4 is an inflammatory caspase that is found in the human, and not mouse, genome. To test whether caspase-4 is involved in endotoxin sensitivity, we developed a transgenic mouse expressing human caspase-4 in its genomic context. Caspase-4 transgenic mice exhibited significantly higher endotoxin sensitivity, as measured by enhanced cytokine secretion and lethality following LPS challenge. Using bone marrow-derived macrophages, we then observed that caspase-4 can support activation of caspase-1 and secretion of IL-1? and IL-18 in response to priming signals (LPS or Pam3CSK4) alone, without the need for second signals to stimulate the assembly of the inflammasome. These findings indicate that the regulation of caspase-1 activity by human caspase-4 could represent a unique mechanism in humans, as compared with laboratory rodents, and may partially explain the higher sensitivity to endotoxins observed in humans. Regulation of the expression, activation, or activity of caspase-4 therefore represents targets for systemic inflammatory response syndrome, sepsis, septic shock, and related disorders. PMID:24879791

Kajiwara, Yuji; Schiff, Tamar; Voloudakis, Georgios; Gama Sosa, Miguel A; Elder, Gregory; Bozdagi, Ozlem; Buxbaum, Joseph D



A Critical Role for Human Caspase-4 in Endotoxin Sensitivity  

PubMed Central

Response to endotoxins is an important part of the organismal reaction to Gram-negative bacteria and plays a critical role in sepsis and septic shock, as well as other conditions such as metabolic endotoxemia. Humans are generally more sensitive to endotoxins when compared with experimental animals such as mice. Inflammatory caspases mediate endotoxin-induced IL-1? secretion and lethality in mice, and caspase-4 is an inflammatory caspase that is found in the human, and not mouse, genome. To test whether caspase-4 is involved in endotoxin sensitivity, we developed a transgenic mouse expressing human caspase-4 in its genomic context. Caspase-4 transgenic mice exhibited significantly higher endotoxin sensitivity, as measured by enhanced cytokine secretion and lethality following LPS challenge. Using bone marrow–derived macrophages, we then observed that caspase-4 can support activation of caspase-1 and secretion of IL-1? and IL-18 in response to priming signals (LPS or Pam3CSK4) alone, without the need for second signals to stimulate the assembly of the inflammasome. These findings indicate that the regulation of caspase-1 activity by human caspase-4 could represent a unique mechanism in humans, as compared with laboratory rodents, and may partially explain the higher sensitivity to endotoxins observed in humans. Regulation of the expression, activation, or activity of caspase-4 therefore represents targets for systemic inflammatory response syndrome, sepsis, septic shock, and related disorders.

Kajiwara, Yuji; Schiff, Tamar; Voloudakis, Georgios; Gama Sosa, Miguel A.; Elder, Gregory; Bozdagi, Ozlem



Coarse particulate matter and airborne endotoxin within wood stove homes.  


Emissions from indoor biomass burning are a major public health concern in developing areas of the world. Less is known about indoor air quality, particularly airborne endotoxin, in homes burning biomass fuel in residential wood stoves in higher income countries. A filter-based sampler was used to evaluate wintertime indoor coarse particulate matter (PM????.?) and airborne endotoxin (EU/mł, EU/mg) concentrations in 50 homes using wood stoves as their primary source of heat in western Montana. We investigated number of residents, number of pets, dampness (humidity), and frequency of wood stove usage as potential predictors of indoor airborne endotoxin concentrations. Two 48-h sampling events per home revealed a mean winter PM????.? concentration (± s.d.) of 12.9 (± 8.6) ?g/mł, while PM?.? concentrations averaged 32.3 (± 32.6) ?g/mł. Endotoxin concentrations measured from PM????.? filter samples were 9.2 (± 12.4) EU/mł and 1010 (± 1524) EU/mg. PM????.? and PM?.? were significantly correlated in wood stove homes (r = 0.36, P < 0.05). The presence of pets in the homes was associated with PM????.? but not with endotoxin concentrations. Importantly, none of the other measured home characteristics was a strong predictor of airborne endotoxin, including frequency of residential wood stove usage. PMID:23551341

McNamara, M; Thornburg, J; Semmens, E; Ward, T; Noonan, C



Lipopolysaccharide Inhibits the Channel Activity of the P2X7 Receptor  

PubMed Central

The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance.

