bluuebunny/crossref_metadata_2025 huggingface dataset:
Red Blood Cell Precursor Mass as an Independent Determinant of Serum Erythropoietin Level
Authors: Cazzola Mario , Guarnone Roberta , Cerani Paola , Centenara Esther , Rovati Andrea , Beguin Yves
Published: October 2019

Serum erythropoietin (sEpo) concentration is primarily related to the rate of renal production and, under the stimulus of hypoxia, increases exponentially as hemoglobin (Hb) decreases. Additional factors, however, appear to influence sEpo, and in this work, we performed studies to evaluate the role of the red blood cell precursor mass. We first compared the relationship of sEpo with Hb in patients with low versus high erythroid activity. The first group included 27 patients with erythroid aplasia or hypoplasia having serum transferrin receptor (sTfR) levels < 3 mg/L (erythroid activity < 0.6 times normal), while the second one included 28 patients with β-thalassemia intermedia having sTfR levels > 10 mg/L (erythroid activity > 2 times normal). There was no difference between the two groups with respect to Hb (8.3 ± 1.6 v 8.0 ± 1.3 g/dL, P > .05), but sEpo levels were notably higher in patients with low erythroid activity (1,601 ± 1,542 v 235 ± 143 mU/mL,P < .001). In fact, multivariate analysis of variance (ANOVA) showed that, at any given Hb level, sEpo was higher in patients with low erythroid activity (P < .0001). Twenty patients undergoing allogeneic or autologous bone marrow transplantation (BMT) were then investigated. A marked increase in sEpo was seen in all cases at the time of marrow aplasia, disproportionately high when compared with the small decrease in Hb level. Sequential studies were also performed in five patients with iron deficiency anemia undergoing intravenous (IV) iron therapy. Within 24 to 72 hours after starting iron treatment, marked decreases in sEpo (up to one log magnitude) were found before any change in Hb level. Similar observations were made in patients with megaloblastic anemia and in a case of pure red blood cell aplasia. These findings point to an inverse relationship between red blood cell precursor mass and sEpo: at any given Hb level, the higher the number of red blood cell precursors, the lower the sEpo concentration. The most likely explanation for this is that sEpo levels are regulated not only by the rate of renal production, but also by the rate of utilization by erythroid cells.
A Common Human β Globin Splicing Mutation Modeled in Mice
Authors: Lewis Jada , Yang Baoli , Kim Ronald , Sierakowska Halina , Kole Ryszard , Smithies Oliver , Maeda Nobuyo
Published: October 2019

The βIVS-2-654 C→T mutation accounts for approximately 20% of β thalassemia mutations in southern China; it causes aberrant RNA splicing and leads to β0 thalassemia. To provide an animal model for testing therapies for correcting splicing defects, we have used the “plug and socket” method of gene targeting in murine embryonic stem cells to replace the two (cis) murine adult β globin genes with a single copy of the human βIVS-2-654 gene. No homozygous mice survive postnatally. Heterozygous mice carrying this mutant gene produce reduced amounts of the mouse β globin chains and no human β globin, and have a moderate form of β thalassemia. The heterozygotes show the same aberrant splicing as their human counterparts and provide an animal model for testing therapies to correct splicing defects at either the RNA or DNA level.
JAK2 and JAK1 Constitutively Associate With an Interleukin-5 (IL-5) Receptor α and βc Subunit, Respectively, and Are Activated Upon IL-5 Stimulation
Authors: Ogata Norihisa , Kouro Taku , Yamada Atsuko , Koike Masamichi , Hanai Nobuo , Ishikawa Takeru , Takatsu Kiyoshi
Published: October 2019

The human interleukin-5 receptor (hIL-5R) consists of a unique α subunit (hIL-5Rα) and a common β subunit (βc) that activate two Janus kinases (JAK1 and JAK2) and a signal transducer and activator of transcription (STAT5). The precise stoichiometry of the hIL-5R subunits and the role of JAK kinases used in IL-5 signaling were investigated. We analyzed the interaction between hIL-5Rα and βc by immunoprecipitation using anti–hIL-5Rα and anti-βc monoclonal antibodies. The binding of JAK1 and JAK2 to each hIL-5R subunit was also evaluated in the hIL-5–responsive cell line, TF-h5Rα. It was observed that IL-5 stimulation induced the recruitment of βc to hIL-5Rα, although in the absence of IL-5 the subunits remain independent. In the absence of IL-5, JAK2 and JAK1 were associated with hIL-5Rα and βc, respectively. IL-5 stimulation resulted in tyrosine phosphorylation of JAK2, JAK1, βc, and STAT5. Moreover, IL-5–induced dimerization of IL-5R subunits caused JAK2 activation and βc phosphorylation even in the absence of JAK1 activation. Furthermore, tyrosine phosphorylation of JAK1 was dependent on the activation of JAK2. Detailed study of the C-terminal truncated cytoplasmic domain of hIL-5Rα revealed that the cytoplasmic stretch at position 346-387, containing the proline-rich region, is necessary for JAK2 binding. These observations suggest that activation of hIL-5Rα–associated JAK2 is indispensable for the IL-5 signaling event.
Natural Killer and B-Lymphoid Potential in CD34+ Cells Derived From Embryonic Stem Cells Differentiated in the Presence of Vascular Endothelial Growth Factor
Authors: Nakayama Naoki , Fang Inghwa , Elliott Gary
Published: October 2019

Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels, differentiation intermediates were investigated. ES cells generated progeny expressing CD34, which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7, the CD34+ cells developed two types of B220+ CD34−lymphocytes: CD3− cytotoxic lymphocytes and CD19+ pre-B cells, and such lymphoid potential was highly enriched in the CD34+ population. Interestingly, the cytotoxic cells expressed the natural killer (NK) cell markers, such as NKR-P1, perforin, and granzymes, classified into two types, one of which showed target specificity of NK cells. Thus, ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells, and both myeloid and lymphoid cells seem to be derived from the CD34+intermediate, on which VEGF may play an important role.
−245 bp of 5′-Flanking Region From the Human Platelet Factor 4 Gene Is Sufficient to Drive Megakaryocyte-Specific Expression In Vivo
Authors: Cui Zheng , Reilly Michael P. , Surrey Saul , Schwartz Elias , McKenzie Steven E.
Published: October 2019

