bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential effects of an infusion of endotoxin on proximal and distal feline jejunal permeability
Authors: Jackman Bradley J. , Eades Susan C. , Moore James N. , Harmon Barry
Published: January 2008

The effects of an intravenous infusion of endotoxin (750 μg/kg) or an equal volume of saline solution (control) on the proximal and distal jejunal permeability and blood flow were evaluated in cats. In 8 cats, proximal and distal jejunal segments were isolated and mucosal clearance of 51Cr-ethylenediaminetetraacetic acid (EDTA) was measured. The mucosal clearance in the distal loop was not altered by endotoxin infusion in 6 cats given endotoxin or 2 cats given saline solution. In 6 cats given endotoxin, the proximal jejunal segment exhibited a 10-fold increase in mucosal permeability. In contrast, mucosal clearance remained stable in the proximal jejunal loop in 2 cats infused with saline solution.

In a second group of 13 cats (7 cats infused with endotoxin; 6 cats infused with saline solution), blood flow to the proximal and distal segments was measured. Either endotoxin (n = 7) or saline solution (n = 6) did not significantly alter blood flow to the proximal and distal jejunum in these experiments.

Samples of proximal and distal jejunum were collected from 12 cats (6 cats infused with endotoxin and 6 cats infused with saline solution). There was significantly more epithelial necrosis in the endotoxin treated cats than in the saline treated cats. Neutrophil infiltration was greater in the jejunal segments of endotoxin treated cats than in the jejunal segments of saline treated cats. In the endotoxin treated cats, there was significantly greater necrosis in the proximal jejunal segment than in the distal jejunal segment. There were no significant differences in numbers of neutrophils in the proximal and distal jejunal segments.

These results demonstrate that the proximal jejunum is more sensitive to endotoxin-induced increases in mucosal permeability than is the distal jejunum. The increases in mucosal permeability in the proximal jejunum were not accompanied by significant reductions in jejunal arterial blood flow. Endotoxemia induced neutrophil infiltration to the proximal and distal jejunum.
In vivo priming of mice with antigen and lipid A bestows proliferative ability on peritoneal exudate T cells in response to antigen or anti-αβ TCR antibody in the absence of macrophages in vitro
Authors: Nitta Toshimasa , Imai Hiroaki , Okamoto Aiko , Matsuura Motohiro , Nakano Masayasu
Published: January 2008

Requirement of macrophages for the in vivo proliferative response of the peritoneal exudate T cells of mice that had been primed in vivo with antigen and lipid A was investigated. Two weeks in advance, C3H/HeN mice (la k) were primed with horse red blood cells (HRBC) and synthetic lipid A. The highly purified T cells [T(HRBC+lipid A)] proliferated in response to HRBC or anti-αβ T cell receptor (TCR) antibody. However, T cells [T(HRBC)] that had been prepared from the mice primed with SRBC did not respond to HRBC and anti-αβ TCR antibody. When T(HRBC) were co-cultured in the presence of HRBC or anti-αβ TCR antibody with an adherent cell population (macrophages) prepared from PEC, the T(HRBC) proliferated. Previous treatment of T(HRBC+lipid A) with anti-CD45 (180 kDa) antibody plus C abolished the response, while the same pretreatment of T(HRBC) did not abolish the response in the presence of macrophages. The previous treatment of these cells with CD45 (220 kDa) antibody plus C eliminated their proliferative response. These results suggest that T(HRBC+lipid A) are different from T(HRBC) on macrophage-dependency, and T(HRBC+lipid A) expressing CD45 (180 kDa) on the membrane are the proliferative cells when their αβ T cell receptors (TCR) are stimulated with the antigen or anti-TCR antibody.
Escherichia coli endotoxin increases cytosolic free Ca2+ by mobilizing intracellular calcium stores in cultured endothelial cells
Authors: Burnier M. , Centeno G.
Published: January 2008

The effects of bacterial lipopolysaccharide (Escherichia coli 0111:B4; LPS) on cytosolic free calcium were examined in cultured bovine endothelial cells. The effect of LPS on the cellular calcium response to vasopressin was also investigated. Intracellular calcium [Ca2+]i was determined using the fluorescent probe Fluo-3/AM and confocal microscopy. LPS caused a dose-dependent increase in [Ca2+]i. The calcium response to LPS was of the same magnitude when cells were studied in the absence of extracellular calcium suggesting that the effect of LPS is due mainly to a mobilisation of intracellular calcium stores. The cellular calcium response to vasopressin was completely abolished by LPS and vice versa. These results show that LPS evokes a rapid increase in intracellular free calcium in endothelial cells. This observation may contribute to an explanation for the early initial phase of the LPS-induced vascular hyporesponsiveness.
Aggregation of serum proteins with lipopolysaccharide (LPS): characterization of the precipitable LPS-protein complex
Authors: Falk M.C. , Fletcher M.A. , Williams T.J. , Ching W.M. , Wu Y. , Morrison T.K.
Published: January 2008

