bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of the interferon regulatory factors, IRF-1 and IRF-2, in LPS-induced cyclooxygenase-2 (COX-2) expression in vivo and in vitro
Authors: Shuling Zhang  , Thomas Karen , Blanco Jorge C.G. , Salkowski Cindy A. , Vogel Stefanie N.
Published: June 2007

Cyclooxygenase (COX) exists as two isoforms: COX-1, which is constitutively expressed in most cell types; and COX-2, which is inducible by lipopolysaccharide (LPS) and cytokines in a variety of cell types. Although previous studies have implicated two DNA binding proteins, interferon regulatory factor (IRF)-1 and IRF-2, in the regulation of LPS- and IFN-γ-induced COX-2, their effects in vivo and in vitro are not well-defined. Using real-time PCR, COX-2 gene expression in the livers and lungs of mice challenged in vivo and in macrophages stimulated with LPS in vitro was investigated i n wild-type and in IRF-1 and IRF-2 knockout mice. In response to 35 mg/kg LPS, IRF-1-, but not IRF-2-deficient mice, exhibited much poorer induction of COX-2 gene expression in both the livers and lungs. In vitro, COX-2 mRNA levels were also poorly induced in IRF-1-deficient macrophages, while IRF-2-deficient macrophages exhibited higher levels than in normal macrophages. IRF-1 and IRF-2 were confirmed to activate and repress expression of the COX-2 promoter, respectively, in a transient transfection system and the role of specific DNA binding sites confirmed by site-specific mutagenesis. Collectively, these data provide evidence for an important role for IRF-1 in vivo and in vitro and for IRF-2 in vitro in the regulation of COX-2 expression by LPS.
Review: The role of the liver in the response to LPS: experimental and clinical findings
Authors: Jirillo E. , Caccavo D. , Magrone T. , Piccigallo E. , Amati L. , Lembo A. , Kalis C. , Gumenscheimer M.
Published: June 2007

The liver plays an important physiological role in lipopolysaccharide (LPS) detoxification and, in particular, hepatocytes are involved in the clearance of endotoxin of intestinal derivation. In experimental shock models, tumor necrosis factor (TNF)-α induces hepatocyte apoptosis and lethal effects are due to secreted TNF-α and not to cell-associated TNF-α. An exaggerated production of TNF-α has been reported in murine viral infections, in which mice become sensitized to low amounts of LPS and both interferon (IFN)-γ and IFN-α/β are involved in the macrophage-induced release of TNF-α. The prominent role of LPS and TNF-α in liver injury is also supported by studies of ethanol-induced hepatic damage. In humans, evidence of LPS-induced hepatic injury has been reported in cirrhosis, autoimmune hepatitis, and primary biliary cirrhosis and a decreased phagocytic activity of the reticulo-endothelial system has been found in these diseases. The origin of endotoxemia in hepatitis C virus (HCV) infected patients seems to be multifactorial and LPS may be of exogenous or endogenous derivation. In endotoxemic HCV-positive patients responsive to a combined treatment with IFN-α/ribavirin (RIB), endotoxemia was no longer detected at the end of the therapeutic regimen. By contrast, 48% of the non-responders to this treatment were still endotoxemic and their monocytes displayed higher intracellular TNF-α and interleukin (IL)-1β levels than responders. Moreover, in responders, an equilibrium between IFN-γ and IL-10 serum levels was attained. In the non-responders, serum levels of IL-10 did not increase following treatment. This may imply that an imbalance between T helper (Th)1 and Th2 derived cytokines could be envisaged in the non-responders.
Mini-review: Enhancement of endotoxin activity by muramyldipeptide
Authors: Takada Haruhiko , Yokoyama Shigekuni , Shuhua Yang
Published: June 2007

Synthetic muramyldipeptide (MDP), the minimum structural moiety of bacterial peptidoglycan for adjuvant and related activities, sensitized mice for two types of lethal shock induced by lipopolysaccharide (LPS): an early anaphylactoid shock and late endotoxin shock. In relation to the late reaction in MDP-primed mice, enhanced production of inflammatory cytokines was induced in response to various bacterial components. MDP showed a priming effect in mice not only when administered parentally but also via the oral route. MDP activated human monocytic THP-1 cells in a CD14-, Toll-like receptor 2 (TLR2)- and TLR4-independent manner to increase expression of MyD88, a common adaptor and signaling molecule for TLRs, and exhibited synergistic cytokine-inducing effects with TLR4 agonists (LPS, synthetic lipid A), TLR2 agonist (synthetic lipopeptide), and TLR9 agonist (bacterial CpG DNA) in THP-1 cells in culture. Consistent with these findings, MDP primed TLR2 knockout mice as well as wild-type controls, but not TLR4-mutated C3H/HeJ mice, to enhance production of tumor necrosis factor-α upon stimulation with synthetic lipid A. In contrast to the BCG- and Propionibacterium acnes-priming system, MDP primed mice in an interferon-γ-independent manner. Further studies are required to elucidate the mechanisms of the synthetic and priming activities of MDP for various bacterial components.
The clinical relevance of endotoxin in human sepsis: a critical analysis
Authors: Opal Steven M.
Published: June 2007

Correlations between circulating endotoxin levels from the blood of septic patients with clinical outcome measures have proven to be rather difficult. The clinical impact of endotoxin in septic patients depends upon the kinetics of LPS release, the concentration of LPS binding proteins, the cellular responsiveness to endotoxin, and numerous other immunogenetic, microbiological and physiological variables. Failure to account for these variables adequately has limited the clinical utility of endotoxin measurement. Several clinical studies have associated high levels of LPS with an excess risk of morbidity and mortality in sepsis that is largely independent of the nature of the micro-organism responsible for the septic episode.
Molecular basis of non-self recognition by the horseshoe crab lectins
Authors: Kawabata Shun-ichiro , Tsuda Ryoko
Published: June 2007

