bluuebunny/crossref_metadata_2025 huggingface dataset:
Epitope Specificity of CD44 for Monoclonal Antibody–Dependent Facilitation of Marrow Engraftment in a Canine Model
Authors: Sandmaier Brenda M. , Storb Rainer , Bennett Kelly L. , Appelbaum Frederick R. , Santos Erlinda B.
Published: October 2019

Primary graft rejection after marrow transplantation occurs more frequently in patients receiving HLA-haploidentical compared with HLA-identical sibling transplants. Both human and experimental animal data suggest that the cells responsible for this phenomenon are either host natural killer (NK) cells, T cells, or both. To investigate the mechanisms of graft rejection, we have developed a canine model of marrow transplantation, which uses DLA-nonidentical unrelated donors in the absence of postgrafting immunosuppression. In this model most animals rejected their marrow grafts after a preparative regimen of 9.2 Gy total body irradiation (TBI). However, engraftment of DLA-nonidentical marrow can be facilitated when the recipients are pretreated with monoclonal antibody (MoAb) S5, which recognizes CD44. In this report, we extended these observations by first cloning the canine CD44 and, next, mapping the epitope recognized by S5, which was located in a region conserved among human and canine CD44 and was distinct from the hyaluronan binding domain. However, in vitro binding of S5 caused a conformational change in CD44, which allowed increased hyaluronan binding. Then, we reexamined the in vivo model of marrow transplantation and compared results with MoAb S5 to those with two other anti-CD44 MoAbs, IM7 and S3. Only MoAb S5 significantly increased the engraftment rate of DLA-nonidentical unrelated marrow, whereas the two other anti-CD44 MoAbs were ineffective. The enhanced in vivo effect was not related to differences in the MoAbs' avidities, since both S5 and IM7 had equivalent binding to CD44, but most likely related to the specific epitope that S5 recognizes. Thus, this study shows that the effect of the anti-CD44 MoAb S5 in facilitating engraftment is epitope specific and if one is to use an anti-CD44 to facilitate engraftment of marrow in humans, one cannot assume that any anti-CD44 would work.
Transgenic Mice for MTCP1 Develop T-Cell Prolymphocytic Leukemia
Authors: Gritti Catherine , Dastot Hélène , Soulier Jean , Janin Anne , Daniel Marie-Thérèse , Madani Ali , Grimber Gisèle , Briand Pascale , Sigaux François , Stern Marc-Henri
Published: October 2019

T-cell prolymphocytic leukemia (T-PLL) is a rare form of mature T-cell leukemia associated with chromosomal rearrangements implicatingMTCP1 or TCL1 genes. These genes encode two homologous proteins, p13MTCP1 and p14TCL1, which share no similarity with other known protein. To determine the oncogenic role of MTCP1, mice transgenic for MTCP1under the control of CD2 regulatory regions (CD2-p13 mice) were generated. No abnormality was detected during the first year after birth. A late effect of the transgene was searched for in a cohort of 48 CD2-p13 mice aged 15 to 20 months, issued from 3 independent founders. Lymphoid hemopathies, occurring in the three transgenic lines, were characterized by lymphoid cells with an irregular nucleus, a unique and prominent nucleolus, condensed chromatin, a basophilic cytoplasm devoid of granules, and an immunophenotype of mature T cells. The molecular characterization of Tcrb rearrangements demonstrated the monoclonal origin of these populations. Histopathological analysis of the cohort demonstrated early splenic and hepatic infiltrations, whereas lymphocytosis and medullar infiltrations were found infrequently. The engraftment of these proliferations in H2-matched animals demonstrated their malignant nature. Cumulative incidence of the disease at 20 months was 100%, 50%, and 21% in F3, F4, and F7 lines, respectively, and null in the control group. The level of expression of the transgene, as estimated by Western blotting in the transgenic lines correlated with the tumoral incidence, with the highest expression of p13MTCP1 being found in F3 mice. CD2-p13 transgenic mice developed an hemopathy similar to human T-PLL. These data demonstrate that p13MTCP1 is an oncoprotein and that CD2-p13 transgenic mice represent the first animal model for mature T-PLL.
Loss of Function of the Homeobox Gene Hoxa-9 Perturbs Early T-Cell Development and Induces Apoptosis in Primitive Thymocytes
Authors: Izon David J. , Rozenfeld Sofia , Fong Stephen T. , Kömüves László , Largman Corey , Lawrence H. Jeffrey
Published: October 2019

Hox homeobox genes play a crucial role in specifying the embryonic body pattern. However, a role for Hox genes in T-cell development has not been explored. The Hoxa-9 gene is expressed in normal adult and fetal thymuses. Fetal thymuses of mice homozygous for an interruption of the Hoxa-9 gene are one eighth normal size and have a 25-fold decrease in the number of primitive thymocytes expressing the interleukin-2 receptor (IL-2R, CD25). Progression to the double positive (CD4+CD8+) stage is dramatically retarded in fetal thymic organ cultures. This aberrant development is associated with decreased amounts of intracellular CD3 and T-cell receptor β (TCRβ) and reduced surface expression of IL-7R and E-cadherin. Mutant thymocytes show a significant increase in apoptotic cell death and premature downregulation of bcl-2 expression. A similar phenotype is seen in primitive thymocytes from adult Hoxa-9−/− mice and from mice transplanted with Hoxa-9−/−marrow. Hoxa-9 appears to play a previously unsuspected role in T-cell ontogeny by modulating cell survival of early thymocytes and by regulating their subsequent differentiation.
Blood Dendritic Cells From Myeloma Patients Are Not Infected With Kaposi's Sarcoma-Associated Herpesvirus (KSHV/HHV-8)
Authors: Yi Qing , Ekman Marianne , Anton Doina , Bergenbrant Susanne , Österborg Anders , Georgii-Hemming Patrik , Holm Göran , Nilsson Kenneth , Biberfeld Peter
Published: October 2019