We have employed an in vitro system to study the interactions between rat serum proteins and lipopolysaccharide (LPS) under conditions similar to those commonly employed in endotoxemia studies. Immediately after mixing, a small fraction of the LPS micelles form a readily precipitable aggregate with serum proteins. Aggregate formation is measurable at LPS concentrations equivalent to maximal serum loads at the lethal threshold in endotoxic shock studies. The amount of aggregate increases with increasing LPS concentration. While the protein content of this aggregate remains stable over the 60 min studied, the LPS content decreases. Only three serum protein species comprise 85-90% of the protein bound in the aggregate: albumin, complement factor C3b and iC3b, and immunoglobulins G and M. Much larger amounts of serum protein-LPS aggregates are formed using serum from animals pre-exposed to sublethal levels of LPS. The results help better characterize the physical-chemical properties of LPS as it is employed in endotoxic shock models in normal and pre-exposed animals and may help explain the altered clearance profiles experienced in these models.
Identification of N-3-hydroxypropionyl-2-O-methyl-D-perosamine as a specific constituent of the lipopolysaccharide from Vibrio bio-serogroup 1875 which has Ogawa antigen factor B of Vibrio cholerae O1
Authors: Isshiki Yasunori , Kondo Seiichi , Haishima Yuji , Iguchi Takehiro , Hisatsune Kazuhito
Published: January 2008

A marine vibrio, designated Vibrio bio-serogroup 1875 Original (1875 Original), has both the Ogawa antigen factor B and the Inaba antigen factor C of Vibrio cholerae 01, in addition to its own O antigen factor. Its variant strain (1875 Variant) has the Inaba antigen factor C. The O polysaccharide chain (O-chain) of the lipopolysaccharide (LPS) isolated from this vibrio has been shown to consist of an ↬ (1 2)-linked homopolymer of N-3-hydroxypropionyl-D-perosamine. The present structural study revealed the presence of N-3-hydroxypropionyl-2- O-methyl-D-perosamine at the non-reducing terminus of the O-chain from the LPS of 1875 Original but not from that of the 1875 Variant. Since 2- O-methyl-D-perosamine is known as an Ogawa-specific constituent of the O-chain from V. cholerae O1 Ogawa O form, it appears that the presence of 2- O-methyl-D-perosamine at the non-reducing terminus of the ↬ (1 2)-linked D-perosamine homopolymer that forms the O-chain is responsible for the expression of the serological specificity of the Ogawa antigen factor B of 1875 Original.
Antibiotic induced bacterial killing activates vascular endothelial cells and whole blood cells
Authors: Arditi Moshe , Jin Zhou  , Sheng He Huang
Published: January 2008

We have combined an ex vivo whole blood and a Transwell insert system to evaluate the biological relevance of antibiotic-induced lipopolysaccharide (LPS) release, and have shown that this event results in a brisk release of TNFα in whole blood (Arditi M., Kabat W., Yogev R. Antibiotic-induced bacterial killing stimulates tumor necrosis factor-α release in whole blood. J Infect Dis 1993; 167: 240-244). We now investigated the activation of vascular endothelial cells in response to antibiotic-induced bacterial killing and free LPS release. We have previously shown that the endothelial cells respond to LPS in a serum- or soluble CD14-dependent manner. We used LPS-induced IL-6 release from human umbilical vein endothelial cells (HUVEC) and human blood cells as two readout systems. Escherichia coli O111:B4 (106 CFU/ml) was placed into the upper chamber of a Transwell filter insert (0.1 μm) and incubated for 6 h with or without ceftriaxone (40x MIC) in 24-well plates containing HUVEC monolayers in the presence of 5% serum, or diluted human blood cells in the lower chamber. Filter inserts were removed at 6 h and plates were incubated for an additional 12 h and supernatants were collected for IL-6 measurement. Bacterial kill curves were generated. Antibiotic-induced killing of E. coli resulted in activation of human endothelial and blood cells as assessed by IL-6 release. Addition of 5 μg/ml of bactericidal permeability factor (BPI) to the media containing ceftriaxone did not influence the kill curve but resulted in over 96% decrease in IL-6 release from both endothelial and blood cells, suggesting that free LPS was the predominant stimulus for IL-6 release. Furthermore, when the endothelial cell experiments were conducted in serum-free conditions, antibiotic-induced bacterial killing did not induce IL-6 release from HUVEC, and addition of recombinant human soluble CD14 into the media reconstituted the serum effect, consistent with the concept that serum or soluble CD14 is absolutely required for LPS-induced endothelial cell responses, including LPS-induced IL-6 release. IL-10 was able to significantly inhibit LPS-induced IL-6 production by human blood cells but had no effect on LPS-mediated IL-6 release from HUVEC. These data suggest that antibiotic-induced free LPS release leads to activation of both human vascular endothelial cells and blood cells, which can be readily inhibited by the addition of BPI. The results support the hypothesis that free endotoxin released from E. coli following bacteriolysis is the major contributor to the enhancement of the inflammation associated with the initiation of antibiotic therapy. The dichotomy of IL-10 effects on LPS-induced cytokine production in blood mononuclear cells versus endothelial cells may play a role in the pathophysiology of TH-2-directed responses.
Biological characterization of Pseudomonas aeruginosa endotoxin released by antibiotic treatment in vitro
Authors: Nakano Masayasu , Kirikae Teruo
Published: January 2008

