bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of PU.1 Induces Growth and Differentiation Inhibition and Apoptotic Cell Death in Murine Erythroleukemia Cells
Authors: Yamada Toshiyuki , Kondoh Nobuo , Matsumoto Mana , Yoshida Midori , Maekawa Akihiko , Oikawa Tsuneyuki
Published: October 2019

PU.1 is a member of the ets family of transcription factors and is expressed in Friend virus-induced murine erythroleukemia (MEL) cells as a consequence of proviral integration into the PU.1/Spi-1 locus. After induction of MEL cell differentiation by treatment with dimethylsulfoxide (DMSO), expression of the PU.1/Spi-1 gene decreased before induction of β-globin gene expression. Overexpression of PU.1 by using a zinc-inducible expression plasmid in MEL cells resulted in unexpected growth inhibition of the transfectants. When PU.1-overexpressing transfectants were treated with DMSO, growth inhibition became much pronounced and apoptosis was induced. Expression of the β-globin gene was not induced under this condition. Neither growth inhibition nor apoptosis was induced in MEL cells after expression of mutant PU.1 proteins with a deletion of the activation domain or the DNA-binding Ets domain irrespective of the presence of DMSO. Interestingly, β-globin gene expression was not induced in the transfectants expressing the former mutant, whereas it was induced in those expressing the latter one in the presence of DMSO. These results indicate that overexpression of PU.1 in MEL cells results in growth and differentiation inhibition and, in conjunction with DMSO treatment, apoptotic cell death. These results also suggest that the activation domain and the Ets domain of PU.1 contribute differently to induction of these effects.
In Vitro Expansion of Hematopoietic Progenitors and Maintenance of Stem Cells: Comparison Between FLT3/FLK-2 Ligand and KIT Ligand
Authors: Yonemura Yuji , Ku Hsun , Lyman Stewart D. , Ogawa Makio
Published: October 2019

The effects of FLT3/FLK-2 ligand (FL) and KIT ligand (KL) on in vitro expansion of hematopoietic stem cells were studied using lineage-negative (Lin−)Sca-1–positive (Sca-1+) c-kit–positive (c-kit+) marrow cells from 5-fluorouracil (5-FU)–treated mice. As single agents, neither FL nor KL could effectively support the proliferation of enriched cells in suspension culture. However, in combination with interleukin-11 (IL-11), both FL and KL enhanced the production of nucleated cells and progenitors. The kinetics of stimulation by FL was different from that by KL in that the maximal expansion by FL of the nucleated cell and progenitor pools required a longer incubation than with KL. We then tested the reconstituting abilities of cells cultured for 1, 2, and 3 weeks by transplanting the expanded Ly5.1 cells together with “compromised” marrow cells into lethally irradiated Ly5.2 mice. Cells that had been expanded with either cytokine combination were able to maintain the reconstituting ability of the original cells. Only cells that had been incubated with KL and IL-11 for 21 days had less reconstituting ability than fresh marrow cells. These results indicate that there can be significant expansion of progenitors in vitro without compromising the reconstituting ability of stem cells. Addition of IL-3 to permissive cytokine combinations significantly reduced the ability of cultured cells to reconstitute the hematopoiesis of irradiated hosts. These observations should provide a basis for a rational approach to designing cytokine combinations for in vitro expansion of hematopoietic stem cells.
Proteolytic Cleavage of Recombinant Type 2A von Willebrand Factor Mutants R834W and R834Q: Inhibition by Doxycycline and by Monoclonal Antibody VP-1
Authors: Tsai Han-Mou , Sussman Ira I. , Ginsburg David , Lankhof Hanneke , Sixma Jan J. , Nagel Ronald L.
Published: October 2019

The susceptibility of recombinant type 2A von Willebrand factor (vWF ) to a recently identified plasma metalloproteinase and the potential application of proteolysis inhibition in the treatment of the disease were investigated. Two recombinant type 2A vWF mutants, R834W and R834Q, were spontaneously cleaved by the partially purified plasma proteinase to smaller forms. When treated with guanidine HCl, both the wild-type and the R834W mutant vWF exhibited a biphasic change in proteolytic susceptibility, reaching the same maximum cleavage at 1.25 mol/L guanidine HCl. Proteolysis of the recombinant vWF generated the same 350-kD and 200-kD species (dimers of the 176-kD and 140-kD fragments, respectively) as those found in normal plasma. The proteinase activity was inhibited by doxycycline, with an IC50 of approximately 0.25 mmol/L. The inhibitory activity of doxycycline was related to its metallic cation binding activity. Susceptibility of the recombinant vWF to the proteinase was inhibited by monoclonal antibody VP-1 (directed against residues 828-842 of the vWF polypeptide), but not by two other monoclonal antibodies M13 and M31. The spontaneous susceptibility to proteolytic cleavage may account for the lack of large multimers in type 2A von Willebrand disease (vWD), and the results with tetracyclines and monoclonal antibody VP-1 offer new strategies for developing specific treatment of type 2A vWD.
Mortality and Causes of Death in Families With the Factor V Leiden Mutation (Resistance to Activated Protein C)
Authors: Hille Elysée T.M. , Westendorp Rudi G.J. , Vandenbroucke Jan P. , Rosendaal Frits R.
Published: October 2019

