bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression pattern of T-cell–associated chemokine receptors and their chemokines correlates with specific subtypes of T-cell non-Hodgkin lymphoma
Authors: Jones Dan , O'Hara Carl , Kraus Madeleine D. , Perez–Atayde Antonio R. , Shahsafaei Aliakbar , Wu Lijun , Dorfman David M.
Published: October 2019

Chemokine receptors mediate the migration of lymphocytes through the binding of soluble ligands, and their expression is differentially regulated in lymphocyte subsets. The pattern of chemokine receptor expression in T-cell non-Hodgkin lymphoma has not been previously studied. Using a panel of mouse monoclonal antibodies, we studied the immunohistochemical expression of the Th1-associated chemokine receptor CXCR3 in 141 patients with T-cell lymphoma, and we studied the receptors CCR4 and CCR5 and some of their ligands in a subset of these tumors. Expression of CXCR3 was typical of the smaller T cells in angioimmunoblastic lymphoma (15 of 18 patients), angiocentric lymphoma (3 of 3 patients), histiocyte-rich tumors (4 of 5 patients), and unspecified T-cell lymphomas (17 of 39 patients). CXCR3 expression was seen in only 1 of 15 patients with anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma. In contrast, all ALK-positive tumors showed diffuse reactivity for the Th2-associated receptor CCR4 (5 of 5 patients). CCR4 expression was also a consistent feature of the large-cell transformation of mycosis fungoides. CCR5 expression showed no consistent association with any T-cell tumor type. The chemokines Mig (CXCR3 ligand), TARC (CCR4 ligand), and MCP-2 (CCR5 ligand) were detected in intratumoral blood vessels and histiocytes. Mig was also coexpressed by a subset of CXCR3-positive tumor cells in 6 of 20 lymphomas. MCP-2 was highly expressed in stromal cells in 3 patients with nodal involvement by cutaneous T-cell lymphoma. As with normal T-cell subsets, we demonstrated that there is frequent differential expression of chemokine receptors in T-cell tumors, which may explain, in part, the distinctive patterns of spread in different tumor subtypes.
ZNF198–FGFR1 transforming activity depends on a novel proline-rich ZNF198 oligomerization domain
Authors: Xiao Sheng , McCarthy Jennifer G. , Aster Jon C. , Fletcher Jonathan A.
Published: October 2019

An acquired chromosomal translocation, t(8;13)(p11;q11-12), observed in a distinctive type of stem cell leukemia/lymphoma syndrome, leads to the fusion of the 5′ portion of ZNF198 and the 3′ portion of FGFR1. ZNF198–FGFR1 fusion transcripts encode 4 to 10 zinc fingers, a proline-rich region, and the intracellular portion of the FGFR1 (fibroblast growth factor receptor 1) receptor tyrosine kinase. We demonstrate that the ZNF198 proline-rich region constitutes a novel self-association domain. When fused to the intracellular domain of FGFR1, the ZNF198 proline-rich region is sufficient to cause oligomerization, FGFR1 tyrosine kinase activation, and transformation of Ba/F3 cells to IL-3 independent growth.
Antileukemic efficacy of 2′-deoxycoformycin in monocytic leukemia cells
Authors: Niitsu Nozomi , Yamamoto-Yamaguchi Yuri , Kasukabe Takashi , Okabe-Kado Junko , Umeda Masanori , Honma Yoshio
Published: October 2019

2′-Deoxycoformycin (dCF) as a single agent has been reported to be less effective against myeloid than against lymphoid malignancies in clinical trials. However, previous studies have shown that in the presence of 2′-deoxyadenosine (dAd), human monocytoid leukemia cell lines are much more sensitive to dCF with regard to the inhibition of cell proliferation. Thus, dCF might be useful for treating monocytoid leukemia with the aid of dAd analogs. The antiproliferative effects of dCF in combination with dAd or its derivatives were examined on normal and malignant blood and bone marrow cells. In the presence of 10 μmol/L dAd, the concentration of dCF required to inhibit the viability of primary monocytoid leukemia cells was much lower than that required to inhibit normal or non-monocytoid leukemic cells. Among the dAd analogs, 9-β-d-arabinofuranosyladenine (AraA) was also effective in combination with dCF. Athymic nude mice were inoculated with human monocytoid leukemia U937 cells and treated with dCF or a dAd analog or both. Although dCF alone slightly but significantly prolonged the survival of mice inoculated with U937 cells, combined treatment with dCF and AraA markedly prolonged their survival. These data suggest that the combination of dCF and AraA may be useful for the clinical treatment of acute monocytic leukemia.
High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia
Authors: Veuger Marjan J. T. , Honders M. Willy , Landegent Jim E. , Willemze Roel , Barge Renée M. Y.
Published: October 2019

Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML.
Reassessment of interactions between hematopoietic receptors using common beta-chain and interleukin-3–specific receptor beta-chain–null cells: no evidence of functional interactions with receptors for erythropoietin, granulocyte colony-stimulating factor, or stem cell factor
Authors: Scott Clare L. , Robb Lorraine , Papaevangeliou Bette , Mansfield Rachel , Nicola Nicos A. , Begley C. Glenn
Published: October 2019

Mice lacking both the gene encoding the shared receptor for granulocyte macrophage–colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 common β-chain (Bc) and the gene for the IL-3 specific receptor (BIL3) were generated. This was achieved by targeting the Bclocus in embryonic stem cells that were heterozygous for a null mutation of BIL3. Cells from mice generated with the doubly targeted embryonic stem cells were unresponsive to all 3 cytokines. Considerable previous data suggested a role for common beta-chain (βc) in modulating signaling of cytokines including erythropoietin (EPO), G-CSF, and stem cell factor (SCF). However, bone marrow cells from mice lacking βc and βIL3 showed normal responsiveness to these cytokines. Thus, there was no evidence for a biologically significant interaction between signaling via βc or βIL3 and signaling by EPO, G-CSF, or SCF. Previously documented biochemical phenomena, including receptor transmodulation, receptor transphosphorylation, and even direct physical interaction, involving the βc/βIL-3 receptor systems do not reflect genuine interactions of physiological significance in primary hematopoietic cells. This study provided results that challenge conclusions previously established using a variety of biochemical assays.
Polarized expression of bone morphogenetic protein-4 in the human aorta-gonad-mesonephros region
Authors: Marshall Caroline J. , Kinnon Christine , Thrasher Adrian J.
Published: October 2019

In the mammal, definitive hematopoietic stem cells (HSCs) are first derived from mesodermal cells within a region of the embryonic para-aortic splanchnopleura known as the aorta-gonad-mesonephros (AGM). Within this region, HSCs are thought to arise from hemangioblast precursors located in the ventral wall of the dorsal aorta. However, the factors that regulate HSC development in vivo are still largely unknown. Bone morphogenetic protein (BMP)-4, a member of the transforming growth factor beta (TGF-β) superfamily of growth factors, is a potent ventralizing factor and has been implicated in the commitment of embryonic mesodermal cells to a hematopoietic fate in a number of systems. In the human AGM, we find that BMP-4 is expressed at high levels, and with striking polarity, in a region of densely packed cells underlying intra-aortic hematopoietic clusters. In contrast, TGF-β1 is expressed predominantly by hematopoietic cells within the clusters. These findings implicate both BMP-4 and TGF-β1 in the initiation and regulation of hematopoiesis in the human AGM. Furthermore, the distribution of BMP-4 expression is highly suggestive of a direct role in the specification of human hematopoietic cells from embryonic mesoderm in vivo.
Carboxy terminal region of the Fanconi anemia protein, FANCG/XRCC9, is required for functional activity
Authors: Kuang Yanan , Garcia-Higuera Irene , Moran Anna , Mondoux Michelle , Digweed Martin , D'Andrea Alan D.
Published: October 2019

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with eight complementation groups. Four of the FA genes have been cloned, and at least three of the encoded proteins, FANCA, FANCC, and FANCG/XRCC9, interact in a nuclear complex, required for the maintenance of normal chromosome stability. In the current study, mutant forms of the FANCA and FANCG proteins have been generated and analyzed with respect to protein complex formation, nuclear translocation, and functional activity. The results demonstrate that the amino terminal two-thirds of FANCG (FANCG amino acids 1-428) binds to the amino terminal nuclear localization signal (NLS) of the FANCA protein. On the basis of 2-hybrid analysis, the FANCA/FANCG binding is a direct protein-protein interaction. Interestingly, a truncated mutant form of the FANCG protein, lacking the carboxy terminus, binds in a complex with FANCA and translocates to the nucleus; however, this mutant protein fails to bind to FANCC and fails to correct the mitomycin C sensitivity of an FA-G cell line. Taken together, these results demonstrate that binding of FANCG to the amino terminal FANCA NLS sequence is necessary but not sufficient for the functional activity of FANCG. Additional amino acid sequences at the carboxy terminus of FANCG are required for the binding of FANCC in the complex.
Langerhans cells develop from a lymphoid-committed precursor
Authors: Anjuère Fabienne , del Hoyo Gloria Martı́nez , Martı́n Pilar , Ardavı́n Carlos
Published: October 2019

