Shotaro Iwamoto

Mesenchymal cells regulate the response of acute lymphoblastic leukemia cells to asparaginase

Because of their low asparagine synthetase (ASNS) expression and asparagine biosynthesis, acute lymphoblastic leukemia (ALL) cells are exquisitely sensitive to asparagine depletion. Consequently, asparaginase is a major component of ALL therapy, but the mechanisms regulating the susceptibility of leukemic cells to this agent are unclear. In 288 children with ALL, cellular ASNS expression was more likely to be high in T-lineage ALL and low in B-lineage ALL with TEL-AML1 or hyperdiploidy. However, ASNS expression levels in bone marrow-derived mesenchymal cells (MSCs), which form the microenvironment where leukemic cells grow, were on average 20 times higher than those in ALL cells. MSCs protected ALL cells from asparaginase cytotoxicity in coculture experiments. This protective effect correlated with levels of ASNS expression: downregulation by RNA interference decreased the capacity of MSCs to protect ALL cells from asparaginase, whereas enforced ASNS expression conferred enhanced protection. Asparagine secretion by MSCs was directly related to their ASNS expression levels, suggesting a mechanism - increased concentrations of asparagine in the leukemic cell microenvironment - for the protective effects we observed. These results provide what we believe to be a new basis for understanding asparaginase resistance in ALL and indicate that MSC niches in the bone marrow can form a safe haven for leukemic cells.

A Pivotal Role of Rho GTPase in the Regulation of Morphology and Function of Dendritic Cells

Dendritic cell (DC) is the most potent activator of CD4+ T cells and has unique dendrites and veils. To explore the function of Rho in DC, exoenzyme C3 from Clostridium botulinum was used as a specific inhibitor of Rho. Treatment of DC with C3 (DC/C3) resulted in profound morphological changes by losing dendrites and emerging of shrunk membrane processes that were in parallel with marked reduction of polymerized actin in the marginal area. Inactivation of Rho-associated coiled coil-containing kinase (p160ROCK) by a specific ROCK inhibitor Y-27632 also led to disappearance of dendrites of DC with retaining large membrane expansions. In scanning electron microscopy, untreated DCs interacted with CD4+ T cells more efficiently than DC/C3. Conjugate formation assay showed that the number of DCs associated with CD4+ T cells was 2-fold higher in untreated DCs than that of DC/C3. Alloantigen-presenting capacity of DC/C3 was significantly suppressed in a dose-dependent manner. Because C3 treatment did not affect the surface expression of HLA, costimulatory, and adhesion molecules of DC, we examined cytokine production of DC and naive CD4+ T cells to further elucidate the inhibitory mechanism of MLR. Unexpectedly, DC/C3 increased IL-12 production after LPS stimulation. Naive CD4+ T cells cocultured with DC/C3 produced the increased percentage of IFN-γ-producing cells, whereas the percentage of IL-2-producing T cells was decreased. These results demonstrate that Rho GTPase in DC controls both characteristic shape and immunogenic capacity.

Down syndrome and GATA1 mutations in transient abnormal myeloproliferative disorder: mutation classes correlate with progression to myeloid leukemia

Twenty percent to 30% of transient abnormal myelopoiesis (TAM) observed in newborns with Down syndrome (DS) develop myeloid leukemia of DS (ML-DS). Most cases of TAM carry somatic GATA1 mutations resulting in the exclusive expression of a truncated protein (GATA1s). However, there are no reports on the expression levels of GATA1s in TAM blasts, and the risk factors for the progression to ML-DS are unidentified. To test whether the spectrum of transcripts derived from the mutant GATA1 genes affects the expression levels, we classified the mutations according to the types of transcripts, and investigated the modalities of expression by in vitro transfection experiments using GATA1 expression constructs harboring mutations. We show here that the mutations affected the amount of mutant protein. Based on our estimates of GATA1s protein expression, the mutations were classified into GATA1s high and low groups. Phenotypic analyses of 66 TAM patients with GATA1 mutations revealed that GATA1s low mutations were significantly associated with a risk of progression to ML-DS (P < .001) and lower white blood cell counts (P = .004). Our study indicates that quantitative differences in mutant protein levels have significant effects on the phenotype of TAM and warrants further investigation in a prospective study.

BK virus-associated fatal renal failure following late-onset hemorrhagic cystitis in an unrelated bone marrow transplantation.

The human polyomavirus BK (BKV)-associated hemorrhagic cystitis (HC) has been a frequent and seldom life-threatening complication after bone marrow transplantation (BMT). The authors report a male with myelodysplastic syndrome, who developed BKV-associated late-onset HC 12 days after HLA-matched unrelated BMT. His urine contained epithelial cells with intranuclear inclusion bodies suggestive of BKV infection and was positive for BKV in polymerase chain reaction. He did not respond to any treatment for HC. In addition, he developed BKV-associated acute renal failure on day 26, followed by hepatic veno-occlusive disease on day 42. This is the first case in which BKV may be associated with fatal progressive renal failure.

