Masahiro Ieko

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.

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.

Tc-99m labeled tissue-type plasminogen activator: preparation, stability and preliminary imaging of thrombus-bearing rats.

Tissue-type plasminogen activator (t-PA) is a thrombolytic agent that directly binds to fibrin formed in clots. In terms of radiolabeling and nuclear imaging, t-PA has several advantages in Tc-99m labeling: it is stable in acidic solution at pH 3, which is suitable for labeling Tc-99m by a method of stannous reduction and blood disappearance after administration is rapid, which is desirable for imaging targets using short-lived radionuclides.Recombinant t-PA was labeled with Tc-99m by a method of stannous reduction without significant degradation of biochemical activity, over 95 % of which was retained after the labeling procedure. Labeling efficiency in paper chromatography was over 98 %. The moiety of hydrolyzed Tc-99m that was not eluted through the Sephadex column was estimated to be less than 10%. Tc-99m labeled t-PA, however, appeared to become unstable when diluted with normal saline. Nevertheless, inin vitro fibrin binding, Tc-99m labeled t-PA showed high affinity with fibrin: 80% of 100 ng/ml of Tc-99m t-PA bound to 10−5 mol of the fibrinogen. Preliminary animal studies also showed a concentration of Tc-99m labeled t-PA at fresh thrombi formed in the inferior vena cava.Tc-99m labeled t-PA appears to have potential for thrombus imaging and the preparation of an instant kit.

Role of physiologic concentrations of stem cell factor in leukemic type growth of myelodysplastic CD34+ cells

The stem cell factor (SCF: a ligand for c-kit) plays a central role in the growth of myelodysplastic (MDS) progenitor cells with leukemic type growth. In this study, the role of physiologic concentrations of SCF on the proliferation and differentiation on MDS progenitor cells was further analyzed in the presence of combined cytokines. For this purpose, marrow CD34+ cells were purified up to 94% for 12 normal individuals and 90% for 18 MDS patients, using monoclonal antibodies and immunomagnetic microspheres. The purified CD34+ cells were cultured for 14 days with saturating doses of cytokines, including recombinant human macrophage colony stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte/macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3) and rSCF. The clonal growth of MDS CD34+ cells supported by a combination of all the above cytokines was then subdivided into the two patterns of leukemic or non-leukemic. The role of various concentrations of rSCF (0, 0.5, 5, 50 and 500 ng ml(-1)), with or without the above cytokines, in proliferation and differentiation of MDS CD34+ cells was analyzed in each group. The physiologic concentration of SCF at 5 ng ml(-1) significantly increased undifferentiated blast cell colonies or clusters in leukemic type growth of MDS CD34+ cells over that seen in normal CD34+ cells. SCF is present in plasma at a level of ng ml(-1). This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blasts.

In vivo kinetics of99mTc labeled recombinant tissue plasminogen activator in rabbits

Our previous studies demonstrated that99mTc labeled recombinant tissue plasminogen activator (rt-PA) retained high affinity with fibrinin vitro but showed unexpectedly low uptake in fresh thrombiin vivo. The present study was performed to determine thein vivo kinetics of radiolabeled t-PA in the rabbit.Sequential images and blood samples after the intravenous administration of99mTc labeled rt-PA in thrombus-bearing rabbits were taken. The radioactivity and immunological level of t-PA and PAI-1 in the solution eluted to each fraction by gel permeation chromatography were measured by means of a well scintillation counter and enzyme-linked immunosorbent assay (ELISA). Most of the radioactivity was eluted in the fraction (Fr. 7) of larger molecular weight than that (Fr. 9) of intact t-PA. The level of intact rt-PA was increased with a regimen involving the preadministration of cold rt-PA which was followed by the administration of hot rt-PA. The level of PAI-1 in plasma showed an increased rebound 15 minutes after the intravenous injection. These results suggest two possible reasons why rt-PA retains high affinity with fibrinin vitro, once radiolabeled, but was ineffective in delineating fresh thrombi with a gamma camera: 1) some plasma components such as PAI-1 combine with circulating radiolabeled rt-PA and form a larger molecule immediately and/or 2) radiolabeled rt-PA is modulated as a consequence of the radiolabeling and forms a larger molecule than intact rt-PA.

Thrombocytopenia Associated with Type IIB von Willebrand’s Disease of Pregnant Identical Twins

Type II B von Willebrand’s disease (vWD) is an inherited hemorrhagic disorder. The disease is characterized by the enhanced platelet aggregation at low ristocetin concentrations in the patient’s platelet-rich plasma (PRP). This characteristic suggests that type II B vWD may induce a marked decrease in the platelet count under some conditions. We found pregnant identical twin mothers with marked thrombocytopenia. The twins also had type IIB vW disease as well as systemic lupus erythematosus (SLE). We will report about these two cases with a recommendation for an appropriate therapy for thrombocytopenia associated with pregnancy in patients with type II B vWD disease.