bluuebunny/crossref_metadata_2025 huggingface dataset:
The t(3;21) Fusion Product, AML1/Evi-1, Interacts With Smad3 and Blocks Transforming Growth Factor-β–Mediated Growth Inhibition of Myeloid Cells
Authors: Kurokawa Mineo , Mitani Kinuko , Imai Yoichi , Ogawa Seishi , Yazaki Yoshio , Hirai Hisamaru
Published: October 2019

The t(3;21)(q26;q22) chromosomal translocation associated with blastic crisis of chronic myelogenous leukemia results in the formation of the AML1/Evi-1 chimeric protein, which is thought to play a causative role in leukemic transformation of hematopoietic cells. Here we show that AML1/Evi-1 represses growth-inhibitory signaling by transforming growth factor-β (TGF-β) in 32Dcl3 myeloid cells. The activity of AML1/Evi-1 to repress TGF-β signaling depends on the two separate regions of the Evi-1 portion, one of which is the first zinc finger domain. AML1/Evi-1 interacts with Smad3, an intracellular mediator of TGF-β signaling, through the first zinc finger domain, and represses the Smad3 activity, as Evi-1 does. We also show that suppression of endogenous Evi-1 in leukemic cells carrying inv(3) restores TGF-β responsiveness. Taken together, AML1/Evi-1 acts as an inhibitor of TGF-β signaling by interfering with Smad3 through the Evi-1 portion, and both AML1/Evi-1 and Evi-1 repress TGF-β–mediated growth suppression in hematopoietic cells. Thus, AML1/Evi-1 may contribute to leukemogenesis by specifically blocking growth-inhibitory signaling of TGF-β in the t(3;21) leukemia.
Molecular Cytogenetic Characterization of a Critical Region in Bands 7q35-q36 Commonly Deleted in Malignant Myeloid Disorders
Authors: Döhner Konstanze , Brown Jill , Hehmann Ute , Hetzel Claudia , Stewart Janet , Lowther Gordon , Scholl Claudia , Fröhling Stefan , Cuneo Antonio , Tsui Lap C. , Lichter Peter , Scherer Stephen W. , Döhner Hartmut
Published: October 2019

Loss of chromosome 7 (−7) or deletion of the long arm (7q−) are recurring chromosome abnormalities in myeloid leukemias. The association of −7/7q− with myeloid leukemia suggests that these regions contain novel tumor suppressor gene(s), whose loss of function contribute to leukemic transformation or tumor progression. Based on chromosome banding analysis, two critical regions have been identified, one in band q22 and another in bands q32-q35. Presently there are no data available on the molecular delineation of the distal critical region. In this study we analyzed bone marrow and blood samples from 13 patients with myeloid leukemia (de novo myelodysplastic syndrome [MDS] , n = 3; de novo acute myeloid leukemia [AML], n = 9; therapy-related (t-) AML, n = 1) which, on chromosome banding analysis, exhibited deletions (n = 12) or in one case a balanced translocation involving bands 7q31-qter using fluorescence in situ hybridization (FISH). As probes we used representative clones from a contig map of yeast artificial chromosome (YAC) clones that spans chromosome bands 7q31.1-qter. In the 12 cases with loss of 7q material, we identified a commonly deleted region of approximately 4 to 5 megabasepairs in size encompassing the distal part of 7q35 and the proximal part of 7q36. Furthermore, the breakpoint of the reciprocal translocation from the patient with t-AML was localized to a 1,300-kb sized YAC clone that maps to the proximal boundary of the commonly deleted region. Interestingly, in this case both homologs of chromosome 7 were affected: one was lost (−7) and the second exhibited the t(7q35). The identification and delineation of translocation and deletion breakpoints provides the first step toward the identification of the gene(s) involved in the pathogenesis of 7q35-q36 aberrations in myeloid disorders.
The Costs and Cost-Effectiveness of Unrelated Donor Bone Marrow Transplantation for Chronic Phase Chronic Myelogenous Leukemia
Authors: Lee Stephanie J. , Anasetti Claudio , Kuntz Karen M. , Patten Jonathan , Antin Joseph H. , Weeks Jane C.
Published: October 2019

Unrelated donor transplantation prolongs survival in some patients with chronic myelogenous leukemia (CML) in chronic phase. However, there are growing concerns about the intensive resources required for this procedure given health care budget constraints. To address this issue, we conducted a study of the costs and cost-effectiveness of unrelated donor transplantation for chronic phase CML. The costs of transplantation were derived from 157 patients from the Brigham and Women’s Hospital (BWH) and the Fred Hutchinson Cancer Research Center (FHCRC). Estimates of the effectiveness of transplantation were taken from our previous work using data from the International Bone Marrow Transplant Registry and the National Marrow Donor Program. The median cost of the first 6 months of care including donor identification, marrow collection, patient hospitalization for transplantation and all outpatient medications and readmissions through 6 months postmarrow infusion was $178,500 (range, $85,000 to $462,400) and the mean was $196,200. Mean costs for patients surviving beyond 6 months posttransplant were significantly lower than for patients dying within that period ($189,700 v $211,000, respectively,P = .03). Posttransplant follow-up costs were high for months 6 to 18, then decreased. The incremental cost-effectiveness of transplantation within 1 year of diagnosis versus -interferon therapy without transplant in the base case of a 35-year-old patient was $51,800/quality-adjusted life year (QALY) gained. Sensitivity analysis showed that most ratios were between $50,000 to $100,000/QALY or within the intermediate zone of acceptable cost-effectiveness ratios.
A Phase II Study of BTI-322, a Monoclonal Anti-CD2 Antibody, for Treatment of Steroid-Resistant Acute Graft-Versus-Host Disease
Authors: Przepiorka Donna , Phillips Gordon L. , Ratanatharathorn Voravit , Cottler-Fox Michele , Sehn Laurie H. , Antin Joseph H. , LeBherz Donna , Awwad Michel , Hope James , McClain J. Bruce
Published: October 2019