The target molecules of innate immunity are not proteins of direct gene products but the molecular arrays or patterns on pathogens, called pathogen-associated molecular patterns (PAMPs). The self/non-self discrimination by innate immunity through simple ligands universally expressed both on pathogens and hosts, such as monosaccharides and the acetyl group, probably depends on the density or clustering patterns of the ligands. The specific recognition by the horseshoe crab lectins, named tachylectins, with a propeller-like fold or a propeller-like oligomeric arrangement is reinforced by the short distance between the individual binding sites that interact with PAMPs. There is virtually no conformational change in the main or side chains of tachylectins upon binding with the ligands. This low structural flexibility of the propeller structures must be very important for specific interaction with PAMPs.
Continuous cardiac output and hemodynamic monitoring: high temporal correlation between plasma TNF-α and hemodynamic changes during a sepsis-like state in cancer immunotherapy
Authors: Caorsi Carlos , Quintana Enrique , Valdés Sergio , Muñoz Carlos
Published: June 2007

Through continuous cardiac output monitoring, we investigated the temporal relationship between hemodynamic changes and plasma cytokines in a cancer patient who developed collateral sepsis to immunotherapy. A 52-year-old male with metastatic renal cell carcinoma received interleukin-2 (IL-2) infusion completing 72 h of administration. The patient developed 3 sepsis-like states including systemic inflammatory response syndrome (SIRS), shock, and multiple organ dysfunction syndrome (MODS). Hemodynamic parameters including systemic vascular resistance index (SVRI), left ventricular stroke work index (LVSWI) and cardiac index (CI) were measured over 60 h. Peripheral blood was drawn when SVRI dropped 20% in the patient and plasma cytokines including TNF-α , IL-6 and IL-1β were measured using ELISA. After 60 h of immunotherapy, the patient showed a 63.4% decrease in SVRI, 54.5% decrease in LVSWI and 65.4% increase in CI. The evaluation of systemic cytokines revealed different kinetic patterns: (i) a sustained increase in TNFα levels through all 3 sepsis-like states; (ii) IL-6 increased preferentially during SIRS and shock, while up/down-responses were found during MODS; (iii) IL-1β was undetectable during the entire study period. A high temporal relationship between hemodynamic changes and plasma TNF-α, but not IL-6, was found. Although there are factors other than cytokines that can alter vascular resistance, this finding could represent an approach to evaluate the course of hemodynamia and probably the systemic cytokine expression after IL-2 administration in renal cancer.
Review: The lipo-oligosaccharides of Haemophilus influenzae: an interesting array of characters
Authors: Swords W. Edward , Jones Paul A. , Apicella Michael A.
Published: June 2007

The composition of the lipo-oligosaccharide (LOS) of Haemophilus influenzae is highly variable, especially in the oligosaccharide region. Many of the biosynthetic and transferase genes involved in LOS biosynthesis vary in seemingly random fashion by means of polymerase stuttering within redundant sequences in the 5'-portion of the genes. This results in a heterogeneous population of individual bacteria expressing literally thousands of LOS glycoforms. The simultaneous variation in the expression and structural context of a large number of individual carbohydrate and lipid structures within the LOS yields a diverse array of LOS glycoforms. The expression of glycoforms that mimic host structures may allow the organism to evade innate defenses and to manipulate host cell biology. We review how this randomly generated bacterial combinatorial chemistry results in the production of a large number of carbohydrate structures, in essentially any conceivable structural context, some of which allow the organism to utilize host cell receptors. By generating a diverse population of bacteria expressing different LOS glycoforms, discrete H. influenzae subpopulations may be adapted for survival of different environmental stresses within the airways. Thus, H. influenzae utilizes a simple and efficient `Monte Carlo' strategy for achieving maximal variation in cell surface structures, which allow the organism to adapt efficiently to environmental stresses with a small genome.
TLR2 and TLR4 agonists stimulate unique repertoires of host resistance genes in murine macrophages: interferon-β-dependent signaling in TLR4-mediated responses
Authors: Toshchakov Vladimir , Jones Bryan W. , Lentschat Arnd , Silva Aristobolo , Perera Pin-Yu , Thomas Karen , Cody M. Joshua , Shuling Zhang  , Williams Bryan R.G. , Major Jennifer , Hamilton Thomas A. , Fenton Matthew J. , Vogel Stefanie N.
Published: June 2007

That TLRs share a common MyD88-dependent signaling pathway which results in the generation of nuclear DNA-binding proteins, such as NF-κB, is a well-accepted paradigm. However, studies from our laboratories and others suggested that TLR4 agonists elicit a more diverse pattern of gene expression in murine macrophages than TLR2 agonists. The data presented show that activation of TLR4 by Escherichia coli LPS results in an MyD88-independent, TIRAP/Mal-dependent signaling pathway that, in turn, leads to early induction of interferon-β (IFN-β). IFN-β , in turn, acts in an autocrine/paracrine fashion on the macrophage to activate STAT1-containing DNA binding complexes that participate in the induction of genes not expressed in response to natural or synthetic TLR2 agonists. These data support the hypothesis that the host response to microbes is controlled by TLRs at two levels: (i) the `sensing' of differences in microbial structures through the TLR extracellular domain; and (ii) signaling pathways that are initiated via interactions through unique intracytoplasmic regions of different TLRs with adaptor proteins.
Coagulation and inflammation
Authors: Esmon Charles T.
Published: June 2007

