Yuichi Akiyama

EFFECT OF MATCHING OF CLASS I HLA ALLELES ON CLINICAL OUTCOME AFTER TRANSPLANTATION OF HEMATOPOIETIC STEM CELLS FROM AN UNRELATED DONOR

BACKGROUND The requirements with respect to HLA compatibility and the relative importance of matching for individual class I and class II HLA alleles in the transplantation of hematopoietic stem cells from unrelated donors have not yet been established. METHODS We performed retrospective DNA typing of alleles at 11 polymorphic loci of HLA genes in 440 recipients of hematopoietic stem cells from unrelated donors who were serologically identical with their respective recipients for HLA-A, B, and DR antigens. Of these recipients, 80 percent had leukemia; the rest had lymphoma, marrow failure, or a congenital disorder. RESULTS Multivariate analysis showed that incompatibility for HLA-A alleles and incompatibility for HLA-C alleles were independent risk factors for severe acute graft-versus-host disease (GVHD) (HLA-A, P=0.006; HLA-C, P=0.001). Mismatching of HLA-A, but not of HLA-C, alleles was an independent risk factor for death (P<0.001). Matching [corrected] of HLA-C alleles was a significant risk factor for relapse of leukemia (P=0.035). HLA-B disparity was a significant risk factor for both GVHD and death in the univariate analysis, but not in multivariate analysis. Disparities in class II HLA alleles of the DRB1, DQA1, DQB1, DPA1, and DPB1 loci were not identified as significant risk factors for acute GVHD or death in the multivariate analysis. CONCLUSIONS Genomic typing of class I HLA alleles adds substantially to the success of transplantation of hematopoietic stem cells from unrelated donors, even if the donors are serologically identical to their recipients with respect to HLA-A, B, and DR antigens.

Nuclear magnetic resonance abnormalities of the cerebral white matter in children with acute lymphoblastic leukemia and malignant lymphoma during and after central nervous system prophylactic treatment with intrathecal methotrexate

Background. A prospective study was conducted to test the feasibility of nuclear magnetic resonance (NMR) imaging in the early diagnosis of treatment‐induced leukoencephalopathy.

Analysis of 500 bone marrow transplants from unrelated donors (UR-BMT) facilitated by the Japan Marrow Donor Program: Confirmation of UR-BMT as a standard therapy for patients with leukemia and aplastic anemia

In December 1991, the Japan Marrow Donor Program (JMDP) was established with the cooperation of the Japanese Red Cross and Japan Marrow Donor Foundation under the auspices of the Ministry of Health and Welfare in Japan. By December 1998, 122365 HLA-A,B typed volunteer marrow donors and 7207 patients had been cumulatively registered in the JMDP. The results of HLA-matching between donors and patients revealed that 5684 out of 7207 (78.9%) patients could have at least one HLA-A,B,DR serologically matched donor. Among these matched pairs, 1829 unrelated bone marrow transplants (UR-BMT) were performed. The initial 500 UR-BMT transplanted from January 1993 to October 1995 were analyzed as of July 1998. Engraftment was achieved in 95% of cases. Probability of the occurrence of grade III and IV acute GVHD was 18.4%. The rate of disease-free survival (DFS) of the patients who had standard-risk leukemia and did not suffer from grade III or IV acute GVHD (n = 154) was 60–71% and the rate of survival of patients with aplastic anemia was 56%. It can be stated that UR-BMT is a modality of treatment which is as effective as related BMT if the occurrence of grade III or IV acute GVHD is predicted and prevented.

Inhibition of both etoposide-induced DNA fragmentation and activation of poly(ADP-ribose) synthesis by zinc ion

Treatment of a human promyelocytic leukemia cell line (HL-60) with etoposide for 3-4 hrs produced an extensive degradation of DNA. Agarose gel electrophoresis showed DNA fragmentation in a nucleosomal ladder pattern. Simultaneous addition of zinc ion (ZnSO4, 1 mM) inhibited DNA fragmentation, although the amount of DNA strand breakage introduced initially by etoposide did not change significantly as measured by the DNA unwinding assay. Furthermore, zinc ion abrogated both the activation of poly(ADP-ribose) synthesis and the morphologic changes characteristic of apoptosis by etoposide. These results suggest that zinc ion inhibits a metabolic process somewhere between initial DNA cleavage through an interference with type II topoisomerase and delayed degradation of cellular DNA to a nucleosome-like pattern.

New Platelet Antigen, Siba, Involved in Platelet Transfusion Refractoriness in a Japanese Man

Abstract. Siba, a new platelet‐specific alloantigen involved in a case of platelet transfusion refractoriness is reported. The IgG platelet alloantibody was detected in a multiply transfused patient of Japanese extraction (Sib), by the presence of HLA antibodies. After transfusion of HLA‐compatible platelets, the patient suffered from refractoriness. Adsorption studies with pooled lymphocytes showed that the serum contained anti‐platelet activity. Family studies indicate that Siba is inherited as an autosomal codominant trait and separate from HLA and Baka. As of this report, segregation from Zw (PlA) and Yuk (Pen) antigen systems have not yet been determined. The gene frequency of Siba in the Japanese population is estimated to be 0.136.

