Effects of an aplastic anemia urinary extract on mouse erythroid progenitor cells in vivo.

We investigated the in vivo effects of a crude extract from the urine of aplastic anemia patients (AA urinary extract) on erythroid precursor cells in the femoral bone marrow and spleens of normal adult mice. A single intraperitoneal injection of AA urinary extract induced a significant increase in the number of splenic erythroid burst-forming units (BFU-e) and erythroid colony-forming units (CFU-e) within 24 h after injection. We then injected pure recombinant erythropoietin (Epo) equivalent to the amount present in the urinary extract. This addition increased the number of splenic CFU-e by almost the same degree as the amount induced by the AA urinary extract 24 h after injection, but failed to elicit any change in the number of splenic BFU-e. In other studies, mice were injected with the same amount of lipopolysaccharide (LPS) and/or pure Epo as that present in the AA urinary extract. Experiments with Limulus amebocyte lysate-adsorbed (endotoxin-depleted) or nonadsorbed (endotoxin-containing) AA urinary extracts showed that endotoxin contamination interfered with the increase in numbers of marrow CFU-e and enhanced the increase in splenic CFU-e numbers induced by pure Epo or Epo activity in the AA urinary extract. The number of splenic BFU-e, however, was not affected by administration of LPS and/or Epo or by adsorbed endotoxin. These data suggest that AA urinary extract contains a stimulating activity for mouse splenic BFU-e, and that this activity is not attributable to the Epo activity or endotoxin contamination within the urinary extract.

Effects of urinary extracts from patients with idiopathic thrombocytopenic purpura or aplastic anemia on rodent platelet production and megakaryocytopoiesis.

Urinary extracts from idiopathic thrombocytopenic purpura (ITP) patients, aplastic anemia (AA) patients and normal subjects were investigated for their effects on in vivo platelet production, and both in vitro and in vivo megakaryocytopoiesis in rodents. Daily intraperitoneal injection of 1.2 absorbance units (AU, A278) of urinary protein for three consecutive days induced statistically significant increases in rat blood platelet numbers. This increase was observed for 1 of 4 ITP urinary extracts and for all 3 AA urinary extracts, and occurred 24 h after the final injection. In vitro levels of megakaryocyte colony-stimulating factor (Meg-CSF) in ITP urinary extracts were similar to those of normal urinary extracts, and were in dramatic contrast to the markedly elevated levels of Meg-CSF in extracts from AA urine. A single intraperitoneal injection of 0.5 AU of AA urinary protein induced a significant increase in spleen-derived megakaryocyte colony-forming cells (CFU-meg) 48 h past injection. In the group injected with ITP urinary extract, CFU-meg levels remained within normal limits. These results provide evidence that urinary extracts of ITP patients do not contain increased levels of Meg-CSF and a factor which directly stimulates in vivo CFU-meg production, and that the decrease in circulating platelet numbers that is characteristic of ITP patients is not a primary in vivo determinant in the elaboration of these factors.

Establishment of two Ph1 chromosome-positive cell lines, KPB-M8 and KPB-M15.

Two new Philadelphia (Ph1) chromosome-positive cell lines, designated KPB-M8 and KPB-M15, were established from the peripheral blood of two patients with chronic myelogenous leukemia in blastic crisis. Both cell lines were characterized by blastic appearance, the presence of acid phosphatase activity, Fc gamma-receptor, and C3-receptor, and reactivity to monoclonal antibodies such as OKM1, MCS2, MY906, MY4 and MY7. These results indicate that KPB-M8 and KPB-M15 cells are of an undifferentiated blast nature. Both cells retained Ph1 chromosome, and showed numerical and structural changes upon chromosomal analysis. These cell lines should provide a useful source for studying differentiation of hemopoietic cells.

Lack of correlation between in vitro corticosteroid effect on hemopoietic colony formation and response to corticosteroid therapy in aplastic anemia.