The supernatants taken from Pseudomonas aeruginosa cultures in human sera or chemically defined M9 medium in the presence of ceftazidime (CAZ) contained high levels of endotoxin, while those of imipenem (IPM) yielded relatively lower levels of endotoxin. The membrane-filtered supernatants were used as a source of endotoxin and the biological activities of the endotoxin were examined in comparison with those of hot-phenol water-extracted LPS. The CAZ-released endotoxin preparation contained large amounts of protein. The protein, however, appeared to lack significant endotoxic activity through the endotoxin-protein component, since the endotoxin did not show any toxic effect in D(+)-galactosamine (GaIN)-sensitized C3H/HeJ mice in vivo or macrophage activation in vitro. The activities of CAZ- and IPM-released endotoxins as assessed by chromogenic Limulus amebocyte coagulation test were fundamentally identical to those of purified P. aeruginosa LPS and Escherichia coli LPS, since their regression lines were parallel. The biological effects of CAZ-released endotoxin, such as lethal toxicity in GaIN-sensitized mice, in vitro induction of TNF and NO production by peritoneal macrophages, and MAP-kinase activation in macrophages of LPS-responsive C3H/He and LPS-low responsive C3H/HeJ mice, were similar to those of the LPS. Macrophage activation by CAZ-released endotoxin as well as LPS was mainly dependent on serum factors and CD14 antigen. Polymyxin B blocked the activity. These findings indicate that the endotoxic activity of CAZ-released endotoxin is due primarily to LPS (lipid A), although such preparations contain a high level of protein released from or produced by the organisms. Finally, the possibility that the treatment of P. aeruginosa infection with some kind of antibiotics may induce endotoxic shock was suggested in a mouse model.
The in vivo significance of antibiotic-induced endotoxin release in experimental Gram-negative sepsis
Authors: Opal S.M. , Horn D.L. , Palardy J.E. , Parejo N. , Jhung J. , Bhattacharjee A. , Young L.D.
Published: January 2008

Considerable evidence now supports the experimental findings that penicillin-binding protein (PBP)-2 specific antimicrobial agents such as imipenem generate less endotoxin than PBP-3 specific agents such as ceftazidime during the process of bacteriolysis of Gram-negative bacteria. To determine if differences in endotoxin release have pathophysiologic significance in vivo, Sprague-Dawley rats were experimentally challenged with intraperitoneal injections of virulent, serum-resistant clinical strains of the following Gram-negative bacilli: Escherichia coli 018:K1, Klebsiella pneumoniae K2, and Pseudomonas aeruginosa 12.4.4 (immuno type 6). After intravenous administration of imipenem (25 mg/kg), ceftazidime (50 mg/kg) or saline control, imipenem and ceftazidime-treated animals had rapid reductions in the quantitative level of bacteremia from all three pathogens. Peritoneal fluid samples revealed spherical forms with imipenem and long, filamentous forms with ceftazidime. Circulating plasma endotoxin levels were consistently higher ( P < 0.05) with ceftazidime than imipenem for 6 h after administration of E. coli or P. aeruginosa intraperitoneal challenge. Endotoxin levels were unchanged to slightly higher with imipenem than ceftazidime following K. pneumoniae intraperitoneal challenge. TNF levels peaked 2 h post-therapy and were consistently higher with ceftazidime-treated animals ( P < 0.05). D-galactosamine-treated animals had LD50 values that were 0.5-2 log higher ( P < 0.001) with imipenem for E. coli and P. aeruginosa but did not differ from ceftazidime in animals challenged with the K. pneumoniae strain. These results indicate that the PBP-2 specific agent imipenem led to significantly less endotoxin release than did ceftazidime with its great affinity to PBP-3. Differential endotoxin release was found after antimicrobial therapy with the E. coli and P. aeruginosa strains but not with the K. pneumoniae strain tested in this study. The clinical relevance of these findings with treatment of systemic Gram-negative infections in humans will require further clinical investigation.
The clinical significance of endotoxin released by antibiotics: what is the evidence?
Authors: Mock C.N. , Jurkovich G.J. , Dries D. , Maier R.V.
Published: January 2008

Basic investigations indicate variation among antibiotics in their propensity to release endotoxin from bacterial cells. Evidence for the clinical significance of this phenomenon includes: anecdotal reports; studies evaluating changes in endotoxin levels in response to antibiotics in a variety of infectious diseases; two studies which have prospectively evaluated changes in endotoxin levels in response to different antibiotics and correlated the changes with the patients' clinical course; and one study which evaluated differences in mortality in injured patients based on the endotoxin-releasing potential of antibiotics they received. These studies suggest: (i) different types of antibiotics do induce differing levels of endotoxin release during treatment of bacterial infections in humans; (ii) such antibiotic-associated endotoxin release probably does have a biologic consequence; and (iii) this biologic consequence is possibly of clinical relevance. However, the data are not sufficient to advocate changes in current antibiotic prescribing or dosing decisions. Rather, a variety of in vitro and in vivo experiments as well as clinical studies are advocated.
Different endotoxin release and IL-6 plasma levels after antibiotic administration in surgical intensive care patients
Authors: Holzheimer R.G. , Hirte J.F. , Reith B. , Engelhardt W. , Horak K.H. , Leppert R. , Aasen A. , Capel P. , Urbaschek R. , Karch H. , Thiede A.
Published: January 2008

Despite the use of broad-spectrum antibiotics, aggressive fluid resuscitation, vasopressor support, the mortality associated with Gram-negative sepsis and septic shock has not decreased significantly in the last two decades. The consequences of host exposure to endotoxin and the relationship of antibiotic administration to endotoxin release have become important areas of intense interest. In vitro studies have demonstrated that there was a difference in endotoxin release between PBP-3 specific antibiotics (β-lactam antibiotics) and PBP-2 specific antibiotics (carbapenems). This is the first clinical report of surgical patients admitted to the surgical and anaesthesiology intensive care unit on the missing endotoxin release after imipenem treatment; however cefotaxime and ceftriaxone showed significantly more positive endotoxin tests in the plasma when compared to imipenem. Ciprofloxacin and vancomycin were intermediate in endotoxin release and tobramycin did not cause endotoxin release. There were also significant differences in endotoxin neutralizing capacity. IL-6 levels were decreased after imipenem faster than after ceftriaxone or cefotaxime; ciprofloxacin seemed to increase IL-6. Endotoxin may be harmful in patients where the immune system has been continuously challenged. Timing, dosage, or combination with other compounds as well as the effect of antibiotics on macrophages need to be tested in larger clinical trials. In this respect a consecutive study was started.
Endotoxin-like properties of an extract from a symbiotic, eukaryotic Chlorella-like green alga
Authors: Royce C.L. , Pardy R.L.
Published: January 2008

