bluuebunny/crossref_metadata_2025 huggingface dataset:
Familial differences in endotoxin-induced TNF release in whole blood and peripheral blood mononuclear cells in vitro; relationship to TNF gene polymorphism
Authors: Derkx H.H.F. , Bruin K.F. , Jongeneel C.V. , de Waal L.P. , Brinkman B.M.N. , Verweij C.L. , Houwing-Duistermaat J.J. , Rosendaal F.R. , van Deventer S.J.H.
Published: January 2008

Healthy volunteers show large interindividual differences in endotoxin-induced TNF release in vitro and certain HLA class II types can be related to phenotypic TNF resporise. To investigate the possibility of a genetic basis for endotoxin responsiveness, we tested TNF release in whole blood and PBMNC after stimulation by endotoxin in 47 relatives of 7 healthy volunteers. All volunteers were HLA-typed and the TNF-gene associated Nco1 and DNA microsatellite polymorphisms were determined.

A significant difference in TNF release by PBMNC and the Nco1 genotype could be established, showing a lower response of TNFB*2 homozygotes than TNFB*1 homozygotes (165 vs 265, 413 vs 703 and 462 vs 832 pg/106 PBMNC for 1, 10 and 100 ng/ml of endotoxin respectively; P < 0.05). The highest endotoxin-induced TNF release was observed in TNFB*1/TNFB*2 heterozygotes (340, 911 and 1,149 pg/10 6 PBMNC respectively; P < 0.05 compared to TNFB*1 homozygotes and P < 0.0005 when compared to TNFB*2 homozygotes). TNFa and TNFb microsatellite typing revealed extensive polymorphism, showing a significantly lower TNF release in whole blood in individuals with TNFa2, -a6 and -a10 alleles than in individuals with TNFa4 and -a11 microsatellite haplotypes.
Lipopolysaccharide activation of human monocytes mediated by CD14, results in a coordinated synthesis of tissue factor, TNF-α and IL-6
Authors: Osnes L.T.N. , Westvik Å.B. , Øvstebø R. , Joø G-B. , Okkenhaug C. , Kierulf P.
Published: January 2008

We have investigated the role of CD14 in lipopolysaccharide (LPS) stimulation of monocytes to tissue factor (TF) synthesis, tumor necrosis factor (TNF-α) antigen and interleukin-6 (IL-6) antigen release. Elutriation-purified monocytes or peripheral blood mononuclear cells (PBMC) preincubated with a blocking monoclonal antibody against CD14, and monocytes with a reduced expression of CD14 (phosphatidyl inositol specific phospholipase C (PI-PLC) treated monocytes and PBMC isolated from a patient suffering from paroxysmal nocturnal hemoglobinuria, PNH), were stimulated with increasing LPS concentrations. TNF-α antigen and IL-6 antigen were measured in the supernatants, and the cells harvested for measurement of procoagulant activity (PCA) as well as TF, TNF-α and IL-6 mRNA. We conclude that LPS stimulation of monocytes to TF, TNF-α and IL-6 synthesis as well as PCA is totally dependent on CD14 at clinically relevant LPS concentrations (1-50 ng/ml). Moreover, we demonstrate a close correlation between induction of PCA and release of TNF-a and IL-6 antigen indicating a common signalling pathway of induction of these mediators.
The release of endotoxin, TNF and IL-6 during the antibiotic treatment of experimental Gram-negative sepsis
Authors: Dofferhoff A.S.M. , Potthoff H. , Bom V.J.J. , Bartels H.L. , de Vries-Hospers H.G. , Bijzet J. , Weits J. , Buurman W. , Bleichrodt R.P.
Published: January 2008

To evaluate the role of different antibiotics in the release of endotoxin and the production of tumor necrosis factor-α (TNF) and interleukin 6 (IL-6) during the treatment of experimental Escherichia coli septical peritonitis, we obtained serial blood samples from septic rats treated with placebo, ceftazidime, aztreonam or imipenem. We also studied the effect of taurolidine, given alone or in combination with aztreonam, on the release of endotoxin and IL-6. Despite decreasing levels of viable counts after treatment with ceftazidime, aztreonam or imipenem, levels of free endotoxin increased in all animals. We did not notice any significant differences in the extent of plasma endotoxin release between the different treatment groups. However, we did find significant differences in the IL-6 production between the different treatment groups. After 2 h of treatment, IL-6 levels had increased in all animals with the highest levels in the imipenem treated animals, whereafter IL-6 levels decreased again in the rats treated with imipenem or ceftazidime, while in the rats treated with placebo or aztreonam IL-6 levels further increased. This increase in IL-6 levels was associated with acute mortality. In all antibiotic treated animals TNF levels significantly decreased during therapy. After 2 h of treatment TNF levels were the highest in the imipenem treated rats. The high levels of TNF and IL-6 at t = 2 in the imipenem group were thought to be the result of early bacterial lysis, while the late increase in IL-6 levels in the aztreonam treated animals was thought to be the result of the formation of long bacterial filaments in the abdominal cavity. In the present study, treatment with taurolidine could not prevent or inhibit the release of endotoxin or IL-6, but taurolidine, alone or in combination with aztreonam, unexpectedly caused a dramatic increase in IL-6 levels which was associated with an increased acute mortality. We conclude that antibiotics can cause the release of endotoxin in spite of decreasing levels of bacteremia in vivo. It is suggested that circumstances in which antibiotic-induced filamentation occurs are also conditions that yield excessive (local) LPS release. Our data also suggest that there is a lack of relationship between plasma free endotoxin levels and mortality and that the most important inflammatory compartment was the abdominal cavity in this model.
Role of macrophage-derived nitric oxide in endotoxin lethality in mice
Authors: Parmely M.J. , Hao S.-Y. , Morrison D.C. , Pace J.L.
Published: January 2008