The protein C anticoagulant pathway is critical for controlling microvascular thrombosis and is initiated when thrombin binds to thrombomodulin (TM) on the surface of the endothelium. Protein C activation is augmented by an endothelial cell protein C receptor (EPCR). EPCR is shed from the vasculature by inflammatory mediators and thrombin. EPCR binds to activated neutrophils in a process that involves proteinase 3 and Mac-1 and appears to inhibit leukocyte extravasation. EPCR can undergo translocation from the plasma membrane to the nucleus where it re-directs gene expression. During translocation, EPCR can carry activated protein C (APC) to the nucleus, possibly accounting for the ability of APC to modulate inflammatory mediator responses in the endothelium. TNF-α and other inflammatory mediators can down-regulate EPCR and TM. Inhibition of protein C pathway function increases cytokine elaboration, endothelial cell injury and leukocyte extravasation in response to endotoxin and infusion of APC reverses these processes. In vitro, APC has been reported to inhibit TNF-α elaboration from monocytes and to block leukocyte adhesion to selectins. Since thrombin can elicit many inflammatory responses in microvascular endothelium, loss of control of microvascular thrombin generation due to impaired protein C pathway function probably contributes to microvascular dysfunction in sepsis.
Bacterial LPS and CpG DNA differentially induce gene expression profiles in mouse macrophages
Authors: Jian Jun Gao  , Diesl Veronica , Wittmann Tatiana , Morrison David C. , Ryan John L. , Vogel Stefanie N. , Follettie Maximillian T.
Published: June 2007

Bacterial DNA containing unmethylated CpG dinucleotides (CpG DNA) is a potent immune stimulating agent that holds strong promise in the treatment of many disorders. Studies have established that CpG DNA triggers an immune response through activated expression of genes in immune cells including macrophages. To dissect further the molecular mechanism(s) by which CpG DNA activates the immune system, we studied macrophage gene expression profiles in response to CpG DNA using microarray technology. Since CpG DNA is reported to use the TLR9 receptor that shares homology with the TLR4 receptor used by bacterial lipopolysaccharide (LPS), we also evaluated gene expression profiles in macrophages stimulated by LPS versus CpG DNA. Both CpG DNA and LPS modulate expression of a large array of genes. However, LPS modulated the expression of a much greater number of genes than did CpG DNA and all genes induced or repressed by CpG DNA were also induced or repressed by LPS. These data indicate that the CpG DNA signaling pathway through TLR9 activates only a subset of genes induced by the LPS TLR4 signaling pathway.
Molecular insights into the assembly and diversity of the outer core oligosaccharide in lipopolysaccharides from Escherichia coli and Salmonella
Authors: Whitfield Chris , Kaniuk Natalia , Frirdich Emilisa
Published: June 2007

In the Enterobacteriaceae, the core oligosaccharide provides the junction between the highly conserved lipid A and the remarkably diverse polysaccharide O antigen. The basic structure of the inner (lipid A-proximal) core is well conserved, perhaps reflecting constraints imposed by its involvement in the structural integrity of the outer membrane. However, non-stoichiometric modifications do create some structural variants. The outer core may show more variation. Efforts to develop immunoprophylactic strategies based on the core oligosaccharide require a detailed understanding of core immunochemistry, the accessibility of specific epitopes in the LPS, and the distribution of specific structures within natural populations. The availability of sequences for the waa (core biosynthesis) loci and functional data for the gene products provide a molecular basis for the known structural diversity in Escherichia coli and Salmonella core oligosaccharide. Surveys of waa-locus organization have established the distribution of these core types in natural populations and have identified genetic variants that provide candidates for additional novel structures.
Inhibition of LPS-responses by synthetic peptides derived from LBP associates with the ability of the peptides to block LBP-LPS interaction
Authors: Araña Manuel de J. , Vallespi Maribel G. , Chinea Glay , Vallespi Guillermo V. , Rodriguez-Alonso Ingrid , Garay Hilda E. , Buurman Wim A. , Reyes Osvaldo
Published: June 2007

The ability of LPS-binding protein (LBP) to greatly potentiate cell responses to lipopolysaccharide (LPS) may largely contribute to LPS toxicity in sepsis. The study of agents with the capacity to block the interaction between LBP and LPS might improve the understanding of the role of LBP in Gram-negative infections as well as offering new therapeutic tools for septic disorders. Here we confirm the ability of synthetic peptides comprising the human LBP amino acid region 86-108 to interfere with the LBP-LPS interaction. The analysis of selected alanine mutants of a blocking peptide corresponding to the LBP region 86-99 suggests the importance of peptide amphipathicity for the inhibitory activity. The potency of the native peptide and a selected analogue at inhibiting in vitro and in vivo LPS-induced responses was associated with their relative activity in blocking LBP-LPS interaction. It was remarkable that these peptides were at least 500-fold more active in vivo than in vitro. Also, the inhibitory activity of peptides LBP86-99 and LBPK95A seems to be independent of LBP concentrations, a behavior that may be relevant for the potential use of these peptides in septic disorders where LBP serum concentrations are considerably elevated.
Hyperlipoproteinemic low-density lipoprotein receptor-deficient mice are more susceptible to sepsis than corresponding wild-type mice
Authors: Lanza-Jacoby Susan , Miller Sheldon , Jacob Shushan , Heumann Didier , Minchenko Alexander G. , Flynn John T.
Published: June 2007