Leiva-Salcedo, Elias; Coddou, Claudio; Rodriguez, Felipe E.; Penna, Antonello; Lopez, Ximena; Neira, Tanya; Fernandez, Ricardo; Imarai, Monica; Rios, Miguel; Escobar, Jorge; Montoya, Margarita; Huidobro-Toro, J. Pablo; Escobar, Alejandro; Acuna-Castillo, Claudio



Interactions of a designed peptide with lipopolysaccharide: Bound conformation and anti-endotoxic activity  

SciTech Connect

Designed peptides that would selectively interact with lipopolysaccharide (LPS) or endotoxin and fold into specific conformations could serve as important scaffolds toward the development of antisepsis compounds. Here, we describe solution structure of a designed amphipathic peptide, H{sub 2}N-YVKLWRMIKFIR-CONH{sub 2} (YW12D) in complex with endotoxin as determined by transferred nuclear Overhauser effect spectroscopy. The conformation of the isolated peptide is highly flexible, but undergoes a dramatic structural stabilization in the presence of LPS. Structure calculations reveal that the peptide presents two amphipathic surfaces in its bound state to LPS whereby each surface is characterized by two positive charges and a number of aromatic and/or aliphatic residues. ITC data suggests that peptide interacts with two molecules of lipid A. In activity assays, YW12D exhibits neutralization of LPS toxicity with very little hemolysis of red blood cells. Structural and functional properties of YW12D would be applicable in designing low molecular weight non-toxic antisepsis molecules.

Bhunia, Anirban; Chua, Geok Lin; Domadia, Prerna N. [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Warshakoon, Hemamali; Cromer, Jens R.; David, Sunil A. [Department of Medicinal Chemistry, University of Kansas, 2030 Becker Drive, Lawrence, KS 66047 (United States); Bhattacharjya, Surajit [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore)], E-mail:



Lipopolysaccharides of Brucella abortus and Brucella melitensis Induce Nitric Oxide Synthesis in Rat Peritoneal Macrophages  

Microsoft Academic Search

Received 28 October 1999\\/Returned for modification 16 November 1999\\/Accepted 6 December 1999 Smooth lipopolysaccharide (S-LPS) and lipid A of Brucella abortus and Brucella melitensis induced the production of nitric oxide (NO) by rat adherent peritoneal cells, but they induced lower levels of production of NO than Escherichia coli LPS. The participation of the inducible isoform of NO synthase (iNOS) was




Lipopolysaccharide-induced Diaphragmatic Contractile Dysfunction in Mice Lacking the Inducible Nitric Oxide Synthase  

Microsoft Academic Search

The goal of this study was to evaluate the importance of the inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-induced diaphragmatic contractile dysfunction. Many investigators have proposed that iNOS induction in the ventilatory and limb muscles of animals injected with Escherichia coli LPS leads to impaired muscle contractility and increased fatigability. We tested this proposal by examining wild-type mice and




Nimesulide prevents lipopolysaccharide-induced elevation in plasma tumor necrosis factor-? in rats  

Microsoft Academic Search

The present study was undertaken to test the hypothesis of possible inhibitory effect of nimesulide (4-nitro-2-phenoxymethane-sulfoxide) on plasma TNF-? level. Male Sprague-Dawley rats were injected intraperitoneally (ip) with E. coli lipopolysaccharide (LPS; 1mgkg), which resulted in a dramatic increase in plasma TNF-? level peaked 60 min post injection (3890 ± 280 pgml, compared to undetectable values in the control group).

Abed Azab; Vadim Fraifeld; Jacob Kaplanski



An Anti-Interleukin-2 Receptor Drug Attenuates T- Helper 1 Lymphocytes-Mediated Inflammation in an Acute Model of Endotoxin-Induced Uveitis  

PubMed Central

The aim of the present study was to evaluate the anti-inflammatory efficacy of Daclizumab, an anti-interleukin-2 receptor drug, in an experimental uveitis model upon a subcutaneous injection of lipopolysaccharide into Lewis rats, a valuable model for ocular acute inflammatory processes. The integrity of the blood-aqueous barrier was assessed 24 h after endotoxin-induced uveitis by evaluating two parameters: cell count and protein concentration in aqueous humors. The histopathology of all the ocular structures (cornea, lens, sclera, choroid, retina, uvea, and anterior and posterior chambers) was also considered. Enzyme-linked immunosorbent assays of the aqueous humor samples were performed to quantify the levels of the different chemokine and cytokine proteins. Similarly, a biochemical analysis of oxidative stress-related markers was also assessed. The inflammation observed in the anterior chamber of the eyes when Daclizumab was administered with endotoxin was largely prevented since the aqueous humor protein concentration substantially lowered concomitantly with a significant reduction in the uveal and vitreous histopathological grading. Th1 lymphocytes-related cytokines, such as Interleukin-2 and Interferon-?, also significantly reduced with related anti-oxidant systems recovery. Daclizumab treatment in endotoxin-induced uveitis reduced Th1 lymphocytes-related cytokines, such as Interleukin-2 and Interferon gamma, by about 60–70% and presented a preventive role in endotoxin-induced oxidative stress. This antioxidant protective effect of Daclizumab may be related to several of the observed Daclizumab effects in our study, including IL-6 cytokine regulatory properties and a substantial concomitant drop in INF?. Concurrently, Daclizumab treatment triggered a significant reduction in both the uveal histopathological grading and protein concentration in aqueous humors, but not in cellular infiltration.