In recent studies, the sequence of Kaposi's sarcoma-associated herpes virus (KSHV) or human herpes virus-8 (HHV-8) was detected in dendritic cells (DC) of patients with multiple myeloma (MM). A concern was raised whether there is an causal association between the viral infection and development of these tumors. In the present study, we have examined DC generated from blood adherent cells from 8 Swedish MM patients at different clinical stages and 2 patients with monoclonal gammopathy of undetermined significance. In addition, 6 myeloma cell lines and bone marrow cells from 2 MM patients were also studied. By polymerase chain reaction (PCR), including nested PCR, no virus DNA was demonstrable in the patients' DC or in myeloma cell lines or fresh bone marrow cells. Moreover, no antibody against KSHV was found in the serum of these 10 patients. Thus, our results indicate that blood-derived DC of MM patients in Sweden usually are not infected with KSHV/HHV-8. This study also suggests that KSHV/HHV-8 is not regularly associated with MM and consequently does not play a primary role in the pathogenesis of these tumors.
Early Intensification of Intrathecal Chemotherapy Virtually Eliminates Central Nervous System Relapse in Children With Acute Lymphoblastic Leukemia
Authors: Pui Ching-Hon , Mahmoud Hazem H. , Rivera Gaston K. , Hancock Michael L. , Sandlund John T. , Behm Frederick G. , Head David R. , Relling Mary V. , Ribeiro Raul C. , Rubnitz Jeffrey E. , Kun Larry E. , Evans William E.
Published: October 2019

Central nervous system (CNS) relapse has been an obstacle to uniformly successful treatment of childhood acute lymphoblastic leukemia (ALL) for many years. We therefore intensified intrathecal chemotherapy (simultaneously administered methotrexate, hydrocortisone, and cytarabine) for 165 consecutive children with newly diagnosed ALL enrolled in Total Therapy Study XIIIA from December 1991 to August 1994. The 64 patients (39%) who had 1 or more blast cells in cytocentrifuged preparations of cerebrospinal fluid at diagnosis, with or without associated higher-risk features, received additional doses of intrathecal chemotherapy during remission induction and the first year of continuation treatment. Patients with higher-risk leukemia, regardless of cerebrospinal fluid findings, also received additional doses of intrathecal chemotherapy during the first year of continuation treatment. Cranial irradiation was reserved for patients with higher-risk leukemia (22% of the total). The 5-year cumulative risk of an isolated CNS relapse among all 165 patients was 1.2% (95% confidence interval, 0% to 2.9%), whereas that of any CNS relapse was 3.2% (0.4% to 6.0%). The probability of surviving for 5 years without an adverse event of any type was 80.2% ± 9.2% (SE). Our results suggest that early intensification of intrathecal chemotherapy will reduce the risk of CNS relapse to a very low level in children with ALL, securing a higher event-free survival rate overall.
The Reliability and Specificity of c-kit for the Diagnosis of Acute Myeloid Leukemias and Undifferentiated Leukemias
Authors: Bene M.C. , Bernier M. , Casasnovas R.O. , Castoldi G. , Knapp W. , Lanza F. , Ludwig W.D. , Matutes E. , Orfao A. , Sperling C. , van't Veer M.B.
Published: October 2019

We document findings on c-kit (CD117) expression in 1,937 pediatric and adult de novo acute leukemia cases, diagnosed in five single European centers. All cases were well characterized as to the morphologic, cytochemical, and immunologic features, according to the European Group for the Immunological Classification of Leukemias (EGIL). The cases included 1,103 acute myeloid leukemia (AML), 819 acute lymphoblastic leukemia (ALL), 11 biphenotypic acute leukemia (BAL), and 4 undifferentiated (AUL). c-kit was expressed in 741 (67%) AML cases, regardless of the French-American-British (FAB) subtype, one third of BAL, all four AUL, but only in 34 (4%) of ALL cases. The minority of c-kit+ ALL cases were classified as: T-cell lineage (two thirds), mainly pro-T–ALL or T-I, and B lineage (one third); cells from 62% of these ALL cases coexpressed other myeloid markers (CD13, CD33, or both). There were no differences in the frequency of c-kit+ AML or ALL cases according to age being similar in the adult and pediatric groups. Our findings demonstrate that c-kit is a reliable and specific marker to detect leukemia cells committed to the myeloid lineage, and therefore should be included in a routine basis for the diagnosis of acute leukemias to demonstrate myeloid commitment of the blasts. c-kit expression should score higher, at least one point, in the system currently applied to the diagnosis of BAL, as its myeloid specificity is greater than CD13 and CD33. Findings in ALL and AUL suggest that c-kit identifies a subgroup of cases, which may correspond to leukemias either arising from early prothymocytes and/or early hematopoietic cells, both able to differentiate to the lymphoid and myeloid pathways.
Epstein-Barr Virus in Nasal Lymphomas Contains Multiple Ongoing Mutations in the EBNA-1 Gene
Authors: Gutiérrez M.I. , Spangler G. , Kingma D. , Raffeld M. , Guerrero I. , Misad O. , Jaffe E.S. , Magrath I.T. , Bhatia K.
Published: October 2019