High circulating concentrations of lipoproteins have been shown to modify the cytokine response and reduce mortality after endotoxin or live bacterial challenge. Sepsis, however, is more complex than endotoxemia, and it is not clear whether elevated plasma lipoproteins will be protective. Previous studies have shown that the low-density-lipoprotein receptor deficient (LDLR —/— ) mice with increased circulating LDL are protected against the lethal effects of endotoxemia and Gram-negative infection. We evaluated whether the LDLR —/— mice would be protected against the effects of sepsis induced by cecal ligation and puncture (CLP). Mortality was greater in LDLR—/ — mice than in control C57Bl/6J mice. At 120 h after inducing sepsis, 20% of the control mice survived whereas none of the LDLR—/— mice were alive. Prior to inducing sepsis, serum concentrations of amyloid A protein and lipopolysaccharide binding protein (LBP) were significantly elevated in the LDLR —/— mice in comparison to the C57Bl/6J mice. Protein expression of sCD14 was also greater in the serum from the LDLR —/ — mice than the C57Bl/6J mice. The elevated serum concentrations of LBP and CD14 were not associated with increases in the levels of liver CD14 mRNA and LBP mRNA. After inducing sepsis, serum concentration of interleukin (IL)-1β was also significantly higher in LDLR — /— mice than in the control C57Bl/6J mice. These findings indicate that the LDLR — /— mice were more susceptible to the lethal effects of sepsis induced by CLP. The LDLR — /— mice also had higher serum concentrations of baseline, acute phase response proteins, SAA and LBP, and increased production of IL-1β in response to CLP.
LPS binding protein does not participate in the pharmacokinetics of E5564
Authors: Kaneko Kazuhiro , Ueda Rika , Kawata Tsutomu , Ishizaka Sally , Yoshimura Tsutomu
Published: June 2007

E5564, a lipid A analogue, is a potent antagonist of lipopolysaccharide (LPS). Clinically, E5564 was developed as a possible therapy for treatment of sepsis and septic shock. Surface plasmon resonance (SPR) analysis indicates that E5564 binds to LPS binding protein (LBP), in a manner similar to LPS. Gel-filtration radioactive chromatograms of [14C]-E5564 in plasma revealed that E5564 initially distributes to the lipoprotein fractions, separated from high-density lipoprotein (HDL); the bound fraction is then released and binds to HDL. Similar results were obtained by heparin-manganese precipitation. At doses of E5564 relevant to its clinical use ( i.e. 6 µg/ml), antibodies against LBP did not influence either the distribution of E5564 to non-HDL lipoprotein fractions or the transfer of E5564 from non-HDLs to HDL. Under these conditions, transfer of E5564 to HDL occurs similarly in the plasma of LBP knockout (KO) mice as in the plasma from wild-type mice. In addition, plasma clearance of E5564 in LBP KO mice is similar to that of wild-type mice. Thus, LBP binds E5564 in a manner similar to LPS, but does not play a role in E5564 redistribution/binding to lipoprotein and plasma clearance.
Detection of endotoxin in sera from children hospitalized for treatment of diarrhea in Bangladesh
Authors: Ahmed Tahmeed , Mohamed Ali Azam  , Armed Nusrat , Jamil Kazi M. , Hassan Ferdaus , Ogura Norihiko , Tamura Hiroshi , Yokochi Takashi
Published: June 2007

The level of circulating endotoxin was determined in the sera from children hospitalized for treatment of diarrhea at the International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh. A significant level of endotoxin was detected in the sera from 23 (54%) of 42 children. On the other hand, in 32 normal controls, endotoxin was below the limits of detection of the assay. Antibiotic and fluid therapy markedly reduced the level of serum endotoxin and improved the general condition of most patients. Non-survivors ( n = 5) had higher levels of circulating endotoxin before treatment than survivors ( n = 37), suggesting a significant correlation between the serum endotoxin level before treatment and mortality. Malnutrition did not affect the serum endotoxin level in the patients with diarrhea. It was suggested that infection of Gram-negative bacteria might be involved in a significant number of patients with diarrhea in Bangladesh and that endotoxin might play a pathogenic role in those patients.
Hemoperfusion with polymyxin B-immobilized fibers reduced the number of CD16+CD14 + monocytes in patients with septic shock
Authors: Tsujimoto Hironori , Ono Satoshi , Hiraki Shuichi , Majima Takashi , Kawarabayashi Nobuaki , Sugasawa Hidekazu , Kinoshita Manabu , Hiraide Hoshio , Mochizuki Hidetaka
Published: June 2007

Background: CD16 +CD14+ monocytes dramatically increase in number in patients with severe infection. Hemoperfusion with PMX-F (direct hemoperfusion with polymyxin B immobilized fibers) has been reported to be a safe and effective treatment for patients with septic shock, although the molecular mechanism that accounts for its effectiveness is still unclear. The purpose of this study was to quantify the number of CD16+CD14+ monocytes in patients with an intra-abdominal infection and to evaluate the effects of PMX-F treatment on clinical parameters and leukocyte surface antigen expression in these patients.

Materials and Methods : Seventeen septic patients who had an intra-abdominal infection were enrolled in this study; 7 of these patients received PMX-F treatment. Peripheral blood samples were obtained immediately after admission, and were also collected from the above 7 patients before, during, and immediately after their PMX-F treatment. The expression of CD14, CD16, and Toll-like receptor (TLR)-4 on these patients' monocytes was evaluated using flow cytometry. In addition, lipopolysaccharide (LPS)-induced production of TNF-α and IL-1β by these cells was measured by ELISA.

Results: Monocytic expression of CD16 and TLR-4 was significantly greater in septic patients than in healthy controls, and their proportion of CD16+CD14 + monocytes was similarly elevated. LPSinduced production of TNF-α and IL-1β by peripheral blood mononuclear cells (PBMCs) of septic patients was significantly reduced compared to controls. Furthermore, there was a reduction in the proportion of CD 16+CD 14+ monocytes during PMX-F treatment, and in the expression of TLR-4 on monocytes after PMX-F treatment.

