Time-dependent distinct roles of Toll-like receptor 4 in a house dust mite-induced asthma mouse model.

House dust mites (HDMs) are a common source of allergens that trigger both allergen-specific and innate immune responses in humans. Here, we examined the effect of allergen concentration and the involvement of Toll-like receptor 4 (TLR4) in the process of sensitization to house dust mite allergens in an HDM extract-induced asthma mouse model.　Intranasal administration of HDM extract induced an immunoglobulin E response and eosinophilic inflammation in a dose-dependent manner from 2.5 to 30 µg/dose. In TLR4-knockout mice, the infiltration of eosinophils and neutrophils into the lung was decreased compared with that in wild-type mice in the early phase of inflammation (total of three doses). However, in the late phase of inflammation (total of seven doses), eosinophil infiltration was significantly greater in TLR4-knockout mice than in wild-type mice. This suggests that the roles of TLR4 signaling are different between the early phase and the later phase of HDM allergen-induced inflammation. Thus, innate immune response through TLR4 regulated the response to HDM allergens, and the regulation was altered during the phase of inflammation.

Predominant role of the substituents on the hydroxyl groups of 3-hydroxy fatty acids of non-reducing glucosamine in lipid A for the endotoxic and antagonistic activity.

The synthetic disaccharide precursor of lipid A (406: identical to lipid IVA) was found to reduce its endotoxic activity in mice by an order of 10(5) or more, by replacing the hydroxyl groups with succinyl or acetyl residues. Both the succinylated and acetylated 406 were also found to antagonize the endotoxic mitogenicity on murine splenocytes. Previous studies demonstrated that the succinylated or acetylated synthetic complete lipid A preparations retained the whole endotoxic activity [1994, Infect. Immunol. 62, 1705]. The drastic contrast in all of these results suggests the importance of the substituents on the hydroxyl groups of 3-hydroxy fatty acids of non-reducing glucosamine of lipid A for the activity and for transformation to the antagonistic structure.

Essential regions of the lipopolysaccharide of Pseudomonas aeruginosa responsible for pyrogenicity and activation of the proclotting enzyme of horseshoe crabs. Comparison with antitumor, interferon-inducing and adjuvant activities.

Regions of lipopolysaccharide derived from Pseudomonas aeruginosa essential for pyrogenicity and activation of the proclotting enzyme of the horseshoe crab were examined. Free lipid A with intact fatty acids showed strong pyrogenicity but showed little activation of the proclotting enzyme. Chemical modification of the polysaccharide portion and deacylation of the lipopolysaccharide diminished activation of the proclotting enzyme. The native-protein portion attached to the lipopolysaccharide also inhibited the activation of proclotting enzyme by lipopolysaccharide, but not pyrogenicity. These results indicate that free lipid A is sufficient for pyrogenicity, whereas the complete lipopolysaccharide is the strongest activator of the proclotting enzyme. The lipopolysaccharide of P. aeruginosa, which showed the strongest activation of proclotting enzyme, showed the weakest pyrogenicity of all the lipopolysaccharides tested here. All these results demonstrate that there is not correlation between pyrogenicity and proclotting enzyme activation induced by lipopolysaccharides.

A compound possessing antitumor and interferon-inducing activities derived from the common antigen (OEP) of Pseudomonas aeruginosa.

OEP, a component consisting mainly of protein with small amounts of lipids and sugars, has been isolated from the autolysate of Pseudomonas aeruginosa and purified by physicochemical methods. It possesses remarkable biological properties, showing antitumor and interferon-inducing activities. As regards the antitumor activity of the sample, the ED50 value against ascites sarcoma-180 was 1 microgram/kg mouse/day, and its interferon-inducing activity amounted to 15 units at a concentration of 0.01 microgram/ml. Both activities increased after protease digestion, reaching about ten times those of the sample which had not undergone digestion. The protease-treated OEP contained 17% protein, 14.5% total sugars, 31% lipids, 12.5% hexosamine, 3.8% KDO, and 2.7% phosphorus. Neutral sugars consisted of 12.4% rhamnose, 2.7% mannose, 66.9% glucose, and other unidentified material. Total lipids derived from OEP consisted of 65% loosely-bound and 35% covalently-bound lipids; the former contained C14:10, C16:0, C16:1, C18:0, and C15:1 acids and the latter, beta-OH C10:0, C12:0, alpha-OH C12:0, beta-OH C12:0, C16:0, and C16:1 acids. The antitumor and interferon-inducing activities of OEP remained after the removal of loosely-bound lipids from OEP.