Hot phenol-water extraction of axenic cultures of the eukaryotic symbiotic green alga, Chlorella, yielded a substance having many of the characteristics of bacterial endotoxin or lipopolysaccharide (LPS). This material caused gelation of extracts of Limulus amoebocyte lysate at concentrations similar to those manifest by LPS from Gram-negative enteric bacteria. Activity was reduced substantially by incubation with polymyxin B sulfate and Limulus endotoxin-neutralizing protein (ENP), both of which have been shown to neutralize the biological activity of LPS from Gram-negative enteric bacteria. Partially purified biologically active material was found to contain 3-deoxy-D-manno-octulosonic acid (KDO), a sugar characteristically found in LPS. Acid hydrolysis of the Chlorella extract yielded a precipitate with characteristics of lipid A. Further hydrolysis followed by methylation yielded products with retention times on gas chromatography indistinguishable from 3-hydroxylauric and 3-hydroxymyristic acids. Results of transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining are consistent with those obtained with purified enteric bacterial LPS. Exhaustive precautions against potential experimental artifacts due to cross contamination by endotoxin from Gram-negative bacteria, either in algal cultures or associated with glassware allowed the conclusion that the green alga, Chlorella (strain NC64A) synthesizes a LPS-like molecule.
Inhibition of LPS-induced systemic and local TNF production by a synthetic anti-endotoxin peptide (SAEP-2)
Authors: Demitri M.T. , Velucchi M. , Bracci L. , Rustici A. , Porro M. , Villa P. , Ghezzi P.
Published: January 2008

Lipopolysaccharide (LPS) exerts its biological activity through the lipid A moiety. We tested the efficiency in inhibiting TNF production in sera and in tissues of mice and in the derma of rabbits challenged with LPS, of a synthetic anti-LPS peptide (SAEP-2) previously shown to specifically detoxify the lipid A region of LPS on the basis of structural similarities with the antibiotic polymyxin B (PMXB). In mice, SAEP-2 (100 μg/mouse, i.v.) injected with various schedules ('-30 to +10 min from LPS at 50 ng/mouse, i.v.) significantly inhibited serum TNF as well as liver, spleen and lung-associated TNF. In rabbits, SAEP-2 significantly inhibited TNF produced in dermal tissue and the resulting local hemorrhagic necrosis. The amount of tissue-associated TNF released by LPS challenge in the mouse was up to 6 times that present in the serum and inhibition by SAEP-2 or PMXB accounted for 75% of the total. Direct measurement of the binding kinetics by surface plasmon resonance and molecular filtration at equilibrium revealed that SAEP-2 and PMXB bind to LPS only in the presence of a significant amount of water but that they are unable to bind LPS in undiluted serum. Altogether these findings strongly suggest that inhibition of LPS-induced TNF by SAEP-2 and PMXB may occur in tissues.
Endotoxin-tolerant mice produce an inhibitor of tumor necrosis factor synthesis
Authors: Schade F.U. , Schlegel J. , Hofmann K. , Brade H. , Flach R.
Published: January 2008

The present study was carried out to investigate a new aspect of early endotoxin tolerance in mice. Evidence was obtained that early endotoxin tolerance is associated with the production of an activity interfering with the synthesis of tumor necrosis factor α (TNFα). LPS-tolerant mice, challenged with lipopolysaccharide (LPS) had lower serum levels of TNF than LPS-treated normal animals. In parallel, whole blood of tolerant mice, treated with LPS ex vivo generated lower levels of TNF than blood of normal animals. Plasma obtained 30 min after challenge with LPS from tolerant mice was inhibitory for TNF synthesis in whole blood of normal mice ex vivo. This seems to be specific for tolerant mice, since plasma prepared from normal, endotoxin-challenged mice was devoid of inhibitory activity for TNF production. The TNF-inhibitor in plasma of endotoxin tolerant mice did not interfere with the cytotoxic activity of murine recombinant TNFα on WEHI cells. Heat treatment of plasma destroyed the activity.
CD14/LPS receptor exhibits lectin-like properties
Authors: Cavaillon J-M. , Marie C. , Caroff M. , Ledur A. , Godard I. , Poulain D. , Fitting C. , Haeffner-Cavaillon N.
Published: January 2008

We had previously shown that the specific binding of endotoxin (lipopolysaccharide, LPS) to human monocytes in the presence of serum was mediated by the polysaccharide moiety of the LPS molecule. CD14 has been identified as the main receptor for endotoxin on monocytes/macrophages. In the present report we demonstrate that the CD14 molecule exhibits lectin-like properties. Anti-CD14 monoclonal antibodies inhibited the binding of [3H]-radiolabeled Neisseria meningitidis LPS as efficiently as the homologous unlabeled LPS. Rough Escherichia coli LPS (Rc- and Re-types) could also inhibit the binding of [3H]-LPS to a similar extent, whereas lipid A had no or very weak inhibitory activity. This suggests a major contribution of the inner-core region within the LPS and particularly the Kdo sugars. The lectin-like nature of CD14 was assessed with polyanionic sugars as well as with uncharged polysaccharides. The relative efficiencies in competition were dextran sulfate > fucoidan > mannan > polygalacturonic acid = heparan sulfate ≥ heparin ≥ chondroitin sulfate. Candida albicans phospholipomannan was far more active in the competition experiment than the mannan, indicating that, besides the osidic residues, anionic charges and/or fatty acids may contribute to the interaction with the CD14 molecule. Binding of polysaccharide to CD14 was not sufficient to trigger TNFα and IL-6 production since phospholipomannan and dextran sulfate were unable to induce cytokine release. Taken together, these results demonstrate that the binding of [3H]-LPS to CD14 involves the contribution of sugars and suggest that the signals for cytokine production require additional interactions.
Bacterial lipopolysaccharide-induced hyporeactivity in perfused rat resistance vessels: modulating effects of dexamethasone
Authors: Serio M. , Potenza M.A. , Montagnani M. , Mansi G. , Rinaldi R. , Pece S. , Fumarola D. , Jirillo E. , Mitolo-Chieppa D.
Published: January 2008