Conclusions: These results showed that the number of peripheral blood CD16+CD14 + monocytes and monocytic TLR-4 expression were markedly increased, and the production of pro-inflammatory cytokines in response to LPS significantly reduced in patients with sepsis. PMX-F treatment was found to be effective in reducing the number of CD 16+CD 14+ monocytes and in decreasing the monocytic expression of TLR-4 in patients with septic shock.
Ligand-dependent Toll-like receptor 4 (TLR4)-oligomerization is directly linked with TLR4-signaling
Authors: Saitoh Shin-ichiroh , Akashi Sachiko , Yamada Takenao , Tanimura Natsuko , Matsumoto Fumi , Fukase Koichi , Kusumoto Shoichi , Kosugi Atsushi , Miyake Kensuke
Published: June 2007

Toll-like receptor 4 (TLR4) and MD-2 recognize lipid A, the active moiety of microbial lipopolysaccharide (LPS). Little is known about mechanisms for LPS recognition by TLR4/MD-2. We here showed, by using in vitro transfectants, ligand-induced TLR4-oligomerization, which required both membrane CD14 and MD-2. We previously reported that lipid IVa, a lipid A precursor, is agonistic on mouse TLR4/MD-2 but antagonistic on human TLR4/MD-2 and chimeric mouse TLR4/human MD-2. Lipid IVa triggered oligomerization of mouse TLR4/MD-2 but not human TLR4/MD-2 or chimeric mouse TLR4/human MD-2. Further, lipid IVa inhibited lipid A-dependent oligomerization of chimeric mouse TLR4/human MD-2. These results demonstrate that ligand-induced TLR4-oligomerization is directly linked with TLR4-signaling and suggest that MD-2 has an important role in regulating TLR4-oligomerization.
Lipoprotein-bound endotoxin exerts an immunomodulatory effect on hepatocytes through the lipid A domain of LPS
Authors: Behzad Kasravi F. , Lee Diana H. , Weisgraber Karl , Harris Hobart W.
Published: June 2007

Background: We have previously shown that chylomicron (CM)-bound lipopolysaccharide (LPS) inhibits the host innate immune response by rendering hepatocytes tolerant to pro-inflammatory cytokine stimulation. However, LPS is a complex macromolecule containing both lipid and carbohydrate domains. We hypothesized that just as lipid A confers the toxicity of LPS, it is also responsible for the immunoregulatory effect on hepatocytes.

Methods: We pretreated primary rat hepatocytes for 2 h with a series of CM-LPS complexes in which the endotoxin moiety varied in its structure and/or toxicity. Subsequently, the cells were stimulated with a mixture of pro-inflammatory cytokines. Nitric oxide production was measured as an indicator of hepatocellular activation.

Results: All pretreatments wherein the CM-bound complex contained the lipid A moiety readily inhibited the hepatocellular cytokine response, including CM bound to lipid A alone. In contrast, CM-LPS complexes containing detoxified LPS, which lacks the lipid A domain, had no effect on the hepatocellular response to cytokines.

Conclusions: The lipid A domain of the LPS macromolecule is both sufficient and essential for the CM-mediated induction of cytokine tolerance in hepatocytes. However, this process is independent of the specific endotoxic activity of the lipid A moiety.
Production and purification of recombinants of mouse MASP-2 and sMAP
Authors: Iwaki Daisuke , Fujita Teizo
Published: June 2007

Small mannose-binding lectin (MBL)-associated protein (sMAP) is a component of the complex consisting of MBL and MBL-associated serine proteases (MASPs) in the lectin complement pathway. sMAP is a truncated form of MASP-2, which is generated by an alternative splicing from a single structural MASP-2 gene. Upon activation of the MBL—MASPs complex, MASP-2 cleaves the complement C4, but the role of sMAP which lacks the serine protease domain is not clear. To clarify the role of sMAP in activation of the lectin pathway, we have generated sMAP-gene deficient mice which are also deficient for MASP-2. In this study, we generated and purified mouse recombinant sMAP (rsMAP) and rMASP-2 using the Drosophila expression system for the reconstitution assay of the deficient mice. In preliminary experiments, these purified recombinants were able to reconstitute the MBL—MASPs—sMAP complexes and the addition of rMASP-2 to deficient serum restored the C4 cleavage activity of the MBL—MASPs complex. From these data, rsMAP and rMASP-2 generated in this study seem to be useful in analysis of the deficient mice.
Role of lipid A palmitoylation in bacterial pathogenesis
Authors: Bishop Russell E. , Kim Sang-Hyun , El Zoeiby Ahmed
Published: June 2007

The presence of palmitate in a minor fraction of lipid A has been known since the chemical structure of lipid A was first elucidated, but the functional importance in bacterial pathogenesis of regulated lipid A palmitoylation has become clear only recently. A palmitate chain from a phospholipid is incorporated into lipid A by an outer membrane enzyme PagP. The isolation of pagP mutants from pathogenic Gram-negative bacteria has revealed that palmitoylated lipid A can both protect the bacterium from certain host immune defenses and attenuate the ability of lipid A to activate those same defenses through the TLR4 signal transduction pathway. The mechanisms by which bacteria regulate the incorporation of palmitate into lipid A strikingly reflect the corresponding organism's pathogenic lifestyle. Variations on these themes can be illustrated with the known pagP homologs from Gram-negative bacteria, which include pathogens of humans and other mammals in addition to pathogens of insects and plants. The PagP enzyme is now lending itself both as a target for the development of anti-infective agents, and as a tool for the synthesis of lipid A-based vaccine adjuvants and endotoxin antagonists.
Inhibition of TLR-4/MD-2 signaling by RP105/MD-1
Authors: Divanovic Senad , Trompette Aurelien , Atabani Sowsan F. , Madan Rajat , Golenbock Douglas T. , Visintin Alberto , Finberg Robert W. , Tarakhovsky Alexander , Vogel Stefanie N. , Belkaid Yasmine , Kurt-Jones Evelyn A. , Karp Christopher L.
Published: June 2007

