Harumi Y. Mukai

Major basic protein binding to thrombomodulin potentially contributes to the thrombosis in patients with eosinophilia

Summary. The contribution of an eosinophil granule protein, major basic protein (MBP), to the pathogenesis of thrombosis seen in patients with eosinophilia was investigated. The sera from eosinophilic patients containing elevated levels of MBP inhibited thrombomodulin (TM) function as a cofactor for the thrombin‐catalysed activation of protein C more significantly than those from normal individuals (means 48.5%v 17.4%, respectively). It was suggested that the binding of mature MBP in the sera to TM was electrostatic, because mature MBP (pi 10.9) bound to TM, whereas pro‐MBP (pi 6.2) did not. The inhibition of TM cofactor activity by eosinophil granule proteins was mainly attributed to the mature MBP, because MBP‐depleted eosinophil granule proteins did not inhibit TM cofactor activity significantly. This inhibition seemed to be due to the specific thrombin‐binding to TM being blocked. We concluded that eosinophil granule proteins, particularly MBP, potentially contribute to the hypercoagulation seen in some conditions of eosinophilia, at least because of the inhibition of TM function as a cofactor of the anticoagulation system.

Transgene insertion in proximity to the c-myb gene disrupts erythroid-megakaryocytic lineage bifurcation.

ABSTRACT The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival, and differentiation of hematopoietic cells. We established three lines of erythropoietin receptor-transgenic mice and found that one of them exhibited anemia, thrombocythemia, and splenomegaly. These abnormalities were independent of the function of the transgenic erythropoietin receptor and were observed exclusively in mice harboring the transgene homozygously, suggesting transgenic disruption of a certain gene. The transgene was inserted 77 kb upstream of the c-myb gene, and c-Myb expression was markedly decreased in megakaryocyte/erythrocyte lineage-restricted progenitors (MEPs) of the homozygous mutant mice. In the bone marrows and spleens of the mutant mice, numbers of megakaryocytes were increased and numbers of erythroid progenitors were decreased. These abnormalities were reproducible in vitro in a coculture assay of MEPs with OP9 cells but eliminated by the retroviral expression of c-Myb in MEPs. The erythroid/megakaryocytic abnormalities were reconstituted in mice in vivo by transplantation of mutant mouse bone marrow cells. These results demonstrate that the transgene insertion into the c-myb gene far upstream regulatory region affects the gene expression at the stage of MEPs, leading to an imbalance between erythroid and megakaryocytic cells, and suggest that c-Myb is an essential regulator of the erythroid-megakaryocytic lineage bifurcation.

Serum thrombopoietin level is mainly regulated by megakaryocyte mass rather than platelet mass in human subjects.

A patient with idiopathic thrombocytopenic purpura (ITP) developed T‐cell lymphoma while undergoing steroid therapy. We examined the relationship between the patients serum thrombopoietin (Tpo) level, platelet count, megakaryocyte number and CFU‐Meg number during the second 5 d course of chemotherapy for lymphoma in which megakaryopoiesis switched from ITP phase to amegakaryocytic phase. The patients platelet count was temporarily elevated but CFU‐Meg numbers were markedly suppressed, and megakaryocyte numbers were decreased in this period, whereas serum Tpo level was not suppressed despite an increased platelet count, indicating that serum Tpo level is mainly regulated by megakaryocyte mass.

Successful treatment of a patient with subcutaneous panniculitis-like T-cell lymphoma with high-dose chemotherapy and total body irradiation.

Abstract: A 24‐yr‐old man was referred for fever, right cheek swelling, subcutaneous tumor and liver dysfunction. Physical examination showed an elastic hard subcutaneous tumor on the right cheek, left axillary lymph node swelling and multiple small subcutaneous tumors in the trunk. Laboratory examinations showed elevated levels of transaminase, soluble interleukin‐2 receptor and ferritin. Biopsy of the subcutaneous tumor showed proliferation of medium‐sized cells with abundant clear cytoplasm and hyperchromatic nuclei among the subcutaneous fat tissues. These cells showed CD3+, CD4−, CD8+, CD56− and CD20− phenotype and possessed cytotoxic molecules such as granzyme B and T‐cell intracellular antigen‐1. Bone marrow aspiration showed proliferation of small numbers of abnormal lymphocytes with severe hemophagocytosis. He was thus diagnosed as having subcutaneous panniculitis‐like T‐cell lymphoma (SPTCL) and treated with dose‐escalated CHOP regimen. After three courses of the chemotherapy, he was further treated with high‐dose chemotherapy and total body irradiation (TBI) with autologous peripheral blood stem cell rescue. Thereafter, he has been in remission for more than 2 yr. We consider that SPTCL with hemophagocytosis is an extremely aggressive disease, and high‐dose chemotherapy and TBI should be included for the choice of the treatment.

