Kazuo Ogami

A combination of stem cell factor and granulocyte colony-stimulating factor enhances the growth of human progenitor B cells supported by murine stromal cell line MS-5

We have developed a long‐term culture system using the murine bone marrow stromal cells MS‐5 to support the growth of progenitor B cells with CD34–, CD10+, CD19+, and cytoplasmic μ chain (Cμ)‐negative surface phenotype from human CD34+ cells purified from umbilical cord blood (CB). When 103 CB CD34+ cells/well were seeded on MS‐5 stromal cells at the beginning of culture in the absence of exogenously added cytokines, progenitor B cells first appeared after 14 days, and the maximal cell production was achieved during the 6th week of culture. Intriguingly, the addition of recombinant human stem cell factor (rhSCF) and granulocyte colony‐stimulating factor (rhG‐CSF), but not rhIL‐7, strikingly enhanced the growth of progenitor B cells from CB CD34+ population cultured on MS‐5 stromal cells. The culture of progenitor B cells could be maintained until the 6th week of culture when some cells were revealed to have a Cμ+ phenotype, and a small number of cells had immunoglobulin μ chain on their cell surface in the presence of both rhSCF and rhG‐CSF. When CD34+ cells were cultured physically separated from the stromal layer by membrane, supportive effects of MS‐5 stromal cells for the growth of progenitor B cells were not observed. These results suggest that the present culture system could generate progenitor B cells to proliferate from CB CD34+ cells, that some of these progenitor B cells could differentiate into immature B cells in conjunction with rhSCF and rhG‐CSF, and that a species‐cross‐reactive membrane‐bound factor(s), which stimulates early human B lymphopoiesis, may exist in MS‐5 stromal cells. Further studies are required to investigate the mechanism how rhG‐CSF acts on progenitor B cells to allow their proliferation and differentiation.

IL-2/LAK therapy for refractory acute monoblastic leukemia relapsing after unrelated allogeneic bone marrow transplantation.

Leukemia relapse is a major cause of treatment failure after allogeneic bone marrow transplantation. We administered recombinant interleukin-2 (rIL-2) to a patient who relapsed after unrelated allogeneic bone marrow transplantation (uBMT). While the number of peripheral blood monoblastic leukemia cells increased after administration of rIL-2, the patient achieved durable remission for 5 months after low-dose chemotherapy followed by adoptive transfer of engrafted graft-derived lymphokine-activated killer (LAK) cells. Following the disappearance of the blast cells, however, both cutaneous and liver GVHD were exacerbated. Administration of rIL-2 and adoptive transfer of graft-derived LAK cells are considered to be possible choices for the treatment of acute leukemia relapsing after uBMT when donor leukocyte transfusion is not available.

Severe autoimmune thrombocytopenia after allogeneic bone marrow transplantation for aplastic anemia

Autoimmune thrombocytopenia (AITP) after bone marrow transplantation (BMT) was suggested to occur by immune dys-regulation mainly in association with graft-versus-host disease (GVHD). Here we present a patient who developed severe AITP after BMT. A 40-year-old woman with severe aplastic anemia received a BMT from a partially HLA-matched brother. Despite myeloid and erythroid engraftments, platelet recovery was delayed. All bone marrow cells were 46,XY and were derived from the donor. Grade I acute GVHD involving skin developed from day 34 posttransplantation, but promptly responded to prednisolone in addition to a prophylactic dose of tacrolimus. With the tapering of prednisolone, thrombocytopenia progressed without substantial changes in the white blood cell count, hemoglobin concentration, or reticulocyte count. On day 188, the patient developed chronic GVHD involving skin and liver, which promptly responded to the readministration of prednisolone and increased tacrolimus. However, the patient’s platelet count decreased to 9 X 109 cells/L on day 222. The platelet-associated immunoglobulin G (PAIgG) values were elevated. Bone marrow examination showed hypercellularity with plentiful megakaryocytes. The number of colony-forming units-megakaryocyte was within the normal range. The elevated PAIgG values and a correlation between thrombocytopenia and the intensity of the immunosuppressive agents strongly suggested a causative role of the autoimmune mechanisms for thrombocytopenia in this patient.

The effect of donor leukocyte infusion on refractory pure red blood cell aplasia after allogeneic stem cell transplantation in a patient with myelodysplastic syndrome developing from Kostmann syndrome.

We describe the clinical course of a patient who experienced refractory pure red cell aplasia (PRCA) after undergoing HLA-matched allogeneic peripheral blood stem cell transplantation (allo-PBSCT) for refractory anemia with an excess of blasts in transformation that had evolved from Kostmann syndrome. The treatment for patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) developing from Kostmann syndrome has not been standardized. We treated this patient with allo-PBSCT using a regimen combining high-dose cytosine arabinoside with granulocyte colony-stimulating factor, in addition to total body irradiation and cyclophosphamide without preceding intensive chemotherapy. The donor was ABO incompatible. Myeloid and platelet recoveries were achieved rapidly. Erythroid engraftment was not evident, however, and the patient was given a diagnosis of PRCA. Regimen-related toxicity and graft-versus-host disease (GVHD) were limited. The PRCA did not respond to various therapies, including the discontinuation of immunosuppressants for the induction of chronic GVHD, human recombinant erythropoietin, immunosuppressive treatment with steroids, cyclosporin A, and human anti-CD20 antibody (rituximab). The patient received transfusions 48 times until the resolution of his anemia by donor leukocyte infusion (DLI) at 25 months after PBSCT. He is now clinically well (performance status, 100%) with normal blood cell counts at 5 years after SCT. An in vitro study demonstrated that serum from the recipient blocked the differentiation of erythroid cells in the bone marrow. The results indicate that the conditioning regimen we describe seems safe and effective for those who have MDS/AML and that DLI might be a valuable approach for refractory PRCA after ABO-incompatible SCT.

Alloimmune hemolysis due to major RhE incompatibility after unrelated cord blood transplantation

Masamichi Isobe, Takaaki Konuma, Yuka Abe-Wada, Kyoko Hirata, Kazuo Ogami, Seiko Kato, Maki Oiwa-Monna, Susumu Tanoue, Tokiko Nagamura-Inoue, Satoshi Takahashi and Arinobu Tojo Department of Hematology/Oncology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Department of Cell Processing and Transfusion, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

One allele deletion of the RB1 gene in a case of refractory anemia with del(13)(q12q14): a fluorescence in situ hybridization study of the RB1 gene

The tumor suppressor gene RB1 is known to be located on chromosome band 13q14. We investigated the involvement of the RB1 gene in a case of refractory anemia with del(13)(q12q14) by florescence in situ hybridization (FISH) analysis using the RB1 locus (13q14) DNA probe. Bone marrow cells derived from this patient exhibited a single signal of the RB1 gene in 58 of 100 bone marrow cells, as determined by interphase FISH analysis. Hematopoietic colony-forming assays showed that the absolute number of erythroid, myeloid, and mixed colonies was comparable to that of normal subjects. FISH analysis of selected colonies revealed that only a single signal for the RB1 gene was detected in five of five granulocyte macrophage-colony-forming units (CFUs), four of five erythroid burst-forming units, and two of four mixed CFUs (total 11/14: 78.6%). Thus, the majority of hematopoietic progenitor cells lacked one allele of the RB1 gene, suggesting that in this particular case the RB1 gene played an important role in abnormal hematopoiesis.