Ryukichi Ryo

Establishment of a human leukaemic cell line (CMK) with megakaryocytic characteristics from a Down's syndrome patient with acute megakaryoblastic leukaemia

Summary. A new megakaryoblastic cell line (CMK), which also exhibits erythroid and myeloid markers, was established from a Downs syndrome patient suffering from acute megakaryoblastic leukaemia. The CMK cells were found to be positive in reactions with anti‐platelet antibodies (anti‐glycoproteins IIb/IIIa and Ib, and Plt‐1). Platelet peroxidase (PPO) reactivity was found to be associated with the nuclear envelope and the endoplasmic reticulum but not with the Golgi apparatus. Some cells possessed cytoplasmic granules with the characteristics of α‐granules and demarcation membranes. Karyotyping revealed near‐tetraploidy (modal chromosome number of 95; ranging 87–98) and a translocation der(17)t(11;17), also found in the original leukaemic cells, confirming that the cells were derived from the patients malignant blasts. The CMK cells were also found to be positive in reaction with anti‐glycophorin A antibody, as well as with anti‐myeloid antibodies (MY4, MY7 and MY9). Treatment of CMK cells with phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) greatly enhanced the reactivity with anti‐platelet antibodies, increased the number of cells in which cytoplasm was dissociated into numerous segments and suppressed the reactivity with anti‐glycophorin A. The proliferation of CMK cells was stimulated by interleukin‐3 (IL‐3) and granulocyte‐macrophage colony stimulation factor (GM‐CSF). This cell line should be a useful tool for analysing the basis of the afferent association between megakaryoblastic leukaemia and Downs syndrome, as well as for further study of megakaryocytic differentiation.

Transfusion-Related Acute Lung Injury in a Patient with Acute Myelogenous Leukaemia Having Anti-IgA2m(1) Antibody

A 69-year-old man with acute myelogenous leukaemia developed a transfusion-related acute lung injury (TRALI). He had anti-IgA2m(1) antibody rather than other antibodies that have previously been reported to be related to TRALI. This case suggests that the pre-existing condition of patients may be important in the development of TRALI.

Platelet release reaction during EDTA-induced platelet agglutinations and inhibition of EDTA-induced platelet agglutination by anti-glycoprotein IIb/IIIa complex monoclonal antibody

To characterize the nature of EDTA-induced platelet agglutination, the spontaneous release of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) was examined during EDTA-induced platelet agglutinations. A slight release of beta-TG and PF4 was observed when EDTA-anticoagulated whole blood from cases with EDTA-induced platelet agglutination was kept for 60 minutes, whereas a high spontaneous release of these proteins was found from normal blood anticoagulated with EDTA. These findings imply that EDTA-dependent platelet agglutinin may stabilize the platelet membrane surfaces. Secondly, we found that pretreatment of fresh blood with anti-glycoprotein (GP) II b/III a complex monoclonal antibody dramatically reduced EDTA-induced platelet agglutinations. This study indicated that the binding sites of EDTA-dependent antibody might be GP II b/III a complex. The use of an anti-GP II b/III a complex monoclonal antibody may be useful in avoiding analytical errors in some cases with EDTA-induced pseudothrombocytopenia.

Treatment of post-transfusion graft-versus-host disease with nafmostat mesilate, a serine protease inhibitor

Background: Cytotoxic T lymphocytes from donors are thought to injure the target organs in post–transfusion graft–versus–host disease (PT–GVHD) through perforin–granzyme– and Fas–dependent cell killings. The protease involved is a serine protease, and nafmostat mesilate (NM), a serine protease inhibitor, has been found to inhibit the in vitro allocytotoxicity of the T cell clone established from a patient with PT–GVHD, thus suggesting the usefulness of NM for treatment of PT–GVHD. Case Report: A 47–year–old male with esophageal cancer, who received 3 units of packed red cells and 20 units of platelet concentrates from 5 unrelated donors, was diagnosed as having PT–GVHD on the basis of typical clinical features, HLA typing of the patient and the responsible donor, and a mixed chimera of CD8+ lymphocytes on microsatellite DNA polymorphism analysis. NM was administered to inhibit the activity of the serine proteases, thought to be granzymes; a liver dysfunction and thrombocytopenia with leukocytopenia simultaneously improved. Subsequently, a high–dose methylprednisolone pulse therapy and monoclonal anti–CD3 were administered to reduce the donor’s proliferating lymphocytes, which resulted in lymphopenia accompanied by elimination of the donor’s lymphocytes and normalization of the CD4/CD8 ratio. However, recurrence of the proliferation of the responsible donor’s lymphocytes developed after cessation of NM administration, probably because of excessive immunosuppression caused by steroids and the monoclonal anti–CD3.Conclusion:This case indicates that administration of a serine protease inhibitor may improve PT–GVHD symptoms by inhibiting cytotoxic T–cell–mediated killing of target cells in fatal PT–GVHD. Steroids and monoclonal anti–CD3 were probably responsible for the transient clinical improvements. More studies are required, however, on mechanisms to eliminate the donor’s lymphocytes.