BTI-322, a rat monoclonal IgG2b directed against the CD2 antigen on T cells and natural killer (NK) cells, blocks primary and memory alloantigen proliferative responses in vitro. We have evaluated the pharmacokinetics and safety of BTI-322 during treatment of 20 transplant recipients with steroid-refractory acute graft-versus-host disease (GVHD). Treatment consisted of BTI-322 by intravenous (IV) bolus or 30-minute infusion at approximately 0.1 mg/kg/d for 10 days in addition to continuing high-dose steroids and tacrolimus or cyclosporine. Pharmacokinetic sampling was performed in 10 patients; the t1/2 ± SE was 9.1 ± 1.3 hours, the Cmaxwas 2,549 ± 291 ng/mL, the Vd was 3.97 ± 0.95 L, and the Vd/kg was 0.05 ± 0.01 L/kg. Ten patients experienced transient dyspnea sometimes accompanied by nausea, vomiting, diarrhea, and tachycardia shortly after the initial bolus dose of drug, but serious drug-related adverse events were not seen during the remainder of the infusions. At the end of treatment (day 11), there were six patients with complete responses and five with a reduction in grade of GVHD for a total response rate of 55% (95% confidence interval [CI], 32% to 77%). Antibodies targeting CD2 may be active in the treatment of acute GVHD, and evaluation of a humanized form of BTI-322 is warranted.
γc Gene Transfer in the Presence of Stem Cell Factor, FLT-3L, Interleukin-7 (IL-7), IL-1, and IL-15 Cytokines Restores T-Cell Differentiation From γc(−) X-Linked Severe Combined Immunodeficiency Hematopoietic Progenitor Cells in Murine Fetal Thymic Organ Cultures
Authors: Hacein-Bey S. , Basile G. De Saint , Lemerle J. , Fischer A. , Cavazzana-Calvo M.
Published: October 2019

X-linked severe combined immunodeficiency (SCID-Xl) is a rare human inherited disorder in which early T and natural killer (NK) lymphocyte development is blocked. The genetic disorder results from mutations in the common γc chain that participates in several cytokine receptors including the interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15 receptors. We have shown in a previous report that γc gene transfer into SCID-Xl bone marrow (BM) cells restores efficient NK cell differentiation. In this study, we have focused on the introduction of the γc gene into SCID-Xl hematopoietic stem cells with the goal of obtaining differentiation into mature T cells. For this purpose, we used the in vitro hybrid fetal thymic organ culture (FTOC) system in which a combination of cytokines consisting of stem cell factor (SCF), Flt-3L, IL-7, IL-1, and IL-15 is added concomitantly. In this culture system, CD34+ marrow cells from two SCID-Xl patients were able to mature into double positive CD4+ CD8+ cells and to a lesser degree into CD4+ TCRβ+ single positive cells after retroviral-mediated γc gene transfer. In addition, examination of the output cell population at the TCR DJβ1 locus exhibited multiple rearrangements. These results indicate that restoration of the γc/JAK/STAT signaling pathway during the early developmental stages of thymocytes can correct the T-cell differentiation block in SCID-Xl hematopoietic progenitor cells and therefore establishes a basis for further clinical γc gene transfer studies.
GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation
Authors: Suwabe Naruyoshi , Takahashi Satoru , Nakano Toru , Yamamoto Masayuki
Published: October 2019

Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.
Cooperative Effects of Growth Factors Involved in the Induction of Hematopoietic Mesoderm
Authors: Huber Tara L. , Zhou Yi , Mead Paul E. , Zon Leonard I.
Published: October 2019

Hematopoietic induction occurs on the ventral side ofXenopus gastrulae and is thought to be triggered by the growth factor bone morphogenetic protein 4 (BMP-4). To characterize this process, we developed a quantitative and sensitive assay for the induction of erythroid cells from totipotent ectoderm of the embryo. When high doses of BMP-4 were used in this explant assay, few erythroid cells were detected. In contrast, large numbers of differentiated erythroid cells were induced when ectoderm was treated with BMP-4 and the mesoderm inducers, activin, or fibroblast growth factor (FGF). Ectopic expression of GATA-1 also induced abundant erythroid cells in ectoderm treated with bFGF. This induction of erythroid cells by GATA-1 was blocked by coexpression with a dominant negative BMP-4 receptor, showing that GATA-1 requires the BMP signaling cascade to function. These results suggest that BMP-4 requires mesoderm induction to generate a program of gene expression, which regulates the specification of hematopoietic mesoderm by GATA factors.
Vascular Endothelial Growth Factor Inhibits the Development of Dendritic Cells and Dramatically Affects the Differentiation of Multiple Hematopoietic Lineages In Vivo
Authors: Gabrilovich Dmitry , Ishida Tadao , Oyama Tsunehiro , Ran Sophia , Kravtsov Vladimir , Nadaf Sorena , Carbone David P.
Published: October 2019

