Takeshi Miyanomae

Role of humoral factors in granulopoiesis: Colony promoting factor (CPF) and its target “pre-CFU-C’ in long-term bone marrow culture

Supernatant of long-term bone marrow cultures contains colony promoting factor (CPF). The factor has no colony stimulating activity itself but augments granulocyte-macrophage colony formation in semi-solid culture of bone marrow cells in the presence of colony stimulating factor (CSF). CPF was specifically absorbed by bone marrow cells but not by thymocytes. Incubation of bone marrow cells with CPF alone resulted in the increase of the number of granulocyte-macrophage progenitor cells (CFUc). CPF-containing supernatant did not stimulate the proliferation of pluripotent stem cells (CFUs) but inhibited CFUs proliferation. These results suggest that CPF is a separate factor from CSF or from CFUs regulators, and that CSF-non-responsive cells (pre-CFUc) in the bone marrow mature into CSF-responsive CFUc by the stimulus of CPF.

Colony promoting activity (CPA) produced in long-term culture of murine bone marrow cells☆

The differences between colony promoting activity (CPA) and colony stimulating activity (CSA) in the culture media of murine long-term bone marrow cultures (LTBMC) were demonstrated and the role of adherent cells and nonadherent cells in the production of CPA was studied in this culture system. Supernatant harvested from intact continuous marrow cultures showed high CPA but contained no CSA. Assayable CSA was detected in concentrated supernatant. However, there was no significant relationship between levels of CPA and CSA in the supernatant. When adherent cells and nonadherent cells from LTBMC were separately cultured, CPA was detected in the conditioned medium of adherent cells but not in that of nonadherent cells. The CPA level in LTBMC was related inversely to the number of nonadherent cells and addition of nonadherent cells to adherent cell cultures reduced the level of CPA. Conditioned medium of nonadherent cells showed no inhibitory activity of CPA. These results indicate that CPA is produced by bone marrow adherent cells and that it may be consumed by myeloid progenitor cells in nonadherent cells.

Presence of colony promoting activity (CPA) in the supernatant of the long-term bone marrow cultures

Colony promoting activity (CPA) was detected in the supernatants of the bone marrow cell cultures. CPA itself had no granulocyte-macrophage colony stimulating activity (CSF); but did increase the number and the size of colonies induced by the optimal dose of CSF. This activity was dose dependent and not specific to CSF sources. CPA-responsive cells (target cells) were found in the bone marrow cell cultures as well as in the freshly isolated bone marrow.

Effects of bacterial lipopolysaccharide and X-irradiation on the production of colony-stimulating factor and the maintenance of granulopoiesis in bone marrow culture

Effects of bacterial lipopolysaccharide (LPS) and X-irradiation on CSF production and granulopoiesis in long-term bone marrow cultures were studied. Levels of colony-stimulating factor (CSF) increased soon after the refeeding of the culture, but the activity was undetectable at day 7. Addition of LPS induced a significant increase in CSF levels in the culture, followed by an elevated granulopoiesis. The increase in CSF levels was suppressed when culture medium that had been harvested at refeeding on day 7 was added. Although irradiation did not increase CSF production, granulopoiesis was markedly stimulated shortly after irradiation. Thus granulopoiesis in long-term bone marrow culture may also be regulated by humoral factors such as CSF, and the culture system may represent the in vivo response to haemopoietic stimuli.

Agranulocytosis following infectious mononucleosis.

A girl developed acute agranulocytosis (45/mm3), 37 days after the onset of infectious mononucleosis. The bone marrow showed myeloid hyperplasia with maturation arrest and erythroid hypoplasia. A normal amount of colony forming units of granulocytes and macrophages (CFU-GM) colonies with a relative high number of clusters was observed. Neither anti-neutrophil antibodies nor circulating inhibitors of colony growth were found in serum. Granulocyte and macrophage colony stimulating factor (GM-CSF) activity in the patients serum rose at this time. The agranulocytosis lasted 5 days and her clinical state soon improved. These results suggested that agranulocytosis was presumably not due to serum factors, including auto-antibodies and/or suppressive substances, and that Epstein-Barr virus (EBV) had some direct or indirect effect on the marrow cells of the myeloid series.

Survival and repopulation of irradiated pre-CFU-c in mice

ABSTRACT Supernatants of murine bone‐marrow cultures contain a colony‐promoting factor (CPF) which increases the number of granulocyte and macrophage colonies in semi‐solid agar cultures in the presence of colony‐stimulating factor (CSF). Incubation of bone‐marrow cells with CPF results in an increase in the number of granulocyte/macrophage progenitor cells (CFU‐c) and the CPF‐responsive cells may be younger than the CFU‐c.

Colony promoting activity and its target 'pre-CFUc' in genetically anemic mice of W/Wv genotype.

Incubation of bone marrow cells with supernatant from long-term cultures of bone marrow cells increases the number of granulocyte-macrophage progenitor cells. This study reveals the presence of target cells of the colony promoting activity (CPA) inW/W v mouse marrow. It is also shown that CPA does not stimulate erythroid colony formation in vitro.

Effects of bacterial lipopolysaccharide on the production of colony-stimulating activity in C3H/HeJ mouse long-term bone marrow cultures

Bacterial lipopolysaccharide (LPS)-induced colony-stimulating activity (CSA) in murine long-term bone marrow culture system was investigated. Bone marrow culture cells of LPS-nonresponsive C3H/HeJ mice responded to LPS in terms of CSA production as efficiently as bone marrow culture cells of LPS-responsive C3H/slc mice. On the other hand, both peritoneal macrophages and bone marrow macrophages from C3H/HeJ mice did not produce CSA in vitro after treatment with LPS. Percoll density gradient separation of adherent layer cells in bone marrow cultures showed that two cell populations were present. One population was nonspecific esterase positive, productive of high CSA to LPS stimulation and light density cells, the other population was nonspecific esterase negative, productive of low CSA to LPS stimulation and high density cells, and CSA production stimulated by LPS in C3H/HeJ mice bone marrow culture cells was mainly attributed to the latter population of cells. These results suggest that CSA production stimulated by LPS in C3H/HeJ mice is regulated by different cell populations, respectively in vivo and in vitro.

Low Burst Promoting Activity Production by Phytohemagglutinin-Stimulated Mononuclear Cells of Cord Blood

ABSTRACT: Burst-promoting activity (BPA) produced by phytohemagglutinin(PHA)-stimulated cord blood mononuclear cells (MNC) was examined using the two-stage cell culture assays. Burst-promoting activity was measured as the increase in the number of early erythroid progenitor cells in 2-day incubation of peripheral blood MNC with or without the conditioned medium of PHA-stimulated MNC (PHA-LCM). Burst-promoting activity in PHA-LCM of cord blood was significantly lower than that of adult blood (37 ± 13 versus 105 ± 19%, mean ± SD, p < 0.01). No elevation of inhibitors to the erythroid colony growth was noted in PHA-LCM of cord blood. In contrary, the response of cord blood MNC to PHA was similar to that of adult blood MNC, as determined by colony-stimulating activity production and cell proliferation. These results showed that burst-promoting activity production by PHAstimulated MNC of cord blood was lower than that of adult blood.

Effect of carbon particles on the recovery of bone marrow stem cells after irradiation in LPS-resistant C3H/HeJ mice

Effect of RES-blockade on bone marrow cells was studied serially after irradiation in LPS-resistant mice. Injection of carbon particles reduced damage and accelerated recovery of marrow hemopoietic stem cells, indicating that LPS-resistant mice can react normally to RES-blockade.