Teruhiko Kozuka

Pregnancy-induced thrombocytopenia and TTP, and the risk of fetal death, in Upshaw-Schulman syndrome: a series of 15 pregnancies in 9 genotyped patients

Upshaw–Schulman syndrome (USS) is a congenital thrombotic thrombocytopenic purpura (TTP) due to mutations in the gene that encodes for ADAMTS13 (ADAMTS13), but its clinical signs may be mild or absent during childhood. We have identified 37 patients with USS (24 females, 13 males) belonging to 32 families. The nine women from six families who were diagnosed during their first pregnancy are the focus of this report. Six of the nine women had episodes of thrombocytopenia during childhood misdiagnosed as idiopathic thrombocytopenic purpura. Thrombocytopenia occurred during the second–third trimesters in each of their 15 pregnancies, with 16 babies (one twin pregnancy), often followed by TTP. Of 15 pregnancies, eight babies were stillborn or died soon after birth, and the remaining seven were all premature except one, who was born naturally following plasma infusions to the mother that had started at 8 weeks’ gestation. All nine USS women had severely deficient ADAMTS13 activity. ADAMTS13 analyses demonstrated that eight women were compound heterozygotes of Y304C/G525D (2 siblings), R125VfsX6/Q1302X (2 siblings), R193W/R349C (2 siblings), I178T/Q929X, and R193W/A606P; one woman was homozygous for R193W. Only the R193W mutation has been previously reported. These observations emphasize the importance of measuring ADAMTS13 activity in the evaluation of thrombocytopenia during childhood and pregnancy.

Elevation of serum hepatocyte growth factor during granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization.

We examined serum levels of the angiogenic factors, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF), in normal donors for allogeneic peripheral blood stem cell (PBSC) transplantation. Granulocyte colony‐stimulating factor (G‐CSF) (filgrastim 400 μg/m2/d) was administered to 23 donors for 5 d and aphereses were performed on days 4 and 5. Although bFGF remained at similar levels after G‐CSF treatment, serum VEGF and HGF levels increased 1·5‐fold (n = 13; P = 0·02) and 6·8‐fold (n = 23; P < 0·0001) respectively. The serum HGF level before G‐CSF administration on day 1 correlated inversely with mobilized CD34+ cell numbers. Time course kinetics of HGF showed that on the day after G‐CSF administration (day 2), serum HGF levels increased to 3678 pg/ml. For auto PBSC mobilization with chemotherapy and G‐CSF 200 μg/m2/d (n = 8), we observed similar HGF elevation, which appeared to be dose‐dependent on the G‐CSF administered.

Overexpression of novel short isoforms of Helios in a patient with T-cell acute lymphoblastic leukemia.

OBJECTIVE In previous studies, we demonstrated overexpression of the dominant-negative isoform of the transcription factor Ikaros, Ik-6, in patients with blast crisis of chronic myelogenous leukemia and B-cell acute lymphoblastic leukemia. In the present study, we analyzed expression of the Ikaros family genes Ikaros, Aiolos, and Helios in a panel of human T-cell leukemia/lymphoma cell lines and bone marrow samples of patients with T-cell acute lymphoblastic leukemia. MATERIALS AND METHODS We performed reverse transcriptase polymerase chain reaction, sequencing analysis, immunoblotting, and Southern blotting. RESULTS We found overexpression of novel short isoforms of Helios (Hel-5 and Hel-6) in the HD-Mar cell line. Southern blot analysis suggested that there might be a small deletion in the Helios locus of HD-Mar. In addition, we observed decreased expression of more than one Ikaros family gene in 3 of 9 patients with T-cell acute lymphoblastic leukemia. Moreover, one of the patients overexpressed novel short isoforms of Helios (Hel-7 and Hel-8). CONCLUSION This study provides the first evidence of an Ikaros family member (other than Ikaros) of which novel short isoforms become overexpressed in human leukemia.

Bone marrow transplantation from unrelated donors for patients with adult T-cell leukaemia/lymphoma

Adult T-cell leukaemia/lymphoma (ATLL) is a highly aggressive haematological malignancy. More than 40 cases of ATLL treated by allogeneic bone marrow transplantation (BMT) from sibling donors have been reported, while there have been only a few cases of unrelated BMT for treatment of this disease. We began performing allogeneic BMT from unrelated donors in 1999 to improve the outcome of ATLL patients with no suitable sibling donors. Eight ATLL patients underwent unrelated BMT; five received the conventional conditioning regimen consisting of cyclophosphamide and total body irradiation, while three received a reduced-intensity preparative regimen. Two patients died due to encephalopathy of unknown aetiology on days 10 and 35, and one patient died due to progression of ATLL 25 months after BMT. Five patients are currently alive and disease-free at a median of 20 months after BMT. Proviral human T-lymphotropic virus type-I (HTLV-I) DNA load in peripheral blood mononuclear cells (PBMCs) was assessed in four cases before and after BMT. HTLV-I proviral DNA load was reduced significantly after transplantation. Unrelated BMT is feasible for treatment of ATLL. Further studies in a larger number of cases are required to determine the optimal conditioning regimen and stem cell source.

Predictive value of circulating immature cell counts in peripheral blood for timing of peripheral blood progenitor cell collection after G–CSF plus chemotherapy-induced mobilization

BACKGROUND: Enumeration of CD34+ cells in peripheral blood (PB) before apheresis predicts the number of CD34+ cells collected, although flow cytometric techniques used are complex and expensive. In an attempt to determine the optimal timing for peripheral blood progenitor cell (PBPC) collection, the usefulness of circulating immature cell (CIC) counts in PB was evaluated.