The present study was carried out on mesenteric vascular bed from LPS-injected rats in order to investigate possible mechanisms underlying hyporesponsiveness in resistance blood vessels in the course of septic shock syndrome. The involvement of L-arginine (L-Arg)/nitric oxide (NO) pathway was evaluated by administration of L-Arg, which produced a decrease in perfusion pressure in LPS-treated rats, whereas it was ineffective in control rats. Of note, dexamethasone (DEX) pretreatment in endotoxaemic rats significantly reduced the vasorelaxation by L-Arg; however, this non selective inhibitor of inducible-NOS expression was not able to prevent noradrenaline (NA) hyporeactivity. Furthermore, in order to evaluate whether hyporesponsiveness could be due to an altered contraction mechanism, the effect of endothelin (ET)-1 was tested. This peptide was able to markedly enhance the contractile response to noradrenaline in LPS-treated rats. Collectively, our findings suggest that vascular hyporesponsiveness during septic shock can only be partially explained by activation of the L-Arg/NO pathway. Other mechanisms, probably related to smooth muscle cell contractility, may be involved.
Effects of radiodetoxified endotoxin on nitric oxide production in J774 macrophages and in endotoxin shock
Authors: Gilad E. , Zingarelli B. , O'Connor M. , Salzman A.L. , Bertok L. , Szabo C.
Published: January 2008

Radiodetoxified lipopolysaccharide (RD-LPS) is a 60Co-gamma-irradiated LPS with a modified structure, which decreases its toxic effects. To obtain a better understanding of the mechanism of the reduced toxicity of RD-LPS, here we studied the effect of RD-LPS on the regulation of nitric oxide (NO) production in vitro and in vivo. In control cells, stimulation by native LPS (10 μg/ml) induced the expression of the inducible NO synthase (iNOS) and production of NO, as measured by increase in the concentration of nitrite, breakdown product of NO. Pre-exposure of the cells for 24 h to a subthreshold concentration of RD-LPS (10 ng/ml) induced a complete desensitization to the LPS-induced NO production in comparison to control cells (P < 0.01). On the contrary, pre-exposure of the cells with native LPS (10 ng/ml) did not reduce LPS-induced NO synthesis. RD-LPS induced a smaller production of tumor necrosis factor (TNF) than native LPS, but did not induce a desensitization against subsequent LPS-induced TNF synthesis. ln in vivo studies, pretreatment of rats with repeated doses of sublethal RD-LPS (1 mg/kg/day i.p. for 4 days) inhibited increase of plasma nitrate/nitrite levels, NO production in peritoneal macrophages ex vivo and induction of lung iNOS activity, in response to a high-dose LPS challenge (15 mg/kg i.p.) given at the fifth day. Pretreatment with repeated sublethal doses of the native LPS (1 mg/kg/day i.p.) did not affect NO production in rats subjected to endotoxic shock. The results demonstrate that RD-LPS induces tolerance to the stimulatory effect of LPS on NO synthesis. Suppression of iNOS induction was only observed with RD-LPS, but not with native LPS in the models used herein. It remains to be further investigated whether suppression of iNOS induction by RD-LPS contributes to the protective effects of this compound in shock and inflammation.
Utilization of a chromogenic Limulus amebocyte lysate blood assay in a multi-center study of sepsis
Authors: Ketchum P.A. , Parsonnet J. , Stotts L.S. , Novitsky T.J. , Schlain B. , Bates D.W. , AMCC SEPSIS Project
Published: January 2008

We conducted a prospective study of a chromogenic LAL assay in 346 patients with sepsis syndrome, as defined by a modification of the Bone criteria, and 131 healthy volunteers at eight member centers of the Academic Medical College Consortium (AMCC). We identified patients with endotoxemia (> 0.40 EU/ml) by measuring LAL-reactive material in whole blood, extracted by the Tamura nitric acid method, with the chromogenic LAL (Pyrochrome®) assay. The mean result in sepsis patients with detectable endotoxemia (n = 241) was 1.07 ± 1.57 EU/ml, and the mean result in 131 volunteers was 0.151 ± 0.113 EU/ml, with 73% of the volunteers' results falling below the detectable limit. The average incidence of endotoxemia in sepsis patients was 33%, but varied 2.7-fold among the clinical centers (range 16-44%). Assay results were repeatable when samples tested frozen at the clinical sites were compared to results on frozen samples tested at Associates of Cape Cod, Inc. (ACC). Multiple samples were obtained from 40 patients at 18-24 h interval(s). Fourteen multidraw patients (35%) were endotoxemic at one or more draw(s) and eight of these patients had two or more draws with endotoxin levels > 1.0 EU/ml. The presence of sulfa drugs gave false positive results in two patient samples. A positive LAL test did not correlate with culture-proven bacterial infection and did not significantly correlate with mortality. There was a correlation ( P = 0.014) between a patient having a positive LAL test and the presence of a fungal infection when mixed fungal and bacterial infections were included. There was no correlation with a positive LAL test when only a fungal infection was present (P = 0.425) or when only a fungal and a Gram-positive infection was present ( P = 0.087).
Endotoxin-induced cytokine release from whole blood - similarities between monocyte dysfunction in septic disease and during postoperative acute phase response
Authors: Berger D. , Bölke E. , Seidelmann M. , Vasilescu C. , Beger H.G.
Published: January 2008

