Ara-C sensitivity test and suicide tests of L-CFU using [3H] deoxycytidine (dCyd) and [3H] thymidine (TdR) were performed in patients with acute nonlymphocytic leukemia (ANLL) and with chronic myeloid leukemia in blastic crisis (CML-BC). We found a correlation between ara-C sensitivity and the [3H] dCyd suicide test of L-CFU (p less than 0.001); and between ara-C sensitivity and the [3H] TdR suicide test (p less than 0.05). These results suggest that the [3H] dCyd suicide test reflects the degree of activity of ara-C metabolism in L-CFUs.

Using in vitro clonal culture assays, we investigated the effects of PSK, a protein-bound polysaccharide derived from the cultured mycelium of CM101, Coriolus versicolor (Fr.) Quél in Basidiomycetes, on human hemopoietic progenitors. PSK alone did not stimulate colony formation by human bone marrow progenitors. Although 1-100 micrograms/ml of PSK had no effects on colony formation stimulated by erythropoietin and medium conditioned by phytohemagglutinin-stimulated leukocytes, more than 1 mg/ml of PSK inhibited all types of colony formation. In contrast, medium conditioned by PSK-stimulated leukocytes significantly stimulated formation of various types of colonies including erythroid bursts, granulocyte and/or macrophage colonies, eosinophil colonies, megakaryocyte colonies and mixed hemopoietic colonies. It is speculated that administration of the optimal dose of PSK can reduce the hematological suppression of antitumor drugs.

Four monoclonal antibodies against human erythrocyte membrane antigens were established. The antigenic determinants of KOR-E1, E3, E6 were Pr1h antigen, Wrb antigen, and the trypsin sensitive portion of glycophorin A (EnaTS) respectively. The antigen recognized by KOR-E4 could not be determined. The reactivities of these antibodies with normal hematopoietic cells, malignant hematopoietic cell lines (N = 31), and fresh leukemic cells obtained from 128 patients with various types of leukemias were studied. All antibodies reacted only with erythrocytes among peripheral blood cells, and also KOR-E6 reacted only with erythroid cells among bone marrow cells. KOR-E3 had no reactivity with any cell lines examined, and KOR-E1 and KOR-E4 were reactive with some lymphoid cell lines. However, KOR-E6 had specific reactivities with erythroid (HEL, K562), megakaryocytic (CMK-1), multiphenotypic (KOPM-28), and basophilic (KU-812) cell lines. The antigen (glycophorin A) recognized by KOR-E6 was expressed on a small population of mononuclear cells separated from acute lymphoblastic leukemia (3/70), acute myelogenous leukemia (2/12), monosomy 7-myeloproliferative disorder (1/1), juvenile CML (1/1), and transient myeloproliferative disorder with Down's syndrome (4/12), although it could not be determined whether these cells were leukemic cells or not. KOR-E6 was reactive with a large population of leukemic blasts in erythroleukemia (2/2) and acute megakaryoblastic leukemia (3/6). Thus, KOR-E6 appears to be an erythroid marker of leukemic cells.

The mossy fiber projections to the uvula of the cerebellum were studied by means of retrograde axonal transport of horseradish peroxidase (HRP) in the cat. Following large and small injections into the uvula, distribution of the labeled cells in the brainstem nuclei was investigated. The results showed different afferent projections between the dorsal and ventral uvula. Major sources projecting to the dorsal uvula were the peduncular, paramedian, and lateral nuclei of the pontine nuclei. Labeled cells found in the pontine nuclei amounted to 81.6% of the total number of labeled cells in cat 1. On the other hand, major sources projecting to the ventral uvula were the caudal aspect of the medial and inferior vestibular nuclei, the x- and f-groups of the vestibular nuclei, the dorsal and central aspect of the superior vestibular nucleus, the rostral dorsomedial aspect of the paramedian nucleus of the pontine nuclei, the caudal aspect of the prepositus hypoglossal nucleus, and the infratrigeminal nucleus. Labeled cells in the vestibular nuclei amounted to 72.1% of the total number of the labeled cells in the cat 40. It was revealed that the lateral aspect of the ventral uvula receives inputs from the pontine nuclei, whereas the medial part of the ventral uvula receives inputs from the vestibular nuclei. Mediolateral differences were not found in the dorsal uvula. These mossy fiber zones were mediolaterally wide, and the dorsal uvula was different from the ventral uvula with regard to mossy fiber projection.

