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PubMed1978

[Immunelectrophoretical evidence on inactivation of neuraminidase by limulus polyphemus-amebocyte-lysate (author's transl)].

Neuraminidase in concentrations up to 0.5 nu/0.1 ml (0.05 nu/0.1 ml) added to Limulus amebocyte lysate (LAL) in equal volumes (each 0.1 ml) releases a gelation reaction. Human AB-serum - subjected to immunelectrophoresis - is used as an indicator system to show that neuraminidase is bond to the LAL (incorporated in the gel). If there is a free amount of neuraminidase immunelectrophoresis of the serum glycoproteins indicates the typical changes previously described by Müller (6, 7). LAL does not cause any immunelectrophoretically detectable changes on serum glycoproteins by itself in the temperature range from +4 degrees C to 37 degrees C. It must be assumed that neuraminidase in a certain amount is releasing the coagulation system.

Enzyme Inhibitors↗
PubMed1994

An amino-terminal fragment of human lipopolysaccharide-binding protein retains lipid A binding but not CD14-stimulatory activity.

LPS-binding protein (LBP) mediates the pro-inflammatory effects of bacterial LPS by enhancing LPS-induced cytokine production by monocytic cells. LBP binds specifically to LPS to generate a complex that interacts with the CD14 receptor on the surface of responsive cells. To identify the biologically active regions of the protein responsible for mediating these activities, we cloned and expressed human rLBP (456 amino acids) as well as a truncated form encoding amino acids 1-197 (rLBP25). Both forms of LBP bound to LPS with the same affinity, and similarly inhibited LPS activity in the Limulus amebocyte lysate assay. These results demonstrate that the LPS-binding domain of LBP resides entirely within the N-terminal 197 amino acids of the protein. rLBP and rLBP25 were compared for their ability to mediate CD14-dependent LPS effects on cells. rLBP was effective in mediating uptake of LPS and stimulation of TNF production by human monocytic THP-1 cells, whereas rLBP25 had no significant activity in these assays. Similarly, rLBP was able to mediate LPS-induced TNF production by human PBMC whereas rLBP25 was essentially inactive. These results suggest that the structural features of LBP required for mediating LPS effects via CD14 are probably located in the C-terminal region of the protein. Thus, the LPS-binding activity of LBP can be separated from the CD14-stimulatory activity, suggesting these activities are mediated by structural elements residing in different regions of the protein.

Acute-Phase Proteins↗
PubMed1995

Inducible nitric oxide synthase in cattle. Differential cytokine regulation of nitric oxide synthase in bovine and murine macrophages.

We assessed bovine bone marrow-derived macrophages and monocyte-derived macrophages for expression of inducible nitric oxide synthase (iNOS) activity. Both cell types expressed iNOS activity upon stimulation with Salmonella dublin (S. dublin) or with LPS. A 372-bp fragment of the bovine iNOS mRNA could be amplified by reverse transcription-PCR from mRNA of stimulated macrophages. Cloning and sequencing of the fragment revealed a high degree of homology to human hepatocyte, rat vascular smooth muscle cell, and mouse macrophage iNOS both at the nucleotide (87 to 92%) and amino acid levels (94 to 97%). iNOS mRNA was expressed maximally 6 h after stimulation with S. dublin, whereas maximal nitrite accumulation in supernatants was measured at 24 to 48 h. Significant differences with regard to cytokine regulation of iNOS were observed between murine and bovine macrophages cultured under identical conditions. The most striking difference was the inability of homologous IFN-gamma to induce iNOS both at the level of nitrite production and of mRNA expression in bovine macrophages. TNF-alpha, IL-2, and IL-1 alone or together with IFN-gamma neither induced iNOS nor primed bovine macrophages for enhanced iNOS expression or activity upon stimulation with S. dublin. RhuIL-4, but not rhuTGF-beta, down-regulated S. dublin-induced iNOS activity and mRNA expression in bovine macrophages. Thus, an enzyme with a high degree of homology to rodent iNOS is inducible by stimulation of bovine macrophages with bacteria, but induction and regulation by cytokines occur under more restricted conditions than in rodent macrophages.

