Ikuta Tanaka

Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.

Ex Vivo Large‐Scale Generation of Human Platelets from Cord Blood CD34+ Cells

In the present investigation, we generated platelets (PLTs) from cord blood (CB) CD34+ cells using a three‐phase culture system. We first cultured 500 CB CD34+ cells on telomerase gene‐transduced human stromal cells (hTERT stroma) in serum‐free medium supplemented with stem cell factor (SCF), Flt‐3/Flk‐2 ligand (FL), and thrombopoietin (TPO) for 14 days. We then transferred the cells to hTERT stroma and cultured for another 14 days with fresh medium containing interleukin‐11 (IL‐11) in addition to the original cytokine cocktail. Subsequently, we cultured the cells in a liquid culture medium containing SCF, FL, TPO, and IL‐11 for another 5 days to recover PLT fractions from the supernatant, which were then gel‐filtered to purify the PLTs. The calculated yield of PLTs from 1.0 unit of CB (5 × 106 CD34+ cells) was 1.26 × 1011−1.68 × 1011 PLTs. These numbers of PLTs are equivalent to 2.5–3.4 units of random donor‐derived PLTs or 2/5–6/10 of single‐apheresis PLTs. The CB‐derived PLTs exhibited features quite similar to those from peripheral blood in morphology, as revealed by electron micrographs, and in function, as revealed by fibrinogen/ADP aggregation, with the appearance of P‐selectin and activated glycoprotein IIb‐IIIa antigens. Thus, this culture system may be applicable for large‐scale generation of PLTs for future clinical use.

Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages

We generated red blood cells (RBC) from cord blood (CB) CD34+ cells using a four-phase culture system. We first cultured CB CD34+ cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34+ cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34+ cells from a different donor using the same co-culture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 × 1012 RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 × 1013 RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.

A Patient with Paroxysmal Nocturnal Hemoglobinuria in whom G-CSF Administration was Remarkably Effective against Recurrent Gastrointestinal Infections and Hemolytic Episodes Caused by Cellular Immunodeficiency

We report an elderly patient with paroxysmal nocturnal hemoglobinuria (PNH), having recurrent gastrointestinal infections and hemolytic episodes caused by cellular immunodeficiency due to a decreased number of T cells. On admission to our hospital, the patient had normal neutrophil count and function, but had decreased T cell count, decreased mitogenic reaction to PHA and ConA, and negative reaction to the tuberculin test. Thus, a cellular immunodeficiency state was suggested and postulated to be due to decreased T cell count caused by the loss of PNH clone of T cells. We previously reported that the administration of G-CSF to normal donors of allogeneic peripheral stem cell transplantation increases the lymphocyte count. Based on this experience, we administered G-CSF to this patient. This resulted in an increase in the T cell count, normalization of the mitogenic response to PHA and ConA, positive reaction to the tuberculin test and increases in the blood levels of Thl cytokines and Th2 cytokines, with an improvement in the gastrointestinal infections and hemolytic episodes. This suggested that G-CSF administration resulted in the recovery of cell-mediated immunity in the gastrointestinal mucosa. At 9 months after the start of G-CSF administration, these complications have not recurred. This is the first report of a patient of this type and suggests that G-CSF administration may be useful in the treatment of elderly PNH patients with cellular immunodeficiency.

C (H) OP refractory chronic lymphocytic leukemia patients in whom salvage chemotherapy chosen by evaluating multiple chemotherapeutic drug-resistant factors was remarkably effective

It is well known that the expression of anticancer drug-resistant factors is elevated in patients with primary refractory or relapsed chronic lymphocytic leukemia (CLL) who have been treated with chemotherapy. We report here two C(H)OP refractory patients with CLL in whom salvage chemotherapy chosen by evaluating anticancer drug-resistant factors (glutathione-S-transferase-Π [GST-Π], glycoprotein [GP]-170, multidrug resistance-associated protein [MRP], and lung resistance protein [LRP]) was remarkably effective. A 71-year-old male patient was refractory to induction therapy with cyclophosphamide, vincristine, and prednisone (COP), and his leukemic cells at diagnosis displayed overexpression of GST-Π and GP-170. A 74-year-old female patient’s condition had been stable; she had received ten courses of COP over 9 years. However, because systemic lymphadenopathies recurred, she was treated with chemotherapy consisting of cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP) or dexamethasone, etoposide, ifosphamide, and carboplatin (DeVIC). However, she did not respond at all, and her leukemic cells at recurrence displayed overexpression of GST-Π. Therefore, we chose for these patients a salvage therapy consisting of dexamethasone and high-dose cytosine arabinoside (Ara C), to which neither GST-Π nor GP-170 show any drug resistance. In both patients, this salvage therapy proved effective.

A patient with paroxysmal nocturnal haemoglobinuria in whom granulocyte colony-stimulating factor administration resulted in improvement of recurrent enterocolitis and its associated haemolytic attacks

Summary. We report an elderly patient with paroxysmal nocturnal haemoglobinuria (PNH), having recurrent enterocolitis and haemolytic attacks associated with cellular immunodeficiency. On admission, the patient had normal neutrophil count and function but a decreased T‐cell count, decreased mitogenic reactions, and a negative tuberculin test. Granulocyte colony‐stimulating factor (G‐CSF) was administered, resulting in an increased T‐cell count, normalization of T‐cell function, increased blood levels of helper T cell (Th)1 and Th2 cytokines and improvement in the enterocolitis and haemolytic attacks. This suggests that G‐CSF may be useful in the treatment of elderly PNH patients with cellular immunodeficiency.