Structure-activity relationship of lipid A: comparison of biological activities of natural and synthetic lipid A's with different fatty acid compositions.

To investigate the structure-activity relationships, various biological activities, including pyrogenicity, lethal toxicity, elicitation of Shwartzman reaction, mitogenicity and tumor necrosis factor (TNF)-inducing activity, were compared among natural and synthetic lipid A's differing in fatty acid composition. In all these tests, natural lipid A's from Escherichia coli and Salmonella minnesota and synthetic LA-15-PP, which carries 3-hydroxy- and 3-acyloxy-tetradecanoyl groups at the 2, 3 and 2', 3' positions, respectively, showed the strongest activities among the tested lipid A's. In contrast, LA-16-PP, in which the amide-bound 3-hydroxytetradecanoic acid at position 2 of LA-15-PP is replaced by 3-hexadecanoyloxytetradecanoic acid, exhibited lower activity than LA-15-PP and natural lipid A's. Although LA-16-PP has been assumed to have a typical Salmonella lipid A structure (and, in fact, it has a structure corresponding to one of the components of Salmonella lipid A), the activity of this synthetic compound was not comparable to that of natural Salmonella lipid A. LA-17-PP, in which tetradecanoic acid is the sole fatty acid component, exhibited relatively strong mitogenicity and TNF-inducing activity, but very low pyrogenicity. The activities of LA-18-PP, which has ester-bound tetradecanoic acid and amide-bound 3-hydroxytetradecanoic acid, were lower than those of LA-17-PP. The results indicate that the differences in fatty acid composition of lipid A's have important influences on the biological activities studied.

Structural requirements of lipid A responsible for the functions: a study with chemically synthesized lipid A and its analogues.

To confirm the revised lipid A structure of Escherichia coli and to establish the structure responsible for its functions, biological activities of the synthetic compounds based on the presented structure of E. coli lipid A were investigated. Compound 506, 2-deoxy-6-O-(2-deoxy-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O [(R)3-tetradecanoyloxytetradecanoyl]-beta-D-glucopyranosyl]-3-O-[(R) -3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-alpha -D-glucopyranose 1,4'-bis(phosphate), exhibited activities identical to those of natural E. coli lipid A in eliciting Shwartzman reaction and tests on lethality, pyrogenicity, interferon- and tumor necrosis factor-inducing activities as well as in B-cell activating activity and Limulus amebocyte lysate gelating activity. With the exception of the Shwartzman reaction the monophosphorylated synthetic compounds at either the 1 or 4' position showed slightly lower activities than the compound with the bisphosphorylated compound (Compound 506). The compound without the phosphate group showed no or only very weak activities. The structural requirements for each activity (i.e. binding position and composition of fatty acids and presence of phosphate groups) are discussed taking into account the results of previous investigations.

Development of a new quantitative method for detection of endotoxin by fluorescence labeling of 3-hydroxy fatty acid.

New quantitative method for the detection of minute amounts of endotoxin has been developed using 3-hydroxytetradecanoic acid as a chemical marker. After converting 3-hydroxytetradecanoic acid to methyl ester, it was coupled with a fluorescent probe, anthracene-9-carboxyl chloride, obtained by chlorization of 9-anthroic acid with oxalyl chloride. The resulting ester was isolated by HPLC on silica column. The purified product, methyl-3-0-(9-carboxy-anthracenyl) tetradecanoate (M/Z 462), was highly responsive to a fluorescence spectrophotometer, showing maximum emission with excitation wavelength at 257 nm and emission wavelength at 458 nm in dichloromethane, the limit of detection being as little as 10 f mol. Using this method it is currently possible to detect Salmonella abortus equi endotoxin in aqueous solution at a level of 100 pg.

Migration of lactic acid, lactide and oligomers from polylactide food-contact materials.