Amino Acid Oxidoreductases↗
PubMed1995

[Component analysis of variability as a method of differentiating simple effect from hazardous effect in toxicological experiments].

The article forms the main requirements for every hazard criterion (valid, definite, statistically correct, differential, invariable, sensitive, integral). The suggested hazard criterion is based on additive components of general dispersion. Those components reflect environmental influence (environmental effect) and influence of substance (effect of substance), then the components are compared according to Fisher criterion (F). The criterion works in two-factor dispersion analysis.

Analysis of Variance↗
PubMed1995

In vitro studies on the immunomodulating effects of polypodium leucotomos extract on human leukocyte fractions.

The in vitro effect of Anapsos, a water based extract of the naturally occurring fern Polypodium leucotomos (calagualine), on human leukocyte fractions was investigated. Calagualine inhibited interleukin-2 secretion and concanavalin A (Con A) stimulated proliferation of T-lymphocytes in a concentration dependent manner. In contrast, a greatly enhanced secretion of IL-1 alpha, IL-1 beta and tumor necrosis factor alpha was induced suggesting a stimulation of monocytes and dendritic cells also present in this system. Endotoxin induced stimulation was excluded. Also in the absence of Con A, calagualine stimulated cytokine production. The presented data show for the first time that calagualine exerts an immunomodulating effect on leukocyte fractions, paving the way for further detailed studies related to possible clinical efficacy.

Adjuvants, Immunologic↗
PubMed1995

Inhibition of TNF-triggering activity of lipopolysaccharide by a proteinaceous factor from normal mouse liver extract.

Recent studies have revealed that animals have evolved a wide range of protective systems against the deleterious effects of LPS, but the molecular mechanisms in the barrier functions of liver against enterobacterial LPS, particularly in mammals, are poorly understood. In this study, we extracted a soluble fraction from the liver of normal mice (normal liver extract, NLE) and examined its effect on biologic activities of LPS. Preincubation of NLE and LPS suppressed serum-dependent TNF induction (TNF-triggering activity) of LPS; the effect was dose-dependent and overcome by increasing LPS concentration, but treatment of macrophages with NLE showed no effect. Separation by ultrafiltration and protease sensitivity demonstrated that the factor(s) in NLE was a protein(s). We tentatively called this liver LPS-inactivating factor (LLIF). LPS inactivation by LLIF was temperature-dependent and required the coexistence of divalent cations. LLIF also suppressed a synthetic lipid A analogue, ONO-4007. Pretreatment of LPS with serum rendered LPS refractory to the action of LLIF. However, LLIF was unable to inhibit limulus amebocyte lysate activation by LPS. Direct interaction of LLIF and lipid A was evident by the method of [1-14C]ONO-4007 binding to solid-phase LLIF, but treatment of [1-14C]ONO-4007 with LLIF generated no degradative products. These results suggest that LLIF probably interacts with the lipid A portion of LPS and interferes with the association of LPS and LBP (LPS-binding protein) in serum, and that LLIF may be one of the protective molecules in liver against the gastrointestine-derived LPS.

Acute-Phase Proteins↗
PubMed1994

Anti-lipid A monoclonal antibodies and anti-LPS antiserum effects on limulus activity of LPS.

Anti-lipid A monoclonal antibodies (A78S1 and A523) and anti-LPS antiserum can decrease the mortality due to endotoxic shock in the newborn rat. However, in vitro LPS detoxification of anti-lipid A monoclonal antibodies is not known. Thus, we studied in vivo effects of A78S1 (IgG), A523 (IgM), and anti-LPS antiserum on Limulus activity. Anti-LPS antiserum decreased Limulus activity of S. enteritidis, E. coli and S. typhosa LPS. However, neither A78S1 nor A523 decreased the Limulus activity of E. coli and S. typhosa LPS. A78S1 or A523 incubation with S. enteritidis LPS at different doses (0.1 to 100 ng/ml) did not alter the Limulus activity. Perchloric acid treatment after LPS incubation with A78S1 or A523 significantly decreased Limulus activity. Therefore, antilipid A monoclonal antibodies (A78S1 and A523) can bind LPS, but do not decrease Limulus activity.

