Kazuo Motoyoshi

Functional CD5+ B cells develop predominantly in the spleen of NOD/SCID/γcnull (NOG) mice transplanted either with human umbilical cord blood, bone marrow, or mobilized peripheral blood CD34+ cells

OBJECTIVE Human CD5+ B cells are the major B cell subset in fetal spleen and umbilical cord blood (CB), and their number gradually diminishes in both spleen and peripheral blood from infancy through childhood while conventional B cells increase. In this study, we investigated whether CD5+ cells differentiate from adult hematopoietic stem cells (HSCs) as well as fetal ones in immunodeficient mice. METHODS In our system, NOD/SCID/gammac(null) (NOG) mice were transplanted with CD34+ cells from CB (hCB model), adult bone marrow (hBM model), and mobilized peripheral blood (hMPB model). RESULTS In these model mice, a high proportion of CD19+IgM+CD5+ mature B cells appeared in the spleen, regardless of the CD34+ cell origin, 4 to 8 weeks after transplantation, while the majority were CD19+IgM-CD5- immature B cells in BM. The CD19+CD5- BM cells showed to express CD5 after the coculture with NOG spleen cells. In the sera of immunized hCB model mice with DNP-KLH, antigen-specific IgM but not IgG was enhanced. CONCLUSION Our results show that adult CD34+ cells develop into functional CD5+ B cells in NOG spleen as much as fetal CD34+ cells do.

Double-blind test of human urinary macrophage colony-stimulating factor for allogeneic and syngeneic bone marrow transplantation: effectiveness of treatment and 2-year follow-up for relapse of leukaemia

Summary A randomized, double‐blind placebo‐controlled phase III clinical trial was performed to study the effects of human urinary macrophage colony‐stimulating factor (hM‐CSF) after allogeneic and syngeneic bone marrow transplantation (BMT) in 60 hM‐CSP treated and 59 placebo control patients. HM‐CSF was administered at a daily dose of 2 ± 105 units/kg from day 1 to day 14 after RMT. Significant differences between hM‐CSF and control patients were found in the recovery time to greater than 0. 5 ± 109 granulocytes/1 and the survival rate during the initial 120 d without retransplantation. There was no difference in the incidence or grade of graft‐versus‐host disease (GVHD). There was no difference in the rate of leukaemic relapse at 24–36 months after BMT in patients with acute lymphocytic. acute non‐lymphocytic, or monocytic leukaemia. The results of this trial show that human M‐CSF improves the outcome of BMT without any influence on the occurrence of leukaemic relapse or GVHD.

Cyclic change of cytokines in a patient with cyclic thrombocytopenia.

The serial change of various cytokines in the serum from a patient with cyclic thrombocytopenia is described. Interleukin 7, stem cell factor, and transforming growth factor β1 synchronized with the platelet count, and there was a significant positive correlation between the three cytokines and the platelet count. Levels of macrophage colony‐stimulating factor, thrombopoietin, platelet‐associated IgG and erythropoietin changed reciprocally with the platelet count, and there was a significant negative correlation between the platelet count and these cytokines except erythropoietin. No cyclic change was observed in IL‐3, IL‐6, IL‐11, granulocyte‐macrophage colony‐stimulating factor, or leukaemia inhibitory factor. These observations suggest that this disease involves two cyclic changes: megakaryocytopoiesis and platelet destruction, in both of which the cytokines play an important role.

Burkitt's acute lymphoblastic leukaemia transformatation after myelodysplastic syndrome

We describe a patient with myelodysplastic syndrome (MDS) that transformed to Burkitts acute lymphoblastic leukaemia (ALL). The leukaemic blasts were negative for peroxidase staining, and expressed CD10, CD19, CD22, CD38, human leucocyte antigen (HLA)‐DR and surface immunoglobulin (sIg) M, but neither sIgD nor sIgG were expressed. Chromosomal study during the ALL phase showed t(8;22)(q24;q11) in addition to the karyotypes determined during the MDS phase. Furthermore, overexpression of c‐myc mRNA was confirmed in ALL blasts. These findings indicate that MDS transformed to Burkitts ALL through multiple cytogenetic evolutions, the final event of which seems to be overexpression of the c‐myc gene.