Defective function of dendritic cells (DC) in cancer has been recently described and may represent one of the mechanisms of tumor evasion from immune system control. We have previously shown in vitro that vascular endothelial growth factor (VEGF), produced by almost all tumors, is one of the tumor-derived factors responsible for the defective function of these cells. In this study, we investigated whether in vivo infusion of recombinant VEGF could reproduce the observed DC dysfunction. Continuous VEGF infusion, at rates as low as 50 ng/h (resulting in serum VEGF concentrations of 120 to 160 pg/mL), resulted in a dramatic inhibition of dendritic cell development, associated with an increase in the production of B cells and immature Gr-1+ myeloid cells. Infusion of VEGF was associated with inhibition of the activity of the transcription factor NF-κB in bone marrow progenitor cells. Experiments in vitro showed that VEGF itself, and not factors released by VEGF-activated endothelial cells, affected polypotent stem cells resulting in the observed abnormal hematopoiesis. These data suggest that VEGF, at pathologically relevant concentrations in vivo, may exert effects on pluripotent stem cells that result in blocked DC development as well as affect many other hematopoietic lineages.
Proteasome Inhibitors Induce Apoptosis in Glucocorticoid-Resistant Chronic Lymphocytic Leukemic Lymphocytes
Authors: Chandra Joya , Niemer Irina , Gilbreath Joyce , Kliche Kay-Oliver , Andreeff Michael , Freireich Emil J. , Keating Michael , McConkey David J.
Published: October 2019

Our previous work showed that the nuclear scaffold (NS) protease is required for apoptosis of both thymocytes and chronic lymphocytic leukemic (CLL) lymphocytes. Because partial sequencing of one of the subunits of the NS protease revealed homology to the proteasome, we tested the effects of classical proteasome inhibitors on apoptosis in CLL cells. Here we report that proteasome inhibition caused high levels of DNA fragmentation in all patients analyzed, including those resistant to glucocorticoids or nucleoside analogs, in vitro. Proteasome inhibitor-induced DNA fragmentation was associated with activation of caspase/ICE family cysteine protease(s) and was blocked by the caspase antagonist, zVADfmk. Analysis of the biochemical mechanisms involved showed that proteasome inhibition resulted in mitochondrial dysregulation leading to the release of cytochrome c and a drop in mitochondrial transmembrane potential (▵Ψ). These changes were associated with inhibition of NFκB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in other systems. Together, our results suggest that drugs that target the proteasome might be capable of bypassing resistance to conventional chemotherapy in CLL.
Identification of New Nonrandom Translocations in Multiple Myeloma With Multicolor Spectral Karyotyping
Authors: Sawyer Jeffrey R. , Lukacs Janet L. , Munshi Nikhil , Desikan K. Raman , Singhal Seema , Mehta Jayesh , Siegel David , Shaughnessy John , Barlogie Bart
Published: October 2019

Multicolor spectral karyotyping (SKY) was performed on bone marrow samples from 50 patients with multiple myeloma (MM) in anticipation of discovering new previously unidentified translocations. All samples showed complex karyotypes with chromosome aberrations which, in most cases, were not fully characterized by G-banding. Patients of special interest were those who showed add(14)(q32), add(8)(q24) and those whose G-banding karyotypes showed poor chromosome morphology. Three new recurring chromosome translocations not previously reported in MM were identified. Two of the translocations involve recurring aberrations at band 14q32.3, the site of the IgH locus, with different exchange partners. The most frequently recurring rearrangement was a subtle translocation at 14q32.3 designated as a t(14;16)(q32;q22∼23), which was identified in six patients. A second and larger translocation at 14q32, identified in two patients, was designated as a t(9;14)(p13;q32), previously associated with Waldenstrom’s macroglobulinemia and lymphoplasmacytoid lymphoma. A third translocation, identified in two patients, involved a whole-arm t(6;8)(p10;q10) translocation. The SKY technique was able to refine the designations of over 156 aberrations not fully characterized by G-banding in this study and resolved additional chromosome aberrations in every patient studied except two. The t(14;16)(q32;q22∼23) identified by SKY in this study suggests this may be a frequent translocation in MM associated with complex karyotypes and disease progression. Therefore, the SKY technique provides a useful adjunct to routine G-banding and fluorescence in situ hybridization studies in the cytogenetic analysis of MM.
Most Acute Myeloid Leukemia Progenitor Cells With Long-Term Proliferative Ability In Vitro and In Vivo Have the Phenotype CD34+/CD71−/HLA-DR−
Authors: Blair A. , Hogge D.E. , Sutherland H.J.
Published: October 2019

