Koichi Nakase

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.

Ikaros expression in human hematopoietic lineages

The Ikaros gene has been implicated in lymphoid development and proliferation from the results of gene targeting studies in mice. Recently we reported that the Ikaros gene may be involved in the disease progression of chronic myelogenous leukemia (CML). In this report, we investigated Ikaros isoforms in human non-lymphoid leukemia cell lines and normal granulocyte/macrophage (CFU-GM) and erythroid (BFU-E)-derived colonies. We evaluated Ikaros gene expression by RT-PCR, Southern blotting, sequencing analysis, Northern blotting, and immunoblotting.Ikaros isoforms Ik-1 and Ik-2, 3 were predominantly expressed in human non-lymphoid leukemia cell lines. Ik-4 and Ik-8 were also detectable as a minor population. In contrast to the previous report in mice, multiple Ikaros isoforms were expressed in human CFU-GM and BFU-E-derived colonies, and the dominant-negative isoform Ik-6 was not detectable. We also showed that human Ikaros isoforms contained an additional coding sequence in the N-terminal region, which was highly homologous to the sequence reported in mice. These observations suggest that the Ikaros gene may play some role in the development of human non-lymphoid lineage hematopoiesis. Moreover, the finding that the dominant-negative isoform Ik-6, which was overexpressed in patients with blast crisis of CML, was rarely detectable in non-lymphoid lineages supports its pathogenetic role in human hematologic malignancies.

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.

Over-expression of the dominant-negative isoform of Ikaros confers resistance to dexamethasone-induced and anti-IgM-induced apoptosis

Summary. In previous studies, we demonstrated an over‐expression of the dominant‐negative isoform of the transcription factor Ikaros, Ik‐6, in patients with B‐cell malignancies, including blast crisis of chronic myelogenous leukaemia and acute lymphoblastic leukaemia. To investigate the consequence of over‐expression of Ik‐6 in B cells, we constructed Ik‐6 transfectants of the FDH‐1 and Ramos cell lines. FDH‐1, which was established from a patient with early pre‐B acute lymphoblastic leukaemia, undergoes apoptosis with dexamethasone treatment, whereas Ramos undergoes apoptosis following anti‐IgM antibody treatment. Compared with the wild type, the over‐expression of Ik‐6 rendered the FDH‐1 and Ramos transfectants resistant to dexamethasone‐induced and anti‐IgM‐induced apoptosis respectively. An immunoblotting study demonstrated bcl‐2 upregulation in anti‐IgM‐induced Ramos Ik‐6 transfectants, but not in FDH‐1 Ik‐6 transfectants. Further investigations of the mechanism of leukaemogenesis associated with the over‐expression of Ik‐6 are warranted.

Characterization of the short isoform of Helios overexpressed in patients with T‐cell malignancies

In an earlier report, we demonstrated overexpression of a short isoform of Helios, Hel‐5, which lacks three of four N‐terminal zinc fingers, in patients with adult T‐cell leukemia/lymphoma. Here, we characterized Hel‐5 using immunoprecipitation, and gel shift and luciferase promoter assays, and found that Hel‐5 lacks the repressor function observed with a full‐length isoform of Helios. Moreover, Hel‐5 associates with the full‐length isoforms of the Ikaros gene family, Ikaros, Aiolos and Helios, and inhibits their DNA binding activity when present in excess, leading to dominant‐negative effects on the full‐length isoforms of the Ikaros gene family. Our results suggest a critical role for Helios in the mechanism of leukemogenesis. (Cancer Sci 2007; 98: 182–188)

FDG-PET can evaluate the treatment for fungal liver abscess much earlier than other imagings

Dear Editor, Fungal liver abscesses (FLA), mainly caused by Candida, are among the most serious complications in patients with acute leukemia [1–4]. Diagnosing FLA by imaging such as ultrasound (US) [5] or computed tomography (CT) [6] is easily performed when multiple nodular lesions in the patients liver can be seen. However, these nodular lesions can still be found by imaging for a long time after clinical symptoms begin to improve with anti-fungal treatments. Therefore, it is very difficult to evaluate the early effectiveness of treatments. We first report here a case in which the use of 18F-Fluorodeoxyglucose (FDG) positron emission tomography (PET) imaging was more beneficial than US or CT in evaluating FLA in the early stage of treatment. A 32-year-old woman with relapsed acute myeloid leukemia was given re-induction chemotherapy and achieved second complete remission. Allogeneic bone marrow transplantation from an HLA-identical unrelated donor was planned, but low-grade fever (37–38°C) appeared before the transplantation. At that time, her C-reactive protein (CRP) level was about 3 mg/dL, but her blood cultures and beta-D-glucan were negative. Abdominal US and CT revealed multiple low-density areas in her liver, which suggested the FLA or leukemic infiltration. FDG-PET imaging examination was performed, and FDG uptake was shown to be increased at these lesions (Fig. 1 (1)). She was diagnosed with FLA when the periodic acid-Schiff staining of the liver biopsy specimens showed granuloma and yeast-like fungus, although the strain of the fungus could not be identified. Liposomal amphotericin B and Micafungin were administered daily, but the low-grade fever continued, and her CRP gradually increased. Consequently, the scheduled transplantation was postponed. After a month, the antifungal treatment was changed to oral Voriconazole, then her fever lowered promptly and her CRP level decreased gradually. At 3 months after the anti-fungal treatment was started, she had no clinical symptoms, and her CRP level was negative. While the US and CT tests at that time Y. Miyazaki (*) :M. Yasukawa Department of Bioregulatory Medicine, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan e-mail: miya2ymy@yahoo.co.jp

Single-dose daily infusion of cyclosporine for prevention of Graft-versus-host disease after allogeneic bone marrow transplantation from HLA allele-matched, unrelated donors.

Peak blood concentration of cyclosporine (CsA) in renal transplantation patients was recently reported to be associated with clinical efficacy.We therefore evaluated the toxicity and efficacy of a regimen of once-daily infusion of CsA plus a short course of methotrexate as prophylaxis of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation from an HLA allele-matched, unrelated donor. Nineteen patients with hematologic malignancies received CsA, 3 mg/kg per day, as a 4-hour intravenous (IV) infusion from day -1. After engraftment, patients received CsA orally at twice the IV dose. The CsA dose was adjusted to maintain the blood trough level between 150 and 200 ng/mL. Methotrexate was administered IV at doses of 10 mg/m2 on day 1 and 7 mg/m2 on days 3, 6, and 11. Bone marrow engraftment occurred in all patients. Grade 1 and grade 2 GVHD occurred in 6 (31.6%) and 7 (36.8%) of the 19 patients, respectively. No patient had grade 3 or 4 GVHD. Acute nephrotoxicity developed in 1 (5.3%) of the 19 patients, and hypertension developed in 3 (15.8%) of the 19 patients.We evaluated the pharmacokinetics of 4-hour CsA infusion in 10 patients.The mean trough concentration, mean peak concentration, mean time to peak concentration, and area under the curve (24 hours) were 161 ± 43 ng/mL, 1498 ± 387 ng/mL, 3.2 ± 1.0 hours, and 10,848 ± 1,991 ng ± h/mL, respectively. This regimen was well tolerated and did not enhance the risk of severe GVHD in patients undergoing allogeneic bone marrow transplantation from an HLA allele-matched, unrelated donor.