We performed hemopoietic colony culture assays in 15 patients with aplastic anemia (AA) in order to test the effect of hydrocortisone (HC) on late erythroid colony (CFU-e) formation of the patients' marrow and to correlate the in vitro culture results with the clinical response to corticosteroid therapy. HC enhanced CFU-e growth in four patients. All four patients failed to respond to corticosteroid, but three improved with with androgens. The addition of HC did not increase CFU-e colony formation in 11 patients. However, two of them responded to corticosteroid therapy. Among the nine patients showing no HC effect in vitro, two subsequently improved with androgens and one each with anti-thymocyte globulin and anti-lymphocyte globulin. The results suggest that the in vitro corticosteroid effect may not necessarily correlate with responsiveness to corticosteroid therapy.

Effects of androgen on transient endogenous spleen colonies and other hemopoietic stem cells in mice.

To clarify the effects of androgen on hemopoiesis in mice, we investigated changes in hemopoietic stem cells at various stages of differentiation after injection of 19-nandrolone decanoate (19-ND). 19-ND induced (a) a marked increase in the population of CFUe, especially in the spleen, and (b) a less significant increase in the number of CFUs, GM-CFC, and BFUe. The number of endogenous spleen colonies developed transiently on day 4 after irradiation (TE-CFU) increased significantly, and the initiation and induction of erythropoietic maturation was enhanced. Growth of the pluripotent stem cells (CFUs) assessed by endogenous spleen colony formation was influenced more than was the population size. Production of erythropoietin (Ep) in mice was not affected by the treatment with 19-ND. These results indicate that 19-ND affects mature precursors in erythroid cell lineage rather than pluripotent stem cells and that the effect is unlikely to be induced through an increase in the production of Ep.

Cell cycle-dependent heat sensitization of murine granulocyte-macrophage progenitor cells in regenerating marrow.

The response of murine granulocyte-macrophage progenitor cells to hyperthermia was examined using normal and regenerating marrow. Hyperthermic exposure was given in vitro at 41-44 degrees C for periods of up to 60 min, and the results were compared between the 2 groups. Although almost no difference in percentage of survival was observed between them at 41 degrees C, murine granulocyte-macrophage progenitor cells of regenerating marrow showed markedly increased thermal sensitivity at and above 42 degrees C in comparison with that of normal marrow. Hydroxyurea suicide experiments revealed that the proportion of in vitro colony-forming units in the DNA-synthetic phase of the cell cycle [S phase] was greatly increased in regenerating marrow [66 +/- 4% (SD)] as compared with that in normal marrow [18 +/- 8%]. These data indicate that heat sensitization of hemopoietic cells occurs because of cell cycle effects, when they apparently mean the relative proportion of cells in S phase.

A novel Ph1 chromosome positive cell line established from a patient with chronic myelogenous leukemia in blastic crisis.

A novel Philadelphia (Ph1) chromosome positive cell line, designated KYO-1, was established from the peripheral blood of a patient with chronic myelogenous leukemia (CML) in blastic crisis. Although this line had a unique capacity to differentiate spontaneously along the erythroid and monocytoid lineages as evidenced by cytochemical analysis for the first several months, the capacity was gradually lost after repeated passages. The results suggest that KYO-1 is an undifferentiated myeloid cell line. This cell line provides a useful source for studying differentiation and proliferation of pluripotent stem cells from CML in blastic crisis.

Presence of colony-promoting activity-responsive cells as a separate compartment of granulocytic cell lineage in mice.

A population of cells that responds to colony-enhancing factor has been reported to constitute the most immature subpopulation in the compartment of granulocyte/macrophage progenitors (GM-CFC). A similar colony-promoting activity (CPA) was found in the supernatant of long-term cultures of murine bone marrow cells. Here, some characteristics of the cells responsive to CPA were studied. The CPA-responsive cells in the spleen and bone marrow of W/WV mice were as numerous as in +/+ litter-mates. The concentration of CPA-responsive cells was independent of those of other cell populations, namely pluripotent stem cells (CFU-S), pluripotent precursor cells in vitro and GM-CFC in the spleen and bone marrow. Seeding efficiency in the spleen of irradiated mice and the cell-cycle state of CPA-responsive cells also differed from those of CFU-S and GM-CFC. Accordingly, the target of CPA appears to constitute a separate compartment in the progenitor populations of granulocytic lineage.