We have described 5 major subtypes of Epstein-Barr virus (EBV) based on variations in EBNA-1 sequences. These include P-ala (identical to the prototype B95.8 virus), P-thr, V-pro, V-leu, and V-val. Normal individuals often carry multiple EBV subtypes, the most common being P-ala, whereas EBV-associated tumors examined to date always contain a single subtype, which only on rare occasion is P-ala. The primary hypotheses that these observations generate are as follows: (1) Each of these EBV subtypes are naturally occurring, and in normal individuals the multiplicity of subtypes results from multiple infections. (2) EBV subtypes in normal individuals are generated in vivo from a single infecting virus subtype by mutations in EBNA-1. The second hypothesis essentially excludes the possibilities that the nonrandom association of certain subtypes with lymphomas is secondary to the geographic distribution of EBV subtypes and, if proven correct, could provide strong support for a direct role of EBV in tumorigenesis. In this report, we provide evidence for the latter hypothesis. We show that the P-ala EBV subtype present in most nasal lymphomas undergoes and accumulates multiple mutations consistent with the generation of variant species of EBNA-1 in vivo. This phenomenon is similar to the generation of quasispecies in RNA viruses and is the first description of in vivo generation of subtypes in DNA viruses. In RNA-based viruses, including human immunodeficiency virus and hepatitis C virus, the emergence of quasispecies is linked to replication infidelity and significantly influences disease processes through its effect on viral tropism, the emergence of viruses resistant to the host defenses or to therapy, and pathogenicity. The present data thus raise important questions relating to the mechanisms whereby these mutations are generated in EBV and their relevance to the pathogenicity of EBV-associated lymphomas.
Molecular Defects of the RHCE Gene in Rh-Deficient Individuals of the Amorph Type
Authors: Chérif-Zahar Baya , Matassi Giorgio , Raynal Virginie , Gane Pierre , Mempel Wolfgang , Perez Carmen , Cartron Jean-Pierre
Published: October 2019

The deficiency of Rh proteins on the red blood cells from individuals of the Rhnull amorph type may be the result of homozygosity for a silent allele at the RH locus. This phenotype is also associated with the lack or reduced expression of glycoproteins (Rh50, CD47, LW, and glycophorin B), which interact with Rh polypeptides to form the multisubunit Rh membrane complex. In this study, we describe two molecular alterations affecting the RHCEgene in two unrelated Rhnull amorph individuals bearing Rh50 and CD47 normal transcripts. The first type of mutation, located at the donor splice-site in intron 4, induced the activation of two cryptic splice-sites within this intron and one such site in exon 4 that all generated aberrant transcripts. The second type of mutation affected the coding region and introduced a frameshift and a premature stop codon resulting in a shorter predicted protein (398 v 417 residues), including a completely different C-terminus of 76 amino acids. This suggests that protein folding and/or protein-protein interaction mediated by the C-terminal domain of the Rh proteins may play a role in the routing and/or stability of the Rh membrane complex.
Entry and Trafficking of Granzyme B in Target Cells During Granzyme B-Perforin–Mediated Apoptosis
Authors: Pinkoski Michael J. , Hobman Marita , Heibein Jeffrey A. , Tomaselli Kevin , Li Feng , Seth Prem , Froelich Christopher J. , Bleackley R. Chris
Published: October 2019

In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin–induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death.

© 1998 by The American Society of Hematology.
Neutralization of Tumor Necrosis Factor Activity Shortly After the Onset of Dendritic Cell Hematopoiesis Reveals a Novel Mechanism for the Selective Expansion of the CD14-Dependent Dendritic Cell Pathway
Authors: Santiago-Schwarz Frances , McCarthy Marguerite , Tucci John , Carsons Steven E.
Published: October 2019

The CD14-dependent and -independent dendritic cell (DC) pathways are instituted simultaneously when CD34+ progenitor cells are treated with granulocyte-macrophage colony-stimulating factor (GM-CSF)/tumor necrosis factor (TNF) ± stem cell factor (SCF) (GTS). If TNF activity is neutralized within 48 hours of cytokine exposure, DC development is halted and myelogranulocytic hematopoiesis takes place. In this study, we show that disruption of TNF activity at a later time point produced a distinct alteration within the DC system. Instead of downregulating DC development, treatment of GTS cultures with antibodies to TNF (anti-TNF) on day 3 provoked the selective expansion of the CD14-dependent (monocyte) DC pathway from progenitor cell populations lacking CD14 and CD1a. After an initial decrease in proliferation, anti-TNF produced a rebound in cell growth that yielded intermediate myeloid progenitors exhibiting CD14-dependent DC differentiation potential and CD14+CD1a+ DC precursors. Cultures enriched in CD14-dependent DCs were more potent stimulators of a mixed leukocyte reaction, compared with control GTS cultures containing both types of DCs. The intermediate progenitors expanded in the presence of anti-TNF were CD115+CD33+DR+, long-lived, and displayed clonogenic potential in methylcellulose. When exposed to the appropriate cytokine combinations, these cells yielded granulocytes, monocytes, and CD14-dependent DCs. Antigen-presenting function was acquired only when DC maturation was induced from these myelodendritic progenitors with GM-CSF + interleukin-4 or GTS. These studies show a novel mechanism by which TNF regulates the DC system, as well as providing a strategy for the amplification of the CD14-dependent DC pathway from immature progenitors. Although TNF is required to ensure the institution of DC hematopoiesis from CD34+ progenitor cells, its activity on a later progenitor appears to limit the development of CD14-dependent DCs.