Platelet factor 4 (PF4) serves as a lineage-specific marker of megakaryocyte development. We previously identified two positively acting sequences in the human platelet factor 4 (hPF4) gene promoter that synergized to drive high-level luciferase reporter gene expression in vitro. Using portions of the hPF4 5′-flanking region linked to the lacZ reporter gene, we observed in this investigation that constructs with −245 bp of 5′-flanking region were more active than constructs with −2 kb of 5′-flanking region in vitro. We created two independent transgenic mouse lines with a −245-bp hPF4/lacZ construct. Cells from these mice were tested for β-galactosidase (β-gal) expression at the mRNA level by Northern blot and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and at the protein level by immunohistochemistry assay. Mice from one line showed β-gal expression specifically in all megakaryocytes of all ploidy classes from bone marrow and in platelets. Expression level was comparable to that driven by the 1.1-kb rat PF4 promoter in other transgenic mouse lines. Those in the second line showed no β-gal expression in megakaryocytes, platelets, or any of the eight organs tested. The −245-bp hPF4 promoter is capable of driving reporter gene expression in a megakaryocyte-specific manner in transgenic mice. The small size of this megakaryocyte-specific promoter is compatible with that required in some viral vectors and may provide a model for targeting gene expression to megakaryocytes.
Rat Neutrophils Express α4 and β1 Integrins and Bind to Vascular Cell Adhesion Molecule-1 (VCAM-1) and Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1)
Authors: Davenpeck Kelly L. , Sterbinsky Sherry A. , Bochner Bruce S.
Published: October 2019

The α4 integrins, which are constitutively expressed on all human leukocyte subtypes except neutrophils, interact with vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule (MAdCAM-1) on endothelium to mediate selective recruitment of leukocyte subpopulations, other than neutrophils, to sites of inflammation. However, here we report that a different paradigm of leukocyte recruitment may exist in the rat. Flow cytometric analysis of rat neutrophils using a panel of monoclonal antibodies which recognize rat α4 and β1 integrins showed consistent, low levels of expression. Although α4 was expressed at lower levels on neutrophils than all other rat leukocytes, this level of expression was sufficient to mediate significant levels of α4- and β1-dependent neutrophil adhesion to rat and human VCAM-1, and α4-dependent, but β1-independent, adhesion to human MAdCAM-1. These data suggest that rat neutrophils, unlike other species, may use α4 integrins to traffic to sites of inflammation in vivo.
Chronic Lymphocytic Leukemic B Cells But Not Normal B Cells Are Rescued From Apoptosis by Contact With Normal Bone Marrow Stromal Cells
Authors: Lagneaux L. , Delforge A. , Bron D. , De Bruyn C. , Stryckmans P.
Published: October 2019

The leukemic B lymphocytes from chronic lymphocytic leukemic (CLL) patients have a long survival in vivo, although ex vivo they rapidly die by apoptosis. To further investigate the mechanism of this, we have studied the influence of bone marrow stromal cells from normal subjects on apoptosis of B-CLL cells and normal umbilical cord blood (UCB) B lymphocytes. After 48 hours of incubation in medium alone, leukemic and normal B cells showed, respectively, 22 ± 3% and 31 ± 5% of apoptosis. Cocultures with stromal cells reduced the percentage of leukemic cells undergoing apoptosis (8 ± 2%, P< .0005) and prevented the loss of bcl-2 protein expression. In contrast, stromal cells slightly increased normal B-cell apoptosis (37 ± 6%). Direct contact between leukemic cells and stromal cells was found to be essential for inhibition of leukemic cell apoptosis; indeed, separation of leukemic cells from stromal cells by microporous membrane increased spontaneous apoptosis, and comparable results were obtained with stromal cell conditioned medium. The difference in behavior observed between normal and leukemic B cells plated on stromal cells can be explained by the fact that only a few normal B cells adhere to stromal cells in comparison with B-CLL cells. B-CLL cell adhesion to stromal cells is mediated by β1 and β2 integrins acting simultaneously. Contact between B-CLL cells and bone marrow stromal cells seems to play a major role in the accumulation and survival of B-CLL cells in the bone marrow.
Somatic Hypermutation, Clonal Diversity, and Preferential Expression of the VH 51p1/VL kv325 Immunoglobulin Gene Combination in Hepatitis C Virus–Associated Immunocytomas
Authors: Ivanovski Martin , Silvestri Federico , Pozzato Gabriele , Anand Shubha , Mazzaro Cesare , Burrone Oscar R. , Efremov Dimitar G.
Published: October 2019

A high prevalence of chronic hepatitis C virus (HCV) infection has recently been shown in a subset of B-cell non-Hodgkin's lymphomas, most of which belong to the lymphoplasmacytoid lymphoma/immunocytoma subtype and are characterized by the production of a monoclonal IgM cryoglobulin with rheumatoid factor activity. To better define the stage of differentiation of the malignant B cell and to investigate the role of chronic antigen stimulation in the pathogenesis of the HCV-associated immunocytomas, we analyzed the variable (V) region gene repertoire in 16 cases with this type of tumor. The lymphoma-derived V gene sequences were successfully determined in 8 cases; 5 of them expressed the 51p1 VH gene in combination with the kv325 VL gene. Moreover, a monoclonal 51p1-expressing B-cell population was detected in 4 of the remaining immunocytomas by an allele-specific Ig gene fingerprinting assay, indicating that HCV-associated immunocytomas represent clonal proliferations of a highly selected B-cell population. Somatic mutations and intraclonal diversity were observed in all of the lymphoma V genes, and clonally related IgM and IgG VH transcripts indicative of isotype switching were present in one case. These findings are consistent with an antigen-driven process and support a role for chronic antigen stimulation in the growth and clonal evolution of HCV-associated immunocytomas.
Idiotype Immunization Combined With Granulocyte-Macrophage Colony-Stimulating Factor in Myeloma Patients Induced Type I, Major Histocompatibility Complex–Restricted, CD8- and CD4-Specific T-Cell Responses
Authors: Österborg Anders , Yi Qing , Henriksson Lotta , Fagerberg Jan , Bergenbrant Susanne , Jeddi-Tehrani Mahmood , Rudén Ulla , Lefvert Ann-Kari , Holm Göran , Mellstedt Håkan
Published: October 2019