Effect of Macrophage Colony-Stimulating Factor (M-CSF) on Mouse Immune Responses in Vivo

We examined the effects of recombinant human M-CSF (rhM-CSF) on mouse macrophages and immune responses in vivo. Intraperitoneal administration of rhM-CSF (20-500 microgram/ml) increased Mac-1+ cell numbers in the peritoneal cavity. The tumoricidal activities of the macrophages from vehicle-administered (V-M phi) and from rhM-CSF-administered (M-M phi) mice were the same as those observed in vitro. However, when activated by lipopolysaccharide (LPS), the tumoricidal activity of M-M phi was stronger than that of V-M phi. Intravenous administration of rhM-CSF (500 micrograms/gk) increased the number of spleen cells. Flow cytometric analysis showed that administration of rhM-CSF increased Mac-1+, B220+ and NK 1.1+ cell counts in the spleen. However, CD4+ and CD8+ cell numbers did not change. Concomitant increases were observed in levels of IL-4 and IL-10 in mouse serum following rhM-CSF administration, but no significant changes were observed in the serum level of IFN-gamma. In experiments involving mouse immune responses, the administration of rhM-CSF reduced the contact sensitivity (CS) reaction against picryl chloride (PC) and augmented IgE production in response to 2,4-dinitrophenyl (DNP), but did not affect the production of either IgM or IgC1. These results suggest that administration of rhM-CSF not only activates murine macrophages, but modulates antigen-specific immune responses in vivo.

Second relapse of acute promyelocytic leukemia (ANLL-M3) with t(15;17) and t(1;3)(p36;q21)☆

We describe herein a patient with acute promyelocytic leukemia (APL)-(ANLL-M3) whose bone marrow cells in the second relapse showed t(1;3)(p36;q21) together with t(15;17) (q22;q11-q12). Although a total of 21 patients with t(1;3) have been reported so far, among which three cases with de novo acute nonlymphocytic leukemia were included, our patient is the first case with APL. The hematologic findings in our case confirmed the previous observations that this anomaly is associated with relatively high platelet count and the multi-myeloid lineage involvement of leukemic cells. Our patient responded well to chemotherapy and achieved first and second remission with 42 months of total survival, contrary to our expectation that patients with this anomaly have a poor prognosis.

Effect of Human Macrophage Colony‐stimulating Factor on Granulopoiesis and Survival in Bone‐marrow‐transplanted Mice

Human macrophage colony‐stimulating factor (hM‐CSF) has been isolated from normal human urine and purified to a homogenous protein. The effect of hM‐CSF on granulopoiesis was investigated in BALB/c mice transplanted with a suboptimal number of bone marrow cells. Lethally irradiated (7.8 Gy) mice were transplanted with 1 × 106 syngeneic mouse bone marrow cells and treated with a daily intraperitoneal dose of 64 μg/kg of hM‐CSF for 5 days following the transplant. The hM‐CSF injection resulted in stimulation of the recovery of blood neutrophils as well as an increase in the number of granulocyte‐macrophage progenitor cells (CFU‐GM) in the femur and spleen. The survival of lethally irradiated mice was dependent on the cell number transplanted; most mice transplanted with 2 × 104 cells died within 2 weeks. The recovery of hematopoiesis in mice transplanted with 2 × 104 cells was modestly but significantly stimulated by hM‐CSF administration initiated from 5 days before or 1 day after transplantation for a 5‐day period. Furthermore, the hM‐CSF administrations markedly reduced the mortality in these mice during the early period after the transplantation. Since anaerobic bacteria were frequently detected in arterial blood immediately before the deaths but were not found in the surviving mice, it is speculated that early deaths occurring within 2 weeks after the transplant may be caused by opportunistic infections, and hM‐CSF injection may prevent these mortal infections through its stimulating effect on monocyte‐macrophage functions that are responsible for the production of hematopoietic regulators.