© 1998 by The American Society of Hematology.
Early Detection of Relapse by Prospective Reverse Transcriptase-Polymerase Chain Reaction Analysis of the PML/RARα Fusion Gene in Patients With Acute Promyelocytic Leukemia Enrolled in the GIMEMA-AIEOP Multicenter “AIDA” Trial
Authors: Diverio Daniela , Rossi Vincenzo , Avvisati Giuseppe , DeSantis Silvia , Pistilli Alessandra , Pane Fabrizio , Saglio Giuseppe , Martinelli Giovanni , Petti Maria Concetta , Santoro Alessandra , Pelicci Pier Giuseppe , Mandelli Franco , Biondi Andrea , Coco Francesco Lo
Published: October 2019

Although the majority of patients with acute promyelocytic leukemia (APL) are potentially cured by treatments combining all-trans retinoic acid (ATRA) and chemotherapy (CHT), a sizable proportion (around 30%) will relapse during follow-up. Retrospective molecular monitoring studies using reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific PML/RARα fusion gene, have shown that a positive test usually precedes the occurrence of hematologic relapse. Prospective RT-PCR analyses were performed since 1993 at diagnosis and at preestablished time intervals during follow-up in bone marrow (BM) samples of 163 patients with PML/RARα+ APL enrolled in the multicenter Gruppo Italiano Malattie Ematologiche Maligne dell' Adulto (GIMEMA) trial AIDA (All-trans retinoic acid plus Idarubicin). Treatment consisted of ATRA and idarubicin for induction followed by three polychemotherapy courses as consolidation. The sensitivity level of the RT-PCR assay for PML/RARα, as assessed by serial dilution experiments, was 10−4. All patients were in hematologic remission and tested PCR− at the end of consolidation. Of 21 who converted to PCR-positive thereafter, 20 underwent hematologic relapse at a median time of 3 months (range, 1 to 14) from the first PCR+ result. Seventeen of these 21 (81%) PCR+ conversions were recorded within the first 6 months postconsolidation. Of 142 who tested persistently PCR− in ≥2 tests after consolidation, 8 had hematologic relapse and 134 remained in complete remission (CR) after a median follow-up of 18 months (range, 6 to 38) postconsolidation. Using a time-dependent Cox model, the relative risk of hematologic relapse of patients who converted to PCR+ was 31.8 (confidence limits 95%, 12.9 to 78.3). Our results indicate that conversion to PCR positivity for PML/RARα during remission is highly predictive of subsequent hematologic relapse and highlight the prognostic value of stringent molecular monitoring during the early postconsolidation phase in APL. As a result of the present study, salvage treatment in patients enrolled in the GIMEMA trial AIDA is now anticipated at the time of molecular relapse, defined as the conversion to PCR positivity in two successive BM samplings during follow-up.

© 1998 by The American Society of Hematology.
Expression of Myeloid Markers Lacks Prognostic Impact in Children Treated for Acute Lymphoblastic Leukemia: Italian Experience in AIEOP-ALL 88-91 Studies
Authors: Putti Maria Caterina , Rondelli Roberto , Cocito Maria Grazia , Aricó Maurizio , Sainati Laura , Conter Valentino , Guglielmi Cesare , Cantú-Rajnoldi Angelo , Consolini Rita , Pession Andrea , Zanesco Luigi , Masera Giuseppe , Biondi Andrea , Basso Giuseppe
Published: October 2019

The importance of coexpression of myeloid antigens in childhood acute lymphoblastic leukemia (ALL) has long been debated; results are conflicting. We studied children with ALL treated at Italian Association for Pediatric Hematology-Oncology (AIEOP) institutions over 6 years with Berlin-Frankfurt-Muenster (BFM)-based protocols and have analyzed the incidence of coexpression of six MyAg (CD11b, CD13, CD14, CD15, CD33, CD65w) to determine its prognostic impact. Criteria for MyAg coexpression (MyAg+ALL) included positivity to one or more MyAg on at least 20% of blasts and confirmation of coexpression at double-fluorescence analysis. A total of 291 of 908 cases were MyAg+ALL (32%). Incidence was similar in B-ALL and T-ALL; among common, pre-B, and pre-pre–B-ALL. CD13 and CD33 were most common. Patients with MyAg+ALL had presenting features similar to MyAg−ALL. They entered standard or intermediate risk protocols more frequently and had better prednisone response, but similar complete remission rates. Six-year event-free survival (EFS) was 69.0% in 291 MyAg+ALL cases and 65.3% in 617 MyAg−ALL cases, without significant difference. Cases expressing two or more MyAg presented similar clinical features and treatment response. MyAg+ALL had worse EFS only in infants (0% v47%) (P = .01). Therefore, in this series of homogeneously diagnosed and treated ALL, coexpression of MyAg was not associated with prognostic significance, without relevance for clinical purposes or for patient stratification, except for infants.

© 1998 by The American Society of Hematology.
Different Adhesive Characteristics and VLA-4 Expression of CD34+ Progenitors in G0/G1 Versus S+G2/M Phases of the Cell Cycle
Authors: Yamaguchi Miki , Ikebuchi Kenji , Hirayama Fumiya , Sato Norihiro , Mogi Yuko , Ohkawara Jun-ichi , Yoshikawa Yoshihiro , Sawada Ken-ichi , Koike Takao , Sekiguchi Sadayoshi
Published: October 2019

We identified the cell cycle status of CD34+ cells of steady-state bone marrow (BM) and peripheral blood (PB) obtained from healthy volunteers, and those of apherasis PB samples collected from healthy donors who had been administered granulocyte colony-stimulating factor (G-CSF). More than 10% of CD34+ cells in BM were in S+G2/M phase. In contrast, regardless of whether G-CSF treatment was performed, less than 2% of CD34+ cells in PB were cycling. BM CD34+ cells showed greater VLA-4 expression and adherence to stromal cells than PB CD34+cells. In addition, when cycling and dormant BM CD34+cells were analyzed separately, the cells in S+G2/M phase expressed more VLA-4 and adhered to the stromal cell monolayer more efficiently than the cells in G0/G1 phase. Furthermore, this adhesion of CD34+ cells to the stromal cell layer was almost completely inhibited by anti-VLA-4 antibody. Taken together, these results suggest that CD34+ progenitors in G0/G1 phase of the cell cycle differ from those in S+G2/M phase in adhesiveness mediated by VLA-4 in the hematopoietic microenvironment.