Navea, Amparo; Almansa, Inmaculada; Muriach, Maria; Bosch-Morell, Francisco



An anti-interleukin-2 receptor drug attenuates T- helper 1 lymphocytes-mediated inflammation in an acute model of endotoxin-induced uveitis.  


The aim of the present study was to evaluate the anti-inflammatory efficacy of Daclizumab, an anti-interleukin-2 receptor drug, in an experimental uveitis model upon a subcutaneous injection of lipopolysaccharide into Lewis rats, a valuable model for ocular acute inflammatory processes. The integrity of the blood-aqueous barrier was assessed 24 h after endotoxin-induced uveitis by evaluating two parameters: cell count and protein concentration in aqueous humors. The histopathology of all the ocular structures (cornea, lens, sclera, choroid, retina, uvea, and anterior and posterior chambers) was also considered. Enzyme-linked immunosorbent assays of the aqueous humor samples were performed to quantify the levels of the different chemokine and cytokine proteins. Similarly, a biochemical analysis of oxidative stress-related markers was also assessed. The inflammation observed in the anterior chamber of the eyes when Daclizumab was administered with endotoxin was largely prevented since the aqueous humor protein concentration substantially lowered concomitantly with a significant reduction in the uveal and vitreous histopathological grading. Th1 lymphocytes-related cytokines, such as Interleukin-2 and Interferon-?, also significantly reduced with related anti-oxidant systems recovery. Daclizumab treatment in endotoxin-induced uveitis reduced Th1 lymphocytes-related cytokines, such as Interleukin-2 and Interferon gamma, by about 60-70% and presented a preventive role in endotoxin-induced oxidative stress. This antioxidant protective effect of Daclizumab may be related to several of the observed Daclizumab effects in our study, including IL-6 cytokine regulatory properties and a substantial concomitant drop in INF?. Concurrently, Daclizumab treatment triggered a significant reduction in both the uveal histopathological grading and protein concentration in aqueous humors, but not in cellular infiltration. PMID:24595020

Mérida, Salvador; Sancho-Tello, María; Navea, Amparo; Almansa, Inmaculada; Muriach, María; Bosch-Morell, Francisco



Über die Stoffwechselwirkung eines Lipopolysaccharids aus Escherichia coli  

Microsoft Academic Search

Summary It has been established that pyrogen causes hyperglycemia and diminution of the lactic acid level in the blood. Inin vitro investigations, oxygen requirements of the liver slices show marked increase. The results are regarded as showing a primary action of the pyrogens on metabolism.

J. Venulet; Anna Desperak



Characteristics of lipid A-protein complex from endotoxin of Shigella dysenteriae type 1 (S and R strains).  

PubMed Central

Mild acetic acid hydrolysis of endotoxin (lipopolysaccharide-protein complex) of Shigella dysenteriae type 1 (S and R forms) yielded a lipid A-protein complex that consisted of amino acids, fatty acids, and sugar and, in terms of chemical composition, displayed no marked differences between the S and R forms. Its protein portion (53 to 56%) consisted of at least 16 amino acids. In the fatty acid portion (14 to 18%), myristic, 3-hydroxymyristic, palmitic, and stearic acids accounted for 50%. The sugar portion (10 to 12%) consisted solely of glucosamine. The remainder was unidentified substances, most of which contained phosphorus. Lipid A-protein complexes derived from both S and R forms were not toxic for mice in doses up to 1,000 microgram/mouse, but their Linulus test activity had increased considerably as compared with the starting lipopolysaccharide-protein complex material: from 10(-6) to 10(-10--10(-12) mg/ml. The lipid A-protein complexes were readily soluble in a water solution of triethylamine, in dimethyl sulfoxide, and in pyridine. Images

Sourek, J; Trnka, T; Levin, J; Roubal, J; Michalec, C



Investigations on the Mechanism of Stimulation of Non-Specific Immunity by Bacterial Lipopolysaccharides  

