Shohei Yamamoto

Wnt3a stimulates maturation of impaired neutrophils developed from severe congenital neutropenia patient-derived pluripotent stem cells

The derivation of induced pluripotent stem (iPS) cells from individuals of genetic disorders offers new opportunities for basic research into these diseases and the development of therapeutic compounds. Severe congenital neutropenia (SCN) is a serious disorder characterized by severe neutropenia at birth. SCN is associated with heterozygous mutations in the neutrophil elastase [elastase, neutrophil-expressed (ELANE)] gene, but the mechanisms that disrupt neutrophil development have not yet been clarified because of the current lack of an appropriate disease model. Here, we generated iPS cells from an individual with SCN (SCN-iPS cells). Granulopoiesis from SCN-iPS cells revealed neutrophil maturation arrest and little sensitivity to granulocyte-colony stimulating factor, reflecting a disease status of SCN. Molecular analysis of the granulopoiesis from the SCN-iPS cells vs. control iPS cells showed reduced expression of genes related to the wingless-type mmtv integration site family, member 3a (Wnt3a)/β-catenin pathway [e.g., lymphoid enhancer-binding factor 1], whereas Wnt3a administration induced elevation lymphoid enhancer-binding factor 1-expression and the maturation of SCN-iPS cell-derived neutrophils. These results indicate that SCN-iPS cells provide a useful disease model for SCN, and the activation of the Wnt3a/β-catenin pathway may offer a novel therapy for SCN with ELANE mutation.

Screening of Drugs to Treat 8p11 Myeloproliferative Syndrome Using Patient-Derived Induced Pluripotent Stem Cells with Fusion Gene CEP110-FGFR1

Induced pluripotent stem (iPS) cells provide powerful tools for studying disease mechanisms and developing therapies for diseases. The 8p11 myeloproliferative syndrome (EMS) is an aggressive chronic myeloproliferative disorder (MPD) that is caused by constitutive activation of fibroblast growth factor receptor 1. EMS is rare and, consequently, effective treatment for this disease has not been established. Here, iPS cells were generated from an EMS patient (EMS-iPS cells) to assist the development of effective therapies for EMS. When iPS cells were co-cultured with murine embryonic stromal cells, EMS-iPS cells produced more hematopoietic progenitor and hematopoietic cells, and CD34+ cells derived from EMS-iPS cells exhibited 3.2–7.2-fold more macrophage and erythroid colony forming units (CFUs) than those derived from control iPS cells. These data indicate that EMS-iPS cells have an increased hematopoietic differentiation capacity, which is characteristic of MPDs. To determine whether a tyrosine kinase inhibitor (TKI) could suppress the increased number of CFUs formed by EMS-iPS-induced CD34+ cells, cells were treated with one of four TKIs (CHIR258, PKC 412, ponatinib, and imatinib). CHIR258, PKC 412, and ponatinib reduced the number of CFUs formed by EMS-iPS-induced CD34+ cells in a dose-dependent manner, whereas imatinib did not. Similar effects were observed on primary peripheral blood cells (more than 90% of which were blasts) isolated from the patient. This study provides evidence that the EMS-iPS cell line is a useful tool for the screening of drugs to treat EMS and to investigate the mechanism underlying this disease.

Successful Treatment of Hepatic Sinusoidal Obstructive Syndrome after Hematopoietic Stem Cell Transplantation in a Child Using Recombinant Thrombomodulin