© 1998 by The American Society of Hematology.
Chemopreventive Agent Resveratrol, a Natural Product Derived From Grapes, Triggers CD95 Signaling-Dependent Apoptosis in Human Tumor Cells
Authors: Clément Marie-Véronique , Hirpara Jayshreekumari L. , Chawdhury Sanaul-Haq , Pervaiz Shazib
Published: October 2019

Resveratrol, a constituent of grapes and other food products, has been shown to prevent carcinogenesis in murine models. We report here that resveratrol induces apoptotic cell death in HL60 human leukemia cell line. Resveratrol-treated tumor cells exhibit a dose-dependent increase in externalization of inner membrane phosphatidylserine and in cellular content of subdiploid DNA, indicating loss of membrane phospholipid asymmetry and DNA fragmentation. Resveratrol-induced cell death is mediated by intracellular caspases as observed by the dose-dependent increase in proteolytic cleavage of caspase substrate poly (ADP-ribose) polymerase (PARP) and the ability of caspase inhibitors to block resveratrol cytotoxicity. We also show that resveratrol treatment enhances CD95L expression on HL60 cells, as well as T47D breast carcinoma cells, and that resveratrol-mediated cell death is specifically CD95-signaling dependent. On the contrary, resveratrol treatment of normal human peripheral blood lymphocytes (PBLs) does not affect cell survival for up to 72 hours, which correlates with the absence of a significant change in either CD95 or CD95L expression on treated PBLs. These data show specific involvement of the CD95-CD95L system in the anti-cancer activity of resveratrol and highlight the chemotherapeutic potential of this natural product, in addition to its recently reported chemopreventive activity.

© 1998 by The American Society of Hematology.
Erythropoietin Receptor and STAT5-Specific Pathways Promote SKT6 Cell Hemoglobinization
Authors: Gregory Richard C. , Jiang Ning , Todokoro Kazuo , Crouse Jill , Pacifici Robert E. , Wojchowski Don M.
Published: October 2019

Erythrocyte production in mammals is known to depend on the exposure of committed progenitor cells to the glycoprotein hormone erythropoietin (Epo). In chimeric mice, gene disruption experiments have demonstrated a critical role for Epo signaling in development beyond the erythroid colony-forming unit (CFU-e) stage. However, whether this might include the possible Epo-specific induction of red blood cell differentiation events is largely unresolved. To address this issue, mechanisms of induced globin expression in Epo-responsive SKT6 cells have been investigated. Chimeric receptors containing an epidermal growth factor (EGF) receptor extracellular domain and varied Epo receptor cytoplasmic domains first were expressed stably at physiological levels in SKT6 cells, and their activities in mediating induced hemoglobinization were assayed. While activity was exerted by a full-length chimera (EE483), truncation to remove 7 of 8 carboxyl-terminal tyrosine sites (EE372) markedly enhanced differentiation signaling. Moreover, mutation of a STAT5 binding site in this construct (EE372-Y343F) inhibited induced globin expression and SKT6 cell hemoglobinization, as did the ectopic expression of dominant-negative forms of STAT5 in parental SKT6 cells. As in normal CFU-e, SKT6 cells also were shown to express functional receptors for stem cell factor (SCF). To further define possible specific requirements for differentiation signaling, effects of SCF on SKT6 cell hemoglobinization were tested. Interestingly, SCF not only failed to promote globin expression but inhibited this Epo-induced event in a dose-dependent, STAT5-independent fashion. Thus, effects of Epo on globin expression may depend specifically on STAT5-dependent events, and SCF normally may function to attenuate terminal differentiation while promoting CFU-e expansion.

© 1998 by The American Society of Hematology.
Effective Treatment of Cobalamin Deficiency With Oral Cobalamin
Authors: Kuzminski Antoinette M. , Del Giacco Eric J. , Allen Robert H. , Stabler Sally P. , Lindenbaum John
Published: October 2019

Because cobalamin deficiency is routinely treated with parenteral cobalamin, we investigated the efficacy of oral therapy. We randomly assigned 38 newly diagnosed cobalamin deficient patients to receive cyanocobalamin as either 1 mg intramuscularly on days 1, 3, 7, 10, 14, 21, 30, 60, and 90 or 2 mg orally on a daily basis for 120 days. Therapeutic effectiveness was evaluated by measuring hematologic and neurologic improvement and changes in serum levels of cobalamin (normal, 200 to 900 pg/mL) methylmalonic acid (normal, 73 to 271 nmol/L), and homocysteine (normal, 5.1 to 13.9 μmol/L). Five patients were subsequently found to have folate deficiency, which left 18 evaluable patients in the oral group and 15 in the parenteral group. Correction of hematologic and neurologic abnormalities was prompt and indistinguishable between the 2 groups. The mean pretreatment values for serum cobalamin, methylmalonic acid, and homocysteine were, respectively, 93 pg/mL, 3,850 nmol/L, and 37.2 μmol/L in the oral group and 95 pg/mL, 3,630 nmol/L, and 40.0 μmol/L in the parenteral therapy group. After 4 months of therapy, the respective mean values were 1,005 pg/mL, 169 nmol/L, and 10.6 μmol/L in the oral group and 325 pg/mL, 265 nmol/L, and 12.2 μmol/L in the parenteral group. The higher serum cobalamin and lower serum methylmalonic acid levels at 4 months posttreatment in the oral group versus the parenteral group were significant, with P < .0005 and P < .05, respectively. In cobalamin deficiency, 2 mg of cyanocobalamin administered orally on a daily basis was as effective as 1 mg administered intramuscularly on a monthly basis and may be superior.