PubMed Central

The stimulation of non-specific immunity by lipopolysaccharides could not be correlated with the serum properdin level at the time of challenge. Zymosan was effective in stimulating an increase in properdin without raising resistance to infection. The development of a low properdin level during Esch. coli infection did not inevitably lead to death. The fate of 32P-labelled lipopolysaccharide (32P-LP) after intravenous injection was followed in mice and guinea pigs. Most of it was accumulated rapidly in the reticulo-endothelial system. About a third was removed from the blood more slowly than the remainder, probably owing to a smaller particle size. Some of the 32P was excreted in the urine as inorganic phosphate. Very little radioactivity was found in the circulating leukocytes. The blood clearance of 32P-LP was slowed by the administration of nonradioactive lipopolysaccharide 30 min. previously. When this interval was 48 hr. the reticulo-endothelial system was found to be hyperactive in terms of rate of clearance and phagocytic capacity. This was accompanied by an increase in the number of active macrophages in the liver and, to a lesser extent, in the spleen. These biphasic changes in the phagocytic activity of the R.E.S. parallel the decreased and increased resistance to infection following the injection of microgram amounts of lipopolysaccharide. Successive depression and stimulation of the R.E.S. also followed the injection of milligram amounts of Thorotrast. The hyperactive phase was accompanied by increased resistance to Esch. coli infection and decreased resistance to Salm. typhimurium infection. The lipid A component of lipopolysaccharide was also taken up by the R.E.S. which it stimulated. Resistance to both Esch. coli and Salm. typhimurium infections was enhanced. The basis of lipopolysaccharide-induced non-specific immunity is discussed in relation to changes in the properdin and reticulo-endothelial systems. The superiority of lipopolysaccharide over other colloids in stimulating immunity is considered to be determined by lipid A. ImagesFIG. 3FIG. 4FIG. 5FIG. 6FIG. 7FIG. 8FIG. 9FIG. 10

Howard, J. G.; Rowley, D.; Wardlaw, A. C.



Effects of the immunomodulator, VGX-1027, in endotoxin-induced uveitis in Lewis rats  

PubMed Central

Background and purpose: VGX-1027 is a novel, low molecular weight, immunomodulatory compound that has shown efficacy against a variety of immuno-inflammatory disease models in animals including autoimmune diabetes in NOD mice, collagen-induced arthritis and chemically induced inflammatory colitis. Here, we have studied the effects of VGX-1027 on the development of endotoxin-induced uveitis (EIU) in male Lewis rats, as a model of inflammatory ocular diseases in humans. Experimental approach: EIU was induced by a single footpad injection of 200??g lipopolysaccharide (LPS). Groups of rats were treated with either VGX-1027 (25?mg?kg?1) or its vehicle at different time points (30?min, 6?h or 12?h) after the challenge with LPS or, as positive control, with dexamethasone. The rats were killed within 16?h after LPS challenge, and the eyes and aqueous humor were collected to study serological, immunological and histological signs of EIU. Key results: The rats treated with VGX-1027 within 6?h after LPS challenge exhibited milder clinical, histological and laboratory signs of EIU than those treated with vehicle. Conclusion and implications: This study provides the first evidence that systemic treatment with VGX-1027 counteracts the uveitis-inducing effect of LPS in rats and suggests that this drug may have potential in the treatment of immuno-inflammatory conditions of the eye in humans.

Mangano, K; Sardesai, N Y; Quattrocchi, C; Mazzon, E; Cuzzocrea, S; Bendtzen, K; Meroni, P L; Kim, J J; Nicoletti, F



Differential Effects of Low-Dose Endotoxin on the Cerebral Circulation During Pregnancy  

PubMed Central

It is well-known that the pregnant state is associated with increased sensitivity to endotoxin in renal and uterine circulations; however, the effects on the cerebral circulation are not known. Intravenous infusion of low-dose lipopolysaccharide ([LPS]; 1.5 ?g/kg) to pregnant Wistar rats on day 15 of pregnancy caused significantly decreased myogenic tone of posterior cerebral arteries on day 20, which was not seen in similarly treated nonpregnant rats. Pregnancy alone was associated with a 2-to 4-fold increase in inducible nitric oxide synthase (iNOS), tumor necrosis factor-? (TNF-?), and interferon-? (IFN-?) messenger RNA (mRNA) in cerebral arteries compared to nonpregnant, suggesting that the cerebral circulation is in a state of inflammation during pregnancy. After LPS treatment, cerebral arteries from pregnant animals had increased iNOS and TNF-? compared to LPS-treated nonpregnant animals, but decreased interleukin 10 (IL-10) and IFN-?. These results demonstrate that pregnancy enhances sensitivity to the effects of LPS in the cerebral circulation, which may be due to an enhanced inflammatory state during pregnancy.

