Haruhiko Ninomiya

Clinical Course and Flow Cytometric Analysis of Paroxysmal Nocturnal Hemoglobinuria in the United States and Japan

Abstract: To determine and directly compare the clinical course of white and Asian patients with paroxysmal nocturnal hemoglobinuria (PNH), data were collected for epidemiologic analysis on 176 patients from Duke University and 209 patients from Japan. White patients were younger with significantly more classical symptoms of PNH including thrombosis, hemoglobinuria, and infection, while Asian patients were older with more marrow aplasia. The mean fraction of CD59-negative polymorphonuclear cells (PMN) at initial analysis was higher among Duke patients than Japanese patients. In both cohorts, however, a larger PNH clone was associated with classical PNH symptoms, while a smaller PNH clone was associated with marrow aplasia. Thrombosis was significantly more prevalent in white patients than Asian patients, and was associated with a significantly higher proportion of CD59-negative PMN. For individual patients, CD59-negative populations varied considerably over time, but a decreasing PNH clone portended hematopoietic failure. Survival analysis revealed a similar death rate in each group, although causes of death were different and significantly more Duke patients died from thrombosis. Japanese patients had a longer mean survival time (32.1 yr vs. 19.4 yr), although Kaplan-Meier survival curves were not significantly different. Poor survival in both groups was associated with age over 50 years, severe leukopenia/neutropenia at diagnosis, and severe infection as a complication; additionally, thrombosis at diagnosis or follow-up for Duke patients and renal failure for Japanese patients were poor prognostic factors. These data identify important differences between white and Asian patients with PNH. Identification of prognostic factors will help the design of prospective clinical trials for PNH. Abbreviations: BMT = bone marrow transplantation, GPI = glycosylphosphatidylinositol, GPI-AP = glycosylphosphatidylinositol-anchored protein, Hb = hemoglobin, PMN = polymorphonuclear cells, PNH = paroxysmal nocturnal hemoglobinuria, RBC = red blood cells, WBC = white blood cells

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.

Priming with G-CSF effectively enhances low-dose Ara-C-induced in vivo apoptosis in myeloid leukemia cells.

We investigated the role of apoptosis in chemotherapy for hematologic malignancies. Twelve consecutive patients with acute myelogenous leukemia (AML) or refractory anemia with excess of blasts in transformation (RAEB-t) who were not tolerable for standard-dose chemotherapy were treated with CAG regimen (low-dose cytosine arabinoside [Ara-C] plus aclarubicin with concurrent administration of granulocyte colony-stimulating factor [G-CSF]). Bone marrow mononuclear cells obtained before the commencement of the chemotherapy were cultured with various concentrations (0-10(-5) M) of Ara-C in the presence or absence of 10 ng/mL of G-CSF, and the resultant cell proliferation/cytotoxicity was assayed. In all but one patient, half killing concentration (LC50) of Ara-C was significantly reduced in the presence of G-CSF (by 400- and 1.45-fold, median: 21-fold). Furthermore, LC(50) values in responders assayed in the presence of 10 ng/mL of G-CSF were significantly lower than those in nonresponders (p = 0.02). In vitro killing tests using a G-CSF-dependent leukemic cell line suggested that addition of G-CSF potentiates Ara-C-induced cytotoxicity through the mechanism of apoptosis. We thus assayed apoptosis in peripheral blood leukemic cells during CAG chemotherapy by flow cytometry using 7-amino-actinomycin D. Peak percentages of apoptosis in responders were significantly higher than those in nonresponders (p = 0.02). These results collectively suggest that apoptosis plays an important role for eradicating leukemic cells by CAG chemo-therapy.

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.

Procoagulant properties of microparticles released from red blood cells in paroxysmal nocturnal haemoglobinuria

Thrombosis in paroxysmal nocturnal haemoglobinuria (PNH) has been suggested to be due to several pathophysiological states: a suppressed fibrinolytic system, increased leucocyte‐derived tissue factor, complement (C′)‐mediated damage to platelets and endothelia, or increased platelet‐ and endothelium‐derived microparticles (MPs). Because haemolytic attack is often accompanied by thrombosis in PNH, we studied the role of C′‐induced release of MPs in the thrombogenesis of PNH. C′ activation induced procoagulant alteration in PNH red blood cells (RBC), when assessed by thrombin generation in the presence of C′‐activated PNH RBC, which was abolished by their subsequent treatment with annexin V. Significant amounts of procoagulant MPs, measured by phosphatidylserine‐binding prothrombinase activity, were released from PNH RBC in association with the formation of C5b‐9, but not significantly before C5b‐8. Generation of procoagulant, annexin V‐binding, MPs from C′‐activated RBC was studied also by flow cytometry. While phorbol 12‐myristate 13‐acetate, an activator of protein kinase C (PKC), induced the release of MPs from normal RBC as well as PNH RBC, C′‐induced release of MPs from PNH RBC was Ca2+‐independent and not associated with the activation of PKC, calpain or caspase. Procoagulant properties of MPs released from PNH RBC could contribute to the thrombogenesis of PNH.

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.