Stimulation of GATA-2 as a mechanism of hydrogen peroxide suppression in hypoxia-induced erythropoietin gene expression

Hydrogen peroxide (H2O2) has previously been shown to inhibit the DNA binding activity of hypoxia inducible factor‐1 (HIF‐1), the accumulation of HIF‐1α protein and erythropoietin (Epo) gene expression. Epo gene expression has been previously shown to be down‐regulated through a GATA binding site at its promoter region. In this study, the effect of H2O2 on Epo gene expression under hypoxic conditions through a GATA transcription factor was investigated. Hypoxic induction was found to be inhibited upon the addition of H2O2, and this effect could be reversed through the addition of catalase. Hypoxic induction was found to be suppressed by co‐transfection with a human GATA‐2 cDNA expression plasmid. Transfection of Hep3B cells with a reporter gene bearing a mutation at the promoter GATA binding site was found to be only mildly affected by the addition of H2O2. Electrophoretic gel mobility shift assays (EMSAs), using the Epo promoter GATA site as a probe and the GATA‐2 protein extracted from Hep3B cells, showed that addition of H2O2 enhanced the binding of GATA‐2 while addition of catalase inhibited this binding. From these results, we conclude that H2O2 increases the binding activity of GATA‐2 in a specific manner, thereby suppressing the activity of the Epo promoter and thus inhibiting Epo gene expression. J. Cell. Physiol. 186:260–267, 2001.

High-Dose Chemotherapy with Peripheral Blood Stem Cell Rescue in Blastoid Natural Killer Cell Lymphoma

A 25-year-old man was referred because of skin rash, lymphadenopathy and anemia. Laboratory examinations revealed severe anemia (Hb, 4.8 g/dl) and elevated levels of GOT, GPT, LDH and soluble interleukin-2 receptor. Work-up studies disclosed the involvement of lymphoma cells in lymph nodes, skin, bilateral kidneys and bone marrow. Lymph node biopsy revealed diffuse proliferation of medium- to large-sized lymphoblastic cells. Bone marrow aspiration showed massive infiltration of large blastic cells with no cytoplasmic granules. The lymphoma cells in bone marrow and lymph node showed surface CD3-, cytoplasmic CD3epsilon+, CD4+, CD8-, CD56+, CD57-, CD16- and CD43 (MT-1)+ phenotype. Analyses of T cell receptor beta and gamma genes showed germ line configurations. EBER-1 was not detectable in the lymphoma cells. He was diagnosed as having blastoid natural killer (NK) cell lymphoma. In spite of several courses of combination chemotherapy, the lymphoma was progressive. He was then treated with high-dose chemotherapy and peripheral blood stem cell rescue, achieving remission which has now lasted for more than 12 months. We consider that blastoid NK cell lymphoma is an extremely aggressive subtype of CD56-positive lymphomas, and high-dose chemotherapy with peripheral blood stem cell rescue should be included for the choice of the treatment.

Hemophagocytic syndrome as the primary clinical symptom of Hodgkin's disease.