Acute myeloid leukemia (AML) occurs as the result of malignant transformation in a hematopoietic progenitor cell, which proliferates to form an accumulation of AML blasts. Only a minority of these AML cells are capable of proliferation in vitro, suggesting that AML cells may be organized in a hierarchy, with only the most primitive of these cells capable of maintaining the leukemic clone. To further investigate this hypothesis, we have evaluated a strategy for purifying these primitive cells based on surface antigen expression. As an in vitro endpoint, we have determined the phenotype of AML progenitor cells which are capable of producing AML colony-forming cells (CFU) for up to 8 weeks in suspension culture (SC) and compared the phenotype with that of cells which reproduce AML in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. AML cells were fluorescence-activated cell sorted (FACS) for coexpression of CD34 and CD71, CD38, and/or HLA-DR and the subfractions were assayed in vitro and in vivo at various cell doses to estimate purification. While the majority of primary AML CFU lacked expression of CD34, most cells capable of producing CFU after 2 to 8 weeks in SC were CD34+/CD71−. HLA-DR expression was heterogeneous on cells producing CFU after 2 to 4 weeks. However, after 6 to 8 weeks in SC, the majority of CFU were derived from CD34+/HLA-DR− cells. Similarly, the majority of cells capable of long-term CFU production from SC were CD34+/CD38−. Most cells that were capable of engrafting NOD/SCID mice were also CD34+/CD71− and CD34+/HLA-DR−. Engraftment was not achieved with CD34+/CD71+ or HLA-DR+subfractions, however, in two patients, both the CD34+and CD34− subfractions were capable of engrafting the NOD/SCID mice. A three-color sorting strategy combining these antigens allowed approximately a 2-log purification of these NOD/SCID leukemia initiating cells, with engraftment achieved using as few as 400 cells in one experiment. Phenotyping studies suggest even higher purification could be achieved by combining lack of CD38 expression with the CD34+/CD71− or CD34+/HLA DR− phenotype. These results suggest that most AML cells capable of long-term proliferation in vitro and in vivo share the CD34+/CD71−/HLA-DR− phenotype with normal stem cells. Our data suggests that in this group of patients the leukemic transformation has occurred in a primitive progenitor, as defined by phenotype, with some degree of subsequent differentiation as defined by functional assays.
CBFβ-SMMHC, Expressed in M4eo Acute Myeloid Leukemia, Reduces p53 Induction and Slows Apoptosis in Hematopoietic Cells Exposed to DNA-Damaging Agents
Authors: Britos-Bray Martin , Ramirez Manuel , Cao Wangsen , Wang Xinping , Liu P. Paul , Civin Curt I. , Friedman Alan D.
Published: October 2019

CBFβ-SMMHC is expressed in M4Eo acute myeloid leukemia (AML) as a result of inv(16), but how it contributes to leukemogenesis is unknown. p53 mutations are rare in de novo AML, but they are common in many malignancies. Expression of CBFβ-SMMHC in Ba/F3 cells reduced p53 induction in response to ionizing radiation or etoposide 3- to 4-fold. However, p53 induction was normal in Ba/F3 cells expressing a CBFβ-SMMHC variant that does not interfere with DNA binding by CBF, indicating that a CBF genetic target regulates p53 induction. The p53 gene may be regulated by CBF, because p53 mRNA levels were reduced by CBFβ-SMMHC. Reduced p53 induction was not caused by slowed cell proliferation, a consequence of CBFβ-SMMHC expression, because p53 was induced similarly in control cultures and in cultures propagated in 10-fold less interleukin-3 (IL-3). CBFβ-SMMHC did not slow apoptosis resulting from IL-3 withdrawal, where p53 induction is minimal, but slowed apoptosis in Ba/F3 cells exposed to 10 Gy of ionizing radiation or 3 to 8 μg/mL etoposide, providing 2-fold protection at 6 or 18 hours. Inhibition of apoptosis was temporary, because all the cells exposed to these doses ultimately died, and clonal survival assays performed using 0.04 μg/mL etoposide did not show protection by CBFβ-SMMHC. p21 levels were increased in cells subjected to DNA damage, regardless of CBFβ-SMMHC expression and attenuated p53 induction. Bcl-2, bcl-xL, bcl-xS, and bax levels were unaffected by CBFβ-SMMHC. Attenuated p53 induction may contribute to leukemogenesis by CBFβ-SMMHC by slowing apoptosis via a p21-independent mechanism.
Characterization of Multiple Isoforms of Protein 4.1R Expressed During Erythroid Terminal Differentiation
Authors: Gascard P. , Lee G. , Coulombel L. , Auffray I. , Lum M. , Parra M. , Conboy J.G. , Mohandas N. , Chasis J.A.
Published: October 2019

In erythrocytes, 80-kD protein 4.1R regulates critical membrane properties of deformability and mechanical strength. However, previously obtained data suggest that multiple isoforms of protein 4.1, generated by alternative pre-mRNA splicing, are expressed during erythroid differentiation. Erythroid precursors use two splice acceptor sites at the 5′ end of exon 2, thereby generating two populations of 4.1 RNA: one that includes an upstream AUG-1 in exon 2′ and encodes high molecular weight isoforms, and another that skips AUG-1 in exon 2′ and encodes 4.1 by initiation at a downstream AUG-2 in exon 4. To begin an analysis of the complex picture of protein 4.1R expression and function during erythropoiesis, we determined the number and primary structure of 4.1R isoforms expressed in erythroblasts. We used reverse-transcription polymerase chain reaction to amplify and clone full-length coding domains from the population of 4.1R cDNA containing AUG-1 and the population excluding AUG-1. We observed an impressive repertoire of 4.1R isoforms that included 7 major and 11 minor splice variants, thus providing the first definitive characterization of 4.1R primary structures in a single-cell lineage. 4.1R isoforms, transfected into COS-7 cells, distributed to the nucleus, cytoplasm, plasma membrane, and apparent centrosome. We confirmed previous studies showing that inclusion of exon 16 was essential for efficient nuclear localization. Unexpectedly, immunochemical analysis of COS-7 cells transfected with an isoform lacking both AUG-1 and AUG-2 documented that a previously unidentified downstream translation initiation codon located in exon 8 can regulate expression of 4.1R. We speculate that the repertoire of primary structure of 4.1R dictates its distinct binding partners and functions during erythropoiesis.
Recombination Breakpoints in the Human β-Globin Gene Cluster
Authors: Smith Rachelle A. , Ho P. Joy , Clegg John B. , Kidd Judith R. , Thein Swee Lay
Published: October 2019