Cipolla, Marilyn J.; Houston, Emily M.; Kraig, Richard P.; Bonney, Elizabeth A.



?-Cyclodextrin-polyurethane copolymer adsorbent for selective removal of endotoxin from DNA solution.  


Copolymer particles for removal of endotoxins (lipopolysaccharides, LPSs) were prepared by suspension copolymerization of ?-cyclodextrin (CyD) and 1,6-hexamethylenediisocyanate. The LPS-removing activity of the copolymer particles was compared with that of poly(?-lysine)-immobilized Cellufine (cationic adsorbent) or polystyrene particles (hydrophobic adsorbent) by a batch method. When DNA was present in solution with LPSs under physiological conditions (pH 6.0, ionic strength of ? = 0.05-0.8), LPS-removing activity of the cationic or hydrophobic adsorbent was unsatisfactory because both the DNA and the LPSs were adsorbed onto each adsorbent. By contrast, the copolymer particles with ?-CyD cavity (CyD content: 14-20 mol%) could selectively remove LPSs from a DNA solution (50 ?g ml(-1), pH 6.0, and ? = 0.05-0.2) containing LPSs (15 EU ml(-1)) without the adsorption of DNA. The residual concentration of LPSs in the treated DNA solution was below 0.1 EU ml(-1), and the recovery of DNA was 99%. PMID:23969015

Sakata, Masayo; Uezono, Koji; Kimura, Kasane; Todokoro, Masami



The Aldosterone-Mineralocorticoid Receptor Pathway Exerts Anti-Inflammatory Effects in Endotoxin-Induced Uveitis  

PubMed Central

We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11?-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-?, IFN-?, MIP-1?) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU.

Ly, Andre; Leroux les Jardins, Guillaume; Goldenberg, Brigitte; Naud, Marie-Christine; Jonet, Laurent; Besson-Lescure, Bernadette; Jaisser, Frederic; Farman, Nicolette; De Kozak, Yvonne; Behar-Cohen, Francine



Endotoxin-tolerant Mice Have Mutations in Toll-like Receptor 4 (Tlr4)  

PubMed Central

Bacterial lipopolysaccharide (LPS) provokes a vigorous, generalized proinflammatory state in the infected host. Genetic regulation of this response has been localized to the Lps locus on mouse chromosome 4, through study of the C3H/HeJ and C57BL/10ScCr inbred strains. Both C3H/HeJ and C57BL/10ScCr mice are homozygous for a mutant Lps allele (Lpsd/d) that confers hyporesponsiveness to LPS challenge, and therefore exhibit natural tolerance to its lethal effects. Genetic and physical mapping of 1,345 backcross progeny segregating this mutant phenotype confined Lps to a 0.9-cM interval spanning 1.7 Mb. Three transcription units were identified within the candidate interval, including Toll-like receptor 4 (Tlr4), part of a protein family with members that have been implicated in LPS-induced cell signaling. C3H/HeJ mice have a point mutation within the coding region of the Tlr4 gene, resulting in a nonconservative substitution of a highly conserved proline by histidine at codon 712, whereas C57BL/ 10ScCr mice exhibit a deletion of Tlr4. Identification of distinct mutations involving the same gene at the Lps locus in two different hyporesponsive inbred mouse strains strongly supports the hypothesis that altered Tlr4 function is responsible for endotoxin tolerance.

Qureshi, Salman T.; Lariviere, Line; Leveque, Gary; Clermont, Sophie; Moore, Karen J.; Gros, Philippe; Malo, Danielle



Dead salmonellae or their endotoxin accelerate the early course of a Salmonella infection in mice.  


The course of a Salmonella infection following a low intravenous dose of virulent organisms was studied in mice. Simultaneous administration of 10(4) S. typhimurium C5 together with c. 10(8) dead salmonellae caused a marked acceleration of early net bacterial growth in the liver and spleen, leading to a rapidly overwhelming infection. Administration of similar numbers of either Staphylococcus albus or Bacillus cereus had no effect, whereas 20 micrograms of S. typhimurium Boivin-type lipopolysaccharide (B-LPS) produced an effect similar to dead organisms; 1 microgram B-LPS had a significant infection-accelerating effect. Both B-LPS and Westphal-type endotoxin (W-LPS) could enhance a salmonella infection in LPS-responsive C3H/HeMg mice, whereas only B-LPS was effective in LPS non-responder C3H/HeJ mice, implying that the infection-enhancing effect of a large bolus of dead organisms may be due in part to its LPS content. The results show that the course of a Salmonella infection following administration of large numbers of salmonellae in mice is different from that of Salmonella infections arising from small inocula. The relevance of these results to studies on the possible intracellular location of salmonellae in vivo is discussed. PMID:2087155

Hormaeche, C E



Lack of neutrophil degranulation in low-dose endotoxin inhalation based on a novel intracellular assay.  