Idiotypic structures expressed on the myeloma Ig protein might be regarded as a tumor-specific antigen. Five patients with IgG myeloma were immunized with the purified serum M-component by repeated intradermal injections together with soluble granulocyte-macrophage colony-stimulating factor (GM-CSF). All patients developed an idiotype (Id)-specific T-cell immunity, defined as blood T cells predominantly secreting interferon-γ (IFN-γ) and interleukin-2 (IL-2) (type I cells). Id-specific DNA synthesis was induced in one patient. Delayed-type hypersensitivity against the Id was not evoked. The specific IFN-γ/IL-2 T-cell response was inhibited (46% to 100%) by a major histocompatibility complex (MHC) class I monoclonal antibody (MoAb) in all five patients. A 5% to 37% inhibition by an MHC class II MoAb was seen in four patients. CD4+ as well as CD8+ T cells enriched by magnetic microbeads contained Id-specific cells. The T cells recognized peptides corresponding to the complementarity-determining regions 1, 2, and 3 of the heavy chain of the Id. There was a transient rise of B cells producing IgM anti-idiotypic antibodies in all patients. The results indicate that immunization of myeloma patients using the autologous M-component and soluble GM-CSF may evoke an Id-specific predominantly MHC class I–restricted type I T-cell response.
Characterization of CKβ8 and CKβ8-1: Two Alternatively Spliced Forms of Human β-Chemokine, Chemoattractants for Neutrophils, Monocytes, and Lymphocytes, and Potent Agonists at CC Chemokine Receptor 1
Authors: Youn Byung-S. , Zhang Shang M. , Broxmeyer Hal E. , Cooper Scott , Antol Kathleen , Fraser Malcolm , Kwon Byoung S.
Published: October 2019

Two new members of human β-chemokine cDNA were isolated based on structural and functional similarities to human leukotactin-1. One of these clones was identical to the previously isolated human β-chemokine, CKβ8, whereas the other is a splicing variant of CKβ8, therefore named CKβ8-1. CKβ8 was short in 51 nucleotides (17 amino acids) compared with CKβ8-1. The mature proteins of CKβ8-1 and CKβ8 consisted of 116 and 99 amino acids with calculated molecular weights of 12,500 and 10,950, respectively. Both CKβ8-1 and CKβ8 were potent agonists at CCR1. These chemokines chemoattracted neutrophils, monocytes, and lymphocytes. They also significantly suppressed colony formation by human bone marrow, granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors. To our knowledge, this is the first example that an alternative splicing produces two active β-chemokines from a single gene.
Loss of Imprinting in Disease Progression in Chronic Myelogenous Leukemia
Authors: Randhawa Gurvaneet S. , Cui Hengmi , Barletta Janet A. , Strichman-Almashanu Liora Z. , Talpaz Moshe , Kantarjian Hagop , Deisseroth Albert B. , Champlin Richard C. , Feinberg Andrew P.
Published: October 2019

The pathophysiologic role of the Philadelphia chromosome translocation in chronic myelogenous leukemia (CML) has been known for nearly 20 years. However, the most significant morbidity and mortality in CML are caused by progression to blast crisis, about which comparatively little is known at the molecular level. Genomic imprinting is a chromosomal modification leading to parental-origin–specific gene expression in somatic cells. Recently, we and others have described loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2), leading to biallelic rather than monoallelic expression in a wide variety of solid tumors. We have now examined the imprinting status of IGF2 in samples from CML patients in stable phase, accelerated phase, and blast crisis. Five of six stable-phase patients showed normal imprinting, but LOI was found in all six cases of advanced disease (three accelerated phase, three blast crisis), which was statistically highly significant (P < .01). Thus, LOI represents a novel type of genetic alteration in CML that appears to be specifically associated with disease progression.
Inhibition of P-Glycoprotein and Recovery of Drug Sensitivity of Human Acute Leukemic Blast Cells by Multidrug Resistance Gene (mdr1) Antisense Oligonucleotides
Authors: Motomura Sayuri , Motoji Toshiko , Takanashi Minoko , Wang Yan-Hua , Shiozaki Hiroko , Sugawara Isamu , Aikawa Eizou , Tomida Akihiro , Tsuruo Takashi , Kanda Naotoshi , Mizoguchi Hideaki
Published: October 2019