The human β-globin gene complex spans a region of 70 kb and contains numerous sequence variants. These variant sites form a 5′ cluster (5′ β-haplotype) and a 3′ cluster (3′ β-haplotype) with strong linkage disequilibrium among the sites within each cluster, but not between the two clusters. The 9-kb region between the 5′ and 3′ clusters has been estimated to have rates of recombination that are 3 to 30 times normal, and the region has therefore been proposed as a ‘hotspot’ of recombination. We describe three families with evidence of meiotic recombination within this ‘hotspot’ of the β-globin gene cluster and in which the cross-over breakpoints have been defined at the sequence level. In one family, the recombination has occurred in the maternal chromosome within a region of 361 bp between positions −911 and −550 5′ to the β-globin gene. In the other two families, the recombination has occurred in the paternal chromosome within a region of approximately 1,100 bp between positions −542 and +568 relative to the β-globin gene cap site. Both regions occur within the 2-kb region of replication initiation (IR) in the β-globin gene domain with no overlap. The IR region contains a consensus sequence for a protein (Pur), which binds preferentially to single-stranded DNA, a role implicated in recombination events.
Correction of the PNH Defect by GPI-Anchored Protein Transfer
Authors: Sloand Elaine M. , Maciejewski Jaroslaw P. , Dunn Daniel , Moss Joel , Brewer Bryan , Kirby Martha , Young Neal S.
Published: October 2019

Hemolytic anemia is a major feature of paroxysmal nocturnal hemoglobinuria (PNH). Intravascular red blood cell (RBC) destruction is caused by increased sensitivity of the abnormal erythrocyte to complement-mediated lysis, due to the GPI absence of a membrane-bound glycosylphosphatidylinositol (GPI)-linked protein, which functions as an inhibitor of reactive lysis (CD59). Both in vivo and in vitro models have suggested the feasibility of cell-to-cell transfer of GPI proteins, and patients with hemolysis could potentially benefit from transfer of CD59 to their deficient erythrocytes. We studied the ability of RBC components prepared from outdated packed RBC collections, as well as high-density lipoprotein (HDL) preparations, rich in CD55 and CD59, to promote protein transfer, as assessed by flow cytometry, immunoblotting, and susceptibility to complement-mediated lysis. By flow cytometry, CD55 and CD59 were present on RBC-derived microvesicles that stained with an antiglycophorin antibody Ab; in addition, soluble CD59 and CD55 were detected by immunoblot in soluble fractions eluated from RBC units stored for more than 35 days, but not in fresh blood. Both commercial HDL preparations and those prepared in our laboratory contained CD55 and CD59, as assayed by immunoblot. When RBC that were deficient (GPI)-anchored protein, obtained from five patients, with PNH were incubated with HDL preparations for 2 to 4 hours, there was significant transfer of both proteins to the cell surface, as demonstrated by flow cytometry. Washed RBC microvesicles, prepared by ultrasonification, also mediated transfer of GPI-linked proteins to deficient RBC. Pretreatment of microvesicles, RBC eluate preparations, and HDL with phosphatidylinositol-specific, phospholipase C, abrogated protein transfer to deficient cells, indicating that increased cell-associated CD55 and CD59 levels were related to insertion of the intact GPI moiety, rather than to simple adhesion. PNH RBC that were exposed to HDL, RBC eluate preparations, or microvesicles demonstrated decreased in vitro complement-mediated hemolysis in the Ham test. Transfer of GPI-linked proteins from soluble preparations containing CD55 and CD59 to PNH erythrocytes is feasible and may have clinical utility.
Stat6 Inhibits Human Interleukin-4 Promoter Activity in T Cells
Authors: Georas Steve N. , Cumberland John E. , Burke Thomas F. , Chen Rongbing , Schindler Ulrike , Casolaro Vincenzo
Published: October 2019

The differentiation of naive T-helper (Th) cells into cytokine-secreting effector Th cells requires exposure to multiple signals, including exogenous cytokines. Interleukin-4 (IL-4) plays a major role in this process by promoting the differentiation of IL-4–secreting Th2 cells. In Th2 cells, IL-4 gene expression is tightly controlled at the level of transcription by the coordinated binding of multiple transcription factors to regulatory elements in the proximal promoter region. Nuclear factor of activated T cell (NFAT) family members play a critical role in regulating IL-4 transcription and interact with up to five sequences (termed P0 through P4) in the IL-4 promoter. The molecular mechanisms by which IL-4 induces expression of the IL-4 gene are not known, although the IL-4–activated transcription factor signal transducer and activator of transcription 6 (Stat6) is required for this effect. We report here that Stat6 interacts with three binding sites in the human IL-4 promoter by electrophoretic mobility shift assays. These sites overlap the P1, P2, and P4 NFAT elements. To investigate the role of Stat6 in regulating IL-4 transcription, we used Stat6-deficient Jurkat T cells with different intact IL-4 promoter constructs in cotransfection assays. We show that, whereas a multimerized response element from the germline IgE promoter was highly induced by IL-4 in Stat6-expressing Jurkat cells, the intact human IL-4 promoter was repressed under similar conditions. We conclude that the function of Stat6 is highly dependent on promoter context and that this factor promotes IL-4 gene expression in an indirect manner.
Adenovirus Vector-Based Purging of Multiple Myeloma Cells
Authors: Teoh Gerrard , Chen Ling , Urashima Mitsuyoshi , Tai Yu-Tzu , Celi Leo A. , Chen Dongshu , Chauhan Dharminder , Ogata Atsushi , Finberg Robert W. , Webb Iain J. , Kufe Donald W. , Anderson Kenneth C.
Published: October 2019