Plasma levels of endotoxin, TNFα, interleukin-1β (IL-1β), and interleukin-6 (IL-6) as well as the endotoxin-induced release of these cytokines from whole blood were determined in 20 patients after elective aseptic surgery and eight patients suffering from septic disease. Blood samples were drawn before surgery as well as once daily over 7 days postoperatively. Endotoxin plasma levels increased on the day of the operation from 0.025 ± 0.005 EU/ml to 0.099 ± 0.022 EU/ml and remained elevated for 1 day. No changes were noted in the plasma levels of TNFα and IL-1. Levels of IL-6 increased to 198 ± 52 pg/ml on the day of the operation, while the release of TNFα and IL-6 markedly declined after whole blood was stimulated with endotoxin. The difference between basal and stimulated TNF values amounted to 505 ± 129 pg/ml preoperatively and 132 ± 41 pg/ml after the operation and 297 ± 28 pg/ml and 137 ± 43 pg/ml for IL-6. From the second day of the postoperative period onwards, the endotoxin-induced release of IL-6 was higher than the preoperative values. The results of whole blood stimulation in patients with sepsis were similar to those achieved directly after elective surgery. The cytokine release of TNFα, IL-1β, and IL-6 was reduced. We conclude that, during the early aseptic acute phase reaction, the changes in the functioning of monocytes resemble - qualitatively - those occurring during septic disease, and that this may be related to endotoxemia originating in the gastrointestinal tract.
Contribution of neutrophils to endotoxin-induced mucosal dysfunction in the feline jejunum
Authors: Mueller P.O.E. , Harmon B.G. , Eades S.C. , Moore J.N.
Published: January 2008

Damage to the intestinal mucosal barrier sustained during the compensated stages of shock may result in bacterial translocation and systemic accumulation of toxins, leading to sepsis and multiple organ failure. Neutrophils are known to be an important component of endotoxin and cytokine-induced tissue injury. The purpose of this study, therefore, was to test the hypothesis that neutrophil infiltration and MPO production accompany endotoxin and TNFα induced intestinal mucosal dysfunction.

To evaluate mucosal dysfunction, jejunal blood-to-lumen clearances of chromium 51-labeled ethylenediaminetetraacetate (51Cr-EDTA) were measured in cats administered fetal calf serum (controls), endotoxin, TNFα, or polyclonal anti-TNFα antibody followed by endotoxin. Neutrophil accumulation in the jejunal mucosa was evaluated by histologic enumeration and measurement of jejunal myeloperoxidase (MPO) activity.

Endotoxin and TNFα induced mucosal dysfunction in the proximal jejunum is accompanied by neutrophil accumulation in the jejunal mucosa as indicated by histologic enumeration and increased MPO activity. Pretreatment with anti-TNFα polyclonal antibody significantly decreased endotoxin-induced increases in jejunal MPO activity. The results of this study support the contribution of neutrophils to endotoxin and TNFα induced mucosal dysfunction.
Expression of full length and deletion homologues of Carcinoscorpius rotundicauda Factor C in Saccharomyces cerevisiae: immunoreactivity and endotoxin binding
Authors: Ding J.L. , Chai C. , Pui A.W.M. , Ho B.
Published: January 2008

Deletion homologues of the cloned Factor C cDNAs from the horseshoe crab Carcinoscorpius rotundicauda were engineered to express in Saccharomyces cerevisiae under the regulation of a galactose-inducible promoter. Expression cassettes were constructed in the vectors: pEMBLyex4 and YEpsec1 to direct, respectively, the intracellular expression, and the secretion of the protein into the culture medium using a heterologous signal sequence. The effect of insert size on the efficiency of expression and the functionality of the resulting recombinant Factor C (rFC) were studied by creating expression constructs bearing various deletion and/or hybrid fragments of Factor C. Removal of the long 5' UTR from the Factor C cDNA improved expression of the rFC. 3' Deletions of up to 84%, or internal deletions of 65% of the Factor C cDNA resulted in either the lack of detectable amounts of Factor C or loss of immunoreactivity. Depending on the construct, full length or partial rFC-related proteins were correspondingly expressed intracellularly, regardless of the vector. The rFC partitioned with the insoluble cell fraction, was solubilised with either SDS or Triton X-100, and found to be immunoreactive. The rFCs were functionally active, being able to bind Gram-negative bacterial endotoxin, provided critical regions of the endotoxin-binding domain were preserved.
Endotoxin binding on capillary endothelium and myocyte plasma membranes in perfused rat heart
Authors: Bikhazl A. , Nahle Z. , Kreydiyyeh S. , Haddad R. , Bitar K. , Haddad G. , Abdelnoor A.
Published: January 2008