To overcome the problem of multidrug resistance, we investigated the effectiveness of phosphrothioate antisense oligonucleotides (MDR1-AS) in suppressing multidrug resistance gene (mdr1) expression in drug-resistant acute myelogenous leukemia (AML) blast cells and the K562 adriamycin-resistant cell line K562/ADM. The percentage of cells with the mdr1 gene product P-glycoprotein (P-gp) was decreased from 100% to 26% by 20 μmol/L MDR1-AS in the K562/ADM cells, and from 48.1% to 10.2% by 2.5 μmol/L MDR1-AS in the AML blast cells. Western blot analysis also showed a decrease in the amount of P-gp in the MDR1-AS–treated K562/ADM cells. This effect was specific to MDR1-AS, and not observed with sense or random control oligonucleotides. The expression of mdr1 mRNA in K562/ADM and AML blast cells treated with MDR1-AS was decreased compared with the random control. Intracellular rhodamine retention and [3H]daunorubicin also increased after antisense treatment. Chemosensitivity to daunorubicin increased in MDR1-AS–treated blast cells up to 5.9-fold in the K562/ADM cells and 3.0- to 6.4-fold in the AML blast cells. The expression of mdr1mRNA derived from colony cells decreased in the MDR1-AS–treated groups. No inhibitory effect of the oligonucleotides on normal bone marrow progenitors was observed. These findings suggest that MDR1-AS is useful to overcome multidrug resistance in the treatment of leukemia.
Ineffective Platelet Production in Thrombocytopenic Human Immunodeficiency Virus–Infected Patients
Authors: Cole James L. , Marzec Ulla M. , Gunthel Clifford J. , Karpatkin Simon , Worford Lydia , Sundell I. Birgitta , Lennox Jeffrey L. , Nichol Janet L. , Harker Laurence A.
Published: October 2019

Thrombocytopenia has been characterized in six patients infected with human immunodeficiency virus (HIV) with respect to the delivery of viable platelets into the peripheral circulation (peripheral platelet mass turnover), marrow megakaryocyte mass (product of megakaryocyte number and volume), megakaryocyte progenitor cells, circulating levels of endogenous thrombopoietin (TPO) and platelet TPO receptor number, and serum antiplatelet glycoprotein (GP) IIIa49-66 antibody (GPIIIa49-66Ab), an antibody associated with thrombocytopenia in HIV-infected patients. Peripheral platelet counts in these patients averaged 46 ± 43 × 103/μL (P = .0001 compared to normal controls of 250 ± 40×  103/μL), and the mean platelet volume (MPV) was 10.5 ± 2.0 fL (P > 0.3 compared with normal control of 9.5 ± 1.7 fL). The mean life span of autologous111In-platelets was 87 ± 39 hours (P = .0001 compared with 232 ± 38 hours in 20 normal controls), and immediate mean recovery of 111In-platelets injected into the systemic circulation was 33% ± 16% (P = .0001 compared with 65% ± 5% in 20 normal controls). The resultant mean peripheral platelet mass turnover was 3.8 ± 1.5 × 105 fL/μL/d versus 3.8 ± 0.4 × 105 fL/μL/d in 20 normal controls (P > .5). The mean endogenous TPO level was 596 ± 471 pg/mL (P = .0001 compared with 95 ± 6 pg/mL in 98 normal control subjects), and mean platelet TPO receptor number was 461 ± 259 receptors/platelet (P = .05 compared with 207 ± 99 receptors/platelet in nine normal controls). Antiplatelet GPIIIa49-66Ab levels in sera were uniformly increased in HIV thrombocytopenic patients (P < .001). In this cohort of thrombocytopenic HIV patients, marrow megakaryocyte number was increased to 30 ± 15 × 106/kg (P = .02 compared with 11 ± 2.1 × 106/kg in 20 normal controls), and marrow megakaryocyte volume was 32 ± 0.9 × 103 fL (P = .05 compared with 28 ± 4.5 × 103 fL in normal controls). Marrow megakaryocyte mass was expanded to 93 ± 47 × 1010 fL/kg (P = .007 compared with normal control of 31 ± 5.3 × 1010 fL/kg). Marrow megakaryocyte progenitor cells averaged 3.3 (range, 0.4 to 7.3) CFU-Meg/1,000 CD34+ cells compared with 27 (range, 0.1 to 84) CFU-Meg/1,000 CD34+ cells in seven normal subjects (P = .02). Thus, thrombocytopenia in these HIV patients was caused by a combination of shortening of platelet life span by two thirds and doubling of splenic platelet sequestration, coupled with ineffective delivery of viable platelets to the peripheral blood, despite a threefold TPO-driven expansion in marrow megakaryocyte mass. We postulate that this disparity between circulating platelet product and marrow platelet substrate results from direct impairment in platelet formation by HIV-infected marrow megakaryocytes.
Constitutive Expression of the Promyelocytic Leukemia–Associated Oncogene PML-RARα in TF1 Cells: Isoform-Specific and Retinoic Acid–Dependent Effects on Growth, bcl-2 Expression, and Apoptosis
Authors: Slack James L. , Yu Min
Published: October 2019

Two major isoforms of PML-RARα are associated with (15;17)-positive acute promyelocytic leukemia (APL); however, functional differences between these isoforms have been difficult to define, and the molecular mechanism by which each isoform contributes to the pathogenesis of APL is not fully understood. To address these issues, the ‘short’ (S) and ‘long’ (L) isoforms of PML-RARα were constitutively expressed in the factor-dependent human erythroleukemia cell line, TF1. Expression of the L, but not the S, isoform inhibited growth of these cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). In the absence of GM-CSF, the S isoform partially protected against apoptosis, while the L isoform accelerated cell death. Treatment with all-trans retinoic acid (ATRA) inhibited cell growth and caused apoptosis only in PML-RARα–expressing cells, and these effects of ATRA were more marked in cells expressing the L isoform. ATRA treatment also led to downregulation of bcl-2 and endogenous RARα in PML-RARα–expressing cells, but had little effect on the level of exogenously expressed PML-RARα. We conclude that (1) subtle differences exist in the biologic activities of the L and S isoforms of PML-RARα, and (2) both isoforms are capable of transducing an ATRA-mediated signal that leads to downregulation of bcl-2 and induction of programmed cell death.
Association Between Pretransplant Interferon-α and Outcome After Unrelated Donor Marrow Transplantation for Chronic Myelogenous Leukemia in Chronic Phase
Authors: Morton A. James , Gooley Ted , Hansen John A. , Appelbaum Frederick R. , Bruemmer Barbara , Bjerke Jean W. , Clift Reg , Martin Paul J. , Petersdorf Effie W. , Sanders Jean E. , Storb Rainer , Sullivan Keith M. , Woolfrey Ann , Anasetti Claudio
Published: October 2019