Adenoviruses are efficient gene delivery agents for a variety of neoplasms. In the present study, we have investigated the use of adenoviruses for the delivery of the thymidine kinase(tk) gene into multiple myeloma (MM) cells. We first demonstrated that MM cell lines and MM patient cells express both adenovirus receptors as well as the DF3/MUC1 protein, thus providing a rationale for using adenoviruses to selectively deliver genes under the control of the DF3 promoter. By using an adenoviral construct containing β-galactosidase (β-gal) gene driven by the DF3 promoter (Ad.DF3-βgal), we demonstrate greater than 80% transduction efficiency in OCI-My5 and RPMI 8226 MM cell lines at a multiplicity of infection of 1 to 100. Importantly, transduction with the tk gene driven by the DF3 promoter (Ad.DF3-tk)followed by treatment with 50 μmol/L ganciclovir (GCV) purged ≥6 log of contaminating OCI-My5 and RPMI 8226 MM cells within bone marrow mononuclear cells. In contrast, normal human hematopoietic progenitor cell number was unaffected under these conditions. Selectivity of DF3/MUC1 promoter was further confirmed, because Ad.DF3-βgalor Ad.DF3-tk did not transduce MUC1-negative HeLa cervical carcinoma cells. In addition, GCV treatment of Ad.DF3-tk–transduced RPMI 8226 MM cells did not induce a significant bystander effect. These findings demonstrate that transduction with Ad vectors using a tumor-selective promoter provides a highly efficient and selective approach for the ex vivo purging of MM cells.
Expression Trapping: Identification of Novel Genes Expressed in Hematopoietic and Endothelial Lineages by Gene Trapping in ES Cells
Authors: Stanford William L. , Caruana Georgina , Vallis Katherine A. , Inamdar Maneesha , Hidaka Michihiro , Bautch Victoria L. , Bernstein Alan
Published: October 2019

We have developed a large-scale, expression-based gene trap strategy to perform genome-wide functional analysis of the murine hematopoietic and vascular systems. Using two different gene trap vectors, we have isolated embryonic stem (ES) cell clones containing lacZreporter gene insertions in genes expressed in blood island and vascular cells, muscle, stromal cells, and unknown cell types. Of 79 clones demonstrating specific expression patterns, 49% and 16% were preferentially expressed in blood islands and/or the vasculature, respectively. The majority of ES clones that expressedlacZ in blood islands also expressed lacZ upon differentiation into hematopoietic cells on OP9 stromal layers. Importantly, the in vivo expression of the lacZ fusion products accurately recapitulated the observed in vitro expression patterns. Expression and sequence analysis of representative clones suggest that this approach will be useful for identifying and mutating novel genes expressed in the developing hematopoietic and vascular systems.
Cloning of the Promoter Region of Human Endoglin, the Target Gene for Hereditary Hemorrhagic Telangiectasia Type 1
Authors: Rı́us Carlos , Smith Joshua D. , Almendro Nuria , Langa Carmen , Botella Luisa M. , Marchuk Douglas A. , Vary Calvin P.H. , Bernabéu Carmelo
Published: October 2019

Endoglin (CD105) is a cell surface component of the transforming growth factor-β (TGF-β) receptor complex highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for the hereditary hemorrhagic telangiectasia type 1 (HHT1), also known as Osler-Weber-Rendu syndrome (OMIM 187300). This is an autosomal dominant vascular disorder probably caused by a haploinsufficiency mechanism displaying low levels of the normal protein. To understand the mechanisms underlying the regulated expression of endoglin, a genomic DNA clone containing 3.3 kb of the 5′-flanking sequence of the human endoglin gene has been isolated. The 5′-flanking region of the endoglin gene lacks consensus TATA and CAAT boxes, but contains two GC-rich regions and consensus motifs for Sp1, ets, GATA, AP-2, NFκB, and Mad, as well as TGF-β–, glucocorticoid-, vitamin D-, and estrogen-responsive elements. As determined by primer extension and 5′ RACE experiments, a cluster of transcriptional start sites was found to be located 350 bp upstream from the translation initiation codon. To analyze the endoglin promoter activity, the upstream −400/+341 fragment was fused to the luciferase gene and transient transfections were conducted in several cell types. This construct displayed a tissue-specific activity in human and bovine endothelial cells. Analysis of various deletion constructs showed the existence of a basal promoter region within the −81/+350 fragment as well as major transcriptional regulatory elements within the −400/−141 fragment. Electrophoretic mobility shift assays demonstrated the specific interaction of a member of the ets family with a consensus motif located at position −68. A promoter construct mutated at this ets sequence showed a much reduced activity as compared with the wild-type construct, supporting the involvement of this ets motif in the basal activity of the promoter. The endoglin promoter exhibited inducibility in the presence of TGF-β1, suggesting possible therapeutic treatments in HHT1 patients, in which the expression level of the normal endoglin allele might not reach the threshold required for its function. Isolation and characterization of the human endoglin promoter represents an initial step in elucidating the controlled expression of the endoglin gene.
Lineage Involvement of Stem Cells Bearing the Philadelphia Chromosome in Chronic Myeloid Leukemia in the Chronic Phase as Shown by a Combination of Fluorescence-Activated Cell Sorting and Fluorescence In Situ Hybridization
Authors: Takahashi Naoto , Miura Ikuo , Saitoh Kohki , Miura Akira B.
Published: October 2019