Polylactide (PLA) is used for manufacturing lunch boxes and for packaging fresh food in Japan. PLA can be hydrolysed relatively easily to produce lactic acid, lactide and oligomers. Different types of PLA sheet were subjected to migration tests under various conditions and the lactic acid, lactide and oligomers contents of the migration solutions were determined using liquid chromatography/mass spectrometry (LC/MS). Furthermore, the change in molecular weight was determined by a migration test. PLA was stable at 40 degrees C for 180 days; the total of lactic acid, lactide and oligomers migration levels were 0.28-15.00 microg cm(-2). PLA decomposed clearly at 60 degrees C for only 10 days, the total migration levels were increased to 0.73-2840 microg cm(-2). PLA sheets with a high D-lactic acid content decomposed particularly rapidly. The amounts of alkali decomposition products, based on the conversion of lactide and oligomers to lactic acid by alkali hydrolysis, corresponded with the total migration levels.

Biological properties of the native and synthetic lipid A of Porphyromonas gingivalis lipopolysaccharide.

INTRODUCTION AND METHODS: A pentaacyl and diphosphoryl lipid A molecule found in the lipid A isolated from Porphyromonas gingivalis lipopolysaccharide (LPS) was chemically synthesized, and its characteristics were evaluated to reconfirm its interesting bioactivities including low endotoxicity and activity against LPS-unresponsive C3H/HeJ mouse cells. RESULTS: The synthesized P. gingivalis lipid A (synthetic Pg-LA) exhibited strong activities almost equivalent to those of Escherichia coli-type synthetic lipid A (compound 506) in all assays on LPS-responsive mice, and cells. LPS and native lipid A of P. gingivalis displayed overall endotoxic activities, but its potency was reduced in comparison to the synthetic analogs. In the assays using C3H/HeJ mouse cells, the LPS and native lipid A significantly stimulated splenocytes to cause mitosis, and peritoneal macrophages to induce tumor necrosis factor-alpha and interleukin-6 production. However, synthetic Pg-LA and compound 506 showed no activity on the LPS-unresponsive cells. Inhibition assays using some inhibitors including anti-human Toll-like receptor 2 (TLR2) and TLR4/MD-2 complex monoclonal antibodies showed that the biological activity of synthetic Pg-LA was mediated only through the TLR4 signaling pathway, which might act as a receptor for LPS, whereas TLR2, possibly together with CD14, was associated with the signaling cascade for LPS and native lipid A of P. gingivalis, in addition to the TLR4 pathway. CONCLUSION: These results suggested that the moderated and reduced biological activity of P. gingivalis LPS and native lipid A, including their activity on C3H/HeJ mouse cells via the TLR2-mediated pathway, may be mediated by bioactive contaminants or low acylated molecules present in the native preparations having multiple lipid A moieties.

Migration of formaldehyde and acetaldehyde into mineral water in polyethylene terephthalate (PET) bottles.

The levels of formaldehyde (FA) and acetaldehyde (AA) in polyethylene terephthalate (PET) bottles and in commercial mineral water are reported. All the water samples bottled in Japan contained detectable levels of FA (10.1-27.9 microg l(-1)) and AA (44.3-107.8 microg l(-1)). Of 11 European bottled water samples, eight did not contain either FA or AA, while the remaining three had detectable levels of FA (7.4-13.7 microg l(-1)) and AA (35.9-46.9 microg l(-1)). In three North American bottled water samples, two contained FA (13.6 and 19.5 microg l(-1)) and AA (41.4 and 44.8 microg l(-1)), and one did not. Regardless of the region of origin, all the sterilized water samples contained FA and AA, whilst in contrast, none of the unsterilized water without carbonate contained FA or AA. Of the carbonated water samples, three contained FA and AA, and one did not. When fortified with FA and AA, the commercial water sample without otherwise detectable FA and AA was able to reduce levels, although the commercial water sample containing FA and AA could not. The presence of bacteria in the commercial water samples was investigated using an ATP-based bioluminescent assay and heterotrophic plate count method. The commercial water without FA and AA contained heterotrophic bacteria, whilst the commercial water with FA and AA did not contain detectable bacteria. It is suggested that in this case both FA and AA migrated from PET materials, but were subsequently decomposed by the heterotrophic bacteria in the unsterilized water.