© 1998 by The American Society of Hematology.
Oct-1 Is Involved in the Transcriptional Repression of the von Willebrand Factor Gene Promoter
Authors: Schwachtgen Jean-Luc , Remacle Jacques E. , Janel Nathalie , Brys Reginald , Huylebroeck Danny , Meyer Dominique , Kerbiriou-Nabias Danièle
Published: October 2019

The negative regulation of transcription of the human von Willebrand factor (vWF) gene was investigated in human umbilical vein endothelial cells (HUVECs) and HeLa cells. A fragment spanning −89 to +244 nucleotides (nt), containing the first exon, is active in HUVECs only but not in HeLa cells. The activity of this promoter is sharply reduced by mutagenesis of the GATA binding site at +221. Extension of the upstream sequences from nt −89 to −142 and to −496 results in progressive reduction of the activity of the −89 to +244 promoter identifying a negative regulatory element between nt −142 and −89. A factor present in nuclear extracts from endothelial and nonendothelial cells binds to an AT-rich sequence located between nt −133 and −125. Mutagenesis of the AT-rich sequence interferes with nuclear protein binding and restores the activity of the −142 to +244 fragment to the level of the −89 to +244 promoter. Binding of the nuclear protein to the vWF AT-rich sequence in mobility shift assays is inhibited by competition with a consensus Oct-1 binding site and with a silencer octamer-like sequence from the vascular cell adhesion molecule-1 (VCAM-1) promoter. Subsequent supershift experiments identified Oct-1 as the transcription factor that binds to vWF and VCAM-1 silencer elements. These results indicate that Oct-1 acts as a transcriptional repressor of promoters of genes expressed in endothelial cells.

© 1998 by The American Society of Hematology.
Mice Deficient for the Ecto-Nicotinamide Adenine Dinucleotide Glycohydrolase CD38 Exhibit Altered Humoral Immune Responses
Authors: Cockayne Debra A. , Muchamuel Tony , Grimaldi J. Christopher , Muller-Steffner Hélène , Randall Troy D. , Lund Frances E. , Murray Richard , Schuber Francis , Howard Maureen C.
Published: October 2019

CD38 is a membrane-associated ecto-nicotinamide adenine dinucleotide (NAD+) glycohydrolase that is expressed on multiple hematopoietic cells. The extracellular domain of CD38 can mediate the catalysis of NAD+ to cyclic adenosine diphosphoribose (cADPR), a Ca2+-mobilizing second messenger, adenosine diphosphoribose (ADPR), and nicotinamide. In addition to its enzymatic properties, murine CD38 has been shown to act as a B-cell coreceptor capable of modulating signals through the B-cell antigen receptor. To investigate the in vivo physiological function(s) of this novel class of ectoenzyme we generated mice carrying a null mutation in the CD38 gene. CD38−/− mice showed a complete loss of tissue-associated NAD+ glycohydrolase activity, showing that the classical NAD+ glycohydrolases and CD38 are likely identical. Although murine CD38 is expressed on hematopoietic stem cells as well as on committed progenitors, we show that CD38 is not required for hematopoiesis or lymphopoiesis. However, CD38−/− mice did exhibit marked deficiencies in antibody responses to T-cell–dependent protein antigens and augmented antibody responses to at least one T-cell–independent type 2 polysaccharide antigen. These data suggest that CD38 may play an important role in vivo in regulating humoral immune responses.

© 1998 by The American Society of Hematology.
Aspirin and Salicylate Induce Apoptosis and Activation of Caspases in B-Cell Chronic Lymphocytic Leukemia Cells
Authors: Bellosillo Beatriz , Piqué Maria , Barragán Montserrat , Castaño Esther , Villamor Neus , Colomer Dolors , Montserrat Emilio , Pons Gabriel , Gil Joan
Published: October 2019

We analyzed the effect of aspirin, salicylate, and other nonsteroidal antiinflammatory drugs (NSAIDs) on the viability of B-chronic lymphocytic leukemia (B-CLL) cells. Aspirin induced a decrease in cell viability in a dose- and time-dependent manner. The mean IC50 for cells from 5 patients was 5.9 ± 1.13 mmol/L (range, 4.4 to 7.3 mmol/L). In some cases, 2.5 mmol/L aspirin produced an important cytotoxic effect after 4 days of incubation. No effect was observed with other NSAIDs, at concentrations that inhibit cyclooxygenase, such as ketorolac (10 μmol/mL), NS-398 (100 μmol/mL), or indomethacin (20 μmol/mL), thus suggesting the involvement of cyclooxygenase-independent mechanisms in aspirin-induced cytotoxicity. Salicylate also produced dose-dependent cytotoxic effects on B-CLL cells and the mean IC50 for cells from 5 patients was 6.96 ± 1.13 mmol/L (range, 5 to 7.8 mmol/L). Both aspirin and salicylate induced DNA fragmentation and the proteolytic cleavage of poly(ADP(adenosine 5′-diphosphate)-ribose) polymerase (PARP), demonstrating that both compounds induce apoptosis of B-CLL cells. Finally, inhibition of caspases by Z-VAD.fmk blocked proteolytic cleavage of PARP, DNA fragmentation, and cytotoxicity induced by aspirin. Mononuclear cells from normal donors showed a lower sensitivity than cells from B-CLL patients to aspirin as determined by analysis of cell viability. B and T lymphocytes from normal donors and T lymphocytes from CLL patients are more resistant to aspirin-induced apoptosis, as determined by analysis of phosphatidylserine exposure. These results indicate that aspirin and salicylate induce apoptosis of B-CLL cells by activation of caspases and that this activation involves cyclooxygenase-independent mechanisms.