To investigate whether resistance to activated protein C (APC resistance) because of a mutation in the factor V gene (factor V Leiden) leads to a decrease in life expectancy, we analyzed overall and cause-specific mortality in 171 parents whose offspring carried this mutation. Compared with the Dutch general population, and after adjustment for age, sex, and calendar period, we found no excess deaths in the parents (standardized mortality ratio [SMR], 1.0; 95% confidence interval [CI], 0.8 to 1.2). The cause-specific SMR for malignant neoplasms (1.0; 95% CI, 0.6 to 1.4), diseases of the circulatory system (1.0; 95% CI, 0.7 to 1.4), and cerebrovascular disease (1.0; 95% CI, 0.4 to 1.9) also did not differ from unity. The SMRs for diseases of the respiratory system (1.4; 95% CI, 0.6 to 2.6) and for ischemic heart diseases (1.1; 95% CI, 0.7 to 1.7) were slightly increased. Under the age of 45 years, there was a ninefold increase of dying from ischemic heart disease. Thromboembolic complications were mentioned only once (venous embolism or thrombosis) as an underlying (“primary”) cause of death (SMR, 2.3; 95% CI, 0.1 to 13.0) and three times (pulmonary embolisms) as a contributing (“secondary”) cause of death (SMR, 1.5; 95% CI, 0.3 to 4.4). We conclude that there is no major effect of APC resistance on life expectancy. Therefore, long-term anticoagulation in carriers of factor V Leiden, on the basis of the carrier state alone, is not indicated.
p53 Abnormalities in B-Cell Prolymphocytic Leukemia
Authors: Lens Daniela , J.J.C. De Schouwer Pierre , Hamoudi Rifat A. , Abdul-Rauf Munah , Farahat Nahla , Matutes Estella , Crook Tim , Dyer Martin J.S. , Catovsky Daniel
Published: October 2019

B-cell prolymphocytic leukemia (B-PLL) is an aggressive disorder of mature B cells with distinct clinical and pathologic features. To determine the incidence of abnormalities of p53, we analyzed 19 cases of B-PLL by DNA blot to assess loss of heterozygosity (LOH) at 17p13.3, by immunocytochemistry to assess p53 expression, and by direct DNA sequencing of polymerase chain reaction-amplified exons 5 to 9 of the p53 gene. LOH was detected in 10 of 19 (53%) cases, p53 expression was detected in 8 of 17 (47%), and p53 mutations were detected in 10 of 19 (53%) cases. The pattern of mutations was distinct from that observed in other B-cell malignancies. Six cases exhibited missense mutations; 4 were transversions and 2 were transitions. The G:C → A:T transition at cathepsin G dinucleotides commonly reported in p53 mutations in chronic lymphocytic leukemia (CLL) and other hematologic malignancies was observed in only 1 case of B-PLL. Three cases exhibited deletions (ranging from 3 to 35 bp in length) and one case exhibited a 2-bp insertion. In 1 case, a 27-bp deletion resulted in the expression of a p53 protein lacking 9 amino acids from the DNA binding region. All samples with p53 mutation showed loss of germline p53 sequences. However, 3 of 10 showed no LOH by Southern blot, indicating a localized deletion around the p53 locus at 17p13.1. Five of the 10 cases with p53 mutation exhibited detectable p53 expression, including 4 cases with p53 missense mutation and 1 case with deletion. Two of 7 cases with no detectable mutation of p53 nevertheless overexpressed p53. Therefore, there was no correlation between protein expression and p53 mutation in B-PLL. Our data indicate that the overall abnormalities of p53 occurred in 14 of 19 (75%) cases of B-PLL. The frequency of p53 mutation (53%) in B-PLL is the highest reported in B-cell malignancies and may be responsible for the frequent resistance to therapy of this disease. In addition, the pattern of p53 mutation was different from that observed in CLL and other hematologic malignancies and may indicate that a distinct pathogenic mechanism operates in B-PLL.
Differences Between Graft-Versus-Leukemia and Graft-Versus-Host Reactivity. I. Interaction of Donor Immune T Cells With Tumor and/or Host Cells
Authors: Rocha Marian , Umansky Victor , Lee Kyeong-Hee , Hacker Hans-Jörg , Benner Axel , Schirrmacher Volker
Published: October 2019

Graft-versus-leukemia (GVL) and Graft-versus-host (GVH) reactions were compared after systemic transfer of allogeneic antitumor immune T lymphocytes from B10.D2 (H-2d; MIsb) into DBA/2 (H-2d; MIsa) mice. Before immune cell transfer, recipient DBA/2 mice were sublethally irradiated with 5 Gy to prevent host-versus-graft reactivity. Recipients were either bearing syngeneic metastatic ESb lymphomas (GVL system) or were normal, non–tumor-bearing mice (GVH system). We previously reported that this adoptive immunotherapy protocol (ADI) had pronounced GVL activity and led to immune rejection of even advanced metastasized cancer. In this study, monoclonal antibodies were used for immunohistochemical analysis of native frozen tissue sections from either spleen or liver to distinguish donor from host cells, to differentiate between CD4 and CD8 T lymphocytes, and to stain sialoadhesin-positive macrophages at different time points after cell transfer. The kinetics of donor cell infiltration in spleen and liver differed in that the lymphoid organ was infiltrated earlier (days 1 to 5 after transfer) than the nonlymphoid organ (days 5 to 20). After reaching a peak, donor cell infiltration decreased gradually and was not detectable in the spleen after day 20 and in the liver after day 30. The organ-infiltrating donor immune cells were mostly T lymphocytes and stained positive for CD4 or CD8 T-cell markers. A remarkable GVL-associated observation was made with regard to a subset of macrophages bearing the adhesion molecule sialoadhesin (SER+ macrophages). In the livers of tumor-bearing mice, their numbers increased between days 1 and 12 after ADI by a factor greater than 30. Double-staining for donor cell marker and SER showed that the sialoadhesin-expressing macrophages were of host origin. The SER+ host macrophages from GVL livers were isolated by enzyme perfusion and rosetting 12 days after ADI, when they reached peak values of about 60 cells per liver lobule, and were tested, without further antigen addition, for their capacity to stimulate an antitumor CD8 T-cell response. The results of this immunologic analysis suggest that these cells in the liver function as scavengers of the destroyed metastases and as antigen-processing and -presenting cells for antitumor immune T cells.
Hematopoietic Stem Cell–Based Gene Therapy for Acquired Immunodeficiency Syndrome: Efficient Transduction and Expression of RevM10 in Myeloid Cells In Vivo and In Vitro
Authors: Su Lishan , Lee Robert , Bonyhadi Mark , Matsuzaki Hajime , Forestell Sean , Escaich Sonia , Böhnlein Ernst , Kaneshima Hideto
Published: October 2019