To study the nature of endotoxin or lipopolysaccharide (LPS) induced inflammation, we developed a method of quantifying intracellular human neutrophil elastase (HNE) in lysed sputum polymorphs as a means to study the degranulation status of LPS-recruited neutrophils. Induced sputum, blood and exhaled nitric oxide (NO) were collected from 10 healthy non-atopic human subjects after inhaling a single 15 microg dose of Escherichia coil LPS in an open study. At 6 hours, LPS inhalation caused significant increase of sputum and blood neutrophils but without parallel increase in myeloperoxidase, HNE or interleukin-8 (IL-8) in sputum sol and blood, or exhaled NO. Intracellular HNE in lysed sputum polymorphs or purified blood neutrophils did not show any significant changes between inhaled LPS and saline, nor was there any appreciable change in percentage HNE release induced by N-Formyl-Met-Leu-Phe (fMLP) in vitro. We concluded that in healthy humans, the transient neutrophilic inflammation induced by a single dose of inhaled 15 microg LPS is mainly characterized by cell recruitment, not enhanced secretion of granular mediators or increased exhaled NO based on our experimental conditions. PMID:17136881

Loh, L C; Lo, W H; Kanabar, V; O'Connor, B J



Matrix metalloproteinases contribute to endotoxin and interleukin-1? induced vascular dysfunction  

PubMed Central

Background and purpose: The acute vascular inflammatory dysfunction associated with endotoxaemia may reflect an imbalance between matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), induced by the endotoxin. This possibility was tested in rat aortic tissue. Experimental approaches: Tone induced by phenylephrine in aortic rings was measured after exposure in vitro to ambient lipopolysaccharide (LPS) or the proinflammatory cytokine interleukin-1? (IL-1?) for 6h, with or without MMP inhibitors (doxycycline or GM6001). Gelatinase and MMP activities, TIMP proteins and contractility were measured in aortae taken from rats 6h after receiving LPS in vivo. Key results: Inhibition of MMP prevented the loss of phenylephrine–induced tone in aortic rings after LPS or IL-1?. IL-1? also increased release of MMP-2 activity from aortic tissue. In aortae exposed in vivo to LPS, net gelatinase, MMP-9 activities and TIMP-1 protein levels were increased, whereas TIMP-4 was reduced. These aortae were hypocontractile to both phenylephrine and KCl. Hypocontractility was partially reversed by doxycycline ex vivo. Conclusions and Implications: MMP inhibitors ameliorate vascular hyporeactivity induced by either LPS or IL-1? in vitro. LPS in vivo alters the balance between MMPs and TIMPs, contributing to vascular dysfunction which is partially reversed by MMP inhibitors. Vascular MMPs are activated as a result of LPS or IL-1?-induced stress and contribute to the hyporeactivity of blood vessels to vasoconstrictors.

Lalu, M M; Cena, J; Chowdhury, R; Lam, A; Schulz, R



Effect of chitinase inhibitors on endotoxin-induced uveitis (EIU) in rabbits.  


The acidic mammalian chitinase (AMCase) is significantly increased in tears of human allergic conjunctivitis. The aim of the study was to investigate the effects of chitinase inhibitors, allosamidin and caffeine versus dexamethasone, in rabbit endotoxin-induced uveitis (EIU). EIU was induced in rabbits by a single intravitreal injection of 100ng/10microl lipopolysaccharide (LPS). Drugs at four different concentrations (0.1, 0.01, 0.001 and 0.0001mM) were topically applied to the rabbit eye five times in 24h. Tears were collected at 0, 6 and 24h after LPS to measure the AMCase activity. The effect of treatment was also evaluated at the same time by slit lamp examination. Tear AMCase activity increased 6 and 24h after LPS injection. The AMCase activity was significantly inhibited in all treated groups with all doses of allosamidin and caffeine except with the lowest concentration. A higher AMCase inhibition at 24h was found with allosamidin and caffeine compared to dexamethasone. Moreover, topical administration of allosamidin, caffeine and dexamethasone produced a remarkable reduction of inflammatory signs, in the order: dexamethasone>caffeine>allosamidin. AMCase inhibitors showed in this rabbit model of uveitis a notable control of inflammatory response with a significant reduction of AMCase activity in tears with caffeine and allosamidin. These results support the key role of AMCase in the pathogenesis of human ocular inflammatory diseases and the therapeutic effect of AMCase inhibitors on experimental uveitis. PMID:18353673