Activation of Toll-like receptor (TLR) signaling by microbial and host molecular signatures is critical to the induction of immune responses. Such signaling is, perforce, kept under tight control. We recently discovered a novel endogenous inhibitor of TLR-4 — RP105. Initially identified as a B-cell-specific molecule with a role in B-cell proliferation in response to RP105 mAb and LPS, RP105 is a TLR-4 homologue. Further, like TLR-4 whose surface expression and signaling depends upon co-expression of the secreted protein MD-2, surface expression of RP105 is dependent upon co-expression of the MD2 homologue, MD-1. Unlike the TLRs, however, RP105 lacks a signaling domain, having the apparent structure of a TLR inhibitor. Further, RP105 is not B-cell-specific; its expression directly mirrors that of TLR-4 on dendritic cells and macrophages. These considerations suggested a role for RP105 as a physiological inhibitor of TLR-4 signaling. Indeed, we have recently found that: (i) RP105 is a specific inhibitor of TLR-4 signaling in HEK293 cells; (ii) RP105/MD-1 interacts directly with TLR-4/MD-2, inhibiting the ability of this signaling complex to bind LPS; (iii) RP105 regulates TLR-4 signaling in dendritic cells and macrophages; and (iv) RP105 regulates in vivo responses to LPS.
GM-CSF priming of human monocytes is dependent on ERK1/2 activation
Authors: Lendemans Sven , Rani Meenakshi , Selbach Christian , Kreuzfelder Ernst , Schade Fritz Ulrich , Flohé Sascha
Published: June 2007

The ability to augment monocyte functions such as TNF-α-producing capacities confers a high immunostimulating potential to GM-CSF. In the present investigation, the mechanism of the GM-CSF-mediated enhancement of monocyte cytokine production was analysed with regard to the involvement of intracellular signalling pathways. GM-CSF primes human monocytes dose- and time-dependently for enhanced LPS-stimulated TNF-α synthesis. Pre-incubation with 10 ng/ml GM-CSF for 6 h before LPS stimulation (10 ng/ml) caused a 3.4 ± 1.9-fold increase in TNF-α release compared to unprimed controls. This was associated with increased phosphorylation of IκBα and elevated nuclear levels of the NF-κB components p50 and p65 and NF-κB binding to DNA. LPS-induced AP-1 binding to DNA was also enhanced in GM-CSF-pre-incubated cells. GM-CSF treatment also caused a slight increase in TLR4 expression on monocytes while CD14 expression remained unchanged. GM-CSF-priming was unaffected by inhibitors of p38 MAPK (SB203580) and lipoxygenase (NDGA). In contrast, the broad-spectrum tyrosine kinase inhibitor genistein and the MEK-1 inhibitor (PD98059) abrogated GM-CSF priming of TNF-α release and activation of both NF-κB and AP-1. It is concluded that a tyrosine kinase of the GM-CSF-triggered ERK1/2 pathway augments the LPS-induced NF-κB and AP-1 activation.
The mRNA expression of fatty acid amide hydrolase in human whole blood correlates with sepsis
Authors: Tanaka Masahiro , Yanagihara Itaru , Takahashi Hitoshi , Hamaguchi Mitsuhide , Nakahira Kumiko , Sakata Ikuhiro
Published: June 2007

An excessive accumulation of anandamide ( N-archidonoylethanolamine, AEA) is associated with septic shock. Results of previous studies have suggested that mRNA coding for the AEA degrading enzyme fatty acid amide hydrolase (FAAH), which converts AEA into arachidonic acid and ethanolamine, might be down-regulated in septic shock. We used real-time reverse transcription PCR assays to measure relative FAAH mRNA concentrations in the whole blood of 30 healthy donors and eight sepsis patients to ascertain whether such down-regulation takes place. Our results suggest that concentrations of FAAH mRNA in male and female samples from healthy donors are similar, but that concentrations are significantly lower in sepsis patients. These findings indicate that mRNA expression of FAAH in human whole blood correlates with sepsis, and may be an interesting biomarker for predicting the onset of septic shock.
LPS-dependent changes in the expression of 57 kDa and 53 kDa cell membrane proteins without participation of CD14
Authors: Kirikae Teruo , Kirikae Fumiko , Iwai Hiroki , Qureshi Nilofer , Fukase Koichi , Kusumoto Shoichi , Nakano Masayasu
Published: June 2007