human leukocyte antigen-mismatched related donor. The interval between administration of GO and BMT was 2 months. His body weight was 8.3 kg at the time of BMT. The conditioning regimen comprised busulfan (19.2 mg/ kg), melphalan (6 mg/kg) and fludarabine (4 mg/kg). Graft-versus-host disease prophylaxis included tacrolimus (0.02 mg/kg/day) and short-term methotrexate. Ursodeoxycholic acid and low-molecular-weight heparin prophylaxis for SOS was administered from day –10 before SCT. On day 10, the antithrombin III (AT III) activity level decreased, which required consecutive administration of AT III concentrate (500 U/day) to maintain an AT III activity 1 80%. His weight began to increase at the same day and showed a 12% increase compared with the pretransplant baseline on day 12 ( fig. 1 ). Simultaneously, the total bilirubin level was elevated to 2.1 mg/dl, with massive abdominal effusion and pain in the right upper quadrant. Serum levels of aspartate aminotransferase, alanine aminotransferase, lactase dehydrogenase and alkaline phosphatase were normal. Abdominal ultrasonography revealed an enlarged liver with moderate ascites and retrograde flow in the right hepatic vein. According to the Seattle criteria [6] , the patient was diagnosed with SOS. The coagulation test showed the following: prothrombin time in international normalized ratio 1.45 (normal 0.9– 1.1); fibrin degradation products 222 mg/l (normal ! 4.0); Hepatic sinusoidal obstructive syndrome (HSOS) is recognized as one of the most common and important fatal complications after hematopoietic stem cell transplantation (SCT). It is a clinical syndrome characterized by painful hepatomegaly, jaundice, ascites, fluid retention and weight gain [1] . The incidence of HSOS in childhood was previously reported to be between 11 and 18%, and mortalities ranged from 15.4 to 38.5% [2–4] . Recombinant human soluble thrombomodulin (rTM) comprises the active, extracellular domain of thrombomodulin and inactivates coagulation by binding to thrombin. In addition, the thrombin-rTM complex activates protein C, which inactivates factors VIIIa and Va in the presence of protein S, thereby inhibiting further thrombin formation [5] . We here describe a pediatric patient with HSOS successfully treated with rTM after SCT. A 1-year-old boy was diagnosed with acute megakaryocytic leukemia in 2009. The patient achieved complete remission after induction therapy. However, he developed a first relapse after two courses of consolidation therapy. Therefore, he received FLAG-IDA-GO (fludarabine, cytarabine, granulcoyte colony-stimulating factor, idarubicin and gemtuzumab ozogamicin) as reinduction therapy and achieved a second complete remission. The dosage of GO was 3 mg/m 2 . He subsequently underwent bone marrow transplantation (BMT) from a one-locus Received: July 4, 2012 Accepted after revision: September 5, 2012 Published online: November 9, 2012

Quantitative polymerase chain reaction detection of CEP110–FGFR1 fusion gene in a patient with 8p11 myeloproliferative syndrome

1 Department of Pediatric Hematology/Oncology, Research Hospital, 2 Division of Stem Cell Processing, Center for Stem Cell Biology and Regenerative Medicine, 3 Department of Hematology/Oncology, Research Hospital, 4 Department of Applied Genomics, Research Hospital and 5 Department of Pathology and Laboratory Medicine, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan and 6 Division of Hematology, Department of Internal Medicine, National Defense Medical College, Saitama, Japan

Fatal hepatic sinusoidal obstruction syndrome in a child with primary CNS lymphoma during induction therapy