Bucolo, Claudio; Musumeci, Maria; Maltese, Adriana; Drago, Filippo; Musumeci, Salvatore



Calpain Activation Contributes to Endotoxin-Induced Diaphragmatic Dysfunction  

PubMed Central

Calpain activation occurs in skeletal muscle in response to infection, but it is unknown if calpain inhibition improves muscle functional capacity. We hypothesized that infection induces diaphragm calpain activation, that calpain activation results in cleavage of important diaphragm cytoskeletal proteins, and that inhibition of calpain attenuates infection-induced diaphragm dysfunction. Mice (n = 4–6/group) were given: (1) saline (intraperitoneal); (2) endotoxin (12 mg/kg intraperitoneal); (3) calpain inhibitor peptide III (12 mg/kg intraperitoneal); and (4) endotoxin (12 mg/kg) plus calpain inhibitor peptide III (12 mg/kg). At 24 hours, diaphragms were removed and the following determined: (1) calpain activity by fluorogenic assay; (2) calpain I and II protein levels; (3) talin protein levels; and (4) the force–frequency relationship. Endotoxin significantly increased diaphragm calpain activity (P < 0.001), active calpain I protein (P < 0.001), active calpain II protein (P < 0.01), levels of a calpain-specific cleavage talin degradation product (P < 0.003), and reduced diaphragm force (P < 0.001). Calpain inhibitor III administration prevented endotoxin-induced increases in calpain activity, reduced talin degradation, and attenuated reductions in diaphragm force. Diaphragm-specific force at 150 Hz stimulation was significantly higher in control, endotoxin plus calpain inhibitor III, and calpain inhibitor III alone groups (23 ± 1, 20 ± 1 and 23 ± 1 N/cm2, respectively) than in the endotoxin alone group (15 ± 1 N/cm2) (P < 0.01). This model of sepsis results in significant diaphragm calpain activation and calpain-dependent diaphragm cytoskeletal protein cleavage. Moreover, calpain inhibition attenuates endotoxin-induced diaphragm weakness, suggesting that such inhibitors may be a potential treatment to improve respiratory muscle function in infected patients.

Supinski, Gerald S.; Callahan, Leigh Ann




EPA Science Inventory

Endotoxin exposure has been associated with both protection against development of TH2-immune responses during childhood and exacerbation of asthma in persons who already have allergic airway inflammation.1 Occupational and experimental inhalation exposures to endotoxin have been...


Adenyl Cyclase Activity of Mouse Liver Membranes after Incubation with Endotoxin and Epinephrine.  

National Technical Information Service (NTIS)

Adenyl cyclase activity was determined in isolated mouse liver cell membranes subsequent to incubation with S. typhosa endotoxin and/or epinephrine. Enzyme activity was stimulated twofold by endotoxin and threefold by epinephrine. When used together, endo...

M. A. Donlon R. I. Walker



Anti-lipopolysaccharide antibodies in acute appendicitis detected by ELISA.  


In acute appendicitis the bowel transmissibility of the intestinal flora increases in relation to inflammation and edema formation. We can therefore observe an immunologic response in patients, which is detectable using different bacteria isolated from the normal intestinal flora. Our aim was to measure this immunologic reaction and to detect the relationship between this response and histologic types of acute appendicitis. Sera from 47 cases, comprising 38 patients suffering from appendicitis and 9 healthy controls, were examined. The sera were taken shortly before appendectomy and 14 days after operation. The antigens were lipopolysaccharides (LPS) extracted from bacteria of normal intestinal flora: Escherichia coli O21, O22, O33, O61, O68, Bacteroides fragilis and an absolute rough mutant: Shigella sonnei Re 4350. Antibodies were detected by ELISA. We showed a direct relationship between severity of appendicitis and registered antibody titer. Both aerobic and anaerobic bacteria play a role in infection in appendicitis. According to our serologic results the synergy of B. fragilis with E. coli from normal flora is more important in the initiation of inflammation, but in the perforation process the role of E. coli seems more important compared to that of B. fragilis. PMID:16689825

Péterfi, Zoltán; Kovács, Krisztina; Antal, András; Kocsis, Béla



Airborne endotoxin associated with particles of different sizes and affected by water content in handled straw.  


