Masakazu Umemoto

Successful hematopoietic reconstitution by transplantation of umbilical cord blood cells in a transfusion-dependent child with Diamond-Blackfan anemia

A 4-year-old boy with Diamond–Blackfan anemia and a history of multiple transfusions underwent umbilical cord blood transplantation from his HLA-identical female sibling born by vaginal delivery at 38 weeks. The patient was prepared with busulfan, cyclophosphamide and antilymphocyte globulin. Methotrexate and cyclosporin A were given for the prophylaxis of GVHD. Regimen-related toxicity was not observed and successful engraftment occurred, including the erythroid series. No evidence of acute or chronic GVHD has been observed for 14 months after transplantation. This is the first case of successful umbilical cord blood transplantation to a patient with Diamond–Blackfan anemia.

Conditioning with cyclophosphamide/antithymocyte globulin for allogeneic bone marrow transplantation from HLA-matched siblings in children with severe aplastic anemia

Graft rejection has been a problem after bone marrow transplantation for patients with severe aplastic anemia (SAA). Ten children with SAA were conditioned for bone marrow transplantation from HLA-identical siblings, using cyclophosphamide (CY, 50 mg/kg) plus antithymocyte globulin (ATG, 15 mg/kg ) for 4 successive days. Marrow was infused 36 h after the last dose of CY. Cyclosporin A and methotrexate were administered as graft-versus-host disease (GVHD) prophylaxis. All patients achieved durable engraftment at follow-up of 7–41+ months (mean, 25) without significant GVHD. Since investigators have used different sources, doses, and time schedules of ATG, we compared our results with other published reports. We conclude that CY/ATG conditioning is well tolerated and effective in children with SAA.

Two cytotoxic pathways of natural killer cells in human cord blood: implications in cord blood transplantation

Using K562 cells as a target we investigated cord blood (CB)–natural killer (NK) cytolytic pathways. The cytotoxicity of fresh CB‐NK cells was significantly lower than that of peripheral blood mononuclear cells (PB MNCs). When CB was incubated with IL‐2, the level of CB‐NK cytotoxicity was increased and boosted to the level observed in PB‐NK cells. Fresh CB‐NK cells induced apoptosis in target cells. Activated CB cells induced apoptosis and necrosis in target cells, at the same level as PB MNCs. CB stem cell transplantation may also induce graft‐versus‐host disease (GVHD)/graft‐versus‐leukaemia (GVL), similar to bone marrow transplantation.

Thrombopoietin level is inversely related to blast count, not platelet number, in Down syndrome neonates with transient myeloproliferative disorder

Transient myeloproliferative disorder (TMD) in neonates with Down syndrome is characterized by increased megakaryoblastic cells in the peripheral blood. Despite their spontaneous regression in weeks, prognosis is not always favorable because of fatal hepatic fibrosis. In this study, blood thrombopoietin (TPO) levels were measured by ELISA in six TMD patients and the expression of c‐Mpl, a ligand for TPO, was examined on the blast cells from four patients by flow cytometer. At the onset, TPO level was undetectable in one patient and significantly lower in five patients than six neonatal controls (mean 0.52 fmol/ml, range 0.30–0.93 vs. 3.70, 1.38–8.33, P < 0.001), although platelet counts were similar (mean 321 × 109/l, range 42–1,040 vs. 253 × 109/l, 124–381). Two patients died of hepatic failure. TPO levels were measured in five patients after regression of the blast cells. With regression of blast cells, TPO levels were remarkably increased in four survived patients. In one patient with hepatic failure, TPO level was poorly elevated and relatively lower compared to the others. TPO levels were inversely correlated with blast numbers (r = −0.85, P < 0.001), but not with platelet counts (r = 0.426). Blast cells from four patients were all positive for c‐Mpl. Our findings suggest that megakaryocyte mass is a major regulator of TPO levels and hepatic failure may affect the TPO level because liver is a major source of TPO production. Am. J. Hematol. 58:267–272, 1998.

Cytokine‐enhanced mixed lymphocyte reaction (MLR) in cord blood

Although in cord blood (CB) transplantation graft‐versus‐host disease (GVHD) is reported to be less severe, GVHD may occur even in patients with HLA‐identical sibling donors. This result shows that HLA typing can not entirely predict GVHD. The standard MLR with CB cells was either normal or slightly reduced compared with adult peripheral blood (PB) cells. We used two manipulations to increase the responses of CB cells to allo‐antigens. The first was to treat the stimulator cells with cytokines, and the second to amplify weak proliferative responses by adding exogenous cytokines to MLR cultures (modified MLR). The stimulator cells were treated with both interferon‐gamma (IFN‐γ) and IL‐4. The responder cells were treated with both IL‐2 and tumour necrosis factor‐alpha (TNF‐α). It is still to be determined whether or not this cytokine‐enhanced MLR could be a possible predictor of GVHD. However, using these cytokines, 90% of CB could recognize allo‐antigens, even if the standard MLR was negative.