Endotoxic lipopolysaccharides (LPS) mediate lethality in mice by a complex inflammatory process involving the production of multiple mediators, including tumor necrosis factor-α (TNF-α) and nitric oxide (NO). The present study had two objectives: (i) to determine the extent to which TNF-α contributes to the induction of NO production by mouse macrophages activated with LPS in vitro; and (ii) to assess the contribution of macrophage-derived NO to the pathogenesis of endotoxin shock in mice. The studies reported here show that the synthetic adenyl carbocyclic nucleoside 9-[(1S,3R)- cis-cyclopentan-3-ol]adenine (cPA) inhibited TNF-α, but not NO, production by thioglycollate-elicited peritoneal macrophages that were activated with either LPS alone, LPS + interferon-γ (IFN-γ) or IFN-γ + TNF-α. The expression of cytoplasmic TNF-α in LPS + IFN-γ-activated cells was similarly inhibited by cPA, whereas the appearance of inducible NO synthase was unaffected by the compound. Of significance, pretreatment of mice with a single injection of cPA protected the animals against subsequent LPS challenge in two models of endotoxin lethality. These results suggest that macrophage-derived NO, induced by LPS, may not be an essential mediator of the lethal effects of endotoxin. Further, the results of these studies suggest that TNF-α-induced NO production by tissue macrophages also may not be an essential contributing factor in the pathogenesis of lethality induced by endotoxin in mice.
Expression of an anti-Pseudomonas aeruginosa lipopolysaccharide core recombinant antibody in Escherichia coli
Authors: Emara M.G. , Malburg S.R.C. , Lam J.S.
Published: January 2008

Antibodies against Pseudomonas aeruginosa LPS are generally protective, however, this protection is usually serotype-specific. Thus the generation of antibodies against the more conserved core-lipid A epitopes has a potential for being more broadly cross reactive. Our laboratory has previously produced several monoclonal antibodies (mAb) against the core region and lipid A region of P. aeruginosa LPS. In this study, we cloned the immunoglobulin genes from mAb 7-4, an antibody with specificity for the inner core region of P. aeruginosa LPS. VH and VL genes of 7-4 were cloned into both of the M13-derived phagemid vectors, pComb3 and pComb8. 14 pComb3/VH/VL and 6 pComb8/VH /VL recombinant clones were isolated. The presence of recombinant F(ab) molecules in the periplasmic extracts of Escherichia coli was confirmed by Western immunoblots of these extracts with goat anti-mouse F(ab')2 and goat anti-mouse kappa light chain antibodies under non-reduced conditions. Recombinant 7-4 antibody also interacted with LPS prepared from a rough mutant P. aeruginosa strain AK43 in Western immunoblotting, and ELISA as well as with whole cells of AK43 in immunofluorescence. Thus, recombinant 7-4 antibodies expressed in E. coli are structurally and functionally correct.
Autoregulation by interferons provides an endogenous 'priming' signal for LPS-responsive macrophages
Authors: Fultz M.J. , Vogel S.N.
Published: January 2008

RT-PCR technology was used to evaluate endogenous levels of IFN-α, β, and γ mRNA in macrophages derived from LPS-responsive (C3H/OuJ) and LPS-hyporesponsive (C3H/HeJ) mice. IFN mRNA levels were found to be consistently higher in the LPS-responsive macrophages. That these IFNs may be part of an autocrine loop is supported by the observations that treatment of macrophages, in vitro, with IFN-α or IFN-β induces IFN-γ mRNA and, conversely, that exogenous IFN-γ treatment results in increased detection of both IFN-a and IFN-β mRNA species. In vitro 'priming' of LPS-hyporesponsive macrophages with either IFN-α or IFN-γ causes these cells to be activated by a polyI:C triggering signal, in a manner equivalent to that seen for LPS-'primed' C3H/OuJ macrophages. Thus, low levels of endogenous IFNs, which induce each other bidirectionally, may provide a replenishable source of 'primed' macrophages, that are more plentiful in Lpsn mice and that are capable of being 'triggered' to functional maturity during an immune response.
Soluble tumor necrosis factor receptors are released independent of TNF in endotoxemia
Authors: Otto F. , Seiz A. , Galanos C. , Mertelsmann R. , Engelhardt R.
Published: January 2008

The pathway by which the in vivo application of LPS induces the release of soluble TNF receptor molecules (sTNF-R) is not yet understood. Earlier findings have demonstrated that the injection of LPS induces the release of TNFα, and injections of TNFα induce sTNF-R I and II. Thus one would assume that sTNF-R are released after LPS application as a result of endogenous production of TNFα. In this study, i.v. bolus injections of Salmonella abortus equi endotoxin (4 ng/kg body weight) once daily for 5 consecutive days were administered to cancer patients. The time course of serum levels of TNFα and sTNF-R I and II were determined. sTNF-R I and II were released after every LPS injection with peak levels being comparable on days 1 through 5. In contrast, the TNFα peak level on day 1 was about 50 times higher than on day 2. On days 3 through 5, no TNFα could be detected, a phenomenon referred to as endotoxin tolerance. Since LPS was able to provoke a comparable release of sTNF-R despite inducing widely differing levels of TNF-α, we conclude that the release of sTNF-R I and II during endotoxemia is not induced via TNF-α.
Lipopolysaccharide-associated resistance to killing of yersiniae by complement
Authors: Porat R. , McCabe W.R. , Brubaker R.R.
Published: January 2008

Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica share ~70 kb low calcium response (Lcr) plasmids encoding virulence factors expressed at 37°C that, except for the adhesin YadA, are repressed by Ca2+ (Lcr+). Virulence factors encoded on both the Lcr plasmid and chromosome have been reported to protect yersiniae against complement-dependent killing. In this study, LPS was isolated from yersiniae of serum-sensitive phenotypes (Lcr+ and Lcr- Y. enterocolitica and Y. pseudotuberculosis grown at 26°C and Lcr - Y. enterocolitica grown at 37°C) and incorporated into liposomes containing radioactive chromium. These vesicles lysed with release of free 51Cr in normal but not decomplemented serum. Liposomes prepared from serum-resistant phenotypes (Lcr+ and Lcr- Y. pestis grown at 26°C or 37°C, Lcr+ and Lcr- Y. pseudotubercu losis grown at 37°C, and Lcr+ Y. enterocolitica grown at 37°C) did not undergo complement-dependent lysis. LPS from serum-resistant Y. pestis and Y. pseudotuberculosis was rough as judged by deficiency of O-groups.
A murine model for studying endotoxemia and the efficacy of anti-LPS agents in an immunocompromised host
Authors: Neely A.N. , Orloff M.M. , Imwalle A.R. , Holder I.A.
Published: January 2008

In most murine models of endotoxemia, an exogenous agent is injected to increase the sensitivity of the mouse to endotoxin (lipopolysaccharide, LPS). Here, a clinically encountered event, a bum, was found to reproducibly decrease the amount of LPS required to kill half of the mice (LD50). In this more clinically relevant model, the anti-LPS agents, monophosphoryl lipid A and polymyxin B sulfate, each increased the LD50 of burned mice challenged with LPS from Klebsiella pneumoniae, while the LPS-directed monoclonal antibody E5 did not. However, E5 did protect burned mice challenged with smooth or rough LPS from Salmonella typhimurium and S. minnesota, respectively. Hence, in vivo protection was dependent upon both the anti-LPS agent and the chemical composition of the LPS used for intoxication. The differences in protection observed in this intoxication model may explain some protection discrepancies reported when these anti-LPS agents have been tested for protection against Gram negative sepsis.
Structures of the O-antigens of Klebsiella serotypes 02 (2a,2e), 02 (2a,2e,2h), and 02 (2a,2f,2g), members of a family of related D-galactan O-antigens in Klebsiella spp
Authors: Kelly R.F. , Perry M.B. , MacLean L.L. , Whitfield C.
Published: January 2008

The structures of the lipopolysaccharide O-antigens from Klebsiella serotypes 02(2a,2f,2g), O2(2a,2e) and O2(2a,2e,2h) have been determined. These O-polysaccharides are part of a family of related structures, which share a D-galactan I backbone. D-galactan I has the repeating unit structure: [→3)-β-D-Gal f-(1→3)-α-D-Gal p-(1→]. The O-polysaccharide of serotype O2(2a,2f,2g) differs from other known O-polysaccharides in Klebsiella spp. Each of the main-chain Gal p residues in the 02(2a,2f,2g) O-polysaccharide is substituted with an α-(1→4)-linked D-Gal p residue, to form a trisaccharide repeating unit. The LPS O-polysaccharides of serotypes O2(2a,2e) and O2(2a,2e,2h) both contain α-(1→2)-linked D-Galp substituents on the main-chain Galp residues, and resemble serotype O9. The only difference between the 09, O2(2a,2e), and O2(2a,2e,2h) carbohydrate structures involves the stoichiometry of addition of side chain α-D-Gal p residues. However, the polymers from serotypes O2(2a,2e), O2(2a,2e,2h) and 09 are all modified by O-acetylation and these modifications may contribute to altered antigenic factors. The structures reported here resolve ambiguities between previous chemical and serological analyses of 02 antigens. Genetic analyses showed that enzymes involved in the addition of α-D-Gal p residues are encoded by genes outside the rfbO9 (O-antigen biosynthesis) region and this provides an explanation for occasional non-stoichiometric addition of side chain α-D-Galp residues.
Blocking interleukin-1 in sepsis
Authors: Dinarello C.A.
Published: January 2008

Interleukin-1 (IL-1) is a polypeptide which possesses a broad spectrum of biological activities, many of which are associated with disease. It has been studied for its ability to induce fever, sleep and anorexia, elevate prostaglandins and nitric oxide, stimulate the release of pituitary hormones, elevate hepatic acute phase proteins, increase gene expression for other cytokines and augment the proliferation of T and B lymphocytes. In addition, IL-1 increases the synthesis of collagenases and decreases the synthesis of proteoglycans, resulting in joint destruction. More recently, IL-1 has been shown to induce hypotension and upregulate endothelial cell adhesion molecules, both important parameters of the septic shock syndrome. IL-1 also possesses host defense properties. For example, pretreatment with IL-1 non-specifically reduces lethality to bacterial and fungal infections, even in the absence of circulating neutrophils. IL-1 has been given to humans in clinical trials; as increasing doses are given, IL-1 is toxic, producing gastrointestinal disturbances and hypotension. It appears that low doses of exogenously administered IL-1 may prove useful as a therapy. On the other hand, blocking the toxic effects of IL-1 may be necessary in the treatment of certain diseases. IL-1 receptor blockade has been studied in human disease including sepsis. Similar to other studies using corticosteroids, antibodies to tumor necrosis factor, bradykinin antagonists and inhibitors of platelet activating factor, clear-cut beneficial effects of IL-1 receptor blockade have not matched those demonstrated in animals.
Current understanding of sepsis: criticism and a proposal
Authors: Rabinovici R. , Neville L.F. , Feuerstein G.
Published: January 2008

