Serum cholesterol-lowering activity of human monocytic colony-stimulating factor.
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Biomedical subjects
Publications and source records attributed to K Motoyoshi.
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Human monocytic colony-stimulating factor (hM-CSF) is a glycoprotein which stimulates monocyte production in the bone marrow. It enhances CSF (such as G- and GM-CSF) production of monocytes and megakaryocyte-potentiating activity (Meg-POT). It also enhances tumor-killing activity of monocytes against several leukemic cell lines such as K562, U937, HL60 and Daudi. In the clinical studies, it was shown that hM-CSF infusions accelerated the recovery from neutropenia as well as thrombopenia after anticancer chemotherapy against hematological, gynecologic and urogenital malignancies. Human M-CSF infusions were tolerable without any serious side effects. It is reported that infusions of G-CSF and GM-CSF cause the increment of leukemic cell counts in some cases, but hM-CSF infusions did not increase leukemic cell counts. These results indicate that hM-CSF may be potentially useful for the treatment of myelosuppression induced by cancer chemotherapy in cancer patients.
Colony-stimulating factor from human urine (CSF-HU) has been purified to a homogeneous protein, and its complementary DNA (cDNA) has been cloned. Recombinant CSF-HU was prepared from a serum-free medium conditioned by Chinese hamster ovary cells transfected with the cDNA and purified by the same method as that for the native protein. Purified CSF-HU stimulated human bone marrow cells to form macrophage colonies. It also stimulated human mature monocytes prepared from peripheral blood of healthy volunteers to produce human active colony-stimulating activity that stimulates human bone marrow cells to form granulocyte and macrophage colonies. This activity was partially neutralized by the addition of both polyclonal antibodies against human granulocyte colony-stimulating factor and against human granulocyte-macrophage CSF, respectively. The stimulation of monocytes by CSF-HU was not inhibited by the addition of polymyxin-B, which is known as a potent inhibitor of endotoxin. On the other hand, CSF-HU did not stimulate monocyte production of interleukin-1 and interferon. These results indicate that recombinant and native CSF-HU stimulates immature cells as well as mature cells in the human monocyte lineage.
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To determine what kinds of CSF modulate human basophil function, recombinant or purified hemopoietic growth factors were tested for the effect on histamine release from basophils. Both granulocyte (G)-macrophage (M) CSF and IL-3 markedly enhanced histamine release upon stimulation with anti-IgE in a dose-dependent manner (maximal enhancement 25.5% by GM-CSF and 30.8% by IL-3 as expressed as percent increase against total cellular histamine content), whereas G-CSF, M-CSF, and IL-4 had no effect. Enhancing action of these factors was still observed in the highly enriched basophil population, suggesting that this action was not via contaminating cells. Enhancement of histamine release by both factors was not necessarily IgE mediated, because they also amplified histamine release upon stimulation with FMLP and ionophore A23187. The enhancement by both factors was temperature dependent, and took place rapidly and reached plateau levels in 15 min. GM-CSF and IL-3 achieved the similar plateau level of augmentation and no additive effects were observed between them. This finding suggests that they enhance histamine release by sharing the same pathway in the release reaction.
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An enzyme-linked immunosorbent assay (ELISA) system for the quantitation of human monocytic colony-stimulating factor (hM-CSF) was established, which was based on the "dual antibody immunometric sandwich" principle using horse and rabbit polyvalent antibodies against human urinary colony-stimulating factor (CSF-HU). The minimal detectable level of hM-CSF was 10 U/mL, and the assays showed good reproducibility. As measured by this method, the average serum hM-CSF level of 20 normal adults was 540 +/- 110 U/mL (range, 300 to 800 U/mL). The peak of hM-CSF measured by ELISA was identical to that measured by bioassay when semipurified CSF-HU was fractionated by reversed-phase high performance liquid chromatography (HPLC). This method detected two types of hM-CSF, which had approximate molecular weights of 85 Kd (CSF-HU) and 45 Kd in human serum and urine; the ratio of 85:45 Kd was very high in serum and the amounts of the two types were nearly equal in urine. After anticancer chemotherapy, the serum hM-CSF level of one half of the patients with hematological malignancy was elevated according to the reduction in neutrophil number, while it was almost in the normal range in the other half of the patients, indicating the possibility that anticancer chemotherapy damaged the hM-CSF-producing cells. This ELISA method may be useful for monitoring the serum hM-CSF level after anticancer chemotherapy.