Survey of formaldehyde, acetaldehyde and oligomers in polyethylene terephthalate food-packaging materials.

Polyethylene terephthalate (PET) is frequently used as a packaging material for beverage bottles, fruit and vegetable trays, and egg crates in Japan. Levels of formaldehyde (FA), acetaldehyde (AA) and PET oligomers in various PET food packaging were determined. PET samples were initially dissolved in trifluoroacetic acid with 2,4-dinitrophenylhydrazine to derivatize formaldehyde and acetaldehyde to their dinitrophenylhydrazones. The stable derivatives along with the oligomers were analysed using HPLC with ultraviolet light detection at 360 and 254 nm, respectively. The PET pellets contained 3.5-12.4 microg g-1 AA and 4.0-7.2 mg g-1 oligomers, while FA was below the determination limit. FA, AA and oligomer levels in Japanese bottles were 0.6-3.0 microg g-1, 8.4-25.7 microg g-1 and 5.0-8.7 mg g-1, ND-1.6 microg g-1, 5.0-13.1 microg g-1 and 4.9-8.2 mg g-1 in French and Italian bottles, and ND-1.2 microg g-1, 9.1-18.7 microg g-1 and 5.6-8.0 mg g-1 in US and Canadian bottles, respectively. Compared with European bottles, Japanese bottles contain higher FA and AA levels. In sheet-moulding products, their contents were determined as ND-1.1 microg g-1, 11.5-43.1 microg g-1 and 4.6-9.2 mg g-1, respectively. The results show that sheet-moulding products contain lower FA and higher AA in comparison with bottles. FA and AA are considered to be generated from PET during the heating process for moulding the pellets to bottles or sheet-moulding articles and de-aeration during the sheet-moulding process is effective in removing FA. In contrast, the level of the oligomers remains unchanged during the moulding process from pellets to bottles or sheet products.

Dissociation of endotoxic activities in a chemically synthesized lipid A precursor after acetylation.

In a previous study, a chemically synthesized disaccharide precursor of lipid A (406; identical to lipid IVA) was shown to have dramatically reduced lethality, B-cell mitogenicity, and tumor necrosis factor induction in macrophages when its hydroxyl groups were replaced with either succinyl or acetyl residues (K. Tanamoto, FEBS Lett. 351:325-329, 1994). Succinylated 406 was found to lose Limulus amoebocyte lysate gelation activity completely as a result of the modification (about 10(5)-fold), too, as expected. However, acetyl 406, surprisingly, exhibited activity comparable to that of the original 406. Both succinylated and acetylated 406 lost pyrogenicity completely. These results indicate that one of the typical endotoxic activities was dissociated from the others and that the ability to induce Limulus amoebocyte lysate gelation is not always representative of endotoxin activity.

Free hydroxyl groups are not required for endotoxic activity of lipid A.

Previous studies demonstrated that lipid A from Salmonella abortusequi loses its B-cell mitogenicity for murine spleen cells as a result of the introduction of succinyl residues on hydroxyl groups and that the inactivated lipid A specifically antagonizes the mitogenicity of endotoxin. Hypothesizing that the hydroxyl groups are essential both for its biological activity and for producing nontoxic preparations having antagonistic activity, I tested the role of the hydroxyl groups in its activities by using well-characterized biologically active lipid A preparations synthesized chemically (Escherichia coli and Salmonella types 506 and 516, respectively) by the introduction of either succinyl or acetyl residues at the hydroxyl groups of each of these lipid A preparations. However, the biological activities of neither lipid A preparation were reduced at all after succinylation; in fact, succinylated 516 became much more potent than the original molecule with respect to most activities tested, i.e., lethal toxicity, Limulus gelation activity, and the induction of tumor necrosis factor release. On the other hand, when the hydroxyl groups were replaced with acetyl residues, the lethality and tumor necrosis factor-inducing activity of both lipid A preparations were decreased, whereas their Limulus gelation activity was increased. Mitogenicity was not affected much by the chemical modifications of either lipid A preparation. These findings indicate that although the residues introduced into the free hydroxyl groups of lipid A modulate its activities, the hydroxyl groups in lipid A need not exist in free form.