Peripheral blood circulating immature cell counts predict CD34+ cell yields in G-CSF-induced PBPC mobilization in healthy donors

BACKGROUND: It has been previously reported that the number of circulating immature cells (CIC) in peripheral blood (PB) estimates the number of CD34+ cells collected in G‐CSF plus chemotherapy‐induced PBPC mobilization. The correlation of CIC counts in PB with CD34+ cell yield and its usefulness was evaluated in G‐CSF‐induced PBPC mobilization for healthy donors.

Molecular characterization of the ERGIC-53 gene in two Japanese patients with combined factor V–factor VIII deficiency

Abstract. Combined deficiency of factor V and factor VIII is a distinct clinical entity and is an autosomal recessive disorder. Recently identification of the gene, the endoplasmic reticulum-Golgi intermediate compartment (ERGIC-53), responsible for combined factor V–factor VIII deficiency and mutations of the ERGIC-53 gene in affected patients have been reported. In this report we analyzed two Japanese patients with combined factor V–factor VIII deficiency by genomic polymerase chain reaction and sequencing analysis. In one patient we found a point mutation of C to T at nucleotide 604 in exon 5, resulting in a transition of arginine to stop codon, which was reported in previous reports. The DdeI digestion study demonstrated that this patient is homozygous for this nonsense mutation. In the other patient we found no mutation in the ERGIC-53 gene in analysis of the entire coding region and the intron/exon junctions, which is also consistent with the previous reports, suggesting the possibility of defects at other genetic loci.

The type 1 CD10/neutral endopeptidase 24.11 promoter: functional characterization of the 5¢-untranslated region

Summary. The cell surface zinc metalloproteinase CD10/neutral endopeptidase 24.11 (NEP) is expressed on normal and malignant lymphoid progenitors, granulocytes and a variety of epithelial cells. Because CD10/NEP functions as part of a regulatory loop that controls local concentrations of peptide substrates and associated peptide‐mediated signal transduction, its role in each tissue is different depending on the availability of substrate. To characterize further how this widely distributed molecule is regulated differentially in each tissue, we analysed the major type 2 CD10/NEP promoter and found three functionally important transcription factor binding sites, one of which was identical to CCAAT‐binding transcription factor/nuclear transcription factor Y. In this report, we analyse the type 1 CD10/NEP promoter and found a functionally important transcription factor binding site in the 5′‐untranslated region. The results of the competition and supershift experiments demonstrated that the functionally important transcription factor was identical to Sp1. Our results suggest that ubiquitously expressed Sp1 may play an important role in differentiation stage‐specific regulation of CD10/NEP expression in lymphoid lineage.

Over-expression of short isoforms of Helios in patients with adult T-cell leukaemia/lymphoma

Summary. In previous studies, we demonstrated an over‐expression of the dominant‐negative isoform of the transcription factor Ikaros in patients with blast crisis of both chronic myelogenous leukaemia and B‐cell acute lymphoblastic leukaemia (ALL). Recently, we reported an over‐expression of the short isoforms of Helios, which is one of the members of the Ikaros gene family, in a patient with T‐cell ALL. In the present study, we found over‐expression of short isoforms of Helios in human T lymphotropic virus‐I (HTLV1)‐infected patients who had developed chronic and acute forms of adult T‐cell leukaemia/lymphoma. In contrast, we could not detect any over‐expression of short isoforms of Helios in healthy HTLV1 carriers. By Southern blotting, we detected a small deletion in the Helios gene locus of adult T‐cell leukaemia/lymphoma patients. The present results suggest that Helios gene abnormalities might be one of the important mechanisms in the disease progression of HTLV1 infection.

Human bone marrow stroma‐dependent cell line MOLP‐5 derived from a patient in leukaemic phase of multiple myeloma

The novel multiple myeloma (MM) cell line MOLP‐5 and its homologous sister cell line B407, a lymphoblastoid cell line (LCL), were established from the peripheral blood of a 71‐year‐old Japanese patient with Bence‐Jones κ‐type multiple myeloma (stage IIIB with hyperammonaemia and hypercalcaemia). The growth of MOLP‐5 cells is constitutively dependent on bone marrow stroma (BST) cells; none of the cytokines tested nor the culture supernatant of the bone marrow stroma cells could support the growth of MOLP‐5. Wright–Giemsa‐stained MOLP‐5 cells showed typical plasma cell morphology with abundant cytoplasm and one to three nuclei. The immunoprofile of MOLP‐5 corresponds to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) κ light chain, CD28, CD29, CD38, CD40, CD44, CD49d, CD54, CD56, CD58, CD71, CD138 and PCA‐1; the cells were negative for surface Ig and various other B‐cell, T‐cell and myelomonocyte‐associated immunomarkers. Interleukin 6 (IL‐6) receptor mRNA was found in the reverse transcriptase polymerase chain reaction (RT‐PCR) analysis. IL‐6 and IL‐10 could induce cellular proliferation in short‐term induction experiments. IL‐6 or IL‐10 production was not detected by specific enzyme‐linked immunoabsorbent assay (ELISA). MOLP‐5 cells expressed parathyroid hormone‐related protein (PTHrP) at the mRNA level. Cytogenetic analysis showed the typical t(11; 14) chromosome abnormality. The novel MOLP‐5 cell line together with the B407 B‐LCL sister line will be useful model systems in the investigation of the biology of MM.