© 1998 by The American Society of Hematology.
Arsenic Trioxide and Melarsoprol Induce Programmed Cell Death in Myeloid Leukemia Cell Lines and Function in a PML and PML-RAR Independent Manner
Authors: Wang Zhu-Gang , Rivi Roberta , Delva Laurent , König Andrea , Scheinberg David A. , Gambacorti-Passerini Carlo , Gabrilove Janice L. , Warrell Raymond P. , Pandolfi Pier Paolo
Published: October 2019

Inorganic arsenic trioxide (As2O3) and the organic arsenical, melarsoprol, were recently shown to inhibit growth and induce apoptosis in NB4 acute promyelocytic leukemia (APL) and chronic B-cell leukemia cell lines, respectively. As2O3 has been proposed to principally target PML and PML-RAR proteins in APL cells. We investigated the activity of As2O3 and melarsoprol in a broader context encompassing various myeloid leukemia cell lines, including the APL cell line NB4-306 (a retinoic acid–resistant cell line derived from NB4 that no longer expresses the intact PML-RAR fusion protein), HL60, KG-1, and the myelomonocytic cell line U937. To examine the role of PML in mediating arsenical activity, we also tested these agents using murine embryonic fibroblasts (MEFs) and bone marrow (BM) progenitors in which the PML gene had been inactivated by homologous recombination. Unexpectedly, we found that both compounds inhibited cell growth, induced apoptosis, and downregulated bcl-2 protein in all cell lines tested. Melarsoprol was more potent than As2O3 at equimolar concentrations ranging from 10−7 to 10−5 mol/L. As2O3 relocalized PML and PML-RAR onto nuclear bodies, which was followed by PML degradation in NB4 as well as in HL60 and U937 cell lines. Although melarsoprol was more potent in inhibiting growth and inducing apoptosis, it did not affect PML and/or PML-RAR nuclear localization. Moreover, both As2O3 and melarsoprol comparably inhibited growth and induced apoptosis of PML+/+ and PML−/− MEFs, and inhibited colony-forming unit erythroid (CFU-E) and CFU granulocyte-monocyte formation in BM cultures of PML+/+ and PML−/− progenitors. Together, these results show that As2O3 and melarsoprol inhibit growth and induce apoptosis independent of both PML and PML-RAR expression in a variety of myeloid leukemia cell lines, and suggest that these agents may be more broadly used for treatment of leukemias other than APL.

© 1998 by The American Society of Hematology.
Control of Erythropoietin Delivery by Doxycycline in Mice After Intramuscular Injection of Adeno-Associated Vector
Authors: Bohl Delphine , Salvetti Anna , Moullier Philippe , Heard Jean Michel
Published: October 2019

We reported previously that controlled expression of a foreign gene in response to tetracycline derivative can be accomplished in mice by the autologous transplantation of retrovirus-modified muscle cells. Although regulated systemic delivery of therapeutic proteins from engineered tissues has potential clinical application, the transplantation of muscle cells is not currently feasible in humans. Several studies have shown that a single injection of adeno-associated virus (AAV) vectors into mouse muscle results in long-term expression of reporter genes as well as sustained delivery of proteins into the serum. Because this method is potentially applicable clinically, we constructed an AAV vector in which the expression of the mouse erythropoietin (Epo) cDNA is modulated in response to doxycycline. The vector was injected intramuscularly in normal mice. We observed that hematocrit and serum Epo concentrations could be modulated over a 29-week period in response to the presence or absence of doxycycline in the drinking water of these animals. Thus, a regulated gene expression cassette can be incorporated into a single AAV vector, such that intramuscular injection of the vector allows sustained and regulated expression of a desired gene.

© 1998 by The American Society of Hematology.
Retroviral Marking of Canine Bone Marrow: Long-Term, High-Level Expression of Human Interleukin-2 Receptor Common Gamma Chain in Canine Lymphocytes
Authors: Whitwam Todd , Haskins Mark E. , Henthorn Paula S. , Kraszewski Jennifer N. , Kleiman Sandra E. , Seidel Nancy E. , Bodine David M. , Puck Jennifer M.
Published: October 2019

Optimization of retroviral gene transfer into hematopoietic cells of the dog will facilitate gene therapy of canine X-linked severe combined immunodeficiency (XSCID) and in turn advance similar efforts to treat human XSCID. Both canine and human XSCID are caused by defects in the common γ chain, γc, of receptors for interleukin-2 and other cytokines. In this study, normal dogs were given retrovirally transduced bone marrow cells with and without preharvest mobilization by the canine growth factors granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). Harvey sarcoma virus and Moloney murine leukemia virus constructs were used, both containing cDNA encoding human γc. The Harvey-based vector transduced into cytokine-primed marrow yielded persistent detectable provirus in bone marrow and blood and expression of human γc on peripheral lymphocytes. In three dogs, human γc expression disappeared after 19 to 34 weeks but reappeared and was sustained, in one dog beyond 16 months posttransplantation, upon immunosuppression with cyclosporin A and prednisone, with up to 25% of lymphocytes expressing human γc. The long-term expression of human γc in a high proportion of normal canine lymphocytes predicts that retrovirus-mediated gene correction of hematopoietic cells may prove to be of clinical benefit in humans affected with this XSCID.