Gene delivery via the hematopoietic stem cell (HSC) offers an attractive means to introduce antiviral genes into both T cells and macrophages for acquired immunodeficiency syndrome (AIDS) gene therapy. An amphotropic retroviral vector encoding a bicistronic gene coexpressing RevM10 and the murine CD8α′ chain (lyt2) was developed to transduce HSC/progenitor cells. After transduction of CD34+ cells isolated from human umbilical cord blood, the lyt2 molecule detected by flow cytometry was used to monitor the level of gene transduction and expression and to enrich RevM10-expressing cells by cell sorting without drug selection. Using this quantitative method, high levels of gene transduction and expression (around 20%) were achieved by high-speed centrifugation of CD34+ cells with the retroviral supernatant (spinoculation). After reconstitution of human bone marrow implanted in SCID mice (SCID-hu bone) with the transduced HSC/progenitor cells, a significant number of donor-derived CD14+ bone marrow cells were found to express the RevM10/lyt2 gene. Finally, replication of a macrophage-tropic human immunodeficiency virus–type 1 (HIV-1) isolate was greatly inhibited in the lyt2+/CD14+ cells differentiated from transduced CD34+ cells after the enrichment of lyt2+ population. Thus, the RevM10 gene did not appear to inhibit the differentiation of HSC/progneitor cells into monocytes/macrophages. The level of retrovirus-mediated RevM10 expression in monocytes/macrophages derived from transduced HSCs is sufficient to suppress HIV-1 replication.
Randomized Comparison of DAT Versus ADE as Induction Chemotherapy in Children and Younger Adults With Acute Myeloid Leukemia. Results of the Medical Research Council's 10th AML Trial (MRC AML10)
Authors: Hann Ian M. , Stevens Richard F. , Goldstone Anthony H. , Rees John K.H. , Wheatley Keith , Gray Richard G. , Burnett Alan K.
Published: October 2019

The relative efficacy and toxicity of the chemotherapeutic agents thioguanine (6TG) and etoposide (VP16) were assessed by a randomized comparison of the DAT (daunorubicin, cytarabine, thioguanine) versus ADE (daunorubicin, cytarabine, etoposide) regimens in the Medical Research Council's 10th acute myeloid leukaemia trial (MRC AML 10), which was open to patient entry from May 1988 to April 1995. In this, the largest reported trial of AML therapy to date, 1,857 eligible patients, mostly less than 56 years old, were randomized: 929 (including 143 children under 15 years old) were allocated to DAT and 928 (143 children) to ADE. The two groups were well matched for presentation features. The complete remission (CR) rate was 81% with DAT and 83% with ADE (P = .3). The percentages of remitters achieving remission after 1, 2, or more than 2 courses were 70%, 22%, and 8% for DAT and 74%, 21%, and 5% for ADE. The percentages failing to achieve a CR due to resistant disease were 11% with DAT versus 9% with ADE (P = .07). There was a slightly higher death rate in CR during consolidation chemotherapy with ADE (9%) than with DAT (6%) (P = .06). Patients receiving DAT took slightly but significantly longer to recover from neutropenia and thrombocytopenia but the median number of days in hospital were similar in each group. ADE patients experienced slightly more severe nonhematologic toxicity. There was also no significant difference between the groups in the longer-term measures of efficacy: disease-free survival at 6 years from CR was 42% (±4) for DAT and 43% (±4) for ADE (P = .8); relapse rate at 6 years was 50% (±4) for DAT and 49% (±5) for ADE (P = .6); survival at 6 years was 40% (±4) for both DAT and ADE (P = .9). Subgroup analysis failed to show any benefit for etoposide in patients with monocytic or myelomonocytic disease, or in any other diagnostic subgroup. In conclusion, DAT and ADE both achieve high remission rates and good long-term survival, and are equally effective chemotherapy regimens for the treatment of AML patients aged up to 55 years.
Clinical Relevance of Point Mutations in the Cytoplasmic Domain of the Granulocyte Colony-Stimulating Factor Receptor Gene in Patients With Severe Congenital Neutropenia
Authors: Tidow N. , Pilz C. , Teichmann B. , Müller-Brechlin A. , Germeshausen M. , Kasper B. , Rauprich P. , Sykora K.-W. , Welte K.
Published: October 2019

Recently, point mutations in the gene of the granulocyte colony-stimulating factor (G-CSF ) receptor have been reported in two patients with severe congenital neutropenia who developed acute myeloid leukemia (AML). We investigated the frequency of these specific G-CSF receptor mutations in patients with congenital neutropenia undergoing treatment with r-metHuG-CSF (Filgrastim) and the clinical relevance of these mutations. Nucleotides 2306 to 2561 including the critical region (nucleotides 2384-2429) from the intracellular domain of the G-CSF receptor gene were amplified by reverse transcriptase-polymerase chain reaction. Detection of point mutations was performed with specific restriction enzyme analysis, as well as sequencing of PCR products. Both genomic DNA and cDNA from neutrophils and mononuclear cells were analyzed from 28 patients with severe congenital neutropenia. Four of 28 patients with congenital neutropenia displayed a point mutation in the tested cytoplasmic region of the G-CSF receptor gene. The point mutations replace a glutamine codon by a stop codon of the G-CSF receptor gene. Among these four congenital neutropenia patients with a mutated G-CSF receptor, two developed AML. All four patients were investigated regularly and no correlation between occurrence of G-CSF receptor mutation and time or dose of r-metHuG-CSF treatment was found. No point mutations in the G-CSF receptor critical domain could be detected in cells from the other 24 congenital neutropenia patients. Furthermore, we tested six family members of the two patients with AML including mothers and fathers, one sister, and one brother who suffers from congenital neutropenia, as well. All family members displayed a normal G-CSF receptor gene. After the acquisition of the G-CSF receptor mutations, the congenital neutropenia patients continued to respond to G-CSF therapy with an increase in absolute neutrophils in the peripheral blood. We conclude that the point mutations in the critical region of the intracellular part of the G-CSF receptor occur spontaneously and are not inherited. From our data, we suggest that the described G-CSF receptor point mutations do not alter the response to treatment with r-metHuG-CSF and are not the cause of severe congenital neutropenia.
HPA-10wb (Laa): Genetic Determination of a New Platelet-Specific Alloantigen on Glycoprotein IIIa and Its Expression in COS-7 Cells
Authors: Peyruchaud O. , Bourre F. , Morel-Kopp M.-C. , Reviron D. , Mercier P. , Nurden A. , Kaplan C.
Published: October 2019