The failure to improve outcome of septic patients may reflect flawed hypotheses pertaining to the pathogenesis of sepsis. Alternatively, it may represent the use of clinically irrelevant experimental animal models. To address these issues, an alternative hypothesis is proposed. It is suggested that leukocyte activation in sepsis is initiated by a priming effect of minuscule doses of endotoxin on leukocytes for enhanced responsiveness to minute doses of inflammatory mediators (or vice versa) as opposed to activation by overwhelming doses of endotoxin per se. The experimental impetus of this hypothesis has been provided by a novel rat model of sepsis precipitated by the co-administration of PAF and LPS at doses 1/1000 lower than those previously used to elicit sepsis in the same species.
Risk assessment in recent clinical trials in sepsis/SIRS: lessons learned and future directions
Authors: Knaus W.A. , Wagner D.P. , Sun X. , Harrell F.E. , Hedstrom J.S.
Published: January 2008

This presentation reviews recent evidence that the design and analysis of clinical trials in sepsis could be improved by using individualized sepsis specific risk assessments. These individual patient risk profiles are derived from independent databases. They describe patients on a continuum of risk. They have correlated very closely to mortality outcomes in recent clinical trials. These individual risk assessments can improve the interpretation of trial data by controlling for potentially confounding patient risk factors, thereby permitting the signal or impact from novel therapy to be more precisely determined.

When these individual risk assessments are combined with data on the degree of the host's specific inflammatory response they may eventually lead to the development of specific patient profiles to describe where immunotherapy is appropriate and other situations where it may either not be indicated or pose a risk. This relationship needs further investigation, however, before it can be confirmed.

Changes in the acute physiology score can also hold substantial promise as an alternative endpoint for clinical trials. Therapies which significantly and consistently reduce the APS during the initial days of sepsis in small, early exploratory Phase I and II trials may be excellent candidates for investigation in larger Phase III studies.
Pulmonary TNFα is a critical mediator in Adult Respiratory Distress Syndrome
Authors: Feuerstein G.Z. , Neville L.F. , Rabinovici R.
Published: January 2008

The development of effective pharmacotherapies to combat the Adult Respiratory Distress Syndrome (ARDS) is critically dependent upon: (1) the development of clinically-relevant animal models; (2) identification of inflammatory mediators centrally involved in eliciting lung injury; (3) understanding the inter-relationships or 'cross-talk' between pro and anti-inflammatory mediators which modulate the lung inflammation; and (4) the application of molecular techniques to isolate potentially novel genes involved in the development of ARDS. In this paper, we will present evidence from a rat model of microvascular lung injury produced by interleukin-2 (IL-2), that pulmonary TNFα is a primary and pivotal mediator of lung injury and that different modes of TNFα inhibition may represent feasible strategies to prevent ARDS. Furthermore, we will describe how the application of Differential Display Reverse Transcriptase Polymerase Chain Reaction (DDRT-PCR) can allow the rapid isolation of partial fragments of potentially new genes involved in ARDS. The products of such genes could represent future target sites for pharmacotherapeutic intervention.
Preclinical and clinical evaluation of carbohydrate immunopharmaceuticals in the prevention of sepsis and septic sequelae
Authors: Williams D.L. , Mueller A. , Browder W.
Published: January 2008

Sepsis and sepsis syndrome are significant causes of morbidity and mortality in critically ill patients. Despite technological and therapeutic advances in critical care, sepsis continues to be a pivotal factor in 20-50 % of deaths in surgical intensive care units. It is clear that alternative approaches to the prevention and/or treatment of sepsis must be found. Preclinical data indicate that macrophage activation with (1→3)-β-D-glucans will ameliorate sequelae associated with Gram-negative septicemia. Recent clinical data indicate that macrophage activation with (1→3)-β-D-glucans will significantly reduce septic morbidity and mortality in trauma and/or high-risk surgical patients. This work reviews the preclinical and clinical evaluation of (1→3)-β-D-glucans in the prevention of sepsis and septic sequelae.
A novel LBP-BPI fusion protein with in vivo efficacy and longer half life
Authors: Au-Young J. , Seilhamer J.J. , Lin L. , McKelligon B.M. , Lane J.C. , Snable J.L. , Kikuchi M.T. , Scott R.W. , Marra M.N.
Published: January 2008

Bactericidal/permeability-increasing protein (BPI) is a neutrophil azurophilic granule protein which binds to and neutralizes the biological activity of Gram-negative bacterial endotoxin both in vitro and in vivo. The therapeutic utility of BPI is hampered by its remarkably short circulating half life. BPI shares 44% sequence identity with lipopolysaccharide binding protein (LBP), an acute phase protein which facilitates endotoxin-mediated cellular responses and has a long circulating half-life. BPI and LBP are members of a family of proteins which regulate LPS activity in vivo. Genetically engineered variants of BPI and LBP were designed to maintain the endotoxin neutralizing properties of BPI but to have an extended circulating half life. One of these, LBP-BPI, is comprised of the N-terminal half of LBP, linked to the C-terminal half of BPI. Like BPI, LBP-BPI had endotoxin neutralizing activity in vitro and in vivo and gave a significant protective effect against lethal endotoxin challenge in mice (90% survival in treated group vs 10% survival in untreated controls P < 0.001). Neither LBP nor the 25 kDa N-terminal fragments of BPI and LBP were protective in this model of lethal endotoxin challenge. Pharmacokinetic studies in CD-1 mice showed that the LBP-BPI variant is cleared 74 times more slowly than BPI. Longer half-life versions of BPI have a broader window of protection and require lower overall doses to maintain therapeutically effective circulating concentrations. Molecules such as the LBP-BPI variant will have greater therapeutic utility than the native BPI molecule by virtue of these properties.
Altered macrophage function in tumor necrosis factor α- and endotoxin-induced tolerance
Authors: Zingarelli B. , Makhlouf M. , Halushka P.V. , Caputi A.P. , Cook J.A.
Published: January 2008