Treatment options for patients diagnosed with chronic myelogenous leukemia (CML) in chronic phase (CP) who lack a suitable related donor for marrow transplantation include hydroxyurea, interferon-α (IFN-α), or transplantation from an unrelated donor (URD). Most studies support the view that treatment with IFN-α results in prolonged survival compared with hydroxyurea therapy. Some patients are offered URD transplantation as a second-line treatment; however, the impact of pretransplant IFN-α on the outcome of URD transplantation is uncertain. To address this question, we evaluated the effect of pretransplant IFN-α therapy in 184 patients undergoing URD transplantation for CML in CP at a single center. Of the 184 patients, 114 did not receive IFN-α, whereas 22, 23, and 25 patients received IFN-α for, respectively, 1 to 5, 6 to 12, and more than 12 months before transplant. Pretransplant IFN-α therapy administered for ≥6 months was associated with an increased risk of severe (grades III-IV) acute graft-versus-host disease (GVHD; relative risk [RR], 3.0; 95% confidence interval [CI], 1.4 to 6.2; P = .004) and mortality (RR, 2.1; 95% CI, 1.3 to 3.5; P = .003) relative to less than 6 months or no IFN-α therapy. Increased mortality occurred between 100 and 365 days after transplant (P = .005), was limited to patients with severe acute GVHD, and was due to chronic GVHD refractory to immunosuppressive therapy. Other variables associated with mortality included HLA-DRB1 or DQB1 (but not HLA-A or B) mismatched donors, age greater than 50 years, weight ≥110% of ideal body weight, and the absence of cytomegalovirus (CMV) or fungal prophylaxis. For patients treated with IFN-α for less than 6 months before transplant, who were ≤50 years of age, received a HLA-A, B, DRB1, and DQB1 matched URD transplant, and received CMV and fungal prophylaxis after transplant (n = 48), survival was 87% ± 5% at 5 years. These data provide a rationale for immediate transplantation in preference to extended treatment with IFN-α when the patient is ≤50 years of age and has an HLA-compatible unrelated volunteer donor.
The Hyperresponsiveness of Cells Expressing Truncated Erythropoietin Receptors Is Contingent on Insulin-Like Growth Factor-1 in Fetal Calf Serum
Authors: Damen Jacqueline E. , Krosl Jana , Morrison Donna , Pelech Steven , Krystal Gerald
Published: October 2019

We demonstrate herein that the well documented hyperresponsiveness to erythropoietin (Epo) of Ba/F3 cells expressing C-terminal truncated erythropoietin receptors (EpoRs) is contingent on these cells being in fetal calf serum (FCS). In the absence of FCS, their Epo-induced proliferation is far poorer than Ba/F3 cells expressing wild-type (WT) EpoRs. This hyporesponsiveness in the absence of serum is also seen in DA-3 cells expressing these truncated EpoRs. In fact, long-term proliferation studies performed in the absence of serum show that even at saturating concentrations of Epo, Ba/F3 cells expressing these truncated receptors die via apoptosis, while cells bearing WT EpoRs do not, and this programmed cell death correlates with an inability of Epo-stimulated Ba/F3 cells expressing truncated EpoRs to induce the tyrosine phosphorylation of MAPK and the activation of p70S6K. Using neutralizing antibodies to insulin-like growth factor (IGF)-1, we show that a major non-Epo factor in FCS that contributes to the hyperresponsive phenotype of Ba/F3 cells expressing truncated EpoRs is IGF-1. Our results suggest that the Epo-hypersensitivity of truncated EpoR expressing Ba/F3 cells is due to the combined effects of these EpoRs not possessing a binding site for the negative regulator, SHP-1, and the triggering of proliferation-inducing/apoptosis-inhibiting cascades, lost through EpoR truncation, by IGF-1.
A Novel Asn344 Deletion in the Core Domain of Coagulation Factor XIII A Subunit: Its Effects on Protein Structure and Function
Authors: Kangsadalampai Sasichai , Chelvanayagam Gareth , Baker Rohan T. , Yenchitsomanus Pa-thai , Pung-amritt Parichat , Mahasandana Chularatana , Board Philip G.
Published: October 2019

In this study a previously undescribed 3 bp deletion, AAT1030-1032, in the factor XIII A subunit gene, has been detected in a Thai patient. The inframe deletion results in the translation of a factor XIII A subunit that lacks Asn344. This is the first inframe deletion to be identified in the factor XIII A subunit gene because six previously reported deletions have all caused frameshifts. The deletion has been introduced into a factor XIII A subunit cDNA and the deleted polypeptide expressed in yeast. The mRNA encoding the mutant enzyme appears to have normal stability but the translated protein is subject to premature degradation. In addition, the mutated enzyme exhibited very little transglutaminase activity compared with the wild-type enzyme. Structural modeling of the deleted enzyme suggests that the absence of Asn344 would have a potent impact on the catalytic activity by reorienting the residues associated with the catalytic center. Thus, the Asn344 deletion strongly confirms the significance of the residues surrounding the catalytic center of the factor XIII A subunit.
Ig Receptor Binding Protein 1 (α4) Is Associated With a Rapamycin-Sensitive Signal Transduction in Lymphocytes Through Direct Binding to the Catalytic Subunit of Protein Phosphatase 2A
Authors: Inui Seiji , Sanjo Hideki , Maeda Kazuhiko , Yamamoto Hideyuki , Miyamoto Eishichi , Sakaguchi Nobuo
Published: October 2019

