Taro Yasukouchi

Proliferation and differentiation of myelodysplastic CD34+ cells in serum-free medium: response to individual colony-stimulating factors.

Summary. The presence of serum or contaminant cells may alter clonal development of haematopoietic progenitor cells in vitro. To investigate the pathogenesis of myelodysplastic syndromes (MDS), marrow progenitor cells from 13 MDS patients were highly purified using monoclonal antibodies including CD34 and immunomagnetic microspheres. The cells positive for CD34 in the purified cells were in the range from 87% to 98%. These purified cells were cultured in serum‐free medium with individual colony stimulating factors (CSFs) to investigate whether CD34+ cells from MDS patients have abnormal responses to individual CSFs. Dose response experiments with the purified CD34+ cells and recombinant human macrophage‐CSF (rM‐CSF), granulocyte‐CSF (rG‐CSF), granulocyte/macrophage‐CSF (rGM‐CSF), interleukin‐3 (rIL‐3) or erythropoietin (rEP) were performed in serum‐free fibrin clots in 11 patients. Five patients showed a diminished response to rG‐CSF and one patient to rEP. In the remaining six patients the purified CD34+ cells did not respond to a stimulation of any individual CSFs. The results indicate that the progenitor cell growth abnormalities in these disorders involve a defect in the capacity of progenitor cells to respond to stimulation with G‐CSF, and present direct evidence for the manner in which myelodysplastic CD34+ cells are impaired.

Efficacy of Delayed Granulocyte Colony- Stimulating Factor after Full Dose CHOP Therapy in Non-Hodgkin's Lymphoma: A Pilot Study for a Leukocyte Count Oriented Regimen

Bone marrow toxicity is a great challenge for physicians treating patients with non-Hodgkins lymphoma (NHL) and prescribed chemotherapy. Granulocyte colony-stimulating factor (G-CSF) prevents myelotoxicity, but the optimal timing and scheduling of G-CSF administration has not been ascertained. We investigated leukocyte count oriented G-CSF administration schedules, as related to full dose administration of cyclophosphamide, adriamycin, vincristine, and prednisolone (CHOP) chemotherapy, with shortened intervals. Thirty-eight Japanese patients with NHL were included in this study. The standard CHOP combination was administered in six cycles. Patients were given G-CSF in a dose of 2 micrograms/kg/day, subcutaneously starting the day when total leukocytes were < 3,000/microliters. When leukocyte count remained at > 3,000/microliters, G-CSF was started 10 days following CHOP. Treatment with G-CSF was discontinued after the leucocyte count reached > 10,000/microliters, and CHOP was started the next day (CHOP-G treatment; CHOP-G). Doses were not modified in any patient. Patients over 70 years of age received 2/3 of the standard dosage. In the first cycle of CHOP, the day of initiation of G-CSF was 9.6 days following CHOP in the first cycle and 7.7 to 8.5 days during 2 to 6 cycles. The mean duration of G-CSF injection was 7.4 days with a range from 6.8 to 8.0 days, in each CHOP cycle. The mean intervals of CHOP-G was 14.7 days during six consecutive courses, and there was no prolongation of the intervals, even in later cycles. In 23 patients who received all six cycles of CHOP-G, the overall response rate was 91.3% (73.9% complete remission; CR and 17.4% partial remission; PR). In 32 patients with intermediate grade NHL, the overall response rate was 84.4% (65.5% CR and 18.8% PR). Thus, the full dose CHOP with G-CSF, based on the leukocyte count oriented schedule, can be achieved with shortened intervals, an approach which will increase the quality of life (QOL) for the patients by reducing the days of treatment as well as the cost of G-CSF.

Protection by α2‐macroglobulin of tissue plasminogen activator against inhibition by plasminogen activator inhibitor‐1

Tissue plasminogen activator (tPA) is widely used in the treatment of acute myocardial infarction (MI). However, its thrombolytic efficacy does not correlate with the dose administered. The interactions between tPA, α2‐macroglobulin (α2‐M), and plasminogen activator inhibitor‐1 (PAI‐1) were investigated both in vitro and in patients undergoing tPA therapy for MI in an attempt to identify variables that might affect the clinical efficacy of tPA.

