c-Myb and GATA-3 cooperatively regulate IL-13 expression via conserved GATA-3 response element and recruit mixed lineage leukemia (MLL) for histone modification of the IL-13 locus.

The c-Myb and GATA-3 transcription factors play important roles in T cell development. We recently reported that c-Myb, GATA-3, and Menin form a core transcription complex that regulates GATA-3 expression and ultimately Th2 cell development in human peripheral blood T cells. However, c-Myb roles for Th2 cytokine expression were not demonstrated. In this article, we report that c-Myb and GATA-3 cooperatively play an essential role in IL-13 expression though direct binding to a conserved GATA-3 response element (CGRE), an enhancer for IL-13 expression. c-Myb and GATA-3 were shown to activate the CGRE-IL-13 promoter by ∼160-fold, and mutation of the canonical Myb binding site completely abrogated CGRE enhancer activity. In contrast, mutation of the GATA binding site partially decreased CGRE enhancer activity. GATA-3 did not bind to CGRE when c-myb expression was silenced. c-Myb, GATA-3, Menin, and mixed lineage leukemia (MLL) bound to CGRE in human primary CD4(+) effector/memory cells. Moreover, c-myb silencing significantly decreased both methylation of histone H3K4 and acetylation of histone H3K9 at the IL-13 locus in CD4(+) effector/memory cells. Therefore, in addition to the strong enhancer effect for the transcription of IL-13, the c-Myb/GATA-3 complex recruits MLL to the CGRE for histone modification of the IL-13 locus during the differentiation of memory Th2 cells.

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.

Primary hepatic marginal zone B cell lymphoma of mucosa-associated lymphoid tissue type: case report and review of the literature.

A primary hepatic marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT) is very rare. We found a solitary mass 27 mm in size in the left lobe of the liver of a 58-year-old Japanese man with a history of hepatitis-C infection. Based on the results of imaging studies, the tumor was diagnosed as a hepatocellular carcinoma (HCC). The left lobe of the liver was lobectomized and microscopic findings showed that the tumor was a hepatic MALT lymphoma, while immunohistochemistry showed it to be positive for CD20 and CD79a. In a fluorodeoxyglucose-positron emission tomography examination integrated with computed tomography scanning (FDG-PET CT) before surgery, the tumor was revealed to have a high standardized uptake value (SUV) for FDG. The patient received chemotherapy after surgery. To the best of our knowledge, 45 cases had been reported with a mean age for all patients of 61.4 years. The pathogenesis remains unclear, although half of the patients had a past history of chronic inflammatory liver disease. Surgical resection was performed in most cases and some patients received postoperative chemotherapy or radiotherapy. The clinicopathologic characteristics and management of this extremely rare disease are also discussed.

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.

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/m(2) on day 1 and 7 mg/m(2) 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.

Factors for PBPC collection efficiency and collection predictors.

Factors affecting collection efficiency of peripheral blood stem cells (PBSCs) include patient's age, diagnosis, preceding chemoradiotherapy, disease invasion of the bone marrow and mobilizing chemotherapy in PBSC collection for autologous transplants. Mobilizing cytokines, timing for apheresis, machines and operating software would affect mobilization and collection of PBSCs both for autologous and allogeneic transplantation. Also donor's age and gender would affect PBSC yield for allogeneic transplantation. Surrogate markers including peripheral blood CD34+ cell counts before mobilization and on day of collection have been reported to predict the yield of PBSC harvest. A number of standard procedures have been developed based on these findings. Newer agents for PBSC mobilization are being evaluated and still other factors affecting mobilization are being sought to better predict and cope with poor mobilization.

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. STUDY DESIGN AND METHODS: CIC counts in PB and CD34+ cell counts in the apheresis product from 122 collections were assessed, and the relationship between these two variables was evaluated with the Pearson rank correlation analysis, the chi-squared test, and the U-test. RESULTS: CIC counts were correlated weakly with the number of CD34+ cells per L of blood processed in the apheresis product (Pearson rank correlation analysis; r=0.357, p<0.0001). When a level of 1.7 x 10(9) CICs per L was selected as a cutoff value, the sensitivity and specificity for collecting more than 20 x 10(6) CD34+ cells per L of blood processed were 63.6 and 77.5 percent, respectively. CONCLUSION: The present study suggests that the number of CICs in PB may estimate the number of CD34+ cells collected. The data indicate that CIC counts above 1.7 x 10(9) per L can be used as a good predictor for PBPC collections containing more than 20 x 10(6) CD34+ cells per L of blood processed in a single apheresis procedure.