Langerhans cells (LCs) are specialized dendritic cells (DCs) strategically located in stratified epithelia, such as those of the skin, oral cavity, pharynx, esophagus, upper airways, urethra, and female reproductive tract, which are exposed to a wide variety of microbial pathogens. LCs play an essential role in the induction of T-lymphocyte responses against viruses, bacteria, and parasites that gain access to those epithelial surfaces, due to their high antigen capture and processing potential and their capacity to present antigen peptides to T cells on migration to the lymph nodes.1Although LCs have been classically considered of myeloid origin, recent reports, which demonstrate the existence of lymphoid DCs derived from multipotent lymphoid precursors devoid of myeloid differentiation potential,2–5 raise the question of the lymphoid or myeloid origin of LCs. The present study shows that mouse lymphoid-committed CD4low precursors, with the capacity to generate T cells, B cells, CD8+ lymphoid DCs, and natural killer cells,26 also generate epidermal LCs on intravenous transfer, supporting the view that LCs belong to the lymphoid lineage.
Cleavage by CD26/dipeptidyl peptidase IV converts the chemokine LD78β into a most efficient monocyte attractant and CCR1 agonist
Authors: Proost Paul , Menten Patricia , Struyf Sofie , Schutyser Evemie , De Meester Ingrid , Van Damme Jo
Published: October 2019

Chemokines are proinflammatory cytokines that play a role in leukocyte migration and activation. Recent reports showed that RANTES (regulated on activation normal T-cell expressed and secreted chemokine), eotaxin, macrophage-derived chemokine (MDC), and stromal cell–derived factor-1 (SDF-1) are NH2-terminally truncated by the lymphocyte surface glycoprotein and protease CD26/dipeptidyl peptidase IV (CD26/DPP IV). Removal of the NH2-terminal dipeptide resulted in impaired inflammatory properties of RANTES, eotaxin, MDC, and SDF-1. The potential CD26/DPP IV substrate macrophage inflammatory protein–1β (MIP-1β) and the related chemokine, LD78α (ie, one of the MIP-1α isoforms), were not affected by this protease. However, CD26/DPP IV cleaved LD78β, a most potent CCR5 binding chemokine and inhibitor of macrophage tropic human immunodeficiency virus–1 (HIV-1) infection, into LD78β(3-70). Naturally truncated LD78β(3-70), but not truncated MIP-1β, was recovered as an abundant chemokine form from peripheral blood mononuclear cells. In contrast to all other chemokines processed by CD26/DPP IV, LD78β(3-70) had increased chemotactic activity in comparison to intact LD78β. With a minimal effective concentration of 30 pmol/L, LD78β(3-70) became the most efficient monocyte chemoattractant. LD78β(3-70) retained its high capacity to induce an intracellular calcium increase in CCR5-transfected cells. Moreover, on CCR1 transfectants, truncated LD78β(3-70) was 30-fold more potent than intact LD78β. Thus, CD26/DPP IV can exert not only a negative but also a positive feedback during inflammation by increasing the specific activity of LD78β. CD26/DPP IV–cleaved LD78β(3-70) is the most potent CCR1 and CCR5 agonist that retains strong anti–HIV-1 activity, indicating the importance of the chemokine-protease interaction in normal and pathologic conditions.
Pyk2 and Syk participate in functional activation of granulocytic HL-60 cells in a different manner
Authors: Miura Yasuo , Tohyama Yumi , Hishita Terutoshi , Lala Amitabha , De Nardin Ernesto , Yoshida Yataro , Yamamura Hirohei , Uchiyama Takashi , Tohyama Kaoru
Published: October 2019