Rapamycin is an immunosuppressant that effectively controls various immune responses; however, its action in the signal transduction of lymphocytes has remained largely unknown. We show here that a phosphoprotein encoded by mouse α4 (mα4) gene transmitting a signal through B-cell antigen receptor (BCR) is associated with the catalytic subunit of protein phosphatase 2A (PP2Ac). The middle region of α4, consisting of 109 amino acids (94-202), associates directly with PP2Ac, irrespective of any other accessory molecule. Rapamycin treatment disrupts the association of PP2Ac/α4 in parallel with the inhibitory effect of lymphoid cell proliferation. The effect of rapamycin was inhibited with an excess amount of FK506 that potentially completes the binding to FKBP. Rapamycin treatment also suppresses the phosphatase activity of cells measured by in vitro phosphatase assay. Introduction of the mα4 cDNA into Jurkat cells or the increased association of PP2Ac/α4 by the culture with low serum concentration confers cells with rapamycin resistance. Moreover, glutathione S-transferase (GST)-α4 augments the PP2A activity upon myelin basic protein (MBP) and histone in the in vitro assay. These results suggest that α4 acts as a positive regulator of PP2A and as a new target of rapamycin in the activation of lymphocytes.
B-Cell Chronic Lymphocytic Leukemia Cells Express a Functional Inducible Nitric Oxide Synthase Displaying Anti-Apoptotic Activity
Authors: Zhao Haixia , Dugas Nathalie , Mathiot Claire , Delmer Alain , Dugas Bernard , Sigaux François , Kolb Jean-Pierre
Published: October 2019

The expression of different isoforms of nitric oxide synthase (NOS) was investigated in B-cell chronic lymphocytic leukemia (B-CLL) to delineate a possible role for nitric oxide (NO) in the control of apoptosis of the tumoral cells. By reverse transcription-polymerase chain reaction (RT-PCR), all B-CLL cells were found to express spontaneously inducible NOS (iNOS) mRNA, whereas endothelial constitutive NOS (ecNOS) mRNA was undetectable. The iNOS protein was detected by immunofluorescence in the cytoplasm of permeabilized leukemic cells and identified by Western blotting, using different anti-iNOS antibodies, as a protein of 135 kD in B-CLL cytoplasmic extracts. B-CLL cell lysates also displayed basal NOS enzymatic activity, as measured by the conversion of14C-labeled L-arginine into 14C-L-citrulline. Ligation of CD23, expressed on the vast majority of B-CLL cells, resulted in increased iNOS expression and activity. The NO released exerted an anti-apoptotic effect on B-CLL cells that was counteracted by NOS inhibitors and engagement of the APO-1/Fas pathway. Therefore, the existence of a functional iNOS in B-CLL cells will provide further insights into the mechanisms that control proliferation and apoptosis in these tumor cells.

© 1998 by The American Society of Hematology.
Release of Polymorphonuclear Leukocytes From the Bone Marrow by Interleukin-8
Authors: Terashima Takeshi , English Dean , Hogg James C. , vanEeden Stephan F.
Published: October 2019

Several studies have shown that interleukin-8 (IL-8) causes a rapid granulocytosis with the release of polymorphonuclear leukocytes (PMN) from the bone marrow (BM) partially responsible for the granulocytosis. This study was designed to quantitate the release of PMN from the BM by IL-8 and measure the transit time of PMN through the marrow after IL-8 administration. The thymidine analogue, 5'-bromo-2'-deoxyuridine (BrdU), was used to label dividing PMN in the marrow and follow their release into the circulation after intravenous IL-8. This allowed us to calculate the transit time of PMN through the mitotic and postmitotic pools of BM. BrdU was infused intravenously into rabbits 24 hours before IL-8 (2.5 μg/kg). IL-8 caused a rapid, transient granulocytopenia (5.9 ± 0.4 at baseline v 0.2 ± 0.06 × 10/9L at 5 minutes, P < .05) followed by granulocytosis (8.4 ± 0.1 at 30 minutes, P < .05) associated with an increased number (0.3 ± 0.1 at baseline v1.2 ± 0.6 × 109/L at 30 minutes, P < .05) and percentage of band cells (P < .05), as well as a rapid increase in the number of BrdU-labeled PMN (PMNBrdU) in the circulation (0.09 ± 0.05 at baseline to 1.5 ± 0.6 × 109/L at 60 minutes, P < .05). The transit time of PMN through both the mitotic and postmitotic pools of BM was not affected by IL-8. To determine the marrow compartment from which the PMN were mobilized by IL-8, we quantitated PMN movement from the hematopoietic and sinusoidal compartments into the circulation. The fraction of PMNBrdU in both compartments was higher than in the circulating blood (P < .05) and the fraction and number of PMNBrdU in the sinusoids decreased with IL-8 treatment (P < .05). We conclude that the pool of PMN residing in the BM venous sinusoids are rapidly released into the circulation after administration of IL-8.

© 1998 by The American Society of Hematology.
Role for Bcl-xL in Delayed Eosinophil Apoptosis Mediated by Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-5
Authors: Dibbert Birgit , Daigle Isabelle , Braun Doris , Schranz Corinna , Weber Martina , Blaser Kurt , Zangemeister-Wittke Uwe , Akbar Arne N. , Simon Hans-Uwe
Published: October 2019

Eosinophils are potent inflammatory cells involved in allergic reactions. Inhibition of apoptosis of purified eosinophils by certain cytokines has been previously shown to be an important mechanism causing tissue eosinophilia. To elucidate the role of Bcl-2 family members in the inhibition of eosinophil apoptosis, we examined the expression of the known anti-apoptotic genes Bcl-2, Bcl-xL, and A1, as well as Bax and Bcl-xS, which promote apoptosis in other systems. We show herein that freshly isolated human eosinophils express significant amounts of Bcl-xL and Bax, but only little or no Bcl-2, Bcl-xS, or A1. As assessed by reverse transcription-polymerase chain reaction, immunoblotting, flow cytometry, and immunocytochemistry, we show that spontaneous eosinophil apoptosis is associated with a decrease in Bcl-xL mRNA and protein levels. In contrast, stimulation of the cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-5 (IL-5) results in maintenance or upregulation of Bcl-xL mRNA and protein levels. Moreover, Bcl-2 protein is not induced by GM-CSF or IL-5 in purified eosinophils. Bcl-2 protein is also not expressed in tissue eosinophils as assessed by immunohistochemistry using two different eosinophilic tissue models. Furthermore, Bcl-xL antisense but not scrambled phosphorothioate oligodeoxynucleotides can partially block the cytokine-mediated rescue of apoptotic death in these cells. These data suggest that Bcl-xL acts as an anti-apoptotic molecule in eosinophils.