Response to Granulocyte Colony-Stimulating Factor in an Autoimmune Neutropenic Adult

Clinical value of granulocyte colony-stimulating factor (G-CSF) for autoimmune neutropenia (AIN) has not been well established. We experienced an adult case of AIN which showed an excellent response to G-CSF. A 75-year-old female was admitted with high-grade fever. Her neutrophil count was remarkably low (neutrophil 0.09 × 109/l). Antigranulocyte autoantibody was demonstrated in her serum by an immunofluorescence method and she was diagnosed as AIN. Administration of G-CSF (filgrastim 5 μg/kg) gave a rapid increase of neutrophils (from 0.11 × 109/l to 2.10 × 109/l on the second day), which has enabled us to preserve the use of G-CSF for emergency, that is, for overt serious infection.

Platelet factor 4 mRNA expression in cells from a patient with megakaryoblastic crisis of chronic myelogenous leukemia.

A 61‐year‐old man with Philadelphia chromosome‐positive chronic myelogenous leukemia developed megakaryoblastic leukemia. In the blast phase, his blast cells showed undifferentiated megakaryoblastic characteristics with no α‐granules or demarcation membranes but with detectable platelet peroxidase (PPO) activity and surface glycoprotein (GP) IIb/IIIa. The patient has remained reasonably well for at least 12 months after blastic crisis, and 6‐mercaptopurine alone has been effective in controlling leukocytosis and megakaryoblast proliferation. The expression of mRNA for platelet‐specific proteins, such as GPIIb and platelet factor 4 (PF4), was studied in the patients blast cells by the Northern blot analysis. Both GPIIb and PF4 mRNA were detected in the blast cells. Cytoplasmic maturation occurs later than the synthesis of the surface GP during megakaryocyte maturation. Therefore, PF4 mRNA expression should be a marker of mature megakaryoblasts. The PF4 mRNA expression in megakaryoblastic leukemia may indicate that a patient will have long survival and a good response to chemotherapy.

Refractory anemia terminating in acute megakaryoblastic leukemia (M7).

A 72-year-old man with refractory anemia (RA) developed overt megakaryoblastic leukemia after the course of RA with excess of blasts. The blasts were positive for platelet peroxidase activity and had platelet glycoproteins (GPs) such as GPIIb/IIIa and GPIIIa. The bone marrow biopsy at terminal stage disclosed marked fibrosis. The nature of the megakaryoblasts was investigated. The blasts did not differentiate morphologically into mature megakaryocytes with TPA addition. In vitro colony assay showed the failure of colony-forming unit, megakaryocyte growth in peripheral blood. The pathogenesis of myelofibrosis in our patient is discussed.

Extramedullary tumor as presentation of leukemia: establishment of a new human GPIIb- and GPIIIa-positive leukemia cell line.

SummaryA 25-year-old man noted swelling of the right cervical lymph nodes in October 1983. Diagnosis of malignant lymphoma was made on the basis of pathological examination of biopsies. Despite both chemotherapy and irradiation treatment, blast cells appeared in the peripheral blood and bone marrow in April 1984. Immunophenotypic analysis demonstrated that the blasts in the patients peripheral blood expressed CD13, CD33, CD41a, and no markers for T or B lymphocytes, suggesting that he had been suffering from megakaryocytic sarcoma. We established a new cell line derived from the blasts in the peripheral blood, designated KH184. KH184 cells expressed glycoprotein (GP) Ib (CD42b) and GPIIb/IIIa (CD41a), while platelet peroxidase (PPO) activity was negative in an ultrastructural study. Both Northern blot and flow cytometric analysis of surface antigens and DNA content revealed that treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) did not induce the maturation of these cells. Various cytokines such as interleukin 3 (IL-3), interleukin 6 (IL-6), and leukemia inhibitory factor (LIF) had no effect in promoting the growth of KH184 cells. KH184 cells expressing CD41a seem to possess unusual characteristics. KH184 cells, human GPIIb- and GPIIIa-positive leukemia cells, which lack response to TPA-induced differentiaton, provide a new and unique model for the characterization of factors that are implicated in the terminal differentiation of megakaryocytes, and should aid in studies of the mechanism underlying the occurrence of megakaryocytic sarcoma.

Estimation of Stem Cell Fractions in Peripheral Blood Stem Cell Harvest by Using an SE-9000 Hematology Analyzer

We inquired whether stem cell fractions in peripheral blood stem cell harvest could be precisely detected by using a hematopoietic progenitor cell (HPC) counting system applied to an automated hematology analyzer, SE-9000. Although there was an apparent increase in the HPCs 20 h after storage in nondiluted conditions, samples diluted with RPMI-1640 containing 1.0 mg/ml EDTA-2K showed a relatively stable number of HPCs. There was a significant relationship between HPCs and CD34 cells (n = 75, r = 0.769). This method may represent the least expensive and most time-effective way for stem cell estimation in harvest products.

Inhibition of natural killer cytotoxicity in vitro by clinical grade serine protease inhibitors

The effects of clinical grade serine protease inhibitors on natural killer (NK) activity were compared. Cytotoxicity was measured with the Calcein-AM release method, using K562, Raji as a target. There is a significant correlation between measurements of NK activity by the Calcein-AM method and the 51Cr release assay. Cytotoxicity was inhibited with a calcium chelating agent or a perform inhibitor. Although up to 65% of cytotoxicity was inhibited by nafamostat mesilate with an E/T ratio of 10:1, and by 55% by ulinastatin, neither gabexate mesilate nor antithrombin III inhibited any cytotoxicity. None of these agents inhibited lymphokine-activated killer cell activity. In clinical applications, it should be noted that some protease inhibitors have been proven to have immunosuppressive effects.