Hepatic sinusoidal obstructive syndrome (HSOS) is a lifethreatening syndrome characterized by painful liver enlargement, weight gain, and jaundice, and generally occurs as a complication of hematopoietic stem cell transplantation (HSCT) [1] and chemotherapy in Wilm’s tumor [2]. HSOS after conventional chemotherapy for malignant disease other than Wilm’s tumor is unusual. Here, we report the first case to our knowledge of a 9-yearold boy with PCNSL who developed fatal HSOS during induction therapy. A 9-year-old boy presented with a 4-month history of hyperphagia. He was admitted to our department because of headache. The patient’s body weight was 40 kg (28 % obesity compared to ideal body weight). He had gained 15 kg in body weight over the previous 4 months. A physical examination revealed left-sided hemiparesis with a decreased mental status. The Eastern Cooperative Oncology Group performance status (ECOG-PS) was 3. Laboratory and coagulation tests showed the following: AST, 20 U/L; ALT, 21 U/L; LDH, 384 IU/L; total bilirubin, 0.3 mg/dl; activated partial thromboplastin time (aPTT), 33.2 s (normal 26–36 s); prothrombin time (PT), 10.8 s (normal 10–15 s), prothrombin time in international normalized ratio (PT-INR), 1.05 (normal 0.9–1.1); AT III activity level, 115 % (normal 80–120 %). Magnetic resonance imaging of the brain showed a mass with a homogeneous enhancement located in the right frontal lobe, and this lesion reached the hypothalamus (Fig. 1a). As the leftsided hemiparesis progressed rapidly, a sub-total excision of the tumor was performed. A pathological examination of the surgical specimen revealed a monotonous population of small-sized mononuclear lymphoblastic cells (Fig. 1b). Flow cytometry analysis revealed that lymphoblasts expressed TdT, cytoplasmic CD79a, CD10, and HLA-DR, leading to the diagnosis of precursor B-cell lymphoblastic lymphoma of the right cerebral hemisphere. A bone marrow examination revealed 6 % lymphoblasts. Cerebrospinal fluid (CSF) revealed 81 cells/mm leukocytes, with 82 % lymphoblasts. He showed no evidence of immunodeficiency, and a serological test for HIV was negative. Although optimal treatment of PCNSL in children has not yet been established, the effectiveness of combination chemotherapy for lymphoblastic lymphoma has been studied [3, 4]. We decided to treat the patient according to the protocol of pediatric advanced lymphoblastic lymphoma (ALBNHL03). An induction therapy regimen based on prednisone (60 mg/m daily for 5 weeks), vincristine (VCR) (1.5 mg/m on days 8, 15, 22, and 29), cyclophosphamide (1.0 g/m on day 8), daunorubicin (40 mg/m on days 8, 15, and 22), L-asparaginase (6,000 U/m on days 9, 11, 13, 16, 18, 20, 23, 25, and 27), and triple intrathecal therapy (methotrexate, cytarabine, and hydrocortisone, 12.5, 25, and 25 mg, respectively, on days 1, 15, and 29) (Fig. 2). Although he had shown rapid-onset obesity, no dose modification was made. His left-sided hemiparesis did not change; however, his mental status improved, and CSF tumor cells became negative on day 15. On day 27 of induction therapy, he developed peripheral neuropathy with pain. We stopped the induction therapy other than prednisone thereafter. However, the total bilirubin level was elevated to 2.1 mg/dl, with massive abdominal effusion and pain in the right upper quadrant on day 29. S. Yamamoto (&) K. Akiyama N. Oyama M. Hayashi Y. Fujimoto H. Ikeda K. Isoyama Division of Pediatrics, Department of Showa University Fujigaoka Hospital, 1-30 Fujigaoka Aoba-ku, Yokohama 227-8501, Japan e-mail: shohei-y@med.showa-u.ac.jp

Efficacy and Safety of Slowly Infused Propofol Sedation in Pediatric OncologyProcedures

Children with hematological malignancies frequently undergo invasive procedures such as bone marrow aspiration and lumbar punctures. Although various methods of sedation are currently used for these procedures, no standard has been established to date. midazolam and ketamine (midazolam/ketamine) have been used for procedural sedation in our department. Since both of these drugs have a long half-life, sedation may persist for an excessively long time after the procedure has been completed. Propofol is an intravenously administered anesthetic that is characterized by the easy control of the depth of anesthesia and rapid recovery. Although side effects such as hypoxia and hypotension have been associated with sedation by propofol, their severities may be reduced using slow infusions. Therefore, we herein used slowly infused propofol (0.5 mg/kg over 20 seconds) for these procedures and retrospectively compared its effects with those of midazolam/ketamine. Recovery time was significantly shorter in the propofol group. Although the frequency of side effects such as hypotension was significantly higher in the propofol group, severe side effects were not observed. This may have been because propofol was slowly infused. Although the sample size is too small, the results of the present study suggest that slowly infused propofol might be effective and safe for invasive procedures on pediatric patients. A prospective study is required to further investigate appropriate methods of sedation

Anti-emetic drug maropitant induces intestinal motility disorder but not anti-inflammatory action in mice