This work uses a novel heart-perfusion technique to measure [3H]-lipopolysaccharide ([3H]-LPS) binding on capillary endothelium and myocyte cell membranes in Sprague-Dawley rats. Free or serum-containing Ringer-Lock buffer was infused at a rate of 1 ml/min and in the presence of 20 mM K+ and [ 3H]-LPS through an aortic cannula, and the effluent was collected through a catheter introduced into the right atrium cavity. The capillary endothelial lining was removed by CHAPS treatment to expose the cardiac myocyte surface. A physical model describing 1:1 binding stoichiometry of [3H]-LPS with its receptors is proposed and the mathematical equations derived allow for the calculation of binding constants (kn), reversal constants (k-n), dissociation constants (kd), and residency time constants (τ). The results showed that the presence of serum in the perfusate, slowed the binding of [3H]-LPS with the endothelial lining and myocytes, but increased the residency time by 3- and 50-fold, respectively. Hence, the endothelium and myofiber may contain LPS receptors that can bind more strongly with the ligand in association with sCD14-like and LBP-like molecules in rat serum. Thus it is postulated that the affinity of LPS to its receptor subtypes is not strictly and specifically dependent on the CD14 binding profile.
Detection of lipid A by monoclonal antibodies in S-form lipopolysaccharide after acidic treatment of immobilized LPS on Western blot
Authors: Pantophlet R. , Brade L. , Brade H.
Published: January 2008

Lipopolysaccharides (LPSs) were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and hydrolyzed under acid conditions known to liberate lipid A from a broad variety of bacterial species. Independent of whether bis- or monophosphorylated lipid was set free, it remained bound to the membrane during subsequent immunostaining with monoclonal antibodies (MAbs) specific for the 1,4'-bis- or 4'-monophosphorylated lipid A backbone. Lipid A could be detected in the smooth (S)-form LPS from Enterobacteriaceae (Salmonella enterica, Citrobacter, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Yersinia enterocolitica) , Pseudomonas aeruginosa, Chromobacterium violaceum, Acinetobacter, Vibrio cholerae and Legionella pneumophila, thus allowing LPS-phenotype determination. The procedure can be used for the majority of Gram-negative bacteria, since LPSs containing 2,3-diamino-2,3-dideoxy-glucose or glucosamine in the lipid A disaccharide backbone could be detected. It was shown to be sensitive (up to 20 ng of hydrolyzed LPS could be detected), and could be easily performed using isolated LPS or whole-cell lysates with or without prior digestion with proteinase K. In addition to opening several other application possibilities, the procedure may be particularly useful in cases where silver-staining of the O-chain in SDSpolyacrylamide gels fails and specific antibodies against the O-antigen are not available.
Endotoxin-induced desensitization of THP-1 cells is not associated with altered G protein binding or content
Authors: Durando M. , Ashton S.H. , Makhlouf M.A. , Simmons-Wagner R. , Halushka P.V. , Cook J.A.
Published: January 2008

In rats endotoxin tolerance is characterized by decreased endotoxin-stimulated peritoneal macrophage arachidonic acid metabolism and decreased GTP binding protein function. The hypothesis that THP-1 cells can be altered in a similar manner by pretreatment with endotoxin was tested. These studies examined endotoxin's ability to stimulate eicosanoid and tumor necrosis factor α (TNFα) in control and desensitized THP 1 cells. Additionally, membrane GTPγ 35S binding and Western blot analyses with specific antisera to G i1,2α, Gi3a, Gαcommon, and the β subunit of G in control and endotoxin-desensitized THP-1 cells were assessed. Endotoxin (10 μg/ml) stimulated thromboxane (Tx) B2 production in THP-1 cells. Pretreatment with pertussis toxin (PT), resulted in significant inhibition of TxB2 production at concentrations not inhibited by equimolar concentrations of PT-B protomer. The latter observations suggest a role of PT-sensitive G protein in endotoxin activation of THP-1 cells. Pre-exposure to endotoxin (1 μg/ml) for 18 h desensitized THP-1 cells to endotoxin-stimulated TxB2 production and endotoxin-stimulated TNFα. To determine if endotoxin pretreatment affects G protein function, THP-1 cell membranes were isolated from endotoxin pretreated and control cells for equilibrium binding with GTPγ35S, a nonhydrolyzable analog of GTP. Neither the total number of binding sites (Bmax) nor the dissociation constant (Kd) for GTPγ35S in desensitized THP-1 cells were significantly different from those of control cells. PT-catalyzed ADP-ribosylation of G proteins in control and LPS-desensitized THP-1 cells demonstrated no difference in the quantity of G protein labelled versus desensitized cells. Immunoblots also showed no difference between control and desensitized cells in the membrane content of specific heterotrimeric G proteins. The data demonstrate that pre-exposure to endotoxin desensitizes the cells subsequent endotoxin stimulation of mediator production. However, unlike the in vivo rat model, this is not associated with a decrease in G protein binding or content.
htrB of Haemophilus influenzae: determination of biochemical activity and effects on virulence and lipooligosaccharide toxicity
Authors: Nichols W.A. , Raetz C.R.H. , Clementz T. , Smith A.L. , Hanson J.A. , Ketterer M.R. , Sunshine M. , Apicella M.A.
Published: January 2008

The htrB mutant of Haemophilus influenzae (strain B29) has been shown to lack secondary (non-hydroxylated) acyl groups in its lipid A. We have determined through in vitro biochemical assays that the HtrB protein acts as a specific acyltransferase in the late stages of lipid A biosynthesis and that the preferred acyl group donor is myristoyl-acyl carrier protein. Under the conditions employed, the Escherichia coli precursor, Kdo2-lipid IVA, functions as a myristate acceptor. Introduction of the Haemophilus htrB gene into an E. coli mutant lacking htrB complements the biochemical and physiological defects associated with the E. coli htrB mutation.