© 1998 by The American Society of Hematology.
Intermittent, Repetitive Corticosteroid-Induced Upregulation of Platelet Levels After Adenovirus-Mediated Transfer to the Liver of a Chimeric Glucocorticoid-Responsive Promoter Controlling the Thrombopoietin cDNA
Authors: Narumi Ko , Suzuki Motoyoshi , Song Wenru , Moore Malcolm A.S. , Crystal Ronald G.
Published: October 2019

For many in vivo gene therapy clinical applications, it is desirable to control the expression of the transferred transgene using pharmacologic agents. To evaluate the feasibility of accomplishing this using corticosteroids, pharmacologic agents widely used in clinical medicine, we constructed replication deficient adenoviral (Ad) vectors containing an expression cassette with a chimeric promoter comprised of five glucocorticoid response elements (GRE) and the chloramphenicol acetyltransferase reporter gene (AdGRE.CAT) or the murine thrombopoietin cDNA (AdGRE.mTPO). In vitro studies showed the vectors functioned as expected, with marked glucocorticoid-induced upregulation of the CAT or mTPO transgenes. To evaluate the inducibility of the GRE promoter in vivo, the AdGRE.CAT vector was administered intravenously to C57B1/6 mice, and CAT activity was quantified in liver before and after intraperitoneal administration of dexamethasone. The GRE promoter activity was dependent on the dexamethasone dose, with a 100-fold increase in CAT expression with 50 μg dexamethasone, similar to the levels observed in vivo with the Rous sarcoma virus long terminal repeat constitutive promoter. After dexamethasone administration, maximum CAT activity was observed at day 2, with a slow decline to baseline levels by 2 weeks. Based on these observations, we hypothesized that a single administration of an Ad vector-mediated transfer of the chimeric GRE inducible promoter driving the mTPO cDNA would enable repetitive administration of corticosteroids to repetitively upregulate platelet levels for 1 to 2 weeks. The data show that this occurs, with dexamethasone administration every 3 weeks associated with 1-week elevations (at each 3-week interval) of serum mTPO levels, megakaryocyte numbers in bone marrow, and platelet levels fourfold to sixfold over baseline. Thus, with the appropriate promoter, it is possible to use a commonly used pharmacologic agent to upregulate the expression of a newly transferred gene on demand.

© 1998 by The American Society of Hematology.
Overexpression of HOX11 Leads to the Immortalization of Embryonic Precursors With Both Primitive and Definitive Hematopoietic Potential
Authors: Keller Gordon , Wall Charles , Fong Andrew Z.C. , Hawley Teresa S. , Hawley Robert G.
Published: October 2019

Primitive and definitive erythropoiesis represent distinct hematopoietic programs that differ with respect to stage of development, transcriptional control, and growth regulation. Although these differences have been recognized for some time, the relationship of the two erythroid lineages to each other is not well established. We have used a model system based on the hematopoietic development of embryonic stem (ES) cells in culture to investigate the origins of the earliest hematopoietic populations. Using ES cells transduced with a retrovirus that overexpresses the HOX11 gene, we have established factor-dependent hematopoietic cell lines that represent novel stages of embryonic hematopoiesis. Analysis of three of these cell lines indicates that they differ with respect to cytokine responsiveness, cell surface markers, and developmental potential. Two of the cell lines, EBHX1 and EBHX11, display the unique capacity to generate both primitive and definitive erythroid progeny as defined by morphology and expression of βH1 and βmajor globin. The third line, EBHX14, has definitive erythroid and myeloid potential, but is unable to generate cells of the primitive erythroid lineage. Analysis of the cytokine responsiveness of the two lines with primitive erythroid potential has indicated that exposure to leukemia inhibitory factor (LIF) results in the upregulation of βH1 and a change in cellular morphology to that of primitive erythrocytes. These findings are the first demonstration of a clonal cell line with primitive and definitive hematopoietic potential and support the interpretation that these lineages may arise from a common precursor in embryonic life. In addition, they suggest that LIF could play a role in the regulation of primitive erythropoiesis.

© 1998 by The American Society of Hematology.
Role of Adhesion Molecules in the Homing and Mobilization of Murine Hematopoietic Stem and Progenitor Cells
Authors: Vermeulen Monica , Le Pesteur Françoise , Gagnerault Marie-Claude , Mary Jean-Yves , Sainteny Françoise , Lepault Françoise
Published: October 2019

Bone marrow (BM) transplantation still must overcome multiple difficulties and should benefit from better understanding of stem-cell homing and mobilization. Here, we analyzed the involvement of several adhesion molecules in the two processes by treating mice with monoclonal antibodies against these molecules. Treatment of lethally irradiated mice grafted with isogeneic BM cells showed that at least two migration pathways are important for stem-cell homing to the BM, whereas only one of them is involved in lodging of colony-forming unit–spleen (CFU-S) in the spleen. We confirm that the VLA-4/VCAM-1 adhesion pathway is important for stem-cell homing to the BM only and show that CD44 is involved in CFU-S lodging in both BM and spleen. These results show that entry of CFU-S into the spleen is regulated. The observation that when one migration pathway is altered, CFU-S do not enter the BM via the other pathway may indicate that the two mechanisms involved in CFU-S homing into the BM are linked. The adhesion molecules VLA-4 and CD44 are also implied in the mobilization of stem cells into the blood stream of mice injected once with anti–VLA-4 or anti-CD44. Anti–VLA-4 administration led to a significant increase in circulating stem cells as early as 8 hours after treatment. Stem cells mobilized by anti–VLA-4 comprise cells with high self-renewal potential and thus may be used for long-term reconstitution of the hematopoietic tissue.