The heterodimeric complex glycoprotein (GP)IIb-IIIa, the fibrinogen receptor of platelets, carries numerous alloantigen systems. These polymorphisms are responsible for the immune response after transfusion or during pregnancy. In the latter case, the mother develops an antibody against an epitope present on fetal platelets, and this results in platelet destruction in the fetus. In this report, we describe the molecular characterization of a new alloantigen (Laa) on GPIIIa responsible for neonatal alloimmune thrombocytopenia (NAIT). Using polymerase chain reaction (PCR)–singlestrand conformation polymorphism (SSCP) and DNA sequencing, we found a point mutation (G to A) in a heterozygous state on the GPIIIa gene leading to amino acid substitution Arg to Gln at position 62 of the mature protein. Transient expression of GPIIb-IIIa complexes in Cos-7 cells using wild-type or mutated GPIIIa cDNA allowed us to demonstrate that this mutation was responsible for expression of the Laa epitope.
Possible Roles of an Adult T-Cell Leukemia (ATL)-Derived Factor/Thioredoxin in the Drug Resistance of ATL to Adriamycin
Authors: Wang Jingxin , Kobayashi Masanobu , Sakurada Keisuke , Imamura Masahiro , Moriuchi Tetsuya , Hosokawa Masuo
Published: October 2019

Chemotherapy for adult T-cell leukemia (ATL) has been reported to fail to induce complete remission because of drug resistance in most patients. We have examined the expression of an ATL-derived factor (ADF)/thioredoxin in relation to resistance to adriamycin (ADM) in various T-cell leukemia cell lines including ATL cell lines. Immunoblot analysis demonstrated that ATL cell lines expressed ADF/thioredoxin at levels 2.8 to 12 times those of other T-cell acute lymphocytic leukemia (T-ALL) cell lines, and that ATL cell lines were 2 to 15 times more resistant to ADM than other T-ALL cell lines. Therefore, we established ADM-resistant cell lines from three different ATL cell lines, and examined the correlation between ADM resistance and expression of ADF/thioredoxin. ADM-resistant ATL cell lines were also found to be resistant to other drugs such as cisplatin and etoposide, and they expressed ADF/thioredoxin at levels 5 to 10 times those of parent ATL cell lines. Diamide and sodium selenite, which have been reported to inhibit ADF/thioredoxin, restored the sensitivity to ADM in ATL and ADM-resistant ATL cell lines. The MDR-1 gene product, a membrane P-glycoprotein (Pgp), was not expressed on ATL cell lines or ADM-resistant ATL cell lines. Topoisomerase II and glutathione peroxidase activities in T-cell leukemia cell lines were not correlated with ADM resistance. These results suggest that ADF/thioredoxin may play an important role in the drug resistance of ATL cells to ADM.
11q Deletions Identify a New Subset of B-Cell Chronic Lymphocytic Leukemia Characterized by Extensive Nodal Involvement and Inferior Prognosis
Authors: Döhner Hartmut , Stilgenbauer Stephan , James Michael R. , Benner Axel , Weilguni Traudel , Bentz Martin , Fischer Konstanze , Hunstein Werner , Lichter Peter
Published: October 2019

Deletions of the long arm of chromosome 11 (11q) are one of the most frequent structural chromosome aberrations in various types of lymphoproliferative disorders. However, in most conventional chromosome banding studies of B-cell chronic lymphocytic leukemia (B-CLL), 11q deletions were not identified as a frequent aberration. The objective of this study was to analyze the frequency and clinical impact of 11q deletions in B-CLL by interphase cytogenetics using fluorescence in situ hybridization (FISH). Mononuclear cells from 214 patients with B-CLL were studied by FISH using the yeast artificial chromosome (YAC) clone 755b11 from chromosome region 11q22.3-q23.1; we previously showed that this clone was contained within a 2- to 3-Mb sized segment of 11q commonly deleted in lymphoproliferative disorders. Forty-three of the 214 (20%) tumors exhibited 11q deletions; 11q deletions were the second most frequent chromosome aberration following 13q14 (RB1 and/or D13S25) deletions (45%); they were more frequent than trisomy 12 (15%) or deletion of 17p (TP53 gene) (10%). Patients with 11q deletions were younger (P = .01) and had more advanced clinical stages (P = .01). 11q deletions were associated with extensive peripheral, abdominal, and mediastinal lymphadenopathy (P < .001). Patients with 11q deletions had a more rapid disease progression as shown by a shorter treatment-free interval (9 months v 43 months; P < .001). The prognostic effect of 11q deletion on survival strongly depended on the age: in patients less than 55 years old, the median survival time was significantly shorter in the deletion group (64 months v 209 months; P < .001), whereas in patients ≥ 55 years old there was no significant difference (94 months v 111 months; P = .82). 11q deletions identify a new clinical subset of B-CLL characterized by extensive lymph node involvement. In younger B-CLL patients, this aberration is an important predictor of survival.
Inappropriately High Iron Regulatory Protein Activity in Monocytes of Patients With Genetic Hemochromatosis
Authors: Cairo Gaetano , Recalcati Stefania , Montosi Giuliana , Castrusini Elisa , Conte Dario , Pietrangelo Antonello
Published: October 2019

