Influence of the immunomodulating compound BM 12.531 (azimexone) on radioresistance and granulopoietic colony forming units in mice.

BM 12.531 (azimexone) increases the survival of mice exposed to 650 rad. Levamisole, isoprenosine or aristolochia acid have no effects in this system. After administration of 25 mg/kg azimexone to C57 Bl/6-mice a statistically significant increase in CFUc was found. Maybe this increase in CFUc can explain the radioprotective effect of azimexone.

Chemical characterization of macrophage cytotoxicity factor, macrophage migration inhibitory factor, T-helper cell-replacing factor and colony-stimulating factor from culture supernatants of concanavalin A-stimulated murine spleen cells.

Supernatants from Concanavalin A-stimulated murine spleen cells were subjected to hydrophobic interaction chromatography on phenyl-Sepharose. Macrophage cytotoxicity factor (MCF), macrophage migration inhibitory factor (MIF), T-helper cell-replacing factor (TRF) and colony-stimulating factor (CSF) were bound at high ionic strength and were released stepwise at low ionic strength. CSF thus could be separated from MCF, MIF and TRF and the bulk of other proteins. Chromatograhy of pools containing MCF, MIF and TRF on Sephadex did not lead to a separation of the three activities which were all found in a molecular weight range of 25.000-55.000. Isoelectric focusing of these pools in pH range from 4 to 9 gave two peaks for MCF in a single sharp peak at pH 5.3. The results demonstrate that the four biological activities can be distinguished on a chemical basis and are accessible for purification and chemical characterization.

Humoral factors modulating growth of granulocyte macorphage progenitor cells.

The kinetic of production of colony-stimulating activity (CSA) inducing mouse and human colony-forming cells (CFU-C) was tested in different human leukocyte culture systems. Stimulated and unstimulated cultures of spleen single cell suspensions, peripheral mononuclear leukocytes and acute monocytic leukemia (AMoL) cells were investigated. With the exception of the AMoL cells, stimulated cultures always revealed higher CSA levels than unstimulated controls. The spleen cell cultures exhibited the highest overall activity showing three molecular species of 70,000, 35,000 and 10,000 daltons activating human CFU-C to form colonies in the agar culture system. Furthermore it could be demonstrated that colony formation could be inhibited by low molecular weight fibrinogen degradation products obtained by digestion of fibrinogen with granulocyte-derived elastase.

The influence of a new antitumor agent on hemopoietic colony forming cells.

The cytostatic and immunsuppressive agent N'-methyl-N'-beta-chloroethylbenzaldehyde hydrazone (B1) in in-vitro experiments has a stimulating effect on colony-forming culture (CFUc) of bone marrow from C57BL mice. This unusual behaviour, which is in contrast to other cytostatics, could also be observed in vitro with CFUc obtained from mice treated with therapeutic doses of B1 for 2 weeks. This stimulation is not a particular effect of B1 alone but seems to depend on a synergistic effect of the combination of B1 and the colony-stimulating activity (CSA) present in the serum from endotoxin-treated mice (MP) in the testing system. The results suggest that the described effect of B1 is due to an interference at the cell membrane of CFUc or their precursor cells.

Colony stimulating factor (CSF) in human mixed lymphocyte cultures: effect on human and mouse progenitor cells.

Supernatants from human two-way MLC, poor and rich in monocytes, were tested for their ability to induce colony growth of human and mouse progenitor cells in semi solid agar. Colony stimulating factor (CSF), with activity in both systems, indicated that allogenic lymphocytes require monocytes to produce CSF. Whereas human marrow showed an early kinetics of production, the liberation of CSF active on mouse marrow cells exhibited a delayed kinetics. These results, combined with other evidence, suggest that human lymphocytes produce two different factors.

Preparation of colony stimulating activity from large batches of human urine and production of antisera against it.

In vitro induction of myelopoetic colonies from mouse bone marrow has been used for measurement of leucopoetic colony stimulating activity (CSA) isolated from large batches of human urine. After high flow dialysis in artificial kidneys and immediate adsorption to DEAE-Cellulose, followed by purification on Con A-Sepharose, treatment with insoluble Papain and gelfiltration on Sephadex G 100, enrichment of CSA was about 6,000-fold. An important step of the enrichment procedure was the separation from a CSA-inhibiting protein, probably combining with CSA. Specific activity was further increased by preparative polyacrylamide gel electrophoresis to 5.3 X 10(6) units per mg protein. The total enrichment exceeded 25,000-fold. The final purification product consisted of a group of closely related proteins with high specific activity. Antisera raised with one of the electrophoretic fractions suppressed bioactivity in each of the different purification steps including the final CSA fractions differing in electrophoretic mobility. The antisera furthermore inhibited CSA in human lung and monocyte conditioned media but had only very little effect on partially purified CSA from stimulated human lymphocytes as well as CSA derived from mouse lung conditioned medium.