Subclinical Alterations in Coagulation and Fibrinolysis in Patients Undergoing Autologous Peripheral Blood Stem Cell Transplantation

We monitored 30 laboratory hemostatic parameters in an attempt to better comprehend alterations in coagulation and fibrinolysis in 10 patients with hematological malignancies subjected to autologous peripheral blood stem cell transplantation (APBSCT). These parameters were assessed before and just after high-dose conditioning chemotherapy, on days 1, 7, 14 and 28. Although, clinical manifestations associated with fibrino-coagulation disorders never occurred, including veno-occlusive disease, a statistically significant increase was seen in 7 of 30 parameters, compared to values seen before conditioning chemotherapy. These were subdivided into early and late phase parameters. The early phase parameters, which increased during the first day after the conditioning chemotherapy was given, then returned to baseline values, included protein C, plasma tissue factor and tissue-plasminogen activator. The late phase parameters, which increased over baseline values during days 7 to 28, included free-protein S, fibrinogen, plasmin-alpha2-plasmin inhibitor complex and soluble-thrombomodulin. The increase of early phase parameters, as produced by the liver and by endothelial cells, may reflect tissue damage by conditioning chemotherapy. Late phase parameters increased in parallel with C-reactive protein, which suggests a correlation with the degree of inflammation, such as the presence of infective disease during neutropenia. These subclinical alterations in coagulation and fibrinolysis which take on a biphasic pattern during the course of APBSCT should be kept in mind by the attending physicians during therapy.

Recombinant human interleukin‐4 inhibits the production of granulocyte‐macrophage colony stimulating factor by blood mononuclear cells

Summary. The effect of recombinant human (rh) interleukin‐4 (rIL‐4) on human blood BFU‐E was investigated using two populations of cells: platelet‐depleted low‐density mononuclear cells (FH, Pl− cells), as unpurified cells, and highly purified BFU‐E. When FH, Pl− cells were cultured with rherythropoietin (rEp), rIL‐4 inhibited BFU‐E growth in a dose‐dependent manner. However, the addition of rIL‐4 did not affect rh‐interleukin‐3 (rIL‐3) supported BFU‐E growth. Limiting dilution analysis (LDA) of FH, Pl− cells showed that rIL‐4 suppressed endogenous production of burst‐promoting activity (BPA) by accessory cells. Highly purified BFU‐E were used as target cells to measure BPA in the conditioned medium (CM) that was prepared by FH, Pl− cells. When 100 purified BFU‐E were cultured in 0·5 ml clots with 20% (vol/vol) of the CM, the number of BFU‐E colonies was increased by the CM. The increase was significantly reduced by the addition of the CM prepared in the presence of rIL‐4, but anti‐IL‐4 blocked the effect of rIL‐4. The concentration of IL‐3 and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) in CM was determined by an enzyme‐linked immunoadsorbent assay (ELISA). The spontaneous production of GM‐CSF but not IL‐3 was detected, and this was significantly decreased in the presence of rIL‐4. Anti‐GM‐CSF but not anti‐IL‐3 inhibited CM supported BFU‐E growth, indicating that the main BPA in the CM is GM‐CSF and that rIL‐4 suppresses the spontaneous production of GM‐CSF by accessory cells. From these studies, we conclude that rIL‐4 has a unique mechanism as a negative regulator on erythropoiesis through the inhibition of BPA production by blood mononuclear accessory cells.

Proliferation and differentiation of myelodysplastic CD34+ cells in serum-free medium: II. Response to combined colony-stimulating factors.

Abstract: To investigate the role of colony stimulating factors (CSFs) in the proliferation and differentiation of progenitor cells from myelodysplastic syndromes (MDS), marrow progenitor cells from 18 MDS patients were highly purified using CD34 monoclonal antibody and immunomagnetic microspheres (MDS CD34+ cells). These cells were cultured in serum‐free medium with various combinations of five colony stimulating factors (CSFs): recombinant human interleukin‐3 (rIL‐3), granulocyte/macrophage‐CSF (rGM‐CSF), granulocyte‐CSF (rG‐CSF), macrophage‐CSF (rM‐CSF), and erythropoietin (rEP). Among the tested CSFs, such as rM‐CSF, rG‐CSF, rGM‐CSF and rIL‐3, a combination of the first three CSFs was the most effective stimulus for the proliferation of non‐erythroid MDS progenitor cells. An increase of undifferentiated “blast” cell colonies in 5/18 MDS patients occurred and these 5 patients belonged to the high‐risk group. In the presence of these three CSFs, rIL‐3 had no effect on the proliferation and differentiation of MDS CD34+ cells; however, IL‐3 was efficient for the proliferation of MDS CD34+ cells to the erythroid lineage. rGM‐CSF or rIL‐3 alone did not efficiently support proliferation and differentiation of CD34+ cells. M‐CSF is present in normal human serum at a concentration of 550 ±110 U/ml, a concentration exceeding that used in this study (100 U/ml). Therefore, in vivo administration of G‐CSF combined with GM‐CSF to MDS patients may be one of the most effective CSF combinations for proliferation of MDS progenitor cells to the non‐erythroid lineage. However, the effect on the capacity for differentiation was minimal, especially in patients belonging to the high‐risk group.