In genetic hemochromatosis (GH), excess iron is deposited in parenchymal cells, whereas little iron is found in reticuloendothelial (RE) cells until the later stages of the disease. As iron absorption is inversely related to RE cells stores, a failure of RE to retain iron has been proposed as the basic defect in GH. In RE cells of GH subjects, we examined the activity of iron regulatory protein (IRP), a reliable indicator of the elusive regulatory labile iron pool, which modulates cellular iron homeostasis through control of ferritin (Ft) and transferrin receptor gene expression. RNA-bandshift assays showed a significant increase in IRP activity in monocytes from 16 patients with untreated GH compared with 28 control subjects (1.5-fold) and five patients with secondary hemochromatosis (SH) with similar iron burden (fourfold). In 17 phlebotomy-treated GH patients, IRP activity did not differ from that of control subjects. In both GH and SH monocyte-macrophages, Ft content increased by twofold and the L subunit-rich isoferritin profile was unchanged as compared with controls. IRP activity was still upregulated in vitro in monocyte-derived macrophages of GH subjects but, following manipulations of iron levels, was modulated normally. Therefore, the sustained activity of monocyte IRP found in vivo in monocytes of GH patients is not due to an inherent defect of its control, but is rather the expression of a critical abnormality of iron metabolism, eg, a paradoxical contraction of the regulatory iron pool. By preventing Ft mRNA translation, high IRP activity in monocytes may represent a molecular mechanism contributing to the inadequate Ft accumulation and insufficient RE iron storage in GH.
Mig, the Monokine Induced By Interferon-γ, Promotes Tumor Necrosis In Vivo
Authors: Sgadari Cecilia , Farber Joshua M. , Angiolillo Anne L. , Liao Fang , Teruya-Feldstein Julie , Burd Parris R. , Yao Lei , Gupta Ghanshyam , Kanegane Chiharu , Tosato Giovanna
Published: October 2019

Mig, the monokine induced by interferon-γ, is a CXC chemokine active as a chemoattractant for activated T cells. Mig is related functionally to interferon-inducible protein 10 (IP-10), with which it shares a receptor, CXCR3. Previously, IP-10 was found to have antitumor activity in vivo. In the present study, murine Mig RNA was found to be expressed at higher levels in regressing Burkitt's lymphoma tumors established in nude mice compared with progressively growing tumors. Daily inoculations of purified recombinant human Mig into Burkitt's tumors growing subcutaneously in nude mice consistently caused tumor necrosis associated with extensive vascular damage. These effects were indistinguishable from those produced by intratumor inoculations of Burkitt's tumors with IP-10. These results support the notion that Mig, like IP-10, has antitumor activity in vivo.
Extensive Amplification and Self-Renewal of Human Primitive Hematopoietic Stem Cells From Cord Blood
Authors: Piacibello Wanda , Sanavio Fiorella , Garetto Lucia , Severino Antonella , Bergandi Daniela , Ferrario Jessica , Fagioli Franca , Berger Massimo , Aglietta Massimo
Published: October 2019

The use of umbilical cord blood as a source of marrow repopulating cells for the treatment of pediatric malignancies has been established. Given the general availability, the ease of procurement, and progenitor content, cord blood is an attractive alternative to bone marrow or growth factor mobilized peripheral blood cells as a source of transplantable hematopoietic tissue. However, there is a major potential limitation to the widespread use of cord blood as a source of hematopoietic stem cells for marrow replacement and gene therapy. There may be enough hematopoietic stem cells to reconstitute children, but the ability to engraft an adult might require ex vivo manipulations. We describe an in vitro system in which the growth of cord blood CD34+ cells is sustained and greatly expanded for more than 6 months by the simple combination of two hematopoietic growth factors. Progenitors and cells belonging to all hematopoietic lineages are continuously and increasingly generated (the number of colony-forming unit–granulocyte-macrophage [CFU-GM] present at the end of 6 months of culture are well over 2,000,000-fold the CFU-GM present at the beginning of the culture). Very primitive hematopoietic progenitors, including long-term culture-initiating cells (LTC-ICs) and blast cell colony-forming units, are also greatly expanded (after 20 weeks of liquid culture, LTC-IC number is over 200,000-fold the initial number). The extremely prolonged maintenance and the massive expansion of these progenitors, which share many similarities with murine long-term repopulating cells, suggest that extensive renewal and little differentiation take place. This system might prove useful in diverse clinical settings involving treatment of grown-up children and adults with transplantation of normal or genetically manipulated hematopoietic stem cells.
Human Immunodeficiency Virus (HIV)-Resistant CD4+ UT-7 Megakaryocytic Human Cell Line Becomes Highly HIV-1 and HIV-2 Susceptible Upon CXCR4 Transfection: Induction of Cell Differentiation by HIV-1 Infection
Authors: Baiocchi Marta , Olivetta Eleonora , Chelucci Cristiana , Santarcangelo Anna Claudia , Bona Roberta , d'Aloja Paola , Testa Ugo , Komatsu Norio , Verani Paola , Federico Maurizio
Published: October 2019

