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P388D1 cells, a macrophage (M luminal diameter) cell line, cultured after stimulation by lipopolysaccharide (LPS), produced a glucocorticoid (GC)-dependent hemopoietic activity. The activity, when coexistent with hydrocortisone (HCS), increased the number of granulocyte (Gr) colonies 2-3 times more than that formed by CSF alone. HCS scarcely increased the number of Gr colonies stimulated with CSF. We designated this activity as GC-dependent myelopotentiator (GDMP). GDMP activity was partially purified by FPLC using Superose 12 and Mono Q columns. The molecular weight of GDMP was calculated as about 55 kD. GDMP activity was distinct from CSF and interleukin 1 (IL 1) found in the culture supernatant. GDMP was associated with the activity inducing differentiation of myeloleukemic M1 cells in the presence of HCS. The activity inducing differentiation of M1 cells and GDMP activity were absorbed with M1 cells preincubated with HCS, but not by the cells preincubated with medium alone. Thus, HCS may have induced GDMP receptor on M1 cells and the M1 cells were differentiated by GDMP. These results suggest that both normal Gr precursor cells and myeloid leukemic cells are differentiated by GDMP and HCS by the same mechanism.

A patient whose tuftsin-activities of serum and PMN had been found to be deficient was reported in 1975 by us. After the elapse of 4.5—5.5 years, tuftsin-activity in addition to chemotactic, bactericidal and NBT-reducing activities of the the patient's PMN were investigated. The tuftsin and bactericidal activities against staphylococcus of the patient's PMN fell within the normal range. However, as to the NBT-test and chemotaxis, the patient and her sister who had suffered from similar symptoms in infancy were found to have lower activities. In addition, the serum IgE level of the patient was slightly higher. It is obscure as to why the patient's tuftsin-activity recovered to a normal level, although she has not received γ-globulin therapy for 5.5 years. It is conceivable that the patient suffers from a dysfunction of PMN, but it is impossible at the present time to make a definite diagnosis.

Our recent studies have suggested that bacterial lipopolysaccharide (LPS) attaches to Pronase-sensitive proteins on the murine erythrocyte membrane. In the present study, in order to identify the LPS-binding protein on the murine erythrocyte membrane, a unique method to detect LPS-binding protein on a nitrocellulose membrane was developed. Murine erythrocyte membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred electrophoretically onto a nitrocellulose membrane. The membrane was incubated with LPS of Salmonella minnesota R595 (Re LPS) in phosphate-buffered saline (PBS), after the remaining sites were blocked with gelatin in PBS. We were able to obtain a non-background stain by adding the nonionic detergent octylglucoside at the low concentration of 0.1% to the Re LPS solution. The Re LPS bound to the protein on the nitrocellulose membrane was exposed to affinity purified anti-Re LPS antibodies (IgG) and then to alkaline phosphatase-conjugated anti-IgG. The alkaline phosphatase was detected on the membrane by an enzymatic reaction. This method demonstrated that Re LPS was bound to an erythrocyte protein of 96 kDa. Treatment of erythrocytes with Pronase led to disappearance of the Re LPS-binding protein on the erythrocyte membrane. There was no difference between LPS-responder and LPS-nonresponder murine erythrocyte membranes in amount and molecular weight of the Re LPS-binding protein.

1. Endotoxin injection caused significant increase in procoagulant activity (PCA) of bone marrow cells. Endotoxin also caused an increase in PCA of supernatant (sup) obtained from bone marrow suspension.2. Both activities were destroyed by heating at over 56°C. The activity of mouse brain thromboplastin used as reference preparation was destroyed at 60°C 'but stable at 80°C.3. PCA of bone marrow cells decreased after incubation with Concanavalin A (Con A). The activity recovered by addition of α-methyl-D-glucoside.4. Ploymyxin (PxB) inhibited the PCA of either cells (20%-inhibition) or sup (50%-inhibition).5. Addition of phospholipid (PL) to the cells or sup caused enhancement of PCA.6. Degrees of heat inactivation, Con A- or PxB-inhibition, and PL-enhancement of PCA were higher in sup than those in the bone marrow cells.Procoagulants of the cells and sup were shown to be tissue thromboplastin (Tp) which consists of glycoprotein-phospholipid complex functioning as active sites. Membrane perturbation due to endotoxin would initiate the increase in exposure and/or contents of the active site (PL) on the membrane surface and would participate in the manifestation of Tp activity. As a result of membrane alteration, release of Tp from the cell surface into circulation may also be caused in endotoxemia.

1. Bone marrow cells of mouse given endotoxin (LPS-cells) accerelated the plasma recalcification time (RCT) and partial thromboplastin time (PTT) of mouse or rabbit plasma. PTT was more sensitive than RCT. 2. A significant procoagulant activity of LPS-cells was demonstrated as early as 4hr and the peak was found 8-18hr after endotoxin. This peak was coincided with those of the cytotoxicity and the decrease of nucleated cell counts in marrow. 3. Either the procoagulant activity of LPS-cells of the mouse or the activity of mouse brain thromboplastin (MBT) was more marked in mouse plasma than in rabbit plasma. There would be a species specificity between the two animals, as to coagulation. 4. The two regression lines of the PTT on either the number of LPS-cells or the dose of MBT were nearly parallel. 5. Dose response regression of cytotoxicity or generation of procoagulant activity on concentration of endotoxin injected was significant. Cytotoxicity and procoagulant activity of LPS-cells were demonstrated even by injection of as little as 5μg endotoxin. 6. There existed a definite correlation between the cytotoxicity and procoagulant activity of LPS-cells (n=20, r=-0.8452, p

Biological activities of two groups of synthesized lipid A analogs, the counterpart of biosynthetic precursor, Lehmann's Ia type, 406, and E. coli lipid A type, 506, as well as their non-phosphorylated, and mono-phosphorylated analogs were investigated. The activities employed included four bone marrow cell reactions in mice, mice skin reaction, leukocytes migration in rabbits' cornea, and hemagglutination. Compound 406 and 506 elicited bone marrow reactions in mice and hemagglutination of mouse RBC, although 406 failed to elicit hemorrhage and necrosis also in mice skin. Compound 406 did not elicit corneal reaction in rabbits. The results suggest that for elicitation of this reaction and mice skin reaction, acyloxyacyl structure is required. Cytotoxicity and thromboplastin production of four bone marrow reactions had been reported by us to be endotoxic reactions, since these had not been elicited by peptidoglycan of Lactobacillus and Staphylococcus (1981) and 300 series synthesized analogs (1984) which did not have endotoxic structures. From these results, it seems that these two marrow reactions and hemagglutination require, as does the limulus test, the lipid A part structure as is present in 406.

Effects of β-lactam antibiotics (piperacillin, cefapirin, cefoperazone, cefbuperazone) on human T lymphocyte colony formation were investigated. Peripheral blood mononuclear cells from six normal volunteers were cultured with phytohemagglutinin-P (PHA) and each antibiotic in semi-solid agar for 7 days. These four antibiotics, especially cefbuperazone, increased the number of colonies of mononuclear cells from five volunteers.The effects of these antibiotics on PHA-induced and direct growth of the mononuclear cells were slight and variable among six volunteers. No relationship was observed between the effects on T lymphocyte colony formation and PHA-induced growth by four antibiotics.Cefbuperazone enhanced PHA-induced interleukin 2 production by mononuclear cells from two volunteers, in which enhanced colony formation by the drug was observed.These results suggest that cefbuperazone stimulates the immunological function of lymphocytes.