Excess treatment reduction including anthracyclines results in higher incidence of relapse in core binding factor acute myeloid leukemia in children

Excess treatment reduction including anthracyclines results in higher incidence of relapse in core binding factor acute myeloid leukemia in children

Clinical characteristics and outcome of refractory/relapsed myeloid leukemia in children with Down syndrome

Myeloid leukemia in Down syndrome (ML-DS) is associated with good response to chemotherapy and favorable prognosis. Because little research has been focused on refractory/relapsed (R/R) cases, we conducted a retrospective analysis for R/R ML-DS. Among ML-DS patients diagnosed between 2000 and 2010 in Japan, 26 relapsed (25 in the BM and 1 in the skin), and 3 refractory patients were enrolled. The male/female ratio was 18/11. The median age at initial diagnosis of ML-DS was 2 years, and the median time to relapse was 8.6 months. Each patient initially had been treated with ML-DS-specific protocols. Thirteen of the 26 patients achieved complete remission with various kinds of reinduction chemotherapies; 2 of 8 survived without further recurrence after receiving allogeneic hematopoietic stem cell transplantation, and 4 of 5 maintained complete remissions with chemotherapy alone. Treatment failures mostly were associated with disease progression rather than treatment-related toxicities. The 3-year OS rate was 25.9% ± 8.5%. A longer duration from initial diagnosis to relapse was a significant favorable prognostic factor (P < .0001). We conclude that clinical outcome for patients with R/R ML-DS generally are unfavorable, even in those receiving hematopoietic stem cell transplantation. Novel methods to identify poor prognostic factors for ML-DS are necessary.

Flow cytometric analysis of de novo acute myeloid leukemia in childhood: report from the Japanese Pediatric Leukemia/Lymphoma Study Group

Although the antigen expression patterns of childhood acute lymphoblastic leukemia (ALL) are well known, little attention has been given to standardizing the diagnostic and classification criteria. We retrospectively analyzed the flow cytometric data from a large study of antigen expression in 1,774 children with newly diagnosed ALL in JPLSG. T- and B-lineage ALL accounted for 13 and 87% of childhood ALL cases, respectively. Cytoplasmic CD3 and CD7 antigens were positive in all T-ALL cases. More than 80% of T-ALL cases expressed CD2, CD5 and TdT. In B-lineage ALL, the frequencies of early pre-B, pre-B, transitional pre-B and B-ALL were 81, 15.5, 0.6 and 2.9%, respectively. More than 90% of early pre-B ALL cases expressed CD19, CD79a, CD22, CD10 and TdT. CD34 was expressed in three-fourths of early pre-B ALL cases. The frequencies of TdT and CD34 expression were lower in pre-B ALL than in early pre-B ALL. B-ALL showed less frequent expression of CD22, CD10, CD34 and TdT than other B-lineage ALL cases. Expression of CD13 and CD33, aberrant myeloid antigens, was significantly more frequently associated with B-lineage ALL than with T-ALL. Based on this retrospective study of antigen expression in 1,774 de novo childhood ALL cases in JPLSG, we propose standardized clinical guidelines for the immunophenotypic criteria for diagnosis and classification of pediatric ALL.

Prediction of Reactivity to Noninherited Maternal Antigen in MHC-Mismatched, Minor Histocompatibility Antigen-Matched Stem Cell Transplantation in a Mouse Model

The immunologic effects of developmental exposure to noninherited maternal Ags (NIMAs) are quite variable. Both tolerizing influence and inducing alloreaction have been observed on clinical transplantation. The role of minor histocompatibility Ags (MiHAs) in NIMA effects is unknown. MiHA is either matched or mismatched in NIMA-mismatched transplantation because a donor of the transplantation is usually limited to a family member. To exclude the participation of MiHA in a NIMA effect for MHC (H-2) is clinically relevant because mismatched MiHA may induce severe alloreaction. The aim of this study is to understand the mechanism of NIMA effects in MHC-mismatched, MiHA-matched hematopoietic stem cell transplantation. Although all offsprings are exposed to the maternal Ags, the NIMA effect for the H-2 Ag was not evident. However, they exhibit two distinct reactivities, low and high responder, to NIMA in utero and during nursing depending on the degree of maternal microchimerism. Low responders survived longer with less graft-versus-host disease. These reactivities were correlated with Foxp3 expression of peripheral blood CD4+CD25+ cells after graft-versus-host disease induction and the number of IFN-γ–producing cells stimulated with NIMA pretransplantation. These observations are clinically relevant and suggest that it is possible to predict the immunological tolerance to NIMA.

Functional immaturity of cord blood monocytes as detected by impaired response to hepatocyte growth factor.

Abstract Background : Monocytes as antigen‐presenting cells play an important role in host defense and transplantation. However, there are little reports on cord blood monocytes, and the role of monocytes in cord blood transplantation is largely unknown.

The reconstitution of CD45RBhiCD4+ naive T cells is inversely correlated with donor age in murine allogeneic haematopoietic stem cell transplantation

A high incidence of opportunistic infections after unrelated bone marrow transplantation has been reported. Delayed lymphocyte recovery may be associated with opportunistic infections. Immune reconstitution is influenced by recipient age and graft‐vs‐host disease (GVHD). In fact, children develop GVHD less frequently than adults. However, the role of donor age is largely unknown. We examined the effect of donor age on lymphocyte reconstitution after transplant. Three‐month‐old BALB/c recipient mice were lethally irradiated and transplanted with allogeneic haematopoietic stem cells from A/J donor mice of different ages, ranging from 0 d to 12 months. The recovery of absolute lymphocyte counts and those of CD3+ T cells, CD4+ T cells and CD45RBhi CD4+ naive T cells in the early post‐transplant period correlated inversely with donor age. Recipient mice transplanted with haematopoietic stem cells from younger donors showed significantly higher survival rates and mitogenic responses than adult donors. As T cells, especially CD4+ naive T cells, play an important role in host defence, faster recovery of CD4+ naive T cells in younger donors may contribute to reduced mortality in the early post‐transplant period. The results suggest that it could be better to choose a younger donor if sufficient cell dose is available.