A single intradermal injection of bacterial lipopolysaccharide (LPS) caused hemorrhagic necrosis in mouse skin (skin reaction). A tissue factor, membrane glycoprotein, is known to activate the extrinsic blood coagulation cascade. In this study, an attempt was made to compare the structural requirements of LPS to induce skin reaction in vivo, and to produce tissue factor by peritoneal macrophages in vitro. The skin reaction was induced by injection of a smooth type (S)-LPS, rough type (R: Ra-Re)-LPS, and lipid A derived from Salmonella typhimurium, Salmonella minnesota and Escherichia coli in a dose-dependent manner, but not by the polysaccha-ride portion of S-LPS. Re-LPS induced the strongest dermal inflammation (activation) in ddY mice. This was followed by Rc-LPS, lipid A (the activity of Rc-LPS was similar to lipid A), Ra-LPS and S-LPS in the order mentioned. In the C3H/HeN mice, Re-LPS and lipid A induced the skin reaction to almost the same levels as in the ddY mice. The activity of synthetic E.coκtype lipid A (#506) which has a double acyl structure was similar to or sHghtly weaker than that by natural lipid A, whereas the activity of a synthetic counterpart of a Iipid A precursor (#406) was considerably weak. In the C3H/HeJ mice, Re-LPS and lipid A did not induce any hemorrhagic response. The macrophage suspension of C3H/HeN mice was stimulated with Re-LPS at 37℃ for 6 hours, and the sonic lysate of the cells was used to estimate the tissue factor activity by the clotting method using a fibrometer. The tissue factor was produced by Re-LPS in ddY and C3H/HeN mouse macrophages. The activity of #406 was the same as that of #506. C3H/HeJ macrophages did not respond to even a high dose of Re-LPS. These results indicate that the lipid A portion of LPS is very important to cause a reaction both in viro. As to the lipid A structure, the double acyl structure is not required for the production of tissue factor by macrophages, but is required for the induction of skin reaction.

抄録全体を表示

はグラム陰性細菌の外膜の主要な構成成分であり、広範な免疫活性化能を示し、炎症性反応に関与する。この10数年間に分子に対する化学的、生物学的な新しい知見が付け加えられた。の部分構成成分についての知識、リピドA類縁体の合成、さらにアンタゴニストの開発は、によって引き起こされる細胞活性化過程に関して莫大な情報を与えた。さらに、と可溶性もしくは細胞性受容体との相互作用、によって活性化されるシグナル伝達経路、そしてに対する細胞の応答に関わる知識に多大な進歩があった。しかしながら、宿主における活性発現の詳細な機構は極めて複雑であることが分かり、まだ完全に理解されていない。それでも、LPSによる単球/マクロファージの活性化の理解に対する最新の進歩は、グラム陰性菌の感染との戦いにおいて、新しくそして願わくばより良い戦略に基づく将来の展開の基礎となるであろう。

抄録全体を表示

歯周疾患における細菌性 (bacterial endotoxin) の役割とその作用機序の一端を解明することを目的として, 先ず全唾液中における量を測定し, つづいて Fusobacterium nucleatum からのの分離と精製およびその生物学的活性, Hela 細胞に対する細胞毒性, 好中球走化活性, 歯周疾患患者における抗抗体価, また頻回注射によるラット歯周組織の病理組織学的変化について検索を実施した。その結果, 全唾液中に含有される量は歯垢蓄積量や歯肉炎の程度と関連のあることが示された。歯肉溝および歯肉盲嚢内に常在する Fusobacterium nucleatum からの分離標品は強い生物学的活性を示し, 好中球に対しても走化活性を示すが, HeLa 細胞に対する毒性は, そのLD₅₀濃度がおよそ500μg/mlと比較的低いことが示された。また本抗体価をELISA法により測定した結果, 特異抗体活性はIgM画分に存在し, しかも歯周疾患の進展程度とこの活性の強さには関連が認められた。さらに標品を非免疫ラットに頻回注射した際には歯肉の高度の炎症, osteoprogenitor cell の変性, 壊死ののちに破骨細胞による骨吸収を認めた。一方, 標品を頻回注射した場合には, 歯肉に Arthus 型の過敏反応を示す組織像がみられ, 非免疫ラットと比較して早期に高度の骨吸収像が認められた。