Correlation between in vitro and in vivo response to androgens in patients with aplastic anemia.

In an attempt to predict the clinical response to androgens, the effects of added fluoxymesterone (FMT) on the in vitro growth of marrow erythroid progenitors (CFU-E) were examined in 15 patients with aplastic anemia and 6 control subjects with normal marrow morphology. In the control group, the addition of FMT enhanced the growth of CFU-E in vitro, the maximum absolute increase being dependent on the basal number of CFU-E. In 10 out of the 11 aplastic anemia patients who responded to androgens in vivo. FMT enhanced the growth of CFU-E in vitro. In this group, there was a positive correlation between the basal number of CFU-E and the maximum absolute increase of CFU-E induced by FMT. In patients who did not respond to androgens in vivo, all except one showed no increase in the growth of CFU-E in vitro by the addition of FMT. Furthermore, in four out of seven patients examined prior to androgen therapy, the addition of FMT enhanced the in vitro growth of CFU-E. Androgen therapy in vivo resulted in marked hematological improvements in all of these four patients. Two out of the remaining three patients in whom FMT did not enhance the in vitro growh of CFU-E failed to respond in androgen therapy. These results indicate that an in vitro CFU-E culture system to the presence of FMT may be helpful in predicting the response to androgens in vivo in patients with aplastic anemia.

Suppression of granulocyte-macrophage colony formation in vitro by conditioned medium derived from human bladder cancer cells.

Media conditioned with 8 cell lines were assayed for stimulating and inhibiting activities on granulocyte-macrophage colony formation from bone marrow cells in soft agar (CFUc). Conditioned media from 3 human bladder cancer cell lines (EJ, T24 and KK47) and a rat bladder cancer cell line (BC50-TC) showed marked inhibition of mouse CFUc. Conditioned medium from normal human bladder cells showed a weak colony-stimulating activity, and only a slight inhibition when added to CFUc culture. Conditioned media from mouse or rat fibroblast cell lines (BLP and 3Y1) showed no colony-inhibiting activity, and the conditioned media from human lymphoblastoid cell line (Raji) and human epithelia-like cell line from amniotic membrane (FL) showed moderate inhibitory activity. No colony-stimulating activity was detected in any of the cell lines tested. EJ-conditioned medium inhibited human CFUc, but not erythroid progenitors. Colony-inhibiting activity in EJ-conditioned medium appears not to be prostaglandins; it needs more than 3 hr to inhibit CFUc, is stable at 56 degrees for 30 min but not at 75 degrees for 20 min, and is susceptible to proteolytic enzymes.

Radioautographic studies on the formation and maturation of bone marrow lymphocytes in mice.

DNA labelling by [3H]thymidine and the sandwich radioimmunolabelling method were used to characterize marrow lymphoid cells and to study the kinetics of production and maturation of small lymphocytes in the bone marrow of adult mice. Marrow lymphoid cells consisted of non-proliferating small lymphocytes, 30-40% of which had detectable surface immunoglobulin (SmIg), and proliferating large lymphoid cells lacking SmIg. Double-labelling experiments employing [3H]thymidine in vivo followed by sandwich radioimmunolabelling in vitro indicated that marrow small lymphocytes lack detectable SmIg when they are formed but develop SmIg within the first few days after production. Marrow lymphocytopoiesis includes; (1) proliferation of large lymphoid cells, which are presumptive small lymphocyte progenitors, which have a cell cycle time of 14-15 hr, and (2) a 3-5 day intramyeloid stage when many newly formed small lymphocytes undergo maturational changes towards the B cell lineage.