A phase II study of the use of colony stimulating factor derived from human urine (CSF-HU) was performed after bone marrow transplantation (BMT). Steady and rapid recovery of leukocyte and granulocyte numbers was observed. In most patients who received CSF-HU from day 1, leukocyte numbers started to increase on day 6 and monocytes and granulocytes on day 11. Significant differences in the days to recovery of leukocytes over 1 x 10(9)/l and of granulocytes over 0.5 x 10(9)/l were observed in comparison with non-randomized control patients. In some patients in whom recovery of leukocytes was delayed, CSF also seemed to be effective in increasing leukocyte numbers from 8 days after the start of administration. There was no significant difference in the rate of relapse of leukemia between the two groups. CSF-HU seems very promising as a treatment of patients after BMT by shortening the period of leukopenia or granulocytopenia.
Simultaneous analysis of the cell morphology and karyotypes on single colonies was carried out in two patients with Philadelphia chromosome (Ph1)-positive chronic myelocytic leukemia in blastic transformation in order to clarify the origin of leukemic cells involved. Patient no. 1 was in a typical myeloblastic transformation and patient no. 2 in "basophilic transformation." Both patients exhibited karyotype evolution in blastic phase (BP), so that we could differentiate BP clones with additional chromosomal abnormalities from chronic phase (CP) clones with only Ph1 among single colonies. The number of single colonies yielding two or more analyzable metaphases was 18 in patient no. 1, and 19 in patient no. 2. Among these colonies, only three in patient no. 1 and none in patient no. 2 were from CP clones and 15 in patient no. 1 and 19 in patient no. 2 were from BP clones. Morphological examination revealed that not only blasts but also mature neutrophils, eosinophils, basophils, macrophages, and erythroblasts were derived from BP clones. These results suggested that (1) BP clones developed at the pluripotent stem cell level, (2) additional chromosomal abnormalities were not restricted to occur in a specific cell line representative in BP; and (3) BP clones, if not all, may retain capacity for maturation and differentiation.
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Human lung cancer that induced marked granulocytosis in both the patient and tumor-transplanted nude mice (G2 mice) and from which conditioned medium (G2-T-CM) exhibited human and mouse active colony-stimulating activity (CSA) has been reported (K. Ikeda et al. Cancer Res 1985; 45:4144-4249). Recently, we found differentiation-inducing activity (DIA) in G2-T-CM, which differentiated human promyelocytic leukemic cells (HL-60) to macrophage-like cells. Differentiated HL-60 cells were considered to be mature macrophages as judged by the positivity of butyrate esterase activity, the acquisition of Fc receptor, and the increment in capacity of phagocytosis and nitroblue tetrazolium reduction. The DIA in G2-T-CM was not attributed to interferons known to have DIA, because interferon activity was not found in G2-T-CM by bioassay (less than 4 U/ml) and by radioimmunoassay for gamma-IFN (less than 0.1 U/ml). Molecular weight of DIA was 36,000 Da and separated from CSA of which molecular weight was 22,000 Da by gel filtration on Sephadex G-150. DIA and CSA were also separated on chromatofocusing chromatography, because isoelectric point of DIA was mainly less than 4.0 and that of CSA was 4.3-5.7. This DIA was stable after heat treatment (56 degrees C for 30 min or 100 degrees C for 10 min) and in acidic condition (pH 2.0 for 24 hr). G2-T-CM is a good source of differentiation-inducing factor for further purification and molecular cloning.
We have found that medium conditioned by a colony-stimulating factor producing tumor derived from a granulocytosis case with lung cancer contained a factor to differentiate a human promyelocytic leukemic cell line (HL-60) to macrophage-like cells that were butyrate esterase-positive and had phagocytosing activity and membrane Fc receptors. This differentiation-inducing factor was not active for a human myeloblastic cell line (KG-1), and was separated from a colony-stimulating factor by its molecular weight and isoelectric point. The conditioned medium did not contain a detectable amount of gamma interferon when tested by bioassay as well as by radioimmunoassay. This is the first report that a human lung cancer tissue produces not only a colony-stimulating factor, but also a differentiation-inducing factor. The conditioned medium is considered to be a good source of differentiation-inducing factor.
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