The roles of the protein tyrosine kinases Pyk2 (also called RAFTK or CAK β) and Syk in the process of functional activation of human myeloid cells were examined. During granulocytic differentiation of HL-60 cells with dimethyl sulfoxide (DMSO), the amounts of Pyk2 and β2 integrin increased, whereas the amount of Syk was abundant before differentiation and did not change during differentiation. When the granulocytic cells were stimulated withN-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), tyrosine phosphorylation of Pyk2 occurred promptly and subsequent association of Pyk2 with β2 integrin was detected. In contrast, Syk was not tyrosine phosphorylated by fMLP stimulation but constitutively associated with β2 integrin. Stimulation with fMLP also caused the alteration of β2 integrin to an activated form, a finding that was confirmed by the observation of fMLP-induced cell attachment on fibrinogen-coated dishes and inhibition of this attachment by pretreatment with anti-β2 integrin antibody. Cell attachment to fibrinogen caused the enhanced tyrosine phosphorylation of Pyk2 and the initial tyrosine phosphorylation of Syk, which was also inhibited by pretreatment with anti-β2 integrin antibody. In vitro kinase assays revealed that Pyk2 and Syk represented kinase activities to induce tyrosine phosphorylation of several molecules in the anti-β2 integrin immunoprecipitates of the attached cells. These results showed that Pyk2 is involved in the functional activation of granulocytic cells in 2 signaling pathways: an fMLP receptor–mediated “inside-out” signaling pathway that might cause β2 integrin activation and a subsequent β2 integrin–mediated “outside-in” signaling pathway. Syk was activated in relation to cell attachment to fibrinogen as a result of “outside-in” signaling, although it was already associated with β2 integrin before fMLP stimulation.
Identification of novel circulating human embryonic blood stem cells
Authors: Gallacher Lisa , Murdoch Barbara , Wu Dongmei , Karanu Francis , Fellows Fraser , Bhatia Mickie
Published: October 2019

Using murine models, primitive hematopoietic cells capable of repopulation have been shown to reside in various anatomic locations, including the aortic gonad mesonephros, fetal liver, and bone marrow. These sites are thought to be seeded by stem cells migrating through fetal circulation and would serve as ideal targets for in utero cellular therapy. In humans, however, it is unknown whether similar stem cells exist. Here, we identify circulating hematopoeitic cells present during human in utero development that are capable of multilineage repopulation in immunodeficient NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice. Using limiting dilution analysis, the frequency of these fetal stem cells was found to be 1 in 3.2 × 105, illustrating a 3- and 22-fold enrichment compared with full-term human cord blood and circulating adult mobilized–peripheral blood, respectively. Comparison of in vivo differentiation and proliferative capacity demonstrated that circulating fetal stem cells are intrinsically distinct from hematopoietic stem cells found later in human development and those derived from the fetal liver or fetal bone marrow compartment at equivalent gestation. Taken together, these studies demonstrate the existence of unique circulating stem cells in early human embryonic development that provide a novel and previously unexplored source of pluripotent stem cell targets for cellular and gene-based fetal therapies.
Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells
Authors: Moulding Dale A. , Giles Richard V. , Spiller David G. , White Michael R. H. , Tidd David M. , Edwards Steven W.
Published: October 2019

Mcl-1 is a member of the Bcl-2 protein family, which has been shown to delay apoptosis in transfection and/or overexpression experiments. As yet no gene knockout mice have been engineered, and so there is little evidence to show that loss of Mcl-1 expression is sufficient to trigger apoptosis. U937 cells constitutively express the antiapoptotic protein Bcl-2; but during differentiation, in response to the phorbol ester PMA (phorbol 12 β-myristate 13 α-acetate), Mcl-1 is transiently induced. The purpose of this investigation was to determine the functional role played by Mcl-1 in this differentiation program. Mcl-1 expression was specifically disrupted by chimeric methylphosphonate/phosphodiester antisense oligodeoxynucleotides to just 5% of control levels. The depletion of Mcl-1 messenger RNA (mRNA) and protein was both rapid and specific, as indicated by the use of control oligodeoxynucleotides and analysis of the expression of otherBCL2 family members and PMA-induced tumor necrosis factor–α (TNF-α). Specific depletion of Mcl-1 mRNA and protein, in the absence of changes in cellular levels of Bcl-2, results in a rapid entry into apoptosis. Levels of the proapoptotic protein Bax remained unchanged during differentiation, while Bak expression doubled within 24 hours. Apoptosis was detected within 4 hours of Mcl-1 antisense treatment by a variety of parameters including a novel live cell imaging technique allowing correlation of antisense treatment and apoptosis in individual cells. The induction of Mcl-1 is required to prevent apoptosis during differentiation of U937 cells, and the constitutive expression of Bcl-2 is unable to compensate for the loss of Mcl-1.
Mcl-1 is a common target of stem cell factor and interleukin-5 for apoptosis prevention activity via MEK/MAPK and PI-3K/Akt pathways
Authors: Huang Huei-Mei , Huang Chang-Jen , Yen Jeffrey Jong-Young
Published: October 2019