Effects of the in vivo Administration of Recombinant Human Granulocyte Colony‐stimulating Factor Following Cytotoxic Chemotherapy on Granulocytic Precursors in Patients with Malignant Lymphoma

We examined the effects of the in vivo administration of recombinant granulocytc colony‐stimulating factor (rhG‐CSF) on granulocytic precursors in the bone marrow of 4 patients with malignant lymphoma who received chemotherapy. Patients were treated with rhG‐CSF at doses of 100–800 μg/ m2/day intravenously for 14 days only in the first course of chemotherapy (G‐CSF course) followed by the second course of chemotherapy without rhG‐CSF which was used as a control course. In the G‐CSF course, white blood cell counts (WBCs) demonstrated a biphasic response consisting of a first peak observed within a few days after the initiation of rhG‐CSF administration, and a second peak observed on the last day of rhG‐CSF injection or the day after. In the second peak, the incidence of granulocyte‐macrophage colony‐forming units (CFU‐GM) in mononucleated bone marrow cells did not change significantly after treatment with rhG‐CSF as compared with a control. However, since the number of nucleated cells in the bone marrow increased, the absolute number of CFU‐GM in the bone marrow increased. The number of mature and immature granulocytes in the bone marrow increased. These findings suggest that G‐CSF stimulates the proliferation and differentiation of granulocytic precursors in the bone marrow in granulocytopenic patients who received cytotoxic drugs and causes mature granulocytes to be released from the bone marrow.

Comparative study and effects of macrophage colony-stimulating factor (M-CSF) administration on NK1.1+ cells in mouse spleen and bone marrow.

ABSTRACT We conducted a comparative study of NK1.1+ cells in spleen and bone marrow and the effects of administration of M-CSF on them. Administration of M-CSF to mice increased the number of NK1.1+ cells in spleen but not in bone marrow. The NK1.1+ cells in spleen (Spl-NK1.1) and bone marrow (BM-NK1.1) were purified by magnetic cell sorter. Their cell surface markers and functions were then examined. The percentage of Mac-1 antigen-positive cells (and F4/80 antigen-positive cells) was higher among BM-NK1.1 than Spl-NK1.1. Moreover, the administration of M-CSF increased the number of Mac-1 and F4/80 antigen-positive cells in both Spl-Nk1.1 and BM-NK1.1. The functions (cytolytic activity and IFN-γ production) of Spl-NK1.1 and BM-NK1.1 were the same and were enhanced by the administration of M-CSF. But Spl-NK1.1 produced more IFN-γ than BM-NK1.1 when M-CSF was administered. BM-NK1.1 showed a greater proliferative response to IL-2 than Spl-NK1.1. Administration of M-CSF augmented this response. BM-NK1.1 proliferated in response to IL-4 and IL-15, but Spl-NK1.1 responded only slightly. However, administration of M-CSF stimulated Spl-NK1.1 to respond to these cytokines. Both Spl-NK1.1 and BM-NK1.1 showed only a weak response to M-CSF in vitro. But the expression of c-fms antigen (M-CSFR) increased after the M-CSF injections in vivo. These results suggested that there are phenotypical and functional differences between Spl-NK1.1 and BM-NK1.1. The administration of M-CSF led to an accumulation of NK1.1+ cells which were mobilized from bone marrow in spleen.

Effect of coadministration of M-CSF and IFN-α on NK1.1+ cells in mice

The purpose of this study was to evaluate the effect of coadministration of macrophage colony-stimulating factor (M-CSF) and interferon- a (IFN-a) on NK1.1 1 cells in mice. Administration of M-CSF, but not IFNa, increased the number of NK1.1 1 cells and CD11b 1 cells in spleen and blood. Coadministration of the two agents induced a greater increase in NK1.1 1 cells than did administration of M-CSF alone. Administration of M-CSF or IFN- a augmented the clearance activity of Yac-1 cells in lung, and coadministration of these agents further augmented this effect. The combination of M-CSF and IFN- a effectively reduced the formation of tumor nodules in lung and liver in an experimental metastasis model using B16 melanoma. The combination of M-CSF and IFN- a induced the increase and activation of NK1.1 1 cells more than either agent alone. These effects may contribute to the antimetastatic reaction by NK1.1 1 cells in vivo.