Animals↗
PubMed1995

Antibacterial proteins of granulocytes differ in interaction with endotoxin. Comparison of bactericidal/permeability-increasing protein, p15s, and defensins.

Bactericidal/permeability-increasing protein (BPI), antibacterial 15-kDa protein isoforms (p15s), and defensins (neutrophil peptides or NPs) are granule-associated antibacterial proteins of polymorphonuclear leukocytes (PMN) that have both direct and synergistic growth inhibitory activity against Gram-negative bacteria. In this study, we have compared in vitro the abilities of these antibacterial proteins, alone and in combination, to inhibit the endotoxic activity of isolated LPS and whole bacteria. All three proteins blocked endotoxin activity in: 1) the Limulus amoebocyte lysate assay, 2) priming of PMN for enhanced arachidonate release, and 3) stimulating leukocyte oxidase activity in 1% blood. However, the proteins differ markedly in both relative potency (BPI >> p15s = NP1) in the presence of the plasma LPS-binding protein and in the range of LPS chemotypes that can be inhibited. BPI potently neutralizes LPS of any chemotype, but p15s and defensins are less active against long-chain (S-type) LPS. In whole blood ex vivo, the p15s and NP1 are approximately 1000-fold less potent than BPI, but at subinhibitory doses act in synergy with BPI to inhibit the TNF-inducing activity of a serum-resistant encapsulated strain of Escherichia coli (K1/r). The anti-endotoxic effects of p15 and NP1 against E. coli K1/r in whole blood appear secondary to growth arrest, because, in marked contrast to BPI, they are not evident against nonviable bacteria (pretreated with antibiotic) nor isolated LPS. Thus, BPI stands out for its ability to inhibit isolated or bacterial LPS under physiologic conditions. However, p15s and defensins may also contribute to suppression of endotoxic signaling by Gram-negative bacteria via synergistic (with BPI) growth inhibition upon extracellular release of these proteins from PMN during inflammation.

Animals↗
PubMed1994

Hemoglobin: a newly recognized binding protein for bacterial endotoxins (LPS).

Administration of purified hemoglobin (Hb) as a cell-free resuscitation fluid is associated with multiple organ toxicities. Many of these toxicities are characteristic of the pathophysiological effects of bacterial endotoxins (lipopolysaccharide, LPS). To better understand the potential role of LPS in the observed in vivo toxicities of Hb, we examined mixtures of Hb and LPS for evidence of LPS-Hb complex formation. LPS-Hb complexes were demonstrated by three techniques: ultrafiltration through 300 kDa cut-off membranes, which distinguished LPS in complexes (87-89% < 300 kDa) from LPS alone (90% > 300 kDa); density centrifugation through sucrose, which distinguished denser LPS alone from LPS-Hb complexes; and precipitation by 67% ethanol, which demonstrated 2-3 fold increased precipitability of Hb in complexes compared to Hb alone. Interaction of LPS with Hb was also associated with markedly increased biological activity of LPS, as manifested by enhancement of LPS activation of Limulus amebocyte lysate (LAL), increased release of human mononuclear cell tissue factor, and enhanced production of human endothelial cell tissue factor. These results demonstrated that hemoglobin can serve as an endotoxin binding protein, and that this interaction results in the alteration of several of the physical characteristics of LPS and enhancement of the biological activities of LPS. These findings suggest that a mechanism for the toxicity of infused Hb in vivo may involve potentiation of the biological effects of LPS. In addition, these observations suggest a mechanism by which LPS-related morbidity during sepsis could be enhanced by erythrocyte hemolysis.

Acute-Phase Proteins↗
PubMed1994

Endotoxin and arachidonic acid metabolites in portal, hepatic and arterial blood of cattle with acute ruminal acidosis.