Abstract. It is now well recognized that hemophagocytic syndrome (HPS) is occasionally associated with malignant lymphomas. However, its association with Hodgkins disease has been only rarely reported. We present here a 72-year-old woman manifesting with HPS as the primary and solitary clinical symptom of Hodgkins disease. She had been suffering from high-grade fever and anemia for more than a month. Based on the findings in bone marrow aspirates, she was diagnosed as having HPS. In spite of extensive surveys including various cultures, serological tests for collagen disease, abdominal and cardiac sonography, chest computed tomography (CT), and renal biopsy, the origin of the fever was not determined. She was treated with steroid pulse therapy and then referred. Radiological studies revealed only mild hepatosplenomegaly and small lymph node swellings around celiac and common hepatic arteries. Reevaluation of the bone marrow specimen revealed the infiltration of small numbers of CD30-, CD15-, and EBER-1-positive large-sized lymphocytes with bizarre nucleus. Under the diagnosis of Hodgkins disease, she was treated with combination chemotherapy containing pirarubicin, cyclophosphamide, vincristine, and prednisolone. However, it was not effective and she died of rapidly progressive hepatic failure on the 5th day of the chemotherapy. Autopsy was performed, which showed proliferation of lymphoma cells in para-aortic lymph nodes. We believe that diagnostic survey to rule out the underlying lymphoma should be vigorously performed for patients with hemophagocytic syndrome of unknown origin.

Disruption of the Hbs1l-Myb Locus Causes Hereditary Persistence of Fetal Hemoglobin in a Mouse Model

ABSTRACT The human β-globin locus is comprised of embryonic, fetal, and adult globin genes, each of which is expressed at distinct stages of pre- and postnatal development. Functional defects in globin proteins or expression results in mild to severe anemia, such as in sickle-cell disease or β-thalassemia, but the clinical symptoms of both disorders are ameliorated by persistent expression of the fetal globin genes. Recent genome-wide association studies (GWAS) identified the intergenic region between the HBS1L and MYB loci as a candidate modifier of fetal hemoglobin expression in adults. However, it remains to be clarified whether the enhancer activity within the HBS1L-MYB regulatory domain contributes to the production of fetal hemoglobin in adults. Here we report a new mouse model of hereditary persistence of fetal hemoglobin (HPFH) in which a transgene was randomly inserted into the orthologous murine Hbs1l-Myb locus. This mutant mouse exhibited typically elevated expression of embryonic globins and hematopoietic parameters similar to those observed in human HPFH. These results support the contention that mutation of the HBS1L-MYB genomic domain is responsible for elevated expression of the fetal globin genes, and this model serves as an important means for the analysis of networks that regulate fetal globin gene expression.

Detection of the STAT5B–RARA fusion transcript in acute promyelocytic leukemia with the normal chromosome 17 on G-banding

Acute promyelocytic leukemia (APL) is characterized by chromosomal rearrangements of 17q21, leading to fusion of the gene‐encoding retinoic acid receptor alpha (RARA) with a number of alternative partner genes. Signal transducer and activator of transcription 5 beta (STAT5B) is one of the alternative partners. We report a rare case of APL with STAT5B–RARA fusion transcript and the normal chromosome 17 on G‐banding. Administration of all trans‐retinoic acid improved disseminated intravascular coagulation without decrease of the leukemia cells in his peripheral blood and bone marrow. The molecular mechanism of fusion between STAT5B and RARA by chromosomal rearrangement is discussed based on the data from genome database. Clinical characteristics of APL with STAT5B–RARA are also discussed.

GATA Suppresses Erythropoietin Gene Expression through GATA Site in Mouse Erythropoietin Gene Promoter

The promoter and enhancer elements of the mouse erythropoietin (mEpo) gene, which have high homology with those of the human erythropoietin (hEpo) gene, were fused withluciferase. The construct was transfected into erythropoietin-producing hepatoma cell line (Hep3B) cells by lipofectin with lacZ as an internal standard. The wild type (TGATA) showed a 39.5-fold increase in induction by hypoxia. Mouse GATA-2 inhibited the hypoxic induction of the wild-type (m3), promoter-luciferase construct but not the hypoxic induction of the mutant (m4, 5) promoter-luciferase constructs. NG-monomethyl L-arginine (L-NMMA) inhibited the hypoxic induction of the m3 promoter-luciferase construct, but this inhibition was recovered by L-arginine. H2O2 also inhibited the hypoxic induction of the m3 promoter-luciferase construct, but this inhibition was recovered by catalase. Gel shift assays performed on nuclear extracts of 293 cells overexpressing mGATA-1, -2, and -3 revealed that mGATA-1, -2, and -3 bind to the TGATA element of the mEpo promoter. These results indicate that mGATA binds to the TGATA site of the mEpo promoter and negatively regulates mEpo gene expression. Negative regulation of mEpo gene by GATA transcriptional factors is discussed.