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 microg/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 microg/m2/d (n = 8), we observed similar HGF elevation, which appeared to be dose-dependent on the G-CSF administered.

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

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

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.

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

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.

Autoimmune neutropenia associated with multiple sclerosis.

A 53-year-old Japanese man with multiple sclerosis developed autoimmune neutropenia. The neutrophil count was consistently less than 0.2 x 10(9)/l, irrespective of the disease activity of multiple sclerosis or the administration of immunosuppressive agents or granulocyte colony-stimulating factor. After high-dose gamma-globulin therapy was started, temporary increases in the neutrophil count were observed. Despite a wide spectrum of clinical manifestations in multiple sclerosis, autoimmune neutropenia has never been reported previously.

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. STUDY DESIGN AND METHODS: CIC counts in PB and CD34+ cell counts in the apheresis product from 249 collections were assessed, and the relationship between these two parameters was evaluated by with the Pearson rank correlation analysis, the Fisher exact test, and the U-test. RESULTS: CIC counts were correlated significantly with the number of CD34+ cells per kg of patient's body weight in the apheresis product (Pearson rank correlation analysis: r = 0.635, p < 0.0001). When a level of 1 x 10(9) CICs per L was selected as a cutoff value, the sensitivity and specificity for collecting more than 1 x 10(6) CD34+ cells per kg of body weight were 75.7 and 85.5 percent, respectively. CONCLUSION: The present study strongly suggests that the number of CICs in PB may estimate the number of CD34+ cells collected. The data indicate that CIC counts above 1 x 10(9) per L can be used as a good predictor for PBPC collections containing more than 1 x 10(6) CD34+ cells per kg of body weight in a single apheresis procedure.

Specificity of polymerase chain reaction-based clonality analysis of immunoglobulin heavy chain gene rearrangement for the detection of bone marrow infiltrate in B-cell lymphoma-associated haemophagocytic syndrome.

As a wide range of disorders underlie haemophagocytic syndrome, a rapid distinction between benign polyclonal and malignant monoclonal lymphoid proliferations is critical. We investigated whether polymerase chain reaction (PCR) amplification of immunoglobulin heavy chain gene rearrangement could efficiently detect clonal B-cell populations in non-diagnostic marrow for B-cell lymphoma-associated haemophagocytic syndrome (B-LAHS). On amplifying two DNA samples per biopsy, no reproducible monoclonal PCR result was found in reactive haemophagocytic marrows. In contrast, four out of nine assessable B-LAHS patients with histomorphologically and immunohistochemically lymphoma-free bone marrow showed a reproducible monoclonal immunoglobulin heavy chain gene rearrangement. At the molecular level, two B-LAHS patients had lymphoma-free marrow as demonstrated by patient-specific PCR, suggesting that haemophagocytic marrow is not always associated with lymphoma involvement. PCR-based demonstration of clonal B-cell populations in marrow would add an extra dimension to B-LAHS diagnosis.

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.

Tandem high-dose chemotherapy supported by autologous peripheral blood stem cell transplantation for recurrent soft tissue sarcoma.

BACKGROUND: Patients with recurrent soft tissue sarcoma (STS) are seldom curable, with 5-year survival rates of less than 10% in all large series. The role of high-dose chemotherapy (HDC) with hematopoietic stem cell support in this disease has not been established. CASE REPORT: We report on two patients with recurrent STS who were treated with tandem HDC supported by autologous peripheral blood stem cell transplantation (PBSCT). One patient with malignant fibrous histiocytoma recurred with multiple lung metastases. This patient achieved a partial response after two cycles of induction chemotherapy consisting of ifosfamide and epirubicin. During four cycles of induction chemotherapy, peripheral blood stem cells (PBSCs) were harvested. Tandem high-dose ICE regimen (ifosfamide 3 g/m2 on days-7 to -3, carboplatin 400 mg/m2 on days-7, -5 and 3, etoposide 500 mg/m2 on days-7, -5 and 3) supported by autologous PBSCT gave rise to further regression of the tumors. Another patient with malignant hemangiopericytoma was treated by tandem high-dose ICE regimen supported by autologous PBSCT after the 3rd removal of abdominal tumors. Relapse-free intervals until the 1st, 2nd and 3rd relapses were 40, 19 and 22 months, respectively. Tandem high-dose ICE regimen might delay the relapse. CONCLUSION: These observations suggest that a tandem high-dose ICE regimen with autologous PBSCT is feasible with some clinical efficacy in the control of refractory STS.