Tetsuya Mori

Induction of Apoptosis in Normal Human Renal Tubular Epithelial Cells by Escherichia coli Shiga Toxins 1 and 2

The cytotoxicity of Shiga toxin (Stx) 1 and Stx2 produced by Escherichia coli to human renal cortical epithelial cells (HRCEC) in primary culture was investigated. HRCEC express CD24, the marker of renal distal tubules, as well as globotriaosyl ceramide/CD77, the receptor for Stxs. Binding of Stxs to HRCEC was confirmed by positive staining with specific antibodies to Stxs. Treatment of HRCEC with Stxs induced rapid cell death, which was reversed in the presence of neutralizing antibody specific for Stx. DNA fragmentation was found to be accompanied by Stx-mediated cell death in HRCEC, indicating that apoptosis was part of the process. These data and previous reports indicate that a variety of renal cell types, including tubular epithelial cells as well as glomerular capillary endothelial cells, may be targets for Stx-mediated apoptosis, which could contribute to the pathogenesis of hemolytic-uremic syndrome caused by Stx-producing E. coli infection.

Effects of methylenetetrahydrofolate reductase and reduced folate carrier 1 polymorphisms on high-dose methotrexate-induced toxicities in children with acute lymphoblastic leukemia or lymphoma

The authors investigated whether high-dose methotrexate-induced toxicity differed according to the presence of methylenetetrahydrofolate reductase (MTHFR) or reduced folate carrier 1 (RFC1) genetic polymorphism. The authors studied 15 children with acute lymphoblastic leukemia or lymphoblastic lymphoma who were treated using protocols that included high-dose methotrexate (3.0 g/m2), for an overall total of 43 courses. Methotrexate-induced toxicities and the plasma methotrexate concentrations were evaluated retrospectively. Hematologic toxicity was the most frequently observed toxicity, appearing in 87% of the patients. In a subset of patients (47%), elevation of liver transaminase levels showed a repeated tendency to develop. High plasma methotrexate concentrations at 48 hours after the methotrexate infusion were not significantly related to methotrexate-induced toxicities except for mucositis. A generalized estimating equation analysis revealed that vomiting during the high-dose methotrexate treatment was more pronounced in patients who had a larger number of G alleles at the RFC1 80G>A polymorphism. No significant differences in the development of other toxicities or in the plasma methotrexate concentrations were observed for the different MTHFR 677C>T or RFC1 80G>A polymorphisms. This study suggests but does not prove that the RFC1 80G>A polymorphism may contribute to interindividual variability in responses to high-dose methotrexate.

Globotriaosyl ceramide (CD77/Gb3) in the glycolipid-enriched membrane domain participates in B-cell receptor–mediated apoptosis by regulating Lyn kinase activity in human B cells

The role of CD77 expressed on a fraction of germinal center B cells, also known as glycosphyngolipid Gb3, and as a functional receptor for Shiga toxins (Stx) in B-cell receptor (BCR)-mediated apoptosis was investigated. Using Stx1-sensitive Burkitts lymphoma Ramos cells as an in vitro model of CD77(+) germinal center B cells, intracellular signaling events mediated by either Stx1 or anti-CD77 antibody were examined immunobiochemically and immunocytologically. We observed prompt activation of Lyn and Syk kinases leading to increased binding of these proteins to surface IgM (sIgM) in Ramos cells after Stx1 treatment. We also observed microscopic colocalization of CD77 and sIgM after stimulation with Stx1. Along with the synergism between the cross-linking of CD77 and that of sIgM in their effect on apoptosis induction, it was highly probable that CD77 cross-linking induces activation of the BCR signaling cascade. Analysis using sucrose density gradient centrifugation suggested that Stx1 binding to CD77 induced recruitment and activation of Lyn in the glycolipid-enriched membrane (GEM) fractions. Once activated, however, Lyn seemed to acquire an increased detergent solubility and moved outside of the GEM fractions. This study describes the participation of the GEM domain in BCR-signaling cascade and suggests a possible role of CD77 as a regulator of BCR-induced apoptosis in human B cells.