Stem cell factor (SCF) has been suggested as essential for optimal production of various hematopoietic lineages mainly because of its apoptosis prevention function when it costimulates with other cytokines. However, the underlying mechanism of this synergism of apoptosis prevention is largely unknown. The present study examined the expression of some Bcl-2 family members, including Bcl-2, Bcl-XL, Mcl-1, and Bax, in response to cytokine stimulation in TF-1 and JYTF-1 cells in which SCF costimulation is differentially required for optimal proliferation. The results revealed that only the expression of Mcl-1 highly correlated with the antiapoptotic activity of interleukin-5 (IL-5) and the synergistic effect of SCF. In TF-1 cells, the defect of IL-5 in apoptosis suppression and Mcl-1 induction was associated with the incapability to highly phosphorylate Janus kinases (JAK1, JAK2), signal transducer and activator of transcription-5 (STAT5), mitogen-activated protein kinase (MAPK), and Akt/PKB, whereas SCF costimulation restored the potent phosphorylation of MAPK and Akt/PKB, but not STAT5. The importance of MAPK and Akt/PKB signaling pathways in regulating the expression of Mcl-1 and cell survival was further supported by the observation that inhibition of MEK by PD98059 or phosphatidylinositol-3 kinase (PI-3K) by LY294002 independently resulted in the reduction of Mcl-1 expression and loss of cell viability. Therefore, the data suggest that Mcl-1 is a common antiapoptotic target of both early-stage cytokine SCF and late-stage cytokine IL-5. Both MEK/MAPK and PI-3K/Akt signaling pathways are essential in the regulation of Mcl-1 expression and apoptosis prevention.
Egr-1 gene is induced by the systemic administration of the vascular endothelial growth factor and the epidermal growth factor
Authors: Liu Lixin , Tsai Jo C. , Aird William C.
Published: October 2019

Egr-1 is a transcription factor that couples short-term changes in the extracellular milieu to long-term changes in gene expression. In cultured endothelial cells, the Egr-1 gene has been shown to respond to a variety of extracellular signals. However, the physiological relevance of these findings remains unclear. To address this question, the growth factor-mediated response of the Egr-1 gene under in vivo conditions was analyzed. To that end, either vascular endothelial growth factor (VEGF) or epidermal growth factor (EGF) was injected into the intraperitoneal cavity of mice. Growth factors were delivered to all tissues examined, as evidenced by the widespread distribution of I125-labeled growth factors and the phosphorylation of their respective receptors. In Western blot analyses of whole-tissue extracts, Egr-1 protein levels were shown to be induced in the heart, brain, liver, and spleen of VEGF-treated mice, and in the heart, lung, brain, liver and skeletal muscle of EGF-treated animals. Changes in Egr-1 levels did not correlate with changes in receptor phosphorylation or ERK1/2 phosphorylation. In Northern blot analyses, VEGF induced Egr-1 mRNA levels in all tissues examined except lung and kidney, whereas EGF led to increased transcripts in all tissues except kidney. In immunofluorescence studies, VEGF induced Egr-1 in microvascular endothelial cells of the heart and liver, and EGF induced Egr-1 in the microvascular bed of skeletal muscle. Taken together, these results suggest that the Egr-1 gene is differentially regulated in response to systemically administered VEGF and EGF.
Influence of cytochrome P-450 CYP2C9 polymorphisms on warfarin sensitivity and risk of over-anticoagulation in patients on long-term treatment
Authors: Taube Janis , Halsall David , Baglin Trevor
Published: October 2019

Cytochrome P-450 2C9 is the principle enzyme that terminates the anticoagulant effect of warfarin. Genetic polymorphisms inCYP2C9 producing variants with altered catalytic properties have been identified. Patients (n = 561) with a target international normalized ratio (INR) of 2.5 who had been treated with warfarin for more than 2 months were anonymously genotyped for the wild-typeCYP2C9*1 allele and the 2C9*2 and2C9*3 variants. The mean maintenance dose of warfarin in patients who were wild-type for both alleles was 5.01 mg. The maintenance dose of warfarin was significantly related to genotype (Kruskall-Wallis, χ2 = 17.985, P = .001) with mean maintenance doses in patients with variant alleles between 61% and 86% of that in wild-type patients. The odds ratio for the2C9*2 allele in patients with a maintenance dose of 1.5 mg or less was 5.42 (95% CI 1.68-17.4). The odds ratio for one or more variant alleles in patients developing an INR of 8.0 or greater was 1.52 (95% CI 0.64-3.58). The SD of the mean INR, percentage of high INRs, and person-time spent in range were determined as parameters of stability. There was no difference between patients grouped according to genotype for any parameter of stability. This study confirmed an association between CYP2C9 genotype and warfarin sensitivity. However, the possession of a variant allele does not increase the likelihood of severe over-anticoagulation or stability of anticoagulation during long-term therapy.
Activation of macrophage cytostatic effector mechanisms during acute graft-versus-host disease: release of intracellular iron and nitric oxide–mediated cytostasis
Authors: Nestel Frederick P. , Greene Robert N. , Kichian Krikor , Ponka Premysl , Lapp Wayne S.
Published: October 2019