Suppression of alloreactivity with γδ T-cells: relevance to increased γδ T-cells following bone marrow transplantation

Summary Several reports have shown that an increase in T-cell receptor gamma/delta-positive T-cells ( γδ T-cells) have been observed following bone marrow transplantation. γδ T-cells expanded from peripheral blood mononuclear cells from normal volunteers were used to investigate the function of γδ T-cells in vitro. Peripheral blood mononuclear cells were cultured with synthetic ligand of γδ T-cells, monoethyl phosphates (MEP), for 7 days. MEP specifically expanded γδ T-cells. Expanded γδ T-cells from subject “B” were added to an A anti-B or A anti-C mixed lymphocyte culture (MLC) containing responder cells from subject “A” and irradiated stimulator cells from subjects “B” or “C”. The cultures were harvested on day 6 and tested for cytotoxicity against stimulator-type Con A blasts. γδ T-cells from subject “B” specifically inhibit generation of allospecific cytotoxic T lymphocytes (CTL) in A anti-B MLC. The results indicate that γδ T-cells exhibit veto-type suppression of alloreaction. If the current experiments are also applicable in vivo, γδ T-cells originating from the donor after bone marrow transplantation may inhibit graft rejection by suppressing recipient anti-donor reactivity. γδ T-cells may be involved in the suppression of allogeneic reaction in vivo following allogeneic bone marrow transplantation.

Cytotoxic T-lymphocytes recognizing P-glycoprotein in murine multidrug-resistant leukemias

Abstract: A multidrug‐resistant murine lymphoid leukemia P388/ADR overexpresses P‐glycoprotein (P‐gp), an active transporter that pumps cytotoxic drugs out of cells and a product of mdr1 gene. Cytotoxic T lymphocytes (CTL) that showed cytotoxicity against P388/ADR were generated from mixed lymphocyte tumor cell culture. CTL do not kill drug‐sensitive parental P388 (P388/parent) that does not express P‐gp. Monoclonal antibody against P‐gp inhibited cytotoxic activity. Similar results were obtained in another multidrug‐resistant cell line P388/VP‐16. Cytotoxic activity was mediated by Thyl+ CD4 CD8+ T‐cells. When P388/ADR was treated with murine IL‐4, expression of P‐gp was downregulated. Monoclonal antibody against interleukin‐4 (IL‐4) abrogated the IL‐4‐induced suppression of P‐gp. Cytolytic activity of CTL against IL‐4‐treated P388/ADR was dose dependently inhibited. These results suggest that P‐gp is immunogenic and can be a target of CTL in this murine leukemia model.

Changes in rotavirus genotypes before and after vaccine introduction: a multicenter, prospective observational study in three areas of Japan

In Japan, monovalent and pentavalent rotavirus (RV) vaccines were approved in 2011 and 2012, respectively. To monitor changes in the RV genotypes before and after vaccine introduction, we performed a prospective observational study among children (＜ 5 years) with gastroenteritis who tested RV-positive on antigen rapid tests. Stool samples were collected from 3 different sites in Japan: Tsu City, Mie Prefecture; Kurashiki City, Okayama Prefecture; and Isumi City, Chiba Prefecture. RV genotypes were determined using reverse transcription-polymerase chain reaction. In Tsu City, G3P[8] was dominant (61.0-77.1%) before vaccine introduction, but decreased after introduction. Meanwhile, in an inverse proportion to the decrease in G3P[8], G1P[8] increased until the 2013/14 season, when a sudden predominance of G2P[4] (100%) occurred. A similar trend was observed in Kurashiki City in terms of the extent of reduction in G3P[8] and the emergence of G2P[4]. In Isumi City, G1P[8] was dominant (70.3%) before vaccine introduction, and G9P[8] became predominant (83.3%) in the 2013/14 season. To determine whether the genotype changes are attributable to vaccines or natural epidemiological changes, ongoing continuous monitoring of the RV genotypes is required.

Characterization of unusual DS-1-like G3P[8] rotavirus strains in children with diarrhea in Japan

The emergence and rapid spread of novel DS‐1‐like intergenogroup reassortant rotaviruses having the equine‐like G3 genotype (DS‐1‐like G3P[8] strains) have been recently reported from several countries. During rotavirus surveillance in Japan in 2015–2016, three DS‐1‐like G3P[8] strains were identified from children with severe diarrhea. In the present study, we sequenced and characterized the full genomes of these three strains. On full‐genomic analysis, all three strains showed a unique genotype constellation including both genogroup 1 and 2 genes: G3‐P[8]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2. Phylogenetic analysis revealed that each of the 11 genes of the three strains was closely related to that of Japanese DS‐1‐like G1P[8] and/or Japanese equine‐like G3P[4] human strains. Thus, the three study strains were suggested to be reassortants that acquired the G3‐VP7 gene from equine G3 rotaviruses on the genetic background of DS‐1‐like G1P[8] strains. Our observations will provide important insights into the evolutionary dynamics of emerging DS‐1‐like G3P[8] strains.

Low R-wave amplitude in the right precordial leads in children with symptomatic doxorubicin cardiomyopathy

SummaryElectrocardiographic (ECG) findings were studied in four patients with doxorubicin cardiomyopathy. In all patients with congestive heart failure (CHF), the ECGs had a low R-wave and low R/S ratio in lead V1. Our study suggests that increased injury to myocardial cells in the regions of the anterior septum and anterior left ventricular wall may be important in the pathogenesis of doxorubicin cardiomyopathy. Eight years later, cardiac recovery from CHF occurred with a normal ECG and left ventricular ejection fraction in one patient, indicating that CHF may be reversible in certain cases.