This is a US government work. There are no restrictions on its use.
Antiphospholipid Antibodies Accelerate Plasma Coagulation by Inhibiting Annexin-V Binding to Phospholipids: A “Lupus Procoagulant” Phenomenon
Authors: Rand Jacob H. , Wu Xiao-Xuan , Andree Harry A.M. , Ross J.B. Alexander , Rusinova Elena , Gascon-Lema Mayra G. , Calandri Cesare , Harpel Peter C.
Published: October 2019

The antiphospholipid syndrome is a thrombophilic condition marked by antibodies that recognize anionic phospholipid-protein cofactor complexes. We recently reported that exposure to IgG fractions from antiphospholipid patients reduces the level of annexin-V, a phospholipid-binding anticoagulant protein, on cultured trophoblasts and endothelial cells and accelerates coagulation of plasma exposed to these cells. Therefore, we asked whether antiphospholipid antibodies might directly reduce annexin-V binding to noncellular phospholipid substrates. Using ellipsometry, we found that antiphospholipid IgGs reduce the quantity of annexin-V bound to phospholipid bilayers; this reduction is dependent on the presence of β2-glycoprotein I. Also, exposure to plasmas containing antiphospholipid antibodies reduces annexin-V binding to phosphatidyl serine-coated microtiter plates, frozen thawed washed platelets, activated partial thromboplastin time (aPTT) reagent and prothrombin time reagent and reduces the anticoagulant effect of the protein. These studies show that antiphospholipid antibodies interfere with the binding of annexin-V to anionic phospholipid and with its anticoagulant activity. This acceleration of coagulation, due to reduced binding of annexin V, stands in marked contrast to the “lupus anticoagulant effect” previously described in these patients. These results are the first direct demonstration of the displacement of annexin-V and the consequent acceleration of coagulation on noncellular phospholipid surfaces by antiphospholipid antibodies.

© 1998 by The American Society of Hematology.
An Assessment of Dried Blood-Spot Technology for Identifying Iron Deficiency
Authors: Cook James D. , Flowers Carol H. , Skikne Barry S.
Published: October 2019

The present study was undertaken to assess the feasibility of using ferritin and transferrin receptor measurements on dried capillary blood spots to identify iron deficiency (ID) in public health surveys. Measurements on serum and blood spots prepared from venous blood were performed in 71 healthy subjects, 41 of whom were iron-replete and 30 who had ID, either without (n = 20) or with (n = 10) anemia. Parallel measurements were performed on hemolyzed whole blood and washed hemolyzed red blood cells to assess the erythrocyte contribution of ferritin and transferrin receptor to dried blood samples. The concentration of ferritin in dried blood samples was threefold higher than serum assays due to the release of ferritin from hemolyzed erythrocytes, which diminished the usefulness of ferritin measurements for detecting ID. On the other hand, there was negligible erythrocyte contribution to the measurement of transferrin receptor in dried blood spots. The most sensitive parameter in dried blood spots was the ratio of receptor/ferritin, which was suitable for identifying iron-deficiency anemia (IDA), but less reliable than serum assays for detecting milder ID without anemia. We conclude that tandem measurements of serum ferritin and transferrin receptor in dried blood spots can be used to facilitate the identification of IDA in epidemiologic studies.

© 1998 by The American Society of Hematology.
Increased Susceptibility in Hp Knockout Mice During Acute Hemolysis
Authors: Lim Sai-Kiang , Kim Hongkyun , Lim Shen Kiat , Ali Azhar bin , Lim Yew Koon , Wang Yanping , Chong Siew Meng , Costantini Frank , Baumman Heinz
Published: October 2019

Haptoglobin, a conserved plasma glycoprotein, forms very stable soluble complexes with free plasma hemoglobin. Hemoglobin binding by haptoglobin is thought to be important in the rapid hepatic clearance of hemoglobin from the plasma and in the inhibition of glomerular filtration of hemoglobin. To evaluate these functions,Haptoglobin knockout (−/−) mice were created. These mice were viable but had a small, significant reduction in postnatal viability. Contrary to popular belief, the lack of haptoglobin did not impair clearance of free plasma hemoglobin in −/− mice. Induction of severe hemolysis by phenylhydrazine caused extensive hemoglobin precipitation in the renal tubular cells of both −/− and +/+ mice, with death occurring in 55% of −/− mice and in 18% of +/+ mice. In general, phenylhydrazine-treated −/− mice suffered greater tissue damage, as evidenced by the induction of hepatic acute phase response resulting in increased plasma alpha 1-acid glycoprotein (AGP) levels. Among −/− and +/+ mice that survived, −/− mice tend to suffer greater oxidative damage and failed to repair or regenerate damaged renal tissues, as indicated by their higher plasma malonaldehyde (MDA) and 4-hydroxy-2(E)-nonenal (HNE) levels and lower mitotic indices in their kidneys, respectively. This study suggested that a physiologically important role of hemoglobin-haptoglobin complex formation is the amelioration of tissue damages by hemoglobin-driven lipid peroxidation.

© 1998 by The American Society of Hematology.