Tumor necrosis factor α (TNFα) assays using murine and human macrophage cells indicated that nontypeable H. influenzae (NtHi) strain 2019 and H. influenzae type b strain A2 elicit levels of expression of TNFα that are 30-40 times greater than levels induced by the isogenic htrB mutants (B29 and A2B29). Studies using cell-free LOS indicated that the LOS from wild type strain 2019 elicits levels of TNFα expression that are 6-8-fold higher than those of B29. In situ hybridization studies of a primary human bronchial epithelial cell line demonstrated a greater increase of TNFα message produced in the presence of 2019 LOS than in the presence of B29 LOS. TNFα levels of the cell supernatant of cells stimulated with 2019 LOS were found to be 7-8-fold higher than levels in B29 stimulated supernatants. Using the Limulus amoebocyte lysate for assessment of endotoxic activity, we found that wild type LOS was 8-fold higher in endotoxic activity compared with the mutant LOS. In virulence assays using intraperitoneal inoculation of infant rats, the htrB isogenic strain caused bacteremia at 50% the frequency of the wild type strain. In intranasal inoculation studies, the htrB mutant strain was unable to cause bacteremia whereas the wild type b parent produced bacteremia in 40-60% of the animals. These findings suggest that the htrB gene of H. influenzae is important for virulence and that host TNFα expression is attenuated in response to htrB mutant strains.
Fundamental differences during Gram-positive versus Gram-negative sepsis become apparent during bacterial challenge of D-galactosamine-treated mice
Authors: Silverstein R. , Norimatsu M. , Morrison D.C.
Published: January 2008

Gram-negative and Gram-positive bacteria have been compared with respect to lethal effects when each is administered to normal and D-galactosamine-sensitized mice, both with and without concomitant dexamethasone treatment. In the case of Escherichia coli, the extent of sensitization by D-galactosamine treatment (10,000-fold) and the relative magnitude of the corresponding protection with dexamethasone (150-fold) are both consistent with an expected significant role of LPS in production of TNFα that then mediates lethal toxicity. With Staphylococcus aureus, however, marginal sensitization by D-galactosamine (5-fold) and a corresponding lack of dexamethasone protection indicate a reduced role for TNFα as a lethal mediator. In vitro comparisons of TNFα release from E. coli and S. aureus stimulated peritoneal macrophages (100-fold difference) add further support to this conclusion. Endotoxin hypo-responsive mice (C3H/HeJ) infected with E. coli are not protected by dexamethasone. Each of these comparisons indicate that the contribution of TNFα to the pathophysiological manifestations of experimental sepsis may vary substantially even among extracellular bacteria and, correspondingly, that differential dexamethasone protection may serve a discriminatory function for the potential involvement of this cytokine.
LPS binding protein (LBP) function in germfree mice
Authors: Le Roy D. , Glauser M.P. , Heumann D.
Published: January 2008

The LPS binding protein (LBP) is an acute phase protein and an integral component of innate immunity. In normal plasma, LBP was shown to be involved in the response of monocytes to LPS. LBP was also shown to play a detrimental role in models of endotoxemia in mice. Levels of LBP increase during infection. To determine whether the presence of an intact bacterial flora is a prerequisite for normal LBP function, we analyzed the serum concentrations and functional activity of LBP in conventional and germfree mice. Baseline LBP levels were similar in both types of mice, as was the production of the pro-inflammatory cytokines TNFα and IL-6 in whole blood stimulated with LPS. Moreover, D-galactosamine sensitized germfree and conventional mice exhibited similar susceptibility to LPS. We thus conclude that the presence of a bacterial flora is not required to support normal levels and function of LBP.
Effects of recombinant soluble CD14 on association of bacterial lipopolysaccharide with bovine alveolar macrophages in vitro
Authors: Yang Z. , Lichenstein H.S. , Bochsler P.N.
Published: January 2008

The CD14 receptor on the cell membrane of macrophages is well-known as a receptor for bacterial lipopolysaccharide (LPS) and contributes to cellular activation, but the potential role of the soluble CD14 receptor (sCD14) in macrophage activation is less understood. In this study, CD14-dependent binding or uptake of [3H]-LPS by bovine alveolar macrophages (AM) was determined in vitro, and LPS-mediated activation of AM was evaluated using the tissue factor-procoagulant assay. In the absence of LPS-binding protein (LBP) or other serum components, recombinant human sCD14 (rsCD14) enhanced binding of [3H]-LPS to AM and also increased LPS-mediated activation of AM in a concentration-dependent manner. These effects were inhibitable by anti-CD14 monoclonal antibodies; the antibodies decreased, but did not completely prevent, association of [3H]-LPS with cells. Binding of [3H]-LPS to AM was greater in the presence of rsCD14 + bovine LBP (5-40 ng/ml), as compared to the moderately lower quantity of bound lipopolysaccharide with [3H]-LPS + LBP alone, or compared to the much lower quantity of lipopolysaccharide bound when [3H]-LPS was used alone. In the presence of whole serum, the effects of rsCD14 were dichotomous and depended upon the serum concentration. rsCD14 (4 μg/ml) added to medium containing ≥ 1% (v/v) fetal bovine serum resulted in reduced binding/uptake of [ 3H]-LPS by AM. However, rsCD14 slightly enhanced binding of [3 H]-LPS to AM at a very low concentration of serum (0.1%) and in the absence of serum. These results suggest that sCD14 enhances binding of [ 3H]-LPS to macrophages when serum-deficient conditions prevail, and sCD14 also enhances binding of LPS in the presence of low concentrations of purified LBP. However, sCD14 decreases association of LPS with AM in the presence of serum (≥ 1 %).