Pretreatment of rats with a sublethal dose of human recombinant tumor necrosis factor-α (hrTNFα, 10 μg/kg i.p.) or Salmonella enteritidis LPS (100 μg/kg, i.p.) prevented death when a lethal dose of S. enteritidis lipopolysaccharide (LPS, 15 mg/kg i.p.) was administered 24 h later. The resistance to the lethal effect of LPS was associated with similar alterations of the functional phenotype of peritoneal macrophages from both groups. In ex vivo studies, peritoneal macrophages were harvested 24 h after vehicle (control), hrTNFα or LPS injection and stimulated in vitro with LPS. In macrophages collected from control rats, LPS stimulated arachidonic acid (AA) metabolism, as assessed by 6-keto-prostaglandin F1α (6-keto-PGF1α) levels, nitric oxide (NO) production, as assessed by nitrite, and interleukin 6 (IL-6) production. In macrophages from hrTNFα-pretreated or LPS pretreated rats, basal and LPS-stimulated 6-keto-PGF1α production were significantly reduced compared to controls, while nitrite production was increased (P < 0.001). LPS induced IL-6 synthesis was not affected in macrophages from hrTNFα-pretreated rats but was significantly reduced in stimulated macrophages from LPS treated rats. Furthermore, the macrophage membrane content of guanine nucleotide binding regulatory (G) protein subunits was determined. Macrophages collected from hrTNFα-pretreated rats exhibited a marked reduction of the membrane content of the Giα3 subunit compared to control macrophages, whereas the Giα1,2 and Gβ subunits were not significantly affected. The decrease in Giα3 in hrTNFα treated rats is similar to that previously observed in macrophages from LPS tolerant rats. The results demonstrate that hrTNFα induces cross tolerance to the lethal effect of LPS, and that tolerance induced by TNF or LPS is associated with differential changes in peritoneal macrophage mediator production. These changes may, in part, be a consequence of altered signal transduction via specific G proteins.
Hyaluronan accumulates around portal vessels in liver after endotoxin treatment of rats
Authors: Pertoft H. , Alston-Smith J. , Fraser J.R.E. , Laurent T.C.
Published: January 2008

Livers of normal rats and rats treated with lipopolysaccharide from Escherichia coli were isolated in situ and tested for capability to extract [3H]-hyaluronan. In control livers, excess unlabeled hyaluronan inhibited extraction of [3H]-hyaluronan both at 4°C and 37°C. Furthermore, part of the [3H]-hyaluronan extracted at 4°C could be released by subsequent perfusion with excess of unlabeled hyaluronan. This behaviour is in accordance with the well-characterized receptor-mediated uptake of hyaluronan in sinusoidal endothelial cells. However, in lipopolysaccharide-treated animals, the uptake of [3H]-hyaluronan was not inhibited by unlabelled polysaccharide, nor could [3H]-hyaluronan bound at 4°C be released from its binding sites. Both types of livers were perfused with 10 μg/ml of hyaluronan and recovered for histology. An accumulation of hyaluronan in areas around portal veins was found in endotoxin treated rats, while only traces of hyaluronan could be visualized in the vessels of normal livers. The location of hyaluronan coincided with cell infiltrates. Thus, there is a different mechanism for liver clearance of hyaluronan in the endotoxic animal.
Endotoxin, interleukin-6 and tumor necrosis factor concentrations in equine acute abdominal disease: relation to clinical outcome
Authors: Steverink P.J.G.M. , Sturk A. , Rutten V.P.M.G. , Wagenaar-Hilbers J.P.A. , Klein W.R. , van der Velden M.A. , Németh F.
Published: January 2008

Serial peri-operative plasma and serum samples from 55 horses suffering from acute abdominal disease and presented for surgical intervention were assayed for the presence of endotoxins (lipopolysaccharides; LPS), tumor necrosis factor (TNF) and interleukin-6 (IL-6). At study entry, venous blood was collected for blood cultures. Intra-operatively collected ascitic fluid samples were assayed for the presence of LPS. The clinical course of the disease was documented. Four horses were excluded from the study. At study entry, 21 of the 51 horses (41%) had increased platelet-rich plasma LPS concentrations, i.e. ≥ 5 ng/l (mean 35.5; range 7-197 ng/l), in 34 horses (67%) IL-6 concentrations were increased, i.e. exceeding 35 ng/l (mean 364; range 36—1762 ng/l). Detectable TNF was present in 5 horses (10%); 3 of them died spontaneously during surgery. In all TNF positive samples, markedly increased LPS and IL-6 concentrations were detected. Study entry IL-6 concentrations were significantly higher in non-surviving (mean 394; range < 20—1762 ng/l) than in surviving horses (mean 116; range < 20—894 ng/l; P < 0.0002). This was most evident when non-surviving horses with inflamed bowel disease were considered (mean 1096; range 650—1762 ng/l; P < 0.0001). Positive ascitic fluid LPS concentrations (≥ 3 ng/l) were encountered in 18 horses. There was no significant correlation between the LPS concentrations in ascites and plasma. Study entry concentrations of IL-6 were significantly correlated with LPS concentrations (r = 0.62; P < 0.001) and were inversely correlated with platelet counts (r = -0.46; P < 0.002). The magnitude of a calculated LPS-cytokine score strongly correlated with mortality (P < 0.001). Receiver operating characteristic (ROC) curve analysis showed that the IL-6 assay had reasonable accuracy for the prediction of unfavorable outcome (i.e. the area under the curve equalled 0.72), in contrast to the LPS assay. The results indicate that LPS, TNF and IL-6 are predominantly released in the systemic circulation of horses suffering from inflamed and ischemic bowel disease. IL-6 concentrations have predictive value for unfavorable outcome and the simultaneous presence of increased LPS, TNF and IL-6 concentrations is especially associated with a poor clinical condition and outcome.
Calculating therapeutic indices and therapeutic advantages for endotoxins and monophosphoryl lipid A: an evaluation of data from the scientific literature
Authors: Rudbach J.A. , Keegan D.S. , Sowell C.G.
Published: January 2008