Clinical course, response to various modes of treatment and changes in in vitro marrow culture assay were studied in two patients with congenital pure red cell aplasia (Diamond-Blackfan syndrome) who were followed up for a long period. Patient 1, whose diagnosis was made at 8 months of age, was refractory to prednisolone and anabolic steroid. Bolus methylprednisolone, cyclophosphamide, ALG and high-dose intravenous immunoglobulin were given but none were effective. Particularly, hemolysis occurred during high-dose intravenous immunoglobulin therapy. In colony assay, CFU-E and BFU-E were found to be extremely decreased throughout the course, and colony formation was not corrected by adding prednisolone to the assay system. However, coculture of normal bone marrow cells with the patient's peripheral mononuclear cells resulted in reduction in CFU-E and BFU-E colonies. It was interesting that CFU-E and BFU-E were normalized after high-dose intravenous immunoglobulin therapy. Patient 2, whose diagnosis was made at 3 months of age, responded to prednisolone treatment at the early phase but became dependent on it thereafter. Thus, bolus methylprednisolone and high-dose intravenous immunoglobulin were given, without effect. Unlike patient 1, bolus methylprednisolone therapy induced reticulocytosis once. During high-dose intravenous immunoglobulin therapy, hemolysis was also observed. In colony assay, CFU-E and BFU-E decreased during the course, but were not corrected by adding prednisolone to the assay system. These findings suggest that in vitro colony assay is not always correlated with response to various therapies, and congenital pure red cell aplasia seems to be a heterogeneous disorder. The indication for high-dose intravenous immunoglobulin therapy for this disorder is limited because of hemolysis complicating the therapy.

To clarify the pathogenesis of fetal-like erythropoiesis including high levels of Hb F observed in trisomy 13 syndrome, we examined the biosynthesis rates of G gamma and A gamma globin chains in the erythropoietic bursts cultured from the peripheral blood mononuclear cells of 3 patients aged 1-month-, 2-month and 7-month-old. Globin chains were labeled with 14C-amino acids, separated by isoelectric focusing and quantitated by autoradiography. The synthesis rates of gamma-chains in the erythropoietic bursts were between 83% and 88%. The Gr:A gamma ratios were similar to the synthesis ratio (0.71) of fetal liver BFU-E (12-wk gestation). Furthermore, the G gamma values in the bursts of the patients were in agreement with the G gamma:A gamma ratios in their circulating red blood cells (0.70, 0.71 and 0.67). These results suggest that the erythropoiesis associated with high levels of Hb F in trisomy 13 syndrome is controlled by erythropoietic precursor cells, in which normal switchings of G gamma:A gamma ratio and from Hb F to Hb A have not occurred.

To investigate the role of erythropoietin in aplastic anemia, the effects of high titers of recombinant human erythropoietin (rh-Ep) on CFU-E and BFU-E in patients with aplastic anemia were studied in vitro. Colony assays were performed by methylcellulose culture methods added with 1 to 500 units of rh-Ep. In normal bone marrow, the maximum CFU-E colony formation was observed at 2 to 5 units of rh-Ep, and BFU-E at 2 to 10 units. Colonies did not increase by addition of higher titer of rh-Ep to the cultures. In aplastic anemia, the numbers of CFU-E and BFU-E were low at 2 units of rh-Ep in culture system. In most patients with aplastic anemia studied, erythroid colonies were increased in accordance with the increase of rh-Ep added to cultures. These results suggest that the administration of high titers of rh-Ep in vivo may be useful for the improvement of anemia in aplastic anemia.