High exposures to endotoxin are observed in environments where organic materials are handled and lower exposures are found in e.g. indoor air. Inhaled endotoxin contributes significantly to the induction of airway inflammation and dysfunction. The size of an inhaled particle influences the deposition in the airways and the following health symptoms. The objective is to characterise the distribution of endotoxin on airborne particles of different sizes in straw storage halls with high exposure and in other environments with lower exposure levels to endotoxin. Furthermore we have studied the influence of water content of handled straw on the size distribution of endotoxin containing particles. Total, inhalable, thoracic and respirable endotoxin and particles have each been quantified in aerosols from boiler rooms and straw storage halls at 24 power plants, including 21 biofuel plants. Inhalable, thoracic and respirable endotoxin have been quantified in aerosols from offices and outdoor air. The endotoxin concentration was higher in airborne thoracic dust than in airborne 'total dust'. The median respirable fraction in the straw storage halls, boiler rooms at biofuel plants, boiler rooms at conventional plants, offices and outdoors was respectively 42%, 9%, 19%, 24% and 34%. Thoracic endotoxin per number of thoracic particles was higher than respirable endotoxin per number of respirable particles at the biofuel plants. In straw storage halls the fraction of endotoxin of respirable size was highest on the days with lowest water content in the received straw. Furthermore the exposures to all endotoxin fractions were highest on days with the lowest water content in the received straw. In conclusion the highest exposures and concentrations of endotoxin occur or tend to occur from thoracic dust. A high variation in endotoxin concentrations and in fractions of respirable or thoracic size is found in the different working areas. This is important in the risk assessment and makes attempts to influence the endotoxin exposure a possibility. Water content in straw affected the concentration, exposure level and size distribution of airborne endotoxin. PMID:20362504

Madsen, A M; Nielsen, S H



Ragweed pollen extract intensifies lipopolysaccharide-induced priming of NLRP3 inflammasome in human macrophages  

PubMed Central

Ragweed pollen extract (RWE) possesses intrinsic NADPH oxidase activity that induces oxidative stress by initiating the production of intracellular reactive oxygen species (ROS). The ROS are important contributors to the manifestation of allergic inflammation; furthermore, concomitant exposure to an allergen and an endotoxin trigger a stronger inflammatory response. One of the main pro-inflammatory cytokines produced in inflammatory responses is interleukin-1? (IL-1?), and its production is associated with caspase-1-containing inflammasome complexes. Intracellular ROS have been implicated in NLRP3 inflammasome-mediated IL-1? production, therefore, we aimed to study whether RWE influences the function of NLRP3 inflammasome. Here we describe that, in the presence of NADPH, RWE significantly elevates lipopolysaccharide-induced IL-1? production of THP-1 cells as well as human primary macrophages and dendritic cells. We also demonstrate that increased IL-1? production is mediated through NLRP3 inflammasome in THP-1 macrophages. We provide evidence that RWE elevates cytosolic ROS level in these cells, and ROS inhibitors abolish IL-1? production. Furthermore, we show that RWE enhances lipopolysaccharide-induced gene transcription/expression of pro-IL-1? and key components of the inflammasome via a ROS-dependent mechanism.

Varga, Aliz; Budai, Marietta M; Milesz, Sandor; Bacsi, Attila; Tozser, Jozsef; Benko, Szilvia



Prevention of cardiac damage induced by formyl-leurosine, a potent cytostatic agent, by radio-detoxified endotoxin (Tolerin) in dogs  

SciTech Connect

Radio-detoxified endotoxin (Tolerin), produced by /sup 60/Co-gamma irradiation of Escherichia coli 089 endotoxin, can protect dogs against the acute cardiotoxic side-effects of formyl-leurosine, a semi-synthetic Vinca derivative with promising antineoplastic potency. Formyl-leurosine induces a rapid decrease in arterial blood pressure and diminishes the contractile force of the myocardium in the anaesthetized dog. These responses indicate a direct pharmacologic relaxant effect of the drug on the heart and vasculature smooth muscle. The early cardiovascular depression is of short duration and is unaffected by Tolerin. Tolerin can prevent, however, the secondary, more dangerous phase of circulatory depression that is associated with the severe cardiotoxic manifestations of the drug, as demonstrated by hemodynamic and morphologic (light and electronmicroscopic) patterns.

Bertok, L.; Juhasz-Nagy, A.; Sotonyi, P.



Prevention of cardiac damage induced by formyl-leurosine, a potent cytostatic agent, by radio-detoxified endotoxin (Tolerin) in dogs.  


Radio-detoxified endotoxin (Tolerin), produced by 60Co-gamma irradiation of Escherichia coli 089 endotoxin, can protect dogs against the acute cardiotoxic side-effects of formyl-leurosine, a semi-synthetic Vinca derivative with promising antineoplastic potency. Formyl-leurosine induces a rapid decrease in arterial blood pressure and diminishes the contractile force of the myocardium in the anaesthetized dog. These responses indicate a direct pharmacologic relaxant e