This review article addresses the question of whether or not monophosphoryl lipid A offers a therapeutic advantage over smaller doses of endotoxin. Biological data were collected from a literature review and these were used to calculate therapeutic indices for monophosphoryl lipid A and endotoxin. Subsequent calculations yielded results which showed that, in humans and in other animals, monophosphoryl lipid A did have a therapeutic advantage over smaller doses of endotoxin.
Effects of protease inhibitors on LPS-mediated activation of a mouse macrophage cell line (J774)
Authors: Muroi M. , Muroi Y. , Ito N. , Rice N.R. , Suzuki T.
Published: January 2008

Pretreatment (1 h) of a mouse macrophage-like cell line, J774, with the protease inhibitor, phenylalanine-chloromethyl ketone (PCK) or its structural analogue, tosylphenylalanine chloromethyl ketone (TPCK) was found to cause substantial inhibition of LPS-triggered activation of NF-κB. Pretreatment of cells with other types of protease inhibitors or their various structural analogues had no effect. PCK or TPCK appeared to exert its inhibitory effect by: (i) partially preventing LPS-triggered degradation of IκBα protein; (ii) preventing LPS-triggered nuclear translocation of NF-κB proteins (p50, RelA and Rel); and (iii) inhibiting the DNA-binding activities of NF-κB proteins. Pretreatment of cells with PCK or TPCK also resulted in the total or partial inhibition of LPS activatable (AP-1 or CREB) or constitutively-existing (Oct-1) transcription factors, but not of another constitutively-expressed transcription factor (SP-1). Pretreatment of J774 cells with PCK was found to substantially suppress LPS-induced expression of mRNAs specific for cytokine genes (TNFα, IL-1α and β, and IL-6), inducible nitric oxide synthase (iNOS) gene and IκBα gene, but not NF-κB1 p105 gene or β-actin gene. Furthermore, PCK pretreatment inhibited, in a dose-dependent manner, LPS-triggered production of nitric oxide production and tumoricidal activity.
Effects of bactericidal/permeability-increasing protein upon mononuclear cell interactions with endotoxin: evidence for cell binding
Authors: Burnett R.J. , Lyden C.A. , Tindal C.J. , Marra M.N. , Neely A.N. , Solomkin J.S.
Published: January 2008

Because of the mortality associated with Gram-negative infections, there is interest in the mechanisms by which bacterial endotoxin is processed by leukocytes. Several lipid A binding proteins, including lipopolysaccharide-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI), mediate the process of LPS activity. Our studies show that BPI was capable of mediating the binding of tritiated LPS to the mononuclear cell line THP-1 in a fashion not affected by the addition of an anti-CD14 antibody, and that such binding did not elicit a TNFa response. Soluble BPI was capable of competing with LBP for LPS and inhibiting the TNFα response associated with LBP-LPS stimulation. In addition, cells pretreated with BPI and then washed were then capable of binding significantly more LPS than controls. These pretreated cells were also capable of inhibiting TNFα production to subsequent LBP-LPS. Western blotting confirmed the uptake of BPI to THP-1 cells both with and without LPS. We conclude that BPI is capable of interacting with THP-1 cells in the free form or complexed to LPS, without eliciting a TNFα response. Cells exposed to BPI are resistant to subsequent stimulation with LBP-LPS, thereby suggesting a novel mechanism for the anti-inflammatory effects of BPI.
Compartmentalization of intravesical and systemic interleukin-6 and tumor necrosis factor α in mice stimulated with porins and lipopolysaccharide from Salmonella typhimurium
Authors: Tufano M.A. , Catalanotti P. , Capasso C. , De Paolis P. , Ranieri M. , Rossano F.
Published: January 2008

Mucosal surfaces represent a natural colonization site for Gram-negative bacteria. We have already demonstrated the biologic role of Salmonella typhimurium porins in vitro and in vivo. In this paper we studied mucosal and systemic interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) production in systematically challenged mice or after intravesical administration of LPS or porins from S. typhimurium. It was found that serum IL-6 levels increased in BALB/c mice 4 h after receiving either i.v. LPS or porins from S. typhimurium. The porin challenge was stronger. Serum IL-6 levels were higher after porins than after LPS. IL-6 was not detected in the urine of i.v. challenged mice. Findings regarding IL-6 urine levels in intravesically treated mice were comparable. In porin-challenged mice they decreased more slowly than the LPS-challenged ones. IL-6 was not detected in the serum of intravesically challenged mice. In i.v. LPS-challenged mice, serum TNFα levels peaked earlier (at 2 h) than the IL-6 levels. A higher 2 h peak was instead seen in porin-challenged mice. TNFα was not detected in the urine of i.v. challenged mice. With intravesical LPS challenge, urinary TNFα levels peaked at 24 h, whereas in the porin-challenged mice the peak occurred 12 h earlier and was higher. Serum samples revealed no detectable TNFα. These findings confirm that both porins and LPS activate the mucosal response, without any systemic involvement, as, for example, in patients with diseases such as pyelonephritis or gastroenteritis.
Antioxidants attenuate glycerol-endotoxin induced acute renal failure in rats
Authors: Zurovsky Y. , Zadok I.
Published: January 2008

