Treatment of four patients with myelodysplastic syndrome with a small dose of aclacinomycin-A.

The effect of a small dose of aclacinomycin-A (ACR) was examined in two patients with refractory anemia (RA) and two with refractory anemia with excess of blasts in transformation (RAEB-t). ACR (7 or 14 mg/m2) was given for 10 days in a 2-h per day drip infusion. Clinical symptoms and laboratory data improved in 3 of these 4 patients. In a patient with RA, marked increase in reticulocytes and elevation of the hemoglobin level from 6 to 9 g/dl was observed after two courses of ACR therapy. In two with RAEB-t, Auer's rod bearing cells disappeared in the bone marrow and megaloblastic change of the erythroblasts was diminished in one patient. Hemoglobin levels rose from 4.7 to 10 g/dl in one, and platelets and WBC increased in another. No effect was seen in a patient with RA. The cytoreductive effect of ACR was minor compared to the therapy with small dose of cytosine arabinoside (Ara-C). Therefore, ACR warrants further consideration for the treatment of patients with MDS.

Levels of serum granulocyte colony-stimulating factor in patients with cerebrovascular diseases.

The role of leukocytes in the pathogenesis of cerebrovascular disease, in particular, cerebral ischemic disease has recently become a focus of research. Several studies have reported that a positive correlation between increased functional activities of neutrophils and the risk of cerebral ischemic disease. Granulocyte colony-stimulating factor (G-CSF) is known to be not only a granulocyte proliferating factor but also a potent activator of mature neutrophils. In this study, we measured the serum G-CSF levels in 143 patients with cerebrovascular diseases and in 100 patients with other diseases, using our established enzyme-linked immunosorbent assay (ELISA) for G-CSF The minimal detection level was 20 pg/ml G-CSF. In patients with cerebral infarction, G-CSF could be detected in 18.3% and in patients with cerebral hemorrhage, it could be detected in 9.8% of analyzed samples. On the other hand, 6% of the patients with other diseases had measurable levels of G-CSF. The differences among these three groups were statistically significant according to the chi 2 test (p < 0.01). Our findings that there was a significantly high frequency of elevated levels of G-CSF among patients with cerebrovascular diseases, may indicate that the action of G-CSF as a potent activator of neutrophils plays some role in the occurrence of cerebrovascular disease, in particular, cerebral infarction.

Stimulatory effect of forphenicinol on normal human bone marrow granulocyte-macrophage progenitor cells mediated by T-lymphocytes.

Forphenicinol, L-2-(4-formyl-3-hydroxymethylphenyl) glycine, is a newly discovered low molecular weight immunomodifier. Its effects on normal human bone marrow granulocyte-macrophage progenitor cells (CFU-C) were studied in vitro. Addition of forphenicinol to cell cultures resulted in a significant (p less than 0.05) increase in the number of, and preservation of the ability to form CFU-C colonies per fixed number of human non-adherent bone marrow cells. This effect was not observed in T-lymphocyte depleted fractions of human non-adherent bone marrow cells. Furthermore, colony stimulating factor was released when T-lymphocytes were incubated with forphenicinol. These data suggested that T-lymphocytes mediated the stimulatory effect of forphenicol on human bone marrow CFU-C.

Direct action of human granulocyte colony-stimulating factor on mature human neutrophils: flow-cytometric analysis.

The direct effects of human granulocyte colony-stimulating factor (G-CSF) on mature neutrophils in vitro were studied using flow cytometry. G-CSF increased the neutrophil forward scatter without recruitment of side scatter, as assessed by flow cytometry. This indicates that G-CSF acts on neutrophils directly and induces the activation of mature neutrophils without degranulation of primary granules.

Granulocyte-macrophage colony formation in vitro using human non-phagocytic bone marrow cells.

A technique employing silica particles was used to remove cells producing endogenous colony-stimulating factor (CSF) to allow measurement of the level of colony-forming units in culture (CFU-C) in human bone marrow cells. In comparison with the glass-adherence technique, this new approach resulted in a more complete degree of removal of CSF-producing cells and formation of more colonies. This method seems to be useful for performing an accurate assay of exogenous CSF.

Activation of neutrophils NADPH oxidase by PMA: cytosol activity is translocated in phorbol-primed neutrophils.

1. Translocation of cytosol activity in phorbol-primed neutrophils was studied. 2. Prior exposure of PMA or FMLP could potentiate the oxidative response by subsequent heterogeneous stimulus, FMLP or PMA. 3. In FMLP-primed neutrophils, the cytosol had almost the same activity as resting one and cytosol activity was not eluted from the membrane. 4. In PMA-primed neutrophils, however, the cytosol had less activity and cytosol activity was correspondingly eluted from the membrane. 5. These observations suggested that cytosol activity was translocated in PMA-primed cells.

Multipotent hemopoietic progenitor cells in patients with systemic lupus erythematosus.

Hematologic abnormalities in patients with systemic lupus erythematosus (SLE) were studied before treatment, using an in vitro bone marrow progenitor cell assay. In 10 patients with SLE, there was a decrease in the number of multipotent hemopoietic colonies. Multipotent colony formation was suppressed by the addition of T cells from the patients with SLE. The culture supernatant of phytohemagglutinin stimulated SLE leukocytes had diminished activity to support the multipotent colony formation. These results suggest that the hematologic abnormalities in SLE occur at the multipotent stem cell level. The T cell mediated suppression of hemopoietic progenitor cells and the diminished activity of humoral factors released from SLE leukocytes may play some role in the pathogenesis of hematologic abnormalities in SLE.