Chronic myeloid leukemia (CML) is thought to arise from a pluripotent hematopoietic stem cell that has undergone a reciprocal translocation between the BCR gene on chromosome 22 and the ABL proto-oncogene on chromosome 9. This rearrangement results in a shortened chromosome 22, designated the Philadelphia (Ph) chromosome. The Ph chromosome has been found in cells from all hematopoietic lineages except mature T lymphocytes. To examine this issue, we combined fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) to study lineage involvement of mature cells and stem cells in 12 patients with CML in the chronic phase. We found Ph chromosomes in myeloid cells and most B lymphocytes (CD19+) but not in mature T cells (CD3+) or natural killer (NK) cells (CD3−56+). Moreover, evidence of BCR/ABL fusion was found in pluripotent stem cells (CD34+Thy-1+), B-progenitor cells (CD34+CD19+), T/NK progenitor cells (CD34+CD7+ cells), and T progenitor cells (CD34+CD7+CD5+) with a frequency equal to that in all CD34+ cells isolated by FACS from bone marrow cells. T lymphocytes showed a marked decrease in Ph+ cells between progenitor cells and mature cells. Moreover, the ratios of Ph+ to Ph− cells in mature T cells and NK cells were below background levels, whereas Ph+ B lymphocytes also decreased during their maturation. These data suggest that Ph+ lymphocytes are eliminated during differentiation. In contrast to FISH of blood and bone marrow, which gives information principally about mature cells, the technique of “sorter FISH (FACS + FISH)” provides a powerful tool to explore the cytogenetic changes in immature cell populations of stem cell diseases based on immunophenotypes. Further clarification of genetic changes in stem cells could be achieved by using sorter FISH with monoclonal antibodies.
Involvement of Caspases in Neutrophil Apoptosis: Regulation by Reactive Oxygen Species
Authors: Fadeel Bengt , Åhlin Anders , Henter Jan-Inge , Orrenius Sten , Hampton Mark B.
Published: October 2019

Human neutrophils have a short half-life and are believed to die by apoptosis or programmed cell death both in vivo and in vitro. We found that caspases are activated in a time-dependent manner in neutrophils undergoing spontaneous apoptosis, concomitant with other characteristic features of apoptotic cell death such as morphologic changes, phosphatidylserine (PS) exposure, and DNA fragmentation. The treatment of neutrophils with agonistic anti-Fas monoclonal antibodies (MoAbs) significantly accelerated this process. However, in cells treated with the potent neutrophil activator phorbol 12-myristate 13-acetate (PMA), caspase activity was only evident after pharmacologic inhibition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Similarily, inhibition of the NADPH oxidase in constitutive and Fas/APO-1–triggered apoptosis resulted in increased rather than suppressed levels of caspase activity, suggesting that reactive oxygen species may prevent caspases from functioning optimally in these cells. Moreover, oxidants generated via the NADPH oxidase were essential for PS exposure during PMA-induced cell death, but not for neutrophils undergoing spontaneous apoptosis. We conclude that caspases are an important component of constitutive and Fas/APO-1–triggered neutrophil apoptosis. However, these redox sensitive enzymes are suppressed in activated neutrophils, and an alternate oxidant-dependent pathway is used to mediate PS exposure and neutrophil clearance under these conditions.
Adhesion-Dependent Release of Elastase From Human Neutrophils in a Novel, Flow-Based Model: Specificity of Different Chemotactic Agents
Authors: Rainger G. Ed , Rowley Andrew F. , Nash Gerard B.
Published: October 2019

Neutrophils must adhere to the vessel wall, migrate, and degranulate in an ordered manner to perform their protective function. Disruption of these processes may be pathogenic. Current knowledge of the degranulation process is derived almost exclusively from studies on neutrophils in suspension, in which priming with the nonphysiological agent cytochalasin B is necessary to obtain elastase release in response to activating agents. To avoid this, we have adopted a different approach. Using a novel flow-based adhesion system, we have been able to quantify the release of elastase from the primary granules of activated neutrophils adherent to immobilized platelets or purified receptors without priming. Comparing stimuli, formyl tripeptide (fMLP), interleukin-8 (IL-8), activated complement fragment C5a, and platelet-activating factor (PAF) all induced rapid conversion to CD11b/CD18 (MAC-1) -mediated stationary adhesion when perfused over neutrophils already rolling on platelet monolayers or purified P-selectin. However, fMLP, C5a, and IL-8, but not PAF, induced release of elastase from the adherent cells in minutes. Neutrophils stimulated in suspension showed little degranulation. Treatment of neutrophils with an inhibitor of 5-lipoxygenase–activating protein (MK886) and thus synthesis of leukotrienes (LTs) or with an antagonist of the LTB4 receptor (LY223982) blocked the release of elastase. This indicated that endogenous synthesis of 5-lipoxygenase products such as LTs and autocrine activation of neutrophils was required for fMLP-driven elastase release. We hypothesize that the differential ability of PAF and fMLP to induce elastase release from surface-adherent neutrophils could arise from differential ability to generate leukotrienes, such as LTB4, and would be an appropriate mechanism for the control of elastase release during inflammation in vivo, where it is important that cytotoxic agents are not released until activated neutrophils have migrated into the extravascular tissues.
Interleukin-15 (IL-15) Induces NF-κB Activation and IL-8 Production in Human Neutrophils
Authors: McDonald Patrick P. , Russo Maria Pia , Ferrini Silvano , Cassatella Marco A.
Published: October 2019