Six patients underwent allogeneic bone marrow transplantation (BMT) for treatment of acute non-lymphocytic leukemia. Hemopoietic reconstitution after BMT was monitored by peripheral blood counts, counts of bone marrow cellularity, bone marrow pictures, and clonal assays for myeloid progenitors (CFU-GM). Although bone marrow samples were markedly hypocellular on day 7 posttransplant, myeloid and erythroid elements were seen in 5 of 6 patients. Peripheral blood recovery of these 5 patients was achieved by third weeks posttransplant. The values of (CFU-GM) per 1 X 10(5) marrow mononuclear cells reached normal values on day 7 in two patients and significantly increased by day 28 in a patient. After day 84 the values of (CFU-GM) were remained almost normal and they had no relation to the occurrence of chronic graft-versus-host disease (GVHD). But in patients with chronic GVHD, marrow (CFU-GM) values were significantly increased on day 7 and day 14. These results suggest that marrow (CFU-GM) values by day 28 may predict the occurrence of chronic GVHD.

Effects of Juzen-taiho-toh (TJ-48) on the recovery of haemopoietic systems from radiation injury are analyzed. Female C57BL/6 mice (6 to 8 week-old) were irradiated at doses of 0, 1, 2, 3, 5 or 7 Gy from a 60Co source. During the 7 days after irradiation, the mice were given TJ-48 solution (1.25g in 100ml distilled water). Seven days after irradiation, the mice were sacrificed, and cell counts of bone marrow (both femurs), thymus, spleen, and peripheral blood were made. The bone marrow cells were used for CFU-f, CFU-GM, BFU-E, and CFU-E assays. No difference were observed between the experimental and control groups. However, the CFU-S. d14 and CFU-S.d9 counts in the TJ-48-treated groups were greater than the respective values in the control groups, especially in the low-dose range of irradiation (1, 2, 3 Gy). In the assay for CFU-S.d14, the mice injected with TJ-48-treated bone marrow cells showed better general condition, heavier spleens with larger and more numerous colonies than the control mice. There was no difference in the number of CFU-f between TJ-48-treated and control groups. Since the CFU-S.d14 assay is thought to reflect the most primitive progenitor cells in the haemopoietic system, these results strongly suggest that TJ-48 acts on stem cells in the G0 phase to manifest recovery-enhancing effects from radiation injury.

Seven patients with myelodysplastic syndromes (M-DS) were treated with 30 mg Bestatin daily. Six of the patients (85.7%) responded. Clonogenic bone marrow cell culture studies in the patients have demonstrated intrinsic hemopoietic stem cell and progenitor cell abnormalities. After Bestatin treatment, these abnormalities involved in differentiation and maturation of hematopoietic progenitors were markedly improved and resulted in the improvement of hematological findings in responders. Addition to the culture system of Bestatin at a concentration of 0.01 microgram/ml and 1 microgram/ml enhanced CD4 positive T cell colony formation without enhancing CFU-GM and CFU-GEMM. These results suggest that the effects on hematopoiesis of Bestatin may be mediated by T cells, not by direct action of the drug.

The in vitro effects of the causative drugs and lymphocytes from the patients with agranulocytosis were tested against granulocyte-macrophage colony formation (CFU-C) of bone marrow cells from normal individuals and the patients in recovery stage. A semisolid culture system was used for CFU-C assay. The drug concentrations were adjusted to the therapeutic levels in sera, and the lymphocytes were obtained from the patient's peripheral blood. Three patients with agranulocytosis and one patient with pancytopenia caused by disopyramide, methimazole, sodium valproate, and Towasaal, respectively, were examined. Each of the four drugs except disopyramide suppressed the CFU-C of normal and patient's bone marrow cells in a dose-dependent manner. When the patient's bone marrow cells were cultured with respective drugs and their own lymphocytes or with the culture supernatant of the drug and lymphocytes, CFU-C suppressions was significantly augmented. Phenacetin, an agent of Towasaal, significantly suppressed CFU-C and also CFU-E. These results indicate that humoral factor(s) produced from patient's lymphocytes by reacting with the drugs may function as an immunological mechanism in the patients with drug-induced agranulocytosis.