Maropitant is a neurokinin 1 receptor (NK1R) antagonist that is clinically used as a new anti-emetic drug for dogs. Substance P (SP) and its receptor NK1R are considered to modulate gastrointestinal peristalsis. In addition, SP works as an inflammatory mediator in gastrointestinal diseases. Aim of this study is to clarify the effects of maropitant on intestinal motility and inflammation in mice. Ex vivo examination of luminal pressure-induced intestinal motility of whole intestine revealed that maropitant (0.1–10 µM) increased frequency of contraction, decreased amplitude of contraction and totally inhibited motility index in a concentration-dependent manner. We measured intestinal transit in vivo by measuring transportation of orally administered luminal content labeled with phenol red. Our results demonstrated that maropitant (10 mg/kg, SC) delayed intestinal transit. Geometric center value was significantly decreased in maropitant-treated mice. Anti-inflammatory effects of maropitant against leukocytes infiltration into the intestinal smooth muscle layer in post-operative ileus (POI) model mice were measured by immunohistochemistry. In POI model mice, a great number of CD68-positive macrophages or MPO-stained neutrophils infiltrated into the inflamed muscle region of the intestine. However, in the maropitant treated mice, the infiltration of leukocytes was not inhibited. The results indicated that maropitant has ability to induce disorder of intestinal motility in mice, but has no anti-inflammatory action in the mouse of a POI model. In conclusion, in mice, maropitant induces disorder of intestinal motility in vivo.

Acute megakaryocytic leukemia (AMKL,FAB;M7) with Beckwith–wiedemann syndrome

Beckwith–Wiedemann syndrome (BWS) is characterized by an accumulation of multiple congenital anomalies. Although patients with BWS are known to have a higher incidence of embryonal tumors, there has been no reports associated with acute leukemia. This report describes the case of a patient with BWS who developed Acute Megakaryocytic Leukemia (AMKL,FAB;M7). Because most patients with BWS present gigantism, the therapy‐related toxicity of chemotherapy can be a very serious problem. This patient exhibited no therapy‐related toxicity after chemotherapy, suggesting that acute leukemia with BWS may not require a reduction in dosage. Pediatr Blood Cancer. 2010;55:733–735.

Open Set Domain Adaptation by Backpropagation

Numerous algorithms have been proposed for transferring knowledge from a label-rich domain (source) to a label-scarce domain (target). Most of them are proposed for closed-set scenario, where the source and the target domain completely share the class of their samples. However, in practice, a target domain can contain samples of classes that are not shared by the source domain. We call such classes the “unknown class” and algorithms that work well in the open set situation are very practical. However, most existing distribution matching methods for domain adaptation do not work well in this setting because unknown target samples should not be aligned with the source. In this paper, we propose a method for an open set domain adaptation scenario, which utilizes adversarial training. This approach allows to extract features that separate unknown target from known target samples. During training, we assign two options to the feature generator: aligning target samples with source known ones or rejecting them as unknown target ones. Our method was extensively evaluated and outperformed other methods with a large margin in most settings.

A theory of centriole duplication based on self-organized spatial pattern formation

In each cell cycle, centrioles are duplicated to produce a single copy of each pre-existing centriole. At the onset of centriole duplication, the master regulator Polo-like kinase 4 (Plk4) undergoes a dynamic change in its spatial pattern on the periphery of the pre-existing centriole, forming a single duplication site. However, the significance and mechanisms of this pattern transition remain largely unknown. Using super-resolution imaging, we found that centriolar Plk4 exhibits periodic discrete patterns resembling pearl necklaces, frequently with single prominent foci. We constructed mathematical models that simulated the pattern formation of Plk4 to gain insight into the discrete ring patterns. The simulations incorporating the self-organization properties of Plk4 successfully generated the experimentally observed patterns. We therefore propose that the self-patterning of Plk4 is crucial for the regulation of centriole duplication. These results, defining the mechanisms of self-organized regulation, provide a fundamental principle for understanding centriole duplication.