Stimulatory effects of bestatin on human B-cell colony formation.

Bestatin, (2S, 3R)-3-amino-2-hydroxy-4-phenylbutyryl-L-leucine, is a small molecular immunomodifier. Effects of this compound on human immune function were studied, in vitro, using the human B-cell colony formation technique. B-cell colonies were obtained from enriched B-cell populations placed in conditioned methylcellulose medium containing stimulators and irradiated T-cells as feeders. Addition to the culture of Bestatin at concentrations of 0.1 microgram/ml and 1 microgram/ml led to a significant increase (P less than 0.05) in the number of B-cell colonies and this effect was abolished when irradiated T-cells were not added to the culture. Bestatin increased soluble factor production induced by phytohemagglutinin (PHA)-stimulated T-cells. Such findings suggest that T-cells probably mediate this stimulatory effect of Bestatin on B-cell colony formation.

Protective effect of recombinant murine granulocyte-macrophage colony-stimulating factor against Pseudomonas aeruginosa infection in leukocytopenic mice.

The effects of recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) against Pseudomonas aeruginosa infection in ICR mice were investigated. Mice were treated with cyclophosphamide (CPA) and were then injected intraperitoneally with rmGM-CSF three times daily, beginning on the day after CPA treatment, for 7 days. The number of peripheral blood leukocytes in both CPA- and rmGM-CSF-treated mice and control CPA-treated mice reached a nadir on day 4, when P. aeruginosa was injected intraperitoneally. The administration of rmGM-CSF significantly increased the proportion of survivors among mice infected with a lethal dose of P. aeruginosa. This effect was further analyzed by monitoring sequential changes in leukocyte count and bacterial growth in various organs. The number of bacteria in the peritoneal cavities, peripheral blood samples, and livers of GM-CSF-treated mice decreased to an undetectable level after a transient increase, and the number was significantly lower than that in control mice. In GM-CSF-treated mice, the neutrophil levels in peripheral blood started to increase 5 days after CPA administration and were consistently higher than those in controls. Furthermore, the neutrophils in GM-CSF-treated mice were more mature morphologically. Thus, the prophylactic effect of rmGM-CSF against P. aeruginosa infection may result from a rapid recovery of myelopoiesis and a partial enhancement of mature neutrophil function.

Aplastic anemia associated with type A viral hepatitis--possible role of T-lymphocytes.

Posthepatitic aplastic anemia (PHAA) is rather uncommon. Most reported cases have developed after non-A, non-B or B type hepatitis. The only case of PHAA occurring after hepatitis A reported so far, was described by Smith et al., who diagnosed it by the long-term elevation of IgG-class antibody in response to hepatitis A virus. Recently, the detection of IgM-class antibody specific against hepatitis A virus (IgM anti-HAV) has been commonly employed for precise diagnosis of hepatitis A. The case reported here is the first case of PHAA occurring after hepatitis A to be diagnosed by radioimmunoassay of IgM anti-HAV. Furthermore, evidence is presented suggesting that the PHAA may have been an immune-related response. Addition of the patient's peripheral T-lymphocytes to cultures of her own bone marrow cells resulted in a reduction in the number of colony-forming units in culture (CFU-C).

Human bladder carcinoma cell line HTB9, which secretes a factor to stimulate clonogenic leukemic blast growth, expresses the granulocyte-macrophage colony-stimulating factor gene.

Media conditioned by the human bladder carcinoma cell line HTB9 contained high leukemic blast growth factor activity and also showed granulocyte-macrophage colony-stimulating factor (GM-CSF) activity. Northern blotting analysis of total RNA from HTB9 cells using GM-CSF cDNA as a probe demonstrated abundant expression of the GM-CSF gene. Thus, this cell line secretes GM-CSF, and this factor contributes to the proliferation of clonogenic leukemic blast cells.

Methylcholanthrene-induced murine fibrosarcoma cell line BMT-11 secretes granulocyte colony-stimulating factor.

Murine fibrosarcoma cell line BMT-11 was induced with 3-methylcholanthrene and maintained in culture. Transplantation of BMT-11 into syngeneic C57BL/6 mice produced leukocytosis consisting of marked increments of neutrophils and monocytes associated with massive splenomegaly. In order to elucidate the mechanisms of this leukemoid reaction, we studied the changes occurring in hematopoietic progenitor cells in BMT-11-transplanted mice. The numbers of granulocyte-macrophage colony-forming units (CFU-GM), erythroid colony-forming units (CFU-E), erythroid burst-forming units (BFU-E), and mixed colony-forming units (CFU-Mix) in the spleen showed dramatic 216-fold, 18-fold, 64-fold, and 80-fold increases, respectively, relative to the value in the control mice 5 weeks after the BMT-11 implantation. In contrast, the levels of progenitor cells in the bone marrow remained within normal limits. The nature of the colony-stimulating factor (CSF) secreted from BMT-11 tumor cells was also studied. BMT-11-conditioned medium (BMT-11-CM), BMT-11 tumor extract, and sera from the mice bearing transplanted BMT-11 tumor contained CSF that stimulated mainly granulocyte and macrophage lineages. Furthermore, the expression of the granulocyte colony-stimulating factor (G-CSF) gene in BMT-11 cells were detected by Northern blot analysis.