Peripheral blood stem cells released following chemotherapy were examined in 21 children with malignancies in early remission. In order to obtain more than 1 x 10(5) CFU-GM per liter which is the minimal concentration to achieve autologous blood stem cell transplantation by cytapheresis, myelosuppressive chemotherapy which reduced leukocyte count below 1,000/microliters or neutrophil count below 200/microliters was necessary. Because the repetition of chemotherapy reduced the release of CFU-GM in peripheral blood, blood stem cells should be collected early after the beginning of chemotherapy. By long term culture method, peripheral blood stem cells seemed to contain pluripotent stem cells. Using our therapeutic protocol, more than 1 x 10(5) CFU-GM per liter could be obtained in malignant lymphoma and acute non-lymphoblastic leukemia, thus autologous blood stem cell transplantation seemed to be possible in these diseases.

Glycosphingolipids (GSLs), which are amphipathic molecules composed of both hydrophobic and hydrophilic moieties and synthesized by a group of Golgi enzymes, glycosyltransferases, are located almost exclusively on the outer leaflet of plasma membranes of mammalian cells in general, and recently have become known to show an unexpectedly vast molecular heterogeneity. Furthermore, GSLs have been considered to be involved in cellular interactions and cell growth regulations, changing characteristically their composition and biosynthetic pathway during cell development, differentiation and oncogenic transformation although they constitute only a small portion of the cell surface glycoconjugates. In addition, acidic GSLs, gangliosides, have been recently shown to exhibit special receptor-functions for exogenous, bioactive factors such as bacterial toxins, hormones and biological response modifiers. We and other investigators have found that human and murine hemopoietic malignant cells show ganglioside-patterns characteristic of their cell lineages and differentiation-stages, serving an differentiation-markers for both normal and malignant hemopoietic cells, and further, discovered that particular ganglioside molecules themselves, which specifically increase during differentiation along particular cell lineages induced by a variety of chemical agents, exhibit remarkably potent differentiation-inducing and growth-inhibitory activities on human myelogenous leukemia cells. On the basis of these findings, some important biological functions of GSLs, especially gangliosides, will be discussed in special reference to hemopoietic cell differentiation.

The chemotherapy of malignant brain tumors has been, only partially successful yet. Recently major concern is drug resistance, one of possible mechanisms of such drug resistance stems from inducible repair enzyme, especially in case of chloroethylnitrosoureas as ACNU or BCNU. We examined the changes of acquired resistance to ACNU in rat glioma cells by pretreatment with O6-methylguanine, which is a substrate for O6-methylguanine methyltransferase. ACNU-resistant (9L/AC) cells had established after 10 times treatments of ACNU. 9L/AC cells were pretreated with 2 mM O6-methylguanine for 2 hours, and subsequently challenged with increasing doses of ACNU for 2 hours. In vitro colony formation assay the survival fraction of 9L and 9L/AC cells ranged from 0.39 to 0.63 by 2-hour reaction of 1-3 mM O6-methylguanine. Based on the dose-response curve for ACNU in 9L/AC cells, by O6-methylguanine pretreatment (2 mM), ACNU-resistance decreased markedly to one-third, one-fifth, and one-two hundredth at 12, 24, 36 microM ACNU, respectively. In contrast, the survival of 9L cells against ACNU was similar under O6-methylguanine pretreatment or nontreatment condition. Therefore, ACNU-resistance is considerably related to DNA repair enzyme induction, and the substrates may potentiate the cell-killing effect of ACNU in the resistant glioma cells.