Interleukin-2 (IL-2) and IL-15 exert similar biological actions, which largely reflect the fact that their receptors share common β and γ subunits; in contrast, distinct  subunits are required for high-affinity binding of either cytokine to a heterotrimeric receptor complex. Human neutrophils are known to express both the β and γ subunits of the IL-2/IL-15 receptor complex, and we now report that they also constitutively express messenger RNA transcripts encoding the IL-15 receptor  chain, suggesting that they possess functional, heterotrimeric IL-15 receptors. Accordingly, we show that in neutrophils, IL-15 elicits several functional responses. In particular, neutrophils synthesize and release IL-8 in response to IL-15, but not to IL-2. Moreover, a nuclear factor-κB (NF-κB) DNA-binding activity was enhanced in nuclear extracts of IL-15–treated neutrophils, which could be supershifted by antibodies to p50 or RelA. Again, no detectable effect of IL-2 was observed on this response. In peripheral blood lymphocytes (PBL), however, both IL-2 and IL-15 were potent inducers of NF-κB activation. Conversely, neither IL-15 nor IL-2 elicited the formation of activator protein-1 (AP-1) DNA-binding complexes in neutrophils, even though both cytokines were found to activate these DNA-binding activities in PBL. Collectively, these observations establish neutrophils as a useful cellular model to discriminate between the actions of IL-15 and IL-2. More importantly, this is the first demonstration that IL-15 has the ability to induce NF-κB and AP-1 activation, which further emphasizes the potential relevance of this newly discovered cytokine to immune and inflammatory processes.
Molecular Typing Shows a High Level of HLA Class I Incompatibility in Serologically Well Matched Donor/Patient Pairs: Implications for Unrelated Bone Marrow Donor Selection
Authors: Scott Iain , O’Shea John , Bunce Mike , Tiercy Jean-Marie , Argüello J. Rafael , Firman Helen , Goldman John , Prentice H. Grant , Little Ann-Margaret , Madrigal J. Alejandro
Published: October 2019

In comparison with HLA-matched sibling bone marrow transplants, unrelated donor transplants are associated with increased graft-versus-host disease and graft failure. This is likely in part due to HLA incompatibilities not identified by current matching strategies. High resolution DNA-based typing methods for HLA class II loci have improved donor selection and treatment outcome in unrelated donor bone marrow transplantation. By using DNA-based typing methods for HLA-A and -B on a cohort of 100 potential bone marrow donor/patient pairs, we find that serological typing for HLA class I is limited in its ability to identify incompatibilities in unrelated pairs. Furthermore, the incompatibilities identified are associated with the presence at high frequency of alloreactive cytotoxic T-lymphocyte precursors. DNA typing also indicates that HLA-C mismatches are common in HLA-A and -B serologically matched pairs. Such mismatches appear to be significantly less immunogenic with respect to cytotoxic T-lymphocyte recognition, but are expected to influence natural killer cell activity. Thus, improved resolution of HLA class I shows many previously undisclosed mismatches that appear to be immunologically functional. Use of high resolution typing methods in routine matching is expected to improve unrelated donor selection and transplant outcome.
A Risk Model for Thrombocytopenia Requiring Platelet Transfusion After Cytotoxic Chemotherapy
Authors: Blay J.Y. , Le Cesne A. , Mermet C. , Maugard C. , Ravaud A. , Chevreau C. , Sebban C. , Guastalla J.-P. , Biron P. , Ray-Coquard I.
Published: October 2019

Severe thrombocytopenia is a rare but life-threatening side effect of cytotoxic chemotherapy for which risk factors are not well known. Our objective was to delineate a risk model for chemotherapy-induced thrombocytopenia requiring platelet transfusions in cancer patients. Univariate and multivariate analysis of risk factors for chemotherapy-induced thrombocytopenia requiring platelet transfusions were performed on the cohort of the 1,051 patients (CLB 1996) treated with chemotherapy in the Department of Medicine of the Centre Léon Bérard (CLB) in 1996. In univariate analysis, performance status (PS) greater than 1, platelet count less than 150,000/μL at day 1 (d1) before the initiation of chemotherapy, d1 lymphocyte count ≤700/μL, d1 polymorphonuclear leukocyte count less than 1,500/μL, and the type of chemotherapy (high risk v others) were significantly associated (P < .01) with an increased risk of severe thrombocytopenia requiring platelet transfusions. Using logistic regression, d1 platelet count less than 150,000/μL (odds ratio [OR], 4.3; 95% confidence interval [CI], 1.9 to 9.6), d1 lymphocyte counts ≤700/μL (OR, 3.37; 95% CI, 1.77 to 6.4), the type of chemotherapy (OR, 3.38; 95% CI, 1.77 to 6.4), and PS greater than 1 (OR, 2.23; 95% CI, 1.22 to 4.1) were identified as independent risk factors for platelet transfusions. The observed incidences of platelet transfusions were 45%, 13%, 7%, and 1.5% for patients with ≥3, 2, 1, or 0 risk factors, respectively. This model was then tested in 3 groups of patients treated with chemotherapy used as validation samples: (1) the series of 340 patients treated in the CLB in the first 6 months of 1997, (2) the prospective multicentric cohort of 321 patients of the ELYPSE 1 study, and (3) the series of 149 patients with non-Hodgkin's lymphoma treated in the CLB within prospective phase III trials (1987 to 1995). In these 3 groups, the observed incidences of platelet transfusions in the above-defined risk groups did not differ significantly (P > .1) from those calculated in the model. This risk index could be useful to identify patients at high risk for chemotherapy-induced thrombocytopenia requiring platelet transfusions.