Sepsis sometimes occurs in traumatic rhabdomyolysis; however, the possibility that injection of endotoxin from Gram-negative bacteria may affect the severity of renal injury during rhabdomyolysis has not been directly examined. The aims of this study were: (i) to examine whether injection of glycerol, which causes rhabdomyolysis, simultaneously with a low dose of endotoxin will result in acute renal failure (ARF) in the rat; and (ii) whether this injury is due to the increased formation of free oxygen radicals. Therefore, a low dose of glycerol (0.375 ml/100 g), which does not cause acute renal failure, and endotoxin at a dose that does not cause renal injury (0.0375 mg/100 g) were simultaneously injected. The extent of renal injury was examined 24 h after the simultaneous injection of these two substances. The parameters measured included creatinine, BUN, urine volume, RPF, GFR, percent absorption of sodium and potassium as well as arterial blood pressure. In order to examine whether renal injury due to the combination of glycerol and endotoxin results from free oxygen radicals, DMTU (50 mg/100 g), SOD (8 mg/100 g) and vitamin E (2 mg/kg each day for 7 days) were injected. Acute renal failure was induced by the simultaneous injection of glycerol and endotoxin. Treatment with DMTU or vitamin E significantly reduced renal injury and returned the values of the measured parameters to their control values. The antioxidant SOD also reduced the renal injury; however, with this antioxidant the reduction was less pronounced and the measured parameters did not return to their control values. This study indicates that the severity of renal injury during rhabdomyolysis may increase if endotoxin is present in the body. Furthermore, this study indicates that this increased renal injury may be due to free oxygen radicals. Traumatic rhabdomyolysis is a frequent cause of ARF. Therefore, it is important to create an experimental model that will also include actual possibilities, such as sepsis, which have not been taken into consideration to date.
Endotoxin tests in patients with sepsis
Authors: Ng K.P. , Bhanumathy M. , Ong G.S.Y. , Yong B.H. , Parasakthi N. , Koh M.T. , Siow C.P. , Bosco J.
Published: January 2008

A new whole blood agglutination assay and a plasma Limulus amoebocyte lysate (LAL) assay for the measurement of endotoxin were evaluated for the diagnosis of sepsis and septic shock in 73 intensive care patients and 50 normal blood donors. All samples were blinded to the operators performing the tests. The endotoxin test results were compared with blood culture and clinical observations of sepsis progression. Endotoxin was detectable in 34 of 38 patients with culture positive Gram-negative bacteraemia, 2 of 11 patients with Gram-positive bacteraemia, 3 of the 7 patients with suspected sepsis, whose blood culture was repeatedly negative, and 1 of 17 non-septic patients. All blood donors were found to be negative for endotoxin. In 3 patients monitored during the course of their illness, the endotoxin assays correlated closely.

We conclude that endotoxin measurement was valuable in the care of patients with sepsis and that the whole blood agglutination assay was rapid and simple to perform.
Detection of lipopolysaccharides blotted on nylon membranes
Authors: Chen W.H. , Balakonis P. , Tsai C-M.
Published: January 2008

A sensitive method for the detection of Gram-negative bacterial lipopolysaccharide (LPS) blotted on nylon membranes is described. LPSs are separated by SDS-PAGE and then electrophoretically transferred to nylon membranes. Immobilized LPS is oxidized with periodate and then reacted with a hydrazide conjugated to the steroid, digoxigenin. LPS is visualized by alkaline phosphatase labelled antibodies against the steroid and the enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. LPS banding patterns of both rough (R-) and smooth (S-) type LPSs from over 15 different bacterial species are similar to those of silver stained companion gels, but without nonspecific staining of proteins. The detection of S-LPS from Pseudomonas aeruginosa F-D type 1 and R-LPS from Escherichia coli K12 is sensitive to 10-20 ng per lane. The use of this detection system in combination with antibody or lectin studies on identical blots can provide an effective tool in locating the precise position of certain epitopes or sequences in both R- and S-type LPSs on the blots.
Activity in the Limulus amebocyte lysate assay and induction of tumor necrosis factor-α by diverse Helicobacter pylori lipopolysaccharide preparations
Authors: Pece S. , Fumarola D. , Giuliani G. , Jirillo E. , Moran A.P.
Published: January 2008

Different chemically characterized H. pylori LPS preparations, such as smooth (S)- and rough (R)-form LPS, a completely dephosphorylated R-LPS, and three lipid A chemotypes, from the S- and R- form LPS (S- and R-lipid A) as well as a dephosphorylated derivative of S-lipid A, respectively, were evaluated for expression of potency in a quantitative chromogenic Limulus amebocyte (CLAL) lysate assay and for release of tumor necrosis factor-α (TNF-α) from activated human mononuclear cells. As far as the CLAL activity is concerned, no statistically significant differences could be observed between S- and R-LPS. Dephosphorylation of both R-LPS and S-lipid A caused a significant decrease of CLAL activity. In general terms, all the lipid A chemotypes were significantly less effective than the native LPS molecule and, in particular, R-lipid A expressed the lowest Limulus activity of all preparations. With regard to TNF-α release, R-LPS was the most potent inducer of this cytokine, even though its dephosphorylation reduced activity. In conclusion, the results show that phosphate groups influence both CLAL activity and, to a lesser extent, TNF-α release, and that the core oligosaccharide synergically cooperates with lipid A for the production of this cytokine, being, however, not essential for the expression of CLAL activity. Furthermore, preliminary structural data show that H. pylori D-glucosamine disaccharide backbone, besides being underphosphorylated at position 4', is also characterized by a reduced number of acyloxyacyl residues in comparison with enterobacterial lipid A. These findings, besides providing useful information on the structure-bioactivity relationships within H. pylori LPS, further support the evidence that this non-invasive, slow bacterium possesses the ability to modulate the local cellular immune response via LPS and related inflammatory cytokines.
Separation of wild-type lipopolysaccharides by centrifugal partition chromatography: isolation of smooth form species
Authors: Suda Yasuo , Shiyama Takaaki , Yasukochi Takashi , Kawano Kazuhisa , Brade Helmut , Rietschel Ernst Th. , Kusumoto Shoichi
Published: January 2008