Long-term results of Tokyo Children's Cancer Study Group trials for childhood acute lymphoblastic leukemia, 1984–1999

We report the long-term results of Tokyo Childrens Cancer Study Groups studies L84-11, L89-12, L92-13, and L95-14 for 1846 children with acute lymphoblastic leukemia, which were conducted between 1984 and 1999. The value of event-free survival (EFS)±s.e. was 67.2±2.2% at 10 years in L84-11, which was not improved in the following two studies, and eventually improved to 75.0±1.8% at 10 years in L95-14 study. The lower EFS of the L89-12 reflected a high rate of induction failure because of infection and delayed remission in very high-risk patients. The L92-13 study was characterized by short maintenance therapy; it resulted in poor EFS, particularly in the standard-risk (SR) group and boys. Females did significantly better than males in EFS in the early three studies. The gender difference was not significant in overall survival, partly because >60% of the males survived after the testicular relapse. Randomized studies in the former three protocols revealed that intermediate- or high-dose methotrexate therapy significantly reduced the testicular relapse rate. In the L95-14 study, gender difference disappeared in EFS. Contrary to the results of larger-scale studies, the randomized control study in the L95-14 reconfirmed with updated data that dexamethasone 8u2009mg/m2 had no advantage over prednisolone 60u2009mg/m2 in the SR and intermediate-risk groups. Prophylactic cranial irradiation was assigned to 100, 80, 44, and 44% of the patients in the studies, respectively. Isolated central nervous system relapse rates decreased to <2% in the last two trials. Secondary brain tumors developed in 12 patients at 8–22 years after cranial irradiation. Improvement of the remission induction rates and the complete omission of irradiation are currently main objectives in our studies.

Influence of MTHFR and RFC1 polymorphisms on toxicities during maintenance chemotherapy for childhood acute lymphoblastic leukemia or lymphoma

We investigated preliminarily whether methylenetetrahydrofolate reductase (MTHFR) 677C/T or reduced folate carrier 1 (RFC1) 80G/A polymorphisms were associated with toxicities during maintenance chemotherapy with mercaptopurine (6MP) and methotrexate (MTX) in children with acute lymphoblastic leukemia or lymphoblastic lymphoma. The clinical records of 20 children (2 to 15-y old) who had received maintenance chemotherapy were reviewed retrospectively and their genomic DNA was genotyped to identify polymorphisms at MTHFR 677C/T, RFC1 80G/A, and thiopurine methyltransferase 719A/G. Maintenance chemotherapy with 6MP and MTX was repeated on a weekly basis, and any week during which 6MP and/or MTX dosing was withheld was counted as an interrupted episode. Associations between the risk of interruptions and polymorphisms were studied using a generalized estimating equation analysis. Patients with an increasing number of T alleles at MTHFR 677C/T experienced interruptions in both 6MP (P<0.01) and MTX (P=0.03) more frequently. Patients with an increasing number of A alleles at RFC1 80G/A experienced interruptions in 6MP (P=0.04) more frequently. This preliminary study does not prove but suggests that MTHFR 677C/T and RFC1 80G/A polymorphisms may serve as predictors of toxicity during maintenance chemotherapy.

Allogeneic haematopoietic cell transplantation from alternative donors with a conditioning regimen of low-dose irradiation, fludarabine and cyclophosphamide in Fanconi anaemia

A pilot study was undertaken using a fludarabine‐based conditioning regimen to improve haematopoietic cell transplantation (HCT) from alternative donors in 27 Fanconi anaemia (FA) patients. Patients were conditioned with 150–180u2003mg/m2 of fludarabine, 40u2003mg/kg of cyclophosphamide, 5–10u2003mg/kg of antithymocyte globulin, and 300–450u2003cGy of thoracoabdominal/total body irradiation. One patient who received unrelated cord blood transplantation failed to engraft, another patient died of sepsis. The 1‐year overall survival was 96·3% (95% CI, 89–100). This conditioning regimen exerted an immunosuppressive effect that enabled durable engraftment in alternative donor HCT without severe toxicity.

Apoptosis of renal tubular cells in Shiga-toxin-mediated hemolytic uremic syndrome.