© 1998 by The American Society of Hematology.
Clonal Diversity of Ig and T-Cell–Receptor Gene Rearrangements Identifies a Subset of Childhood B-Precursor Acute Lymphoblastic Leukemia With Increased Risk of Relapse
Authors: Green Elaine , McConville Carmel M. , Powell Judith E. , Mann Jillian R. , Darbyshire Philip J. , Taylor A. Malcolm R. , Stankovic Tatjana
Published: October 2019

Current prognostic indicators such as age, sex, and white blood cell count (WBC) fail to identify all children with more aggressive forms of B-precursor acute lymphoblastic leukemia (ALL), and a proportion of patients without poor prognostic indicators still relapse. Results obtained from an analysis of 65 pediatic B-precursor ALL patients indicated that subclone formation leading to clonal diversity, as detected by Ig and T-cell receptor (TCR) gene rearrangements, may represent a very useful prognostic indicator, independent of age, sex, and WBC. Disease-free survival was significantly shorter in those patients showing clonal diversity at presentation. Furthermore, clonal diversity was detected not only in the majority of high-risk patients who relapsed but was also associated with a high probability of relapse in standard-risk patients. Sixty-five percent (13/20) of standard-risk patients who also showed clonal diversity subsequently relapsed, whereas the percentage of relapses among standard-risk patients without clonal diversity was much lower at 19% (7/36). Continued clonal evolution during disease progression is an important feature of aggressive B-precursor ALL. All 5 patients with clonal diversity who were followed up in our study showed a change in the pattern of clonality between presentation and relapse. This implies an important role for clonal diversity as a mechanism of disease progression through the process of clonal variation and clonal selection.

© 1998 by The American Society of Hematology.
Triggering of CD40 Antigen Inhibits Fludarabine-Induced Apoptosis in B Chronic Lymphocytic Leukemia Cells
Authors: Romano Maria Fiammetta , Lamberti Annalisa , Tassone Pierfrancesco , Alfinito Fiorella , Costantini Silvia , Chiurazzi Federico , Defrance Thierry , Bonelli Patrizio , Tuccillo Franca , Turco Maria Caterina , Venuta Salvatore
Published: October 2019

We analyzed the effect of CD40 triggering on the fludarabine-induced apoptosis of B chronic lymphocytic leukemia (B-CLL) cells. Peripheral blood samples obtained from 15 patients were incubated with fludarabine in the absence or the presence of the anti-CD40 monoclonal antibody (MoAb) G28-5. In 12 patients a significant proportion of apoptotic cells, ranging from 22% to 38% (mean ± SE: 28.5 ± 1.6), were detected after 3 days of culture. In 9 of these samples, the addition of G28-5 reduced apoptosis by at least 30.1% and by 57.1% ± 7.8% on average (P = .0077). Because the CD40 antigen activates NF-κB/Rel transcription factors in B cells, and NF-κB/Rel complexes can inhibit cell apoptosis, we investigated whether the antiapoptotic effect of G28-5, in our system, could be related to modulation of NF-κB/Rel activity. As expected, B-CLL cells displayed significant levels of nuclear NF-κB/Rel activity; p50, RelA, and c-Rel components of the NF-κB/Rel protein family were identified in these complexes. After exposure to fludarabine, NF-κB/Rel complexes were decreased in the nuclei. The addition of G28-5 upregulated the NF-κB/Rel levels. To determine the involvement of NF-κB/Rel activity in the G28-5–mediated inhibition of apoptosis, we blocked the transcription factor with a decoy oligonucleotide, corresponding to the NF-κB/Rel consensus sequence. Cells incubated with the anti-CD40 MoAb in the presence of the decoy oligonucleotide but not a control oligonucleotide displayed a complete impairment of the G28-5 antiapoptotic effect, indicating that NF-κB/Rel activity was required for the inhibition of apoptosis. These results suggest that CD40 triggering in vivo could counteract the apoptotic effect of fludarabine on B-CLL cells and that its neutralization, or the use of NF-κB/Rel inhibitors, could improve the therapeutic effect of fludarabine.

© 1998 by The American Society of Hematology.
Synergistic Activation of MAP Kinase (ERK1/2) by Erythropoietin and Stem Cell Factor Is Essential for Expanded Erythropoiesis
Authors: Sui Xingwei , Krantz Sanford B. , You Min , Zhao Zhizhuang
Published: October 2019

Stem cell factor (SCF) and erythropoietin (EPO) work synergistically to support erythropoiesis, but the mechanism for this synergism is unknown. By using purified human erythroid colony-forming cells (ECFC), we have found that SCF and EPO synergistically activate MAP kinase (MAPK, ERK1/2), which correlates with the cell growth and thus may be responsible for the synergistic effects. Treatment of the cells with PD98059 and wortmannin, inhibitors of MEK and PI-3 kinase, respectively, inhibited the synergistic activation of MAPK and also the cell growth, further supporting this conclusion. Wortmannin only inhibits MAPK activation induced by EPO but not that by SCF, suggesting that SCF and EPO may activate MAPK through different pathways, which would facilitate synergy. Furthermore, EPO, but not SCF, led to activation of STAT5, whereas SCF and wortmannin had no effect on the EPO-induced STAT5 activation, suggesting that STAT5 is not involved in the synergistic action of SCF and EPO. Together, the data suggest that synergistic activation of MAPK by SCF and EPO is essential for expanded erythropoiesis.

© 1998 by The American Society of Hematology.