Clinical significance of elevated soluble interleukin-6 receptor levels in the sera of patients with plasma cell dyscrasias

Summary .We investigated the clinical significance of the serum soluble interleukin‐6 receptor (sIL‐6R) in 42 patients with plasma cell dyscrasias (27 with multiple myeloma (MM), 13 with monoclonal gammopathy of undetermined significance (MGUS), and two with plasma cell leukaemia (PCL)). Serum levels of sIL‐6R in normal individuals were 77 ± 21 ng/ml (mean ± SD, n= 18); those in patients with MGUS and with MM were elevated (102 ± 33 ng/ml, mean ± SD, P < 0–05 and 126 ± 60ng/ml, mean ± SD, P < 0–01, respectively). Significant correlations were not found between the serum levels of sIL‐6R and known prognostic factors (C‐reactive protein, haemoglobin levels, calcium, creatinine, β2‐microglobulin, amounts of M‐protein, or percentages of plasma cells in bone marrow). Elevated serum sIL‐6R did not affect the survival of the patients with MM. Serial measurements of sIL‐6R together with the clinical course of patients with plasma cell neoplasias revealed a good correlation between the sIL‐6R level and disease activity. We conclude that sIL‐6R can be used as a clinical factor correlated with the disease activity, at least in some patients with plasma cell neoplasias.

c-Maf plays a crucial role for the definitive erythropoiesis that accompanies erythroblastic island formation in the fetal liver

c-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the activated protein-1 super family of basic leucine zipper proteins. Despite its overexpression in hematologic malignancies, the physiologic roles c-Maf plays in normal hematopoiesis have been largely unexplored. On a C57BL/6J background, c-Maf(-/-) embryos succumbed from severe erythropenia between embryonic day (E) 15 and E18. Flow cytometric analysis of fetal liver cells showed that the mature erythroid compartments were significantly reduced in c-Maf(-/-) embryos compared with c-Maf(+/+) littermates. Interestingly, the CFU assay indicated there was no significant difference between c-Maf(+/+) and c-Maf(-/-) fetal liver cells in erythroid colony counts. This result indicated that impaired definitive erythropoiesis in c-Maf(-/-) embryos is because of a non-cell-autonomous effect, suggesting a defective erythropoietic microenvironment in the fetal liver. As expected, the number of erythroblasts surrounding the macrophages in erythroblastic islands was significantly reduced in c-Maf(-/-) embryos. Moreover, decreased expression of VCAM-1 was observed in c-Maf(-/-) fetal liver macrophages. In conclusion, these results strongly suggest that c-Maf is crucial for definitive erythropoiesis in fetal liver, playing an important role in macrophages that constitute erythroblastic islands.

Inhibition of complement-mediated haemolysis in paroxysmal nocturnal haemoglobinuria by heparin or low-molecular weight heparin.

Complement (C′)‐mediated haemolysis in paroxysmal nocturnal haemoglobinuria (PNH) is mainly due to the deficiency of glycosyl phosphatidylinositol‐anchored membrane proteins with C′‐regulatory activities CD55 and CD59 in PNH‐affected red blood cells (RBCs). Hydrophobic insertion of C5b‐7 to RBC membranes, initiating the formation of a membrane attack complex, readily results in lysis of PNH RBCs due to the deficiency of CD59. We studied the significance of the electrostatic interactions between C5b‐6 and RBC membranes preceding the insertion of C5b‐7. In vitro, C′‐mediated lysis of PNH RBCs (assessed by sucrose haemolytic assay) was inhibited by heparin, low‐molecular weight heparin (LMWH) or protamine, indicating the significance of the electrostatic interactions between C′ components and RBC membranes in the process of C′‐mediated haemolysis. Neuraminidase‐treated PNH RBCs became resistant to C′ activation, suggesting that the sialic acid moieties on RBC membranes are involved in the interactions of RBC with C′ components. By using biotin‐labelled C7, we demonstrated that LMWH as well as heparin inhibited the insertion of C5b‐7 to RBCs, although they did not inhibit the incorporation of C7 into membrane‐associated C5b‐6. Neither heparin nor LMWH could inhibit the procoagulant alteration of PNH RBC membranes induced by C′ activation even at concentrations which inhibited the haemolysis completely. Because LMWH inhibited the C′‐mediated lysis of PNH RBCs in vitro at the range which induced a limited prolongation of activated partial thromboplastin time of normal plasma, we consider that LMWH may be useful for both the inhibition of haemolysis and the prevention of thrombosis, which often follow a haemolytic attack in PNH.

Immature megakaryocytes undergo apoptosis in the absence of thrombopoietin

We examined withdrawal effects of recombinant mouse Tpo (rm-Tpo) on the apoptosis of mature and immature megakaryocytes in in vitro experiments. Apoptotic megakaryocytes were detected by double staining for acetylcholinesterase and by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. When the purified mature megakaryocytes were cultured with or without rm-Tpo, the numbers of viable megakaryocytes, apoptotic megakaryocytes, and megakaryocytes with cytoplasmic processes were not significantly different between the two groups. In contrast, purified immature megakaryocytes underwent apoptosis when rm-Tpo was absent from the culture system. Murine bone marrow cells were cultured with rm-Tpo (50 U/mL) on days 1-7 to generate immature megakaryocytes and subsequently were cultured with different concentrations of rm-Tpo (0-50 U/mL) on days 8-14. The number of viable megakaryocytes was decreased and that of apoptotic megakaryocytes was increased by rm-Tpo in a dose-dependent manner. These results indicated a clear relation between the rm-Tpo level and the apoptosis of immature megakaryocytes.