抄録全体を表示

Acid soluble protein (ASP) isolated from bone marrow or spleen cells prolonged the clotting times of human or mouse plasma in proportion to the amount added. The anticoagulant property of ASP was demonstrated in both APTT and PT, and the property was thought to be due to the inhibition of Xa generation or to the inhibition of prothrombinase (Xa·V·phospholipid·Ca complex) formation.
ASP had an anticoagulant activity, as heparin did, however, ASP and heparin neutralized the activity with each other.
In a modified APTT system, the anticoagulant effect of 1 unit of heparin was neutralized by 300-900μg ASP.
ASP also neutralized the antithrombin activity of heparin in the thrombinplasma system.
On the contrary, ASP significantly enhanced plasma or fibrinogen clotting caused by small amounts of thrombin (0.1unit/ml).
ASP could precipitate soluble fibrin monomer complexes (SFMC), as protamine sulfate did.
Endotoxin has been shown to cause strong cytotoxic and necrotic damages of bone marrow cells, therf ore, these findings suggest that DIC manifestation in endotoxemia and also in other inflammatory diseases accompanied by destruction of tissues or cells are controlled by the ASP.

抄録全体を表示

1. Procoagulant activity (P-activity) in the intact cells of bone marrow or spleen cells was higher than that in sonic lysate of these cells.
2. Sonic lysate of bone marrow or spleen cells had the anticoagulant activity (A-activity), which was stable against heating at 70°C for 30-60min.
3. A-activity was mainly found in the 40, 000xg supernatant of sonic lysate of the spleen cells.
4. With heat-treated 40, 000xg supernatant prepared from post-LPS-cells or normal-cells, A-activity was also detected in the unactivated partial thromboplastin time (PTT), activated partial thromboplastin time (APTT), prothrombin time (PT) and the thrombin-fibrinogen clotting time (TT).
5. A-activity (prolongation of PTT or TT) of bone marrow or spleen cells was also enhanced by endotoxin injection compared to saline injection.
6. With acid soluble protein (ASP, cationic protein) from bone marrow or spleen cells, A-activity was also detected in the PTT, APTT, and PT, however, was not in the TT.
7. ASP was also stable against heating at 80°C for 30min.
8. In ASP, endotoxin-detoxifying activity was observed.
ASP appears to exist in cytoplasma and to inhibit clotting by blocking the activation of factor X or the formation of plasma thromboplastin. These findings also suggest that the enhancements of P-activity and A-activity of bone marrow or spleen cells in endotoxemia would also regulate the manifestation of DIC.

抄録全体を表示

A single dose of endotoxin caused an increase in cytotoxic damages of bone marrow cells, an increase in procoagulant activity (p-activity) of the cells, decrease in nucleated cell counts in marrow due to the migration into circulation, and an increase in serum FDP levels. The increase in cytotoxicity of bone marrow cells correlated well with the increase in p-activity of the cells. Most of the p-activity was found in the membrane fragments in the cells and p-activity was thought to be tissue thromboplastin.
Endotoxin induced an increase in p-activity in monocytes (6.9-fold) and granulocytes (2.9-fold).
The ddY mice were more responsive than C3H/He mice as to increase in cytotoxicity and also a decrease in nucleated cell counts in marrow. In contrast, none of these reactions were induced in C3H/HeJ mice. Endotoxin caused strain-dependent production of p-activity in mice. ddY mice and C3H/He mice responded to endotoxin with 7.0 and 2.9-fold increase of p-activity in whole nucleated bone marrow cells. In contrast, C3H/HeJ mice did not respond to endotoxin. Endotoxin also caused strain-dependent increase in serum FDP levels. ddY mice were more responsive than C3H/He mice, however, no increase in FDP was found in C3H/HeJ mice even by injection of 500μg of endotoxin. In C3H/HeJ mice, none of three endotoxin preparations which contained different amounts of protein could increase in serum FDP levels.
In endotoxemia, bone marrow cells migrate to circulation, therefore, the cells with an enhanced p-activity would participate in the manifestation of DIC. This possibility is further supported from the findings that endotoxin induced strain-dependent responses as to all of the 4 parameters and that these parameters correlated well with each other.

抄録全体を表示