It is widely presumed that in addition to CD14, other molecules are necessary for lipopolysaccharide (LPS)-induced cell activation. In order to shed light on some of the biological and biochemical properties of these molecules, we examined the LPS responsiveness of CD14-negative, ST2 cells. Although ST2 cells do not express CD14 mRNA, they, nonetheless, expressed IL-6 mRNA and synthesized IL-6 protein when incubated with LPS in serum-free medium ( i.e. without soluble CD14). Paxlitacel (Taxol™) also induced IL-6 mRNA expression in ST2 cells, while Rhodobacter sphaeroides diphoshoryl lipid A (RsDPLA) inhibited both LPS- and Taxol-induced expression of IL-6 mRNA. Collectively, these data suggest that LPS, RsDPLA, and Taxol all recognize the same receptor complex on ST2 cells and do not require the participation of CD14. In addition, using antibody raised against the ST2 cell membrane fraction, we detected a set of LPS-specific membrane antigens in murine peritoneal macrophages, including two designated p57 (57 kDa) and p53 (53 kDa). There was no qualitative difference in the expression of p57 and p53 in LPS-responsive, C3H/HeN and LPS-hyporesponsive, C3H/HeJ macrophages. However, after stimulating the macrophages with LPS or Taxol, expression of p57 and p53 was diminished in C3H/HeN macrophages, but not in C3H/HeJ macrophages. Phorbol ester (PMA) and A23187 calcium ionophore did not suppress p57 or p53 expression, and the lipid A precursor, PE406, did not bind to either protein. Thus, p57 and p53 may play important roles in LPS-evoked responses, but they do not appear to serve as LPS receptors.
Purinergic receptor modulation of LPS-stimulated signaling events and nitric oxide release in RAW 264.7 macrophages
Authors: Sommer J.A. , Fisette P.L. , Hu Y. , Denlinger L.C. , Guerra A.N. , Bertics P.J. , Proctor R.A.
Published: June 2007

Purinergic receptors of the P2 class are cell surface receptors which are sensitive to extracellular adenine nucleotides, such as ATP and ADP. This class of receptors is divided into the P2Y family of G protein-coupled receptors and the P2X family of ligand-gated ion channels. The P2X receptors, seven of which have been cloned, are thought to possess two transmembrane domains and function as multimeric complexes. Numerous studies have suggested a role for P2 receptors in activation of macrophages by Gram-negative bacterial endotoxin (lipopolysaccharide; LPS). LPS is thought to exert its toxic effects, in large part, by inducing macrophages to release inflammatory mediators such as tumor necrosis factor α (TNFα), interleukin-1 (IL-1) and nitric oxide (NO). Although multiple signal transduction pathways are activated by LPS in macrophages, the proximal mechanisms by which LPS exerts these effects remain unclear. The current study examines the role of the P2X7/P2Z purinergic receptor in LPS signaling events and in nitric oxide (NO) production. The results indicate that the P2X7 receptor is required for maximal LPS activation of the mitogenactivated protein (MAP) kinases extracellular signal-regulated kinase (ERK)1 and ERK2, for activation of nuclear factor (NF)-κB, as well as for upregulation of the inducible form of nitric oxide synthase (iNOS). These results are fortified by our recent observation that the C-terminus of the P2X7 receptor is homologous to conserved LPS binding domains of proteins critical to host responses to Gram-negative bacterial infection, such as LPS-binding protein (LBP) and bactericidal permeability-increasing protein (BPI). Taken together, these observations suggest that the P2X 7 receptor plays a fundamental role in LPS signal transduction and activation of macrophages, and thus may represent a therapeutic target for Gram-negative bacterial septicemia.
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: accessory function of NK cells
Authors: Nitta Toshimasa , Shoji Hiroe , Nakano Masayasu
Published: June 2007

Highly purified T cells of mice that have been primed in vivo with horse red blood cells (HRBC) and lipid A, but not with HRBC alone, can proliferate in vitro in response to HRBC or anti-αβ T cell receptor (TCR) antibody without macrophages. The mechanism of proliferation without macrophages was investigated. Mice were primed with HRBC and lipid A together or with HRBC alone 10 days in advance, and the peritoneal exudate cells (PEC) were obtained. Purified T cells were obtained by treating the PEC by the following series of procedures: nylon fiber column-passage, antibody (anti-Ia, anti-Mac-1, and LR-1) treatments with complement (C), and a G-10 column-passage. Cell proliferation was assessed by [3H]-thymidine (TdR) incorporation into the cultured T cells in response to HRBC or anti-αβ TCR antibody. The proliferation of T cells [T(HRBC+lipid A)] that had been prepared from the PEC of the mice primed with HRBC and lipid A increased dose-dependently to these stimulants, but T cells [T(HRBC)] prepared from PEC of mice primed with HRBC showed little or no proliferation in response to them. The expression pattern of a memory cell marker, CD44, on the cell surface of T(HRBC+lipid A) was obviously different from that on T(HRBC). The proliferation of T(HRBC+lipid A) was suppressed when the cells were cultured in the wells coated with hyaluronate, a ligand for CD44, or cultured after a previous treatment with anti-CD44 (IgG2a) and anti-IgG2a antibodies. In contrast, the proliferation of T(HRBC) was up-regulated in culture under the same conditions. Proliferative responses of T(HRBC+lipid A) to anti-αβ TCR antibody were enhanced in the wells coated with anti-CD28 antibody. These findings indicate that the proliferation of T(HRBC+lipid A) was not only supported by a stimulation of TCR but also regulated by another stimulation via ligands such as CD44 and CD28. The proliferation of T(HRBC+lipid A) was abolished when the cells were pretreated with AsGM1 antibody or anti-CD80 antibody and C. These findings indicate that the in vivo priming of mice with HRBC and lipid A generates a memory T cell population that is capable of proliferating without help of macrophages, but with NK cells, when stimulated their TCR.
A biophysical approach towards an understanding of endotoxin-induced signal transduction
Authors: Schromm A.B. , Brandenburg K. , Blunck R. , Fukase K. , Kusumoto S. , Rietschel E.Th. , Seydel U.
Published: June 2007