The smooth (S)-form lipopolysaccharides (LPSs) having long O-polysaccharides were successfully isolated by centrifugal partition chromatography from the co-existing rough (R)-form species from the LPSs of wild-type strains of Salmonella enterica, serovars abortus equi and friedenau, and from Citrobacter freundii. Separation is based on different partition behaviors of the R- and S- form LPS species between aqueous and organic phases. The procedure described proved to be convenient, reproducible and readily applicable to large amounts of various LPS. It is another tool to study the relationship between chemical structure and biological activity of LPS.
Differential mechanisms of LPS-induced NFκB activation in macrophages and fibroblasts
Authors: Wu Chaoqun  , Wanleng Deng  , Ohmori Yoshihiro , Hamilton Thomas A.
Published: January 2008

Lipopolysaccharide is a prototypic stimulus of inflammatory gene expression which can act on a variety of cell types to produce different patterns of response. In the present report, the ability of LPS to stimulate NFKB activity was investigated in a fibroblast cell line (NIH3T3) and compared to LPS-induced response in a macrophage like cell line (RAW264.7). LPS was a potent stimulus of KB binding activity in both cell types though the protein composition of such binding activity varied. LPS caused nuclear translocation of KB binding activity in RAW 264.7 cells which contained NFKB1 (p50), RelA (p65), and high levels of c-Rel. Nuclei from LPS-stimulated NIH3T3 cells contained only NFKB1 and RelA but little c-Rel. Both cell types contain comparable levels of total c-Rel protein. Using two structurally distinct KB sequence motifs, LPS was shown to produce a different pattern of transacting activity in fibroblasts as compared to macrophages; both KB motifs were sensitive to LPS in RAW264.7 cells while only one of the two was functional in LPS-stimulated NIH3T3 cells. Thus LPS appears to utilize the NFKB family of transcription factors differentially depending upon the cell type being stimulated. Such differential activation of transcription factor family members may be an important determinant of the diverse nature of inflammatory response seen in different tissue settings.
Regulation of gene expression and nitric oxide production in murine macrophages by the serine/threonine phosphatase inhibitor okadaic acid
Authors: Barber S.A. , Salkowski C.A. , Fultz M.J. , Perera P-Y. , McNally R. , Vogel S.N.
Published: January 2008

LPS-stimulated macrophages produce cytokines which, at appropriate levels, direct successful immune responses against harmful pathogens. However, excessive cytokine production, as seen in endotoxemia, results in pathophysiological damage to the host. Therefore, understanding mechanisms of cytokine regulation may aid the development of strategies designed to control cytokine production during an ongoing immune response. We have examined the role of okadaic acid-sensitive phosphatases in the production of cytokines and nitric oxide by macrophages. Okadaic acid induces TNFα, IL-1β, IL-6, IFN-β, and IP-10, but not IL-10 or IL-12 (p40) mRNA. Okadaic acid differentially regulates the expression of LPS-inducible IL-10 and IL-12 (p40) mRNA. These findings suggest that okadaic acid-sensitive phosphatases are key regulators of cytokine production in unstimulated and immune-activated macrophages. Finally, okadaic acid inhibits iNOS mRNA and nitric oxide production by macrophages activated by LPS and IFN-γ.
The protein kinase C activator PMA modulates LPS lethality in normal mice and protects against LPS lethality in D-galactosamine-sensitized mice
Authors: Silverstein R. , Johnson W.M. , Bucklin S.E. , Johnson D.C.
Published: January 2008

Pretreatment for 5 h with 10 μg phorbol-12-myristate-13-acetate (PMA), a well established activator of protein kinase C (PKC) in many kinds of cells including macrophages, was found to either (a) delay, or (b) potentiate, lethal endotoxin shock in mice, depending upon the dose of LPS. The latter occurred despite a marked attenuation (>90%) of the TNFα response to LPS. In mice sensitized with D-galactosamine the same PMA pretreatment offered protection against challenge from either LPS or TNFα. This protection, coupled with the ability of PMA to reduce serum TNFα while increasing serum corticosterone in response to LPS, adds in vivo support for a possible role for PKC activation in early endotoxin tolerance. A phorbol ester (4α-phorbol) that is not a PKC activator was found ineffective. PMA produced a prompt and profound decrease in body temperature which reached a nadir at 3 h. However, the protective effect produced by PMA was not dependent upon a decrease in body temperature per se, but was dependent upon administration of PMA at the same time or prior to LPS.
TALF peptide-immunoglobulin G conjugates that bind lipopolysaccharide
Authors: Fletcher Mark A. , Kloczewiak Marek , Loiselle Paul M. , Amato Steve F. , Black Kerry M. , Warren H. Shaw
Published: January 2008

Several peptides mimicking the amino acid sequence of Tachypleus anti-LPS factor (TALF) bind LPS with high affinity and some neutralize LPS in vitro and in vivo (Kloczewiak M., Black K.M., Loiselle P., Cavaillon J-M., Wainwright N., Warren H.S. Synthetic peptides that mimic the binding site of horseshoe crab anti-lipopolysaccharide factor. J Infect Dis 1994; 170: 1490-1497). Two such peptides, TALF29-59 and TALF41-53, were covalently coupled to human IgG via a disulfide bond using the heterobifunctional linker, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). The resulting peptide-lgG conjugates contained 4-8 moles peptide per mole IgG and were evaluated for the ability to bind and neutralize LPS. Both conjugates bound LPS in a LPS capture Western blot assay. In a fluid-phase radioimmunoassay, half-maximal binding of 5 μg/ml LPS by many different Escherichia coli strains occurred at 50-100 μg/ml for both conjugates. Coagulation of Limulus amoebocyte lysate was only minimally inhibited by 5 μg/ml of each conjugate. Our data suggest that TALF peptide-lgG conjugates bind LPS with high affinity, but only weakly neutralize LPS. These studies provide an initial step towards the development of peptide-lgG preparations that might be useful for the treatment of Gram-negative sepsis by binding and clearing LPS.