Recent findings have shown that the expression of the seven trans-membrane G-protein–coupled CXCR4 (the receptor for the stromal cell-derived factor [SDF]-1 chemokine) is necessary for the entry of T-lymphotropic human immunodeficiency virus (HIV) strains, acting as a coreceptor of the CD4 molecule. In the human system, the role of CXCR4 in HIV infection has been determined through env-mediated cell fusion assays and confirmed by blocking viral entry in CD4+/CXCR4+ cells by SDF-1 pretreatment. We observed that the human megakaryoblastic CD4+ UT-7 cell line fails to express CXCR4 RNA and is fully resistant to HIV entry. Transfection of an expression vector containing the CXCR4 c-DNA rendered UT-7 cells readily infectable by different T-lymphotropic syncytium-inducing HIV-1 and HIV-2 isolates. Interestingly, HIV-1 infection of CXCR4 expressing UT-7 cells (named UT-7/fus) induces the formation of polynucleated cells through a process highly reminiscent of megakaryocytic differentiation and maturation. On the contrary, no morphologic changes were observed in HIV-2–infected UT-7/fus cells. These findings further strengthen the role of CXCR4 as a molecule necessary for the replication of T-lymphotropic HIV-1 and HIV-2 isolates and provide a useful model to study the functional role of CD4 coreceptors in HIV infection.
Megakaryocytic Progenitors Can Be Generated Ex Vivo and Safely Administered to Autologous Peripheral Blood Progenitor Cell Transplant Recipients
Authors: Bertolini Francesco , Battaglia Manuela , Pedrazzoli Paolo , Antonio Da Prada Gian , Lanza Annalisa , Soligo Davide , Caneva Lorenza , Sarina Barbara , Murphy Scott , Thomas Terry , Robustelli della Cuna Gioacchino
Published: October 2019

We evaluated different culture conditions to obtain a lineage-selected proliferation of clonogenic megakaryocytic progenitors (MP). In low-density (LD) or CD34+ cell cultures, the best results were obtained in serum-free medium in the presence of megakaryocyte growth and development factor, stem cell factor, interleukin-3 (IL-3), IL-6, IL-11, FLT-ligand, and macrophage inflammatory protein-1α. In paired studies, expansion of LD cells was less effective than expansion of CD34+ cells, and pre-enrichment of CD34+ cells using negative depletion of lineage-positive cells produced significantly larger quantities of MP than pre-enrichment using positive selection. MP proliferation peaked on day 7 in culture, and an 8- ± 5-fold expansion of CD34+/CD61+ cells, a 17- ± 5-fold expansion of colony-forming units-megakaryocytes, and a 58- ± 14-fold expansion of the total number of CD61+ cells was obtained. In a feasibility clinical study, 10 cancer patients (8 with breast cancer and 2 with non-Hodgkin's lymphoma) undergoing autologous peripheral blood progenitor cell (PBPC) transplant received MP generated ex vivo (range, 1 to 21 × 105/kg CD61+ cells) together with unmanipulated PBPC. Eight patients received a single allogeneic platelet transfusion, whereas platelet transfusion support was not needed in 2 of the 4 patients receiving the highest doses of cultured MP. This result compares favorably with a retrospective control group of 14 patients, all requiring platelet transfusion support. Adverse reactions or bacterial contamination of cell cultures have not been observed. In conclusion, MP can be expanded ex vivo and safely administered to autologous transplant recipients. Further clinical trials will indicate the reinfusion schedule able to consistently abrogate the need for allogeneic platelet transfusion support in autologous transplantation.
Human Immunodeficiency Virus gp120 Inhibits Interleukin-12 Secretion by Human Monocytes: An Indirect Interleukin-10–Mediated Effect
Authors: Taoufik Y. , Lantz O. , Wallon C. , Charles A. , Dussaix E. , Delfraissy J.F.
Published: October 2019

Interleukin-12 (IL-12), a cytokine with in vitro and in vivo immunomodulatory effects, is produced mostly by activated monocytes and macrophages. To study the effect of human immunodeficiency virus (HIV) infection on IL-12 production, we investigated the expression of IL-12 at mRNA and protein levels by human monocytes preincubated with HIV-gp120. In these conditions, we show that monocytes have a decreased ability to express IL-12 mRNA subunits and to produce IL-12 p40 and bioactive p70 proteins in response to Staphylococcus aureus strain cowan I (SAC). We showed that in human monocyte cultures, HIV-gp120 induces a significant IL-10 synthesis, which in turn inhibits IL-12 subunits mRNA accumulation and protein secretion after SAC-activation. Similar data were obtained with human macrophages. These results suggest that, during HIV infection, gp120 induces in uninfected monocytes and macrophages IL-10/IL-12 disregulation, which can alter immune response.
Deficiency of the Fas Apoptosis Pathway Without Fas Gene Mutations in Pediatric Patients With Autoimmunity/Lymphoproliferation
Authors: Dianzani Umberto , Bragardo Manuela , DiFranco Daniela , Alliaudi Carla , Scagni Paola , Buonfiglio Donatella , Redoglia Valter , Bonissoni Sara , Correra Antonio , Dianzani Irma , Ramenghi Ugo
Published: October 2019