The purpose of our studies is to define the physicochemical parameters involved in the activation of host cells by endotoxin and to characterize the processes operative during endotoxin/membrane interaction with the aim of understanding transmembrane signal transduction mechanisms. To this end, we determined the molecular conformation of the lipid A component of various endotoxins (endotoxic conformation) using X-ray small angle diffraction, their intercalation into reconstituted macrophage membranes with fluorescence resonance energy transfer spectroscopy, and their IL-6 inducing capacity in whole blood. We also investigated their influence on ion channels as a possible primary event in cell activation applying patch-clamp techniques to macrophages. We found a strong influence of the molecular charge on the molecular conformation, and we could show that the presence of charged groups and a cone- or wedge-like molecular conformation of lipid A are prerequisites for the expression of bioactivity. We also obtained strong evidence supporting the idea that the interaction of endotoxin with ion channels is one of the very early events in the interaction with the cell and, most likely, in signal transduction.
Bacteria-induced hypersensitivity to endotoxin
Authors: Freudenberg Marina A. , Merlin Thomas , Gumenscheimer Marina , Sing Andreas , Galanos Chris
Published: June 2007

Endotoxin (LPS) hypersensitivity may be induced in mice by live or killed Gram-negative and Gram-positive bacteria. It is characterized by an overproduction of pro-inflammatory cytokines in response to LPS and by an enhanced susceptibility of the mice to the lethal activity of LPS and TNFα. The induction of LPS hypersensitivity by bacteria is mediated by IFN-γ. Consequently, its development can be inhibited by anti-IFN-γ antibodies and is absent in mice with an impaired IFNγ receptor. In sensitized mice, the enhanced activity of LPS is strictly LBP-dependent. Bacteria-treated, LBP-deficient mice exhibit only a marginally enhanced LPS responsiveness. In such mice, the administration of exogenous LBP at the time of LPS challenge restores the LPS hyper-response. Mice hypersensitive to purified LPS are also hypersensitive to whole Gram-negative bacteria. Such mice, however, are only moderately sensitized to Gram-positive bacteria or to the bacterial superantigen SEB. This sensitization, in contrast to the LPS and Gram-negative bacteria hypersensitivity, is IFN-γ independent. The role of LPS hypersensitivity for the outcome of Gram-negative infection is a dual one. At the early stages of infection, hypersensitivity enables the host to sense minute quantities of bacterial antigens and react against the infection. A failure of this early protective mechanism to eliminate the invading bacteria may have disastrous consequences, since the threshold of development of endotoxin shock is considerably lower in the hypersensitive host.
Surface presentation of LPS is sufficient for initiation of signaling events
Authors: Ingalls Robin R. , Monks Brian G. , Golenbock Douglas T.
Published: June 2007

The activation of phagocytic leukocytes by LPS has been implicated in the pathogenesis of Gram-negative sepsis. The identification of CD14 as a mediator of LPS-inducible signal transduction was a crucial event in understanding the mechanism by which LPS-induced cellular activation occurs. CD14, a 55 kDa glycosyl phosphatidylinositol (GPI)-linked protein present on the surface of phagocytic leukocytes, has been shown to bind LPS and to mediate cellular activation. While it is generally agreed that the interaction between lipid A and CD14 is central to cellular activation by LPS, details of the downstream events which follow remain obscure. The CD11/CD18 integrins are a second group of LPS receptors. The observation that CD11/CD18 could enable LPS responsiveness independent of CD14 when expressed on the surface of CHO cells suggested that the ability of CD14 to participate in the LPS signaling cascade was not unique. We hypothesized that the common function shared by these two LPS receptors was their ability to bind LPS and bring it in close proximity to the plasma membrane, where it could then interact with a signal transducer. To further investigate the role of LPS binding in cellular signaling, we decided to exploit the activity of two other proteins known to bind LPS: lipopolysaccharide binding protein (LBP) and bactericidal/permeability-increasing protein (BPI). We found that when these normally soluble proteins were expressed in a CHO-K1 fibroblast as a GPI-anchored receptor, both could substitute for CD14 in initiating the nuclear translocation of NF-κB in response to LPS stimulation. Pharmacological studies with an LPS antagonist demonstrated that these artificial LPS receptors had characteristics that were qualitatively identical to CD14. These data support the hypothesis that a receptor distinct from CD14 functions as the actual signal transducer, and suggests that surface binding of LPS to the cell membrane is the crucial first step in initiation of the downstream signaling events. We conclude that any LPS binding protein that can focus LPS on the cell surface is sufficient to activate cells.
Antibodies against bacterial membrane proteins
Authors: Hellman Judith , Warren H. Shaw
Published: June 2007

Prior studies have shown that antiserum raised to the Escherichia coli J5 vaccine binds to three bacterial outer membrane proteins (OMPs) that are present on serum-exposed smooth Enterobacteriaceae and in LPS-containing fragments that are released by bacteria incubated in human serum. The present studies were performed to further analyze release of the three OMPs into human serum. Normal human serum was incubated with E. coli O18K+ bacteria and then filtered to remove intact bacteria. Lipopolysaccharide (LPS) was affinity-purified from sterile filtrates using murine monoclonal IgG directed against the O-polysaccharide chain of E. coli O18 LPS (monoclonal anti-O IgG). Components of sterile filtrates were also separated by ultracentrifugation on sequential potassium bromide and cesium chloride gradients. Samples were analyzed by immunoblotting, using anti-J5 IgG and polyclonal anti-O IgG as primary antibodies. Three OMPs, of MWs 5—9, 18, and 35 kDa were detected in samples that were affinity-purified using monoclonal anti-O IgG, indicating that complexes containing at least these three OMPs and LPS (OMP/LPS complexes) are released into human serum. The 18 kDa OMP was detected in low density fractions of the density gradients, indicating that this OMP is also released separately from the OMP/LPS complexes in a form that floats at the same density as serum lipoproteins.