Ruminal acidosis was induced experimentally with 70 g barley/kg body weight in 2 rumen fistulated cows with chronic indwelling catheters in the portal vein, in a hepatic vein and the carotid artery. The cows were followed for 24 and 20h after the overfeeding and evaluated clinically and by clinical chemistry. The 2 cows exerted different responses to the treatment. Both cows showed signs of severe ruminal acidosis. Both cows had endotoxin in portal and hepatic vein blood, but only 1 of the cows convincingly developed a systemic endotoxaemia. A pre-hepatic release of the stable prostacyclin and thromboxane metabolites, 6-ketoprostaglandin F1 alpha and thromboxane B2 was demonstrated in this cow. The results of the present study show that endotoxin and arachidonic acid metabolites of pre-hepatic origin may be factors involved in the pathogenesis of ruminal acidosis, and that investigation of the factors affecting translocation of ruminal endotoxin and subsequent clearing in the liver, will be of importance.

6-Ketoprostaglandin F1 alpha↗
PubMed1994

Lipopolysaccharides from various Capnocytophaga strains possess potent Limulus amoebocyte lysate clotting activity.

Lipopolysaccharides (LPSs) were extracted by the hot phenol-water method from three ATCC strains (C. sputigena ATCC 33612, C. ochracea ATCC 33596, and C. gingivalis ATCC 33624) and three clinical isolates (C. sputigena TE-1, C. ochracea ONO-26, and C. gingivalis M-12). Endospecy was used to determine the Limulus amoebocyte lysate clotting activities. The activities of LPSs from Capnocytophaga strains were stronger than those of Escherichia coli, with the exception of the LPS from C. gingivalis M-12. Except for the LPS from C. sputigena TE-1, the SDS-PAGE analyses of these preparations showed slow-migrating and repeating ladder bands similar to the LPSs of E. coli and Salmonella typhimurium (wild type) and included both the core-lipid A region and various lengths of O-antigen. The LPS from C. sputigena TE-1 possessed fast-migrating bands and did not have an O-side chain.

Capnocytophaga↗
PubMed1994

Hemoglobin, a newly recognized lipopolysaccharide (LPS)-binding protein that enhances LPS biological activity.

Cell-free hemoglobin (Hb) is a purified preparation of human hemoglobin that is being developed as a resuscitation fluid. In vivo administration of hemoglobin has resulted in significant toxicity, due in part to contamination with bacterial endotoxin (lipopolysaccharide (LPS)). To better understand this toxicity, we have studied the interaction between Hb and LPS. Mixtures of each of three different Hb preparations (cross-linked alpha alpha Hb, cross-linked carbon monoxy-alpha alpha HbCO, and non-cross-linked (native) HbAo) and LPS (Escherichia coli O26:B6 or Proteus mirabilis S1959) were examined by several independent methods for evidence of Hb.LPS complex formation. Binding assays in microtiter plates demonstrated saturable binding of LPS to immobilized Hb, with a kD of 3.1 x 10(-8) M. Binding of LPS to Hb also was demonstrated wiht a radiolabeled LPS photoaffinity probe. Ultrafiltration of Hb/LPS mixtures by 300- and 100-kDa cut-off membranes showed that the majority of LPS in these mixtures (87-97 and 64-72%, respectively) was detected in the filtrates, in contrast to the lack of filterability of LPS in the absence of Hb. Density centrifugation demonstrated that LPS co-migrated with each of the three Hbs, whereas unbound LPS had a distinctly greater sedimentation velocity than Hb or Hb.LPS complexes. Nondenaturing polyacrylamide gel electrophoresis demonstrated that in the presence of Hb, LPS migrated into the gel and co-electrophoresed with Hb, whereas LPS alone did not appreciably enter the gel. Finally, precipitation by ethanol of each of the three Hb preparations was increased in the presence of LPS compared with precipitation in the absence of LPS. Interaction of LPS with each of the three Hb preparations was also associated with altered biological activity of LPS, as shown by enhancement of LPS activation of Limulus amebocyte lysate. Therefore, our data provide several lines of independent evidence for Hb-LPS complex formation and indicated that LPS exhibited altered physical characteristics and enhanced biological activity in the presence of Hb.

Acute-Phase Proteins↗