Fas (CD95) is a transmembrane molecule that induces programmed cell death (PCD) of lymphocytes. We examined its function in children with chronic thrombocytopenia, serum autoantibodies, and lymphadenopathy and/or splenomegaly. We found that T-cell lines from six of seven patients with this autoimmune/lymphoproliferative disease (ALD) were relatively resistant to PCD induced by monoclonal antibodies to Fas. By contrast, Fas function was normal in control patients with typical chronic idiopathic thrombocytopenic purpura (ITP) without lymphadenopathy. The defect was not due to decreased Fas expression, nor to over-production of soluble forms of Fas. Moreover, it specifically involved the Fas system because PCD was induced in the normal way by methylprednisolone. Complementary DNA sequencing of the Fas gene did not identify any causal mutation in patients with ALD. This distinguished them from patients with the human autoimmune lymphoproliferative syndrome (ALPS), who carry mutations of the Fas gene. Moreover, patients with ALD did not show the peripheral expansion of CD4/CD8 double-negative T cells that characterizes the ALPS phenotype. Fas signaling involves activation of a sphingomyelinase-catalyzing production of ceramide. We found that ceramide-induced PCD was defective in patients with ALD and not in patients with typical chronic ITP. These data suggest that the ALD patient defect involves the Fas signaling pathway downstream from the sphingomyelinase and that Fas gene mutations and double-negative T-cell expansion are not the only signs of a defective Fas system.
Monoclonal Antibodies Specific to the Acute Lymphoblastic Leukemia t(1; 19)-Associated E2A/pbx1 Chimeric Protein: Characterization and Diagnostic Utility
Authors: Sang Bi-Ching , Shi Liangru , Dias Peter , Liu Li , Wei Jia , Wang Zhi-Xue , Monell Craig R. , Behm Fred , Gruenwald Stefan
Published: October 2019

Nonrandom chromosomal abnormalities are found in most human malignancies, particularly leukemias and lymphomas. A characteristic t(1; 19) (q23; p13.3) chromosomal translocation is detected in 5% of childhood acute lymphoblastic leukemia (ALL) cases. This translocation results in the formation of a fusion gene, which leads to the expression of an oncogenic E2A/pbx1 protein. Breakpoints in the E2A gene almost invariably occur within a single intron, and the identical portion of PBX1 is joined consistently to exon 13 of E2A in fusion mRNA. In this article, we report the development of monoclonal antibodies against E2A/pbx1 fusion protein using a specific peptide that corresponds to the junction region of the protein. The obtained antibodies recognize specifically the chimeric E2A/pbx1 fusion protein and lack cross-reactivities with E2A and pbx1. Immunohistochemical staining and flow cytometric studies show that these antibodies can distinguish t(1; 19)-positive from t(1; 19)-negative leukemic cells. These results indicate that the obtained E2A/pbx1-specific monoclonal antibodies might prove to be valuable diagnostic reagents and important tools for elucidating the mechanisms involved in oncogenesis and progression of t(1; 19)-positive childhood ALL.
In Vitro Drug Sensitivity Testing Can Predict Induction Failure and Early Relapse of Childhood Acute Lymphoblastic Leukemia
Authors: Hongo Teruaki , Yajima Shuhei , Sakurai Minoru , Horikoshi Yasuo , Hanada Ryoji
Published: October 2019

It is vital to develop effective therapy for children with acute lymphoblastic leukemia (ALL), in whom no remission occurs or who suffer relapse with current protocols. Cellular drug resistance is thought to be an important cause of induction failure and relapse. We performed in vitro tests of bone marrow samples in 196 children with newly diagnosed ALL with a 4-day culture and a methyl-thiazol-tetrazolium assay. We tested 16 drugs and calculated the 70% lethal dose (LD70) for 14 drugs and the leukemic cell survival (LCS) rate for dexamethasone and prednisolone. For each single drug, patients were classified into two groups, sensitive or resistant, by median concentration of LD70 or LCS. When patients were classified into three groups by sensitivity to four drugs of DPAV (dexamethasone, prednisolone, L-asparaginase, and vincristine), 3-year event-free survival (EFS; 95% confidence intervals) of the super sensitive group (SS; sensitive to all 4 drugs) was 0.833 (0.690 to 0.976), that of the intermediate sensitive group (IS; sensitive to 2 or 3 drugs) was 0.735 (0.609 to 0.863), and that of the relatively resistant group (RR; sensitive to no drugs or to 1 drug) was 0.541 (0.411 to 0.670; P = .0008). We then investigated the relationship between the above four-drug sensitivity and the time of relapse. The SS and IS patients tended to maintain continuous complete remission, and RR patients tended to undergo induction failure and early and late relapse (P = .004). Initial white blood cell count, immunologic classification, and age were also predictive factors, but the patient numbers showed no statistical correlation between these factors and the four-drug sensitivity groups (SS, IS, and RR). When we took three groups SS/IS/RR and investigated the EFS for various clinical groups, DPAV sensitivity strongly influenced EFS in the standard-risk ALL (P = .016). In vitro drug sensitivity testing provides additional prognostic information about childhood ALL, and early detection of drug resistance at the time chemotherapy commences may provide a successful strategy for individualizing treatment, as the results indicate de novo resistance to front-line drugs and suggest alternative, second-line drugs.
CD3−CD56+ Non-Hodgkin's Lymphomas With an Aggressive Behavior Related to Multidrug Resistance
Authors: Drénou Bernard , Lamy Thierry , Amiot Laurence , Fardel Olivier , Caulet-Maugendre Sylvie , Sasportes Maryline , Diebold Jacques , Le Prisé Pierre-Yves , Fauchet Renée
Published: October 2019

CD56 expression has been reported previously in some non-Hodgkin's lymphoma (NHL) characterization. They principally involve the nasopharynx, are related to Epstein-Barr virus (EBV), and may be classified as either T- or non–T-natural killer (NK) cells according to CD3/T-cell receptor (TCR) status at the genomic or protein level. The present study reports three cases of non-nasal NK-NHL with the following characteristics: an agressive clinical behavior, heterogenous morphological data evoking pleomorphic T-cell malignant lymphoma, a non–T-NK phenotype using flow cytometry, and immunochemistry. The three cases were CD56+ without membrane expression of specific T markers (CD3, CD5, and TCR). Heterogenous results were observed concerning different antigens: CD2, CD4, CD8, CD16, CD94, CD122, TiA1, perforin, and granzyme B. There was no evidence of detectable clonal TCR gene rearrangement with polymerase chain reaction. No NK activity was detected in the two tested cases, and no relation was found with EBV. Multidrug resistance investigations suggest that agressive clinical findings could be related to MDR1 gene expression as confirmed by MDR1 mRNA detection, MDR1 gene product (Pgp) expression, and a functional multidrug resistance study using rhodamine efflux by flow-cytometry.
Expression and Regulation of Bcl-2, Bcl-xl, and Bax Correlate With p53 Status and Sensitivity to Apoptosis in Childhood Acute Lymphoblastic Leukemia
Authors: Findley Harry W. , Gu Lubing , Yeager Andrew M. , Zhou Muxiang
Published: October 2019