In order to clarify the mechanism of unusual renal tubular dysfunction seen in a child with Shiga toxin (Stx)-mediated hemolytic uremic syndrome (HUS), we studied the renal biopsy specimens for Stx binding and apoptosis of renal tubular cells. A 7-year-old boy with Stx-2-mediated HUS demonstrated extensive renal tubular damage characterized by nonoliguric acute renal failure, increased urinary tubular enzymes and defective urine-concentrating capacity. His renal biopsy specimens were analyzed for Stx binding and apoptotic cell death. Seven kidney tissue specimens obtained from patients without HUS served as controls. Detection of Stx binding to renal sections and apoptotic cells were performed using mouse monoclonal anti-Stx 2 antibody and the TUNEL method, respectively. Positive staining was observed predominantly in renal tubular cells, while the 7 kidney tissue specimens from control patients did not show positive staining. To the best of our knowledge, this is the first case to show Stx binding and apoptotic cell death in renal tubules on biopsy specimens obtained from a child with Stx-mediated HUS. In conclusion, this case suggests that vascular endothelial cells are not the sole nor the consistent target for Stx-mediated cell injury and that significant renal tubular damage other than glomerular damage might occur in some children with Stx-mediated HUS.

Activation of the caspase cascade during Stx1‐induced apoptosis in Burkitt's lymphoma cells

Shiga toxin 1 (Stx1) produced by Escherichia coli has been reported to induce apoptosis in many different cell types, including Burkitts lymphoma (BL) cells. Since it has been established that the caspases play essential roles as the effector molecules in the apoptotic process in most cases, we examined the kinetics of caspase activation during the process of Stx1‐mediated apoptosis of BL cells. Using Ramos BL cells that are highly sensitive to Stx1‐mediated cytotoxicity, we observed that multiple caspases, including caspase‐3, ‐7, and ‐8 were promptly activated following Stx1 treatment, as indicated by both the procaspase cleavages and enhancement of cleavage of the tetrapeptide substrates of the caspases. In addition, the inhibition assay revealed that caspase‐8 is located upstream of both caspase‐3 and ‐7, suggesting that Stx1‐mediated apoptosis utilizes a similar caspase cascade to that involved in Fas‐mediated apoptosis. Neither anti‐Fas mAb nor TNF‐α, however, affected the Stx1‐mediated apoptosis of Ramos cells. Although the precise mechanism of Stx1‐mediated activation of caspase‐8 is still unclear, we have demonstrated that crosslinkage of CD77, a functional receptor for Stx1, with specific antibody is sufficient to induce activation of caspase‐8. Our findings should provide new insight into the understanding of the molecular basis of Stx1‐mediated cell injury. J. Cell. Biochem. 81:128–142, 2001.

Recurrent childhood anaplastic large cell lymphoma: a retrospective analysis of registered cases in Japan

This report presents a retrospective study of 26 Japanese children with recurrent anaplastic large cell lymphoma. The first relapses were documented at a median of 10·5u2003months after the initial diagnosis. Twenty‐four patients achieved a second remission. After a median follow‐up period of 47u2003months, 18 patients are still alive: 15 patients are in second complete remission (CR), three patients are in third CR or later. The 5u2003year overall and relapse‐free survival rates were 61u2003±u200312% and 51u2003±u200312% respectively. The patients who received allogeneic haematopoietic stem cell transplantation during second CR showed a superior outcome to other patients.

First case of aplastic anemia in a Japanese child with a homozygous missense mutation in the NBS1 gene (I171V) associated with genomic instability

The NBS1 gene is strongly linked to several factors involved in genome integrity. Functional disruption of NBS1 could therefore induce genomic instability and carcinogenesis. Four children with acute lymphoblastic leukemia have been reported to be heterozygous for a germline and/or somatic missense mutation in NBS1, leading to the I171V substitution. We screened healthy controls and pediatric patients with hematological malignancies and aplastic anemia (AA) for the presence of I171V. Of the 62 patients, one individual with AA was confirmed to harbor a homozygous I171V mutation. Genetic analysis of NBS1 in this patient and her healthy parents indicated that she inherited the germline I171V mutation from her father and the wild-type allele from her mother, and that the second I171V hit occurred on the wild-type allele early in embryonic development. Furthermore, cytogenetic analysis of lymphoblastic cell lines from the patient indicated a remarkable increase in numerical and structural chromosomal aberrations in the absence of clastogens, suggesting that she potentially carried genomic instability. This is the first report of AA with a homozygous I171V mutation. We hypothesize that NBS1 may play an important role in the pathogenesis of AA.