In vivo administration of granulocyte colony-stimulating factor promotes neutrophil survival in vitro

Abstract: We recently showed that recombinant human granulocyte‐colony stimulating factor (rhG‐CSF) maintained the viability of human neutrophils in incubation for up to 72 hours. However, it is not known whether rhG‐CSF can enhance neutrophil survival in in vivo situations. To clarify this issue, we investigated neutrophil survival in vitro following in vivo injection of rhG‐CSF. Neutrophils were obtained from 4 pediatric patients with malignancies and healthy adult volunteers before and after rhG‐CSF administration. Neutrophils obtained before rhG‐CSF treatment started to undergo apoptosis after 24 h of incubation. In contrast, the survival of neutrophils drawn after rhG‐CSF administration increased by approximately 24 h. Concomitantly, the appearance of typical ladder‐like DNA fragmentation was delayed. Such an increase in neutrophil survival was inhibited by coincubation with either H 7 (10 μmol/1) or H 8 (20 μmol/1), which worked as protein kinase C inhibitors. Although our study did not measure neutrophil survival in vivo directly, it provides us with further evidence that rhG‐CSF may function to prolong neutrophil life expectancy in vivo.

Induction of apoptosis in childhood acute leukemia by chemotherapeutic agents: Failure to detect evidence of apoptosis in vivo

Abstract:  This study is designed to investigate whether apoptosis occurs in vivo in pediatric patients with acute leukemia during induction therapy. When patients with common acute lymphoblastic leukemia (cALL) and acute myeloblastic leukemia (AML) were treated with prednisolone (60 mg/m2/day, p.o. or i.v.) and etoposide (150 mg/m2/day, i.v.), respectively, the blast cell counts fell to below 30% and 5%, respectively, in 1 week. However, during this cytoreduction phase, neither morphologically apoptotic cells nor fragmentation of DNA derived from peripheral blast cells were detected at any preparations. On the other hand, cALL but not AML cells spontaneously undergo apoptosis following their culture in vitro. The addition of autologous serum instead of fetal calf serum substantially prevented apoptosis from occurring spontaneously in cALL cells. When cALL and AML cells freshly obtained from patients before therapy were treated in vitro with 10 μmol/1 prednisolone and 20 μg/ml etoposide, respectively, these cells underwent apoptosis within 6 hours, as determined by a morphological and DNA fragmentation assay. These in vivo and in vitro findings suggest that, although anticancer drugs may induce apoptosis in vivo, these apoptotic cells cannot be detected due to their rapid removal from the circulation.

ASSOCIATION OF HIGH MOLECULAR WEIGHT DNA FRAGMENTATION WITH APOPTOTIC OR NON-APOPTOTIC CELL DEATH INDUCED BY CALCIUM IONOPHORE

Calcium ionophore (A23187)‐induced high molecular weight (HMW) and internucleosomal DNA fragmentation were investigated in human leukemia cell lines. An apoptosis‐sensitive cell line, HL‐60, showed HMW, internucleosomal DNA fragmentation and morphological changes of apoptosis by A23187. MOLT‐4, which is resistant to apoptosis, exhibited only HMW DNA fragmentation and died of necrosis under the same conditions. Autodigestion experiments suggested the endonucleolytic activity to cause HMW fragmentation in the cytoplasm of both cell lines. The activity was more dependent on Mg2+ than Ca2+ in HL‐60, whereas it was Ca2+‐dependent in MOLT‐4. These results suggest that HMW DNA fragmentation is not specific to apoptosis.

Somatic mutations at T-cell antigen receptor and glycophorin a loci in pediatric leukemia pateints following chemotherapy: comparison with HPRT locus mutation

Frequencies of somatic mutations in pediatric patients with leukemia were evaluated following intensive treatment at three different loci: the hypoxanthine-guanine phosphoribosyl transferase (HPRT), T-cell antigen receptor (TCR), and glycophorin A (GPA) gene. Thirty-two children with acute lymphoblastic leukemia (ALL), nine children with acute myelogenous leukemia (AML), and 20 age-matched healthy controls were included in the study of mutant frequencies (Mfs) at the HPRT and TCR loci. Among these patients and controls, individuals with heterozygous MN blood type, i.e., 14 children with ALL, three children with AML, and nine healthy controls, served for the further assessment of variant frequency (Vf) at the GPA locus. In ALL patients, geometric mean Mfs and Vfs at these loci were significantly higher than in healthy controls. The high Mf value at the HPRT locus persisted for up to 8 years after the end of chemotherapy. On the other hand, the Mf values at the TCR locus and Vf values at the GPA locus declined gradually with time. In AML patients, on the other hand, the geometric mean Mf only at the TCR locus was significantly higher than in the controls, albeit to a lesser degree than in ALL patients. These data suggest that anti-cancer therapy induces somatic mutations at various loci and that ALL patients are more susceptible to mutagenic intervention than are AML patients.

Differential modulation of 1-β-d-arabinofuranosylcytosine metabolism by hydroxyurea in human leukemic cell lines

The ability of hydroxyurea (HU) to modulate 1-beta-D-arabinofuranosylcytosine (Ara-C) metabolism was investigated in human leukemic cell lines. Exposure of HL-60 cells to 1 mM HU enhanced the accumulation of Ara-CTP up to 2.5-fold, whereas HU did not have significant effects on Ara-C metabolism in CEM cells. In addition, two adenine nucleosides, deoxyadenosine (dAdo) and 9-beta-D-arabinofuranosyladenine (Ara-A), which are known to be activated by deoxycytidine (dCyd) kinase as Ara-C, were more effectively phosphorylated after the addition of HU only in HL-60 cells. However, the changes of intracellular dCTP and TTP pools induced by HU, i.e. decrease in dCTP and increase in TTP, were the same in both cell lines. Finally, dCyd production under normal culture conditions was at least 3- to 4-fold higher in HL-60 cells and was inhibited significantly by HU administration. These results suggest that the modulation of Ara-C metabolism by HU occurs at the level of dCyd kinase through the regulation of de novo dCyd generation.