Bcl-2 and its homologue, Bcl-xl, encode membrane-associated proteins that protect neoplastic cells from DNA damage-induced apoptosis, whereas Bax is a Bcl-2 antagonist that promotes cell death. In the present study, we examined the expression and regulation of these genes at both the mRNA and protein level in 22 pediatric acute lymphoblastic leukemia (ALL) cell lines, as well as their sensitivity to apoptosis after exposure to ionizing radiation (IR). Eleven of 22 lines expressed wild-type (wt) p53, 4 expressed mutant p53, and 7 did not express p53 (p53-null). Nine of 22 (41%) lines expressed Bcl-2; of these, 8 were wt-p53+ and 1 expressed mutant p53. Bcl-2 was not expressed in any p53-null lines. In contrast, all 22 lines were positive for Bcl-xl and Bax, although expression level varied. Treatment with IR (10 Gy) induced both downregulation of Bcl-2 and upregulation of Bax at 2 to 5 hours post-IR in 5 of 8 (63%) wt-p53+ lines, leading to apoptosis. Conversely, lines that failed to both downregulate Bcl-2 and upregulate Bax after IR were resistant to apoptosis. Although levels of Bcl-xl expression varied among the 22 lines, high levels of Bcl-xl were observed in 5 of 7 (71%) p53− lines. There were no obvious changes in the expression of Bcl-xl in these lines after IR. However, among the p53-null lines, resistance to IR was observed only in those expressing high levels of Bcl-xl. These results suggest that expression of Bcl-2 but not Bcl-xl is p53-dependent and that IR-induced downregulation of Bcl-2 and upregulation of Bax occur in most wt-p53+ lines and are associated with radiosensitivity. Furthermore, high-level expression of Bcl-xl occurs predominantly in p53-null lines and is associated with resistance to IR-induced apoptosis in these lines, indicating differential expression and regulation of Bcl-2 and Bcl-xl in pediatric ALL.
Microphthalmia (mi) in Murine Mast Cells: Regulation of Its Stimuli-Mediated Expression on the Translational Level
Authors: Nechushtan Hovav , Zhang Zhaocheng , Razin Ehud
Published: October 2019

Mice harboring a mutation in the microphthalmia (mi ) gene display a variety of abnormalities, including microphthalmia, depletion of skin melanocytes, deafness, a defect in osteoclasts, and a major decrease in mast cell number and function. However, despite the possible critical role played by this protein in mast cell development and function, characterization of its mRNA and protein synthesis in these cells has not yet been performed. In this study, we investigated the regulation of the synthesis of mi in murine mast cells activated by various physiologic stimuli. Using a specific rabbit polyclonal anti-mi antibody, we found that interleukin-3, interleukin-4, or aggregation of the mast cell high-affinity receptor for IgE (FcεRI) induced the synthesis of mi protein in these cells. None of these stimuli significantly affected the level of mi mRNA in the mast cells at any of the time points tested. Also, using this specific anti-mi antibody, an increase in mi protein synthesis was shown during differentiation of mast cells from their bone marrow cell precursors. Moreover, a complex containing mi bound to upstream stimulating factor 2 was detected only in activated mast cells. We conclude that the regulation of mi expression is on the translational level. Thus, stimulation of mast cells by a variety of stimuli elicits a signaling pathway that regulates mi expression.
Marrow Transplantation for Chronic Myeloid Leukemia: The Influence of Plasma Busulfan Levels on the Outcome of Transplantation
Authors: Slattery J.T. , Clift R.A. , Buckner C.D. , Radich J. , Storer B. , Bensinger W.I. , Soll E. , Anasetti C. , Bowden R. , Bryant E. , Chauncey T. , Deeg H.J. , Doney K.C. , Flowers M. , Gooley T. , Hansen J.A. , Martin P.J. , McDonald G.B. , Nash R. , Petersdorf E.W. , Sanders J.E. , Schoch G. , Stewart P. , Storb R. , Sullivan K.M. , Thomas E.D. , Witherspoon R.P. , Appelbaum F.R.
Published: October 2019

The influence of busulfan (BU) plasma concentration on outcome of transplantation from HLA identical family members for the treatment of chronic myelogenous leukemia (CML) was examined in 45 patients transplanted in chronic phase (CP) (n = 39) or accelerated phase (AP) (n = 6). All patients received the same regimen of BU, 16 mg/kg orally and cyclophosphamide (CY), 120 mg/kg intravenously. Plasma concentrations of BU at steady state (CSSBU) during the dosing interval were measured for each patient. The mean CSSBU was 917 ng/mL (range, 642 to 1,749; median, 917; standard deviation, 213). Of patients with CSSBU below the median, seven (five of 18 in CP and two of four in AP) developed persistent cytogenetic relapse and three of these patients died. There were no relapses in patients with CSSBU above the median. The difference in the cumulative incidence of relapse between the two groups was statistically significant (P = .0003). CSSBU was the only statistically significant determinant of relapse in univariable or multivariable analysis. The 3-year survival estimates were 0.82 and 0.64 for patients with CSSBU above and below the median (P = .33). There was no statistically significant association of CSSBU with survival or nonrelapse mortality, although the power to detect a difference in survival between 0.82 and 0.64 was only 0.24, similarly CSSBU above the median was not associated with an increased risk of severe regimen-related toxicity. We conclude that low BU plasma levels are associated with an increased risk of relapse.