Chemically detoxified lipid A precursor derivatives antagonize the TNF-alpha-inducing action of LPS in both murine macrophages and a human macrophage cell line.

The chemically synthesized disaccharide precursor of lipid A (406) has been demonstrated to reduce its activity in mice by an order of 10(5) or more by replacing the hydroxyl groups with succinyl or acetyl residues. In the present study, these chemically detoxified synthetic lipid A precursors were found to antagonize the LPS action of inducing TNF-alpha in both murine peritoneal macrophages and in human monocyte-macrophage cell line THP-1. These preparations were found to antagonize the induction of TNF-alpha in murine macrophages by both stimulants of LPS and untreated 406, which acts as an agonist in the murine system, in a dose-dependent manner. Complete inhibition by succinylated 406 occurred at 10-fold excesses of the antagonist. Succinylated precursor also antagonized the TNF-alpha-inducing action of LPS in human cell line THP-1. The precursor itself also exhibited antagonism in this assay. The activity of the succinylated 406, however, was much more potent than that of the unmodified precursor. These derivatives do not inhibit either the Limulus gelation activity of LPS, or the induction of TNF-alpha in macrophages by zymosan, indicating that the inhibition is not caused by molecular interaction and that the inhibition is specific to LPS action. These findings suggest common features in murine macrophage and human monocytic cell receptors. They also suggest the importance of the substituents on the hydroxyl groups of 3-hydroxy fatty acids of the nonreducing glucosamine of lipid A for the activity and for transformation to the antagonistic structure.

Induction of lethal shock and tolerance by Porphyromonas gingivalis lipopolysaccharide in D-galactosamine-sensitized C3H/HeJ mice.

Lipopolysaccharide (LPS) obtained from Porphyromonas gingivalis was found to exhibit marked lethal toxicity in galactosamine-sensitized C3H/HeJ mice. Although no lethality was observed in mice intraperitoneally challenged with 1 mg of P. gingivalis LPS without galactosamine, when they were sensitized with 30 mg of galactosamine, challenge with 1 and 10 micrograms of LPS resulted in 67 and 100% lethality, respectively. The lethal dose of LPS was almost the same in LPS-responsive C57BL/6 mice and non-LPS-responsive C3H/HeJ mice. Furthermore, when 1 microgram of P. gingivalis LPS was administered to each mouse 90 min before the challenge with the same LPS with galactosamine, tolerance to the lethal action of LPS was induced, and the mice were completely protected from death, even at a dose 100-fold greater than the lethal dose of LPS. Neither a lethal effect nor induction of tolerance to the lethality of P. gingivalis LPS was exhibited by Salmonella LPS in galactosamine-sensitized C3H/HeJ mice. A protein-LPS complex derived from Pseudomonas aeruginosa, which exhibited strong lethality and induced tolerance to a subsequent challenge with a lethal dose of LPS in galactosamine-sensitized LPS-responsive mice, did not exhibit lethal toxicity in galactosamine-sensitized C3H/HeJ mice and failed to induce tolerance in these mice to the lethality of P. gingivalis LPS. These results indicate that P. gingivalis LPS plays the central role in the activation of non-LPS-responsive C3H/HeJ mice.

A quantitative microanalysis of bacterial endotoxin using [3H]-labeled L-glycero-D-mannoheptitol as a marker.

Quantitative microanalysis of bacterial endotoxin was performed using [3H]-labeled L-glycero-D-mannoheptitol (LD-Heptitol) as a marker. Several different amounts of authentic L-glycero-D-mannoheptose (LD-Heptose) were reduced with 20 micrograms of cold NaBH4 containing 2 micrograms of NaB3H4 (40 Ci/mmol) in 20 microliters of 1 mM NaOH at 4 C for 48 hr. The product, [1-3H]-labeled LD-Heptitol, has high specific activity, and was purified by HPLC and detected using a liquid-scintillation counter. As little as 50 pg of LD-Heptose was detectable, and the radioactivity increased dose-dependently in the 100 pg to 80 ng range tested. More than 2 ng of Salmonella abortus equi endotoxin could be accurately determined by this method. It is possible to detect 50 pg of endotoxin by this method, if 100% hot material (NaB3H4) is used for [3H]-labeling.