During acute graft-versus-host disease (GVHD) the activation of macrophages (Mφ) is mediated by 2 signals, interferon (IFN)-γ and bacteria-derived lipopolysaccharide (LPS). A cascade of inflammatory responses that includes the release of mediators of tissue injury follows Mφ activation. Among the tissues characteristically targeted during acute GVHD are epithelial tissues of the skin and gastrointestinal tract that normally undergo continuous proliferation and are therefore sensitive to cytostatic processes. We have investigated whether Mφ can mediate cytostatic mechanisms capable of interrupting cell proliferation during acute GVHD. GVHD was induced in nonirradiated C57BL/6XAF1 (B6AF1) mice by the injection of 60 × 106 (acute GVHD) or 30 × 106 (nonlethal GVHD) C57BL/6 (B6) lymphoid cells. Mφ from animals undergoing acute GVHD could be triggered by normally insignificant quantities of LPS to mediate a cytostatic effect on target cells, resulting in the complete shutdown of cellular proliferation. The same amounts of LPS had no effect on Mφ from normal or syngeneically transplanted animals. Mφ mediated the release of significant quantities of intracellular iron from target cells undergoing cytostasis. Reversal of cytostasis occurred following inhibition of nitric oxide (NO) production by NG-monomethyl-L-arginine (NMMA). Production of NO by LPS-triggered Mφ reflected the severity of GVHD. NO release increased significantly during acute GVHD but was only transiently increased during nonlethal GVHD. The results provide evidence that, as a result of activation during acute GVHD, Mφ produce NO and induce the release of iron from target cells, resulting in a potent cytostatic effect that inhibits cellular proliferation.
Acute myeloblastic leukemic cells acquire cellular cytotoxicity under genotoxic stress: implication of granzyme B and perforin
Authors: Bruno Alain Pierre , Lautier Dominique , d'Orgeix Aurélie de Thonel , Laurent Guy , Quillet-Mary Anne
Published: October 2019

Granzyme B (GrB) and perforin (PFN) are the major components of cytoplasmic granules contained in immune cellular effectors. The granule secretory pathway is one of the mechanisms by which these cells exert their cellular cytotoxicity. Recently, it has been reported that GrB and PFN are also present in circulating hemopoietic CD34+ progenitor cells mobilized by chemotherapy and granulocyte-colony stimulating factor, whereas these proteins are undetected in steady-state peripheral CD34+ cells. In this study, we hypothesized that anticancer agents may increase GrB and PFN expression in immature myeloid leukemic cells and that these treated leukemic cells become cellular effectors through a granule-dependent mechanism. Our results show that KG1a, HEL, and TF-1 CD34+acute myeloblastic leukemia cells expressed both GrB and PFN. Moreover, ionizing radiation, aracytine, and etoposide not only increase GrB and PFN expression but also conferred potent cellular cytotoxicity to these cells toward various cellular targets. Cellular cytotoxicity required cell-cell contact, was not influenced by anti-tumor necrosis factor α or anti-Fas blocking antibodies, and was abrogated by GrB inhibitors or antisense. These results suggest that, when exposed to genotoxic agents, immature leukemic cells acquire potent GrB- and PFN-dependent cellular cytotoxicity that can be potentially directed against normal residual myeloid progenitors or immune effectors.
Stromal cells regulate survival of B-lineage leukemic cells during chemotherapy
Authors: Mudry Ryan E. , Fortney James E. , York Teresa , Hall Brett M. , Gibson Laura F.
Published: October 2019

Approximately 20% of B-lineage acute lymphoblastic leukemias are not cured by traditional chemotherapy. The possibility was examined that residual leukemic cells that potentially contribute to relapse are harbored in association with fibroblastic stromal cells in the bone marrow. Modulation of cytarabine (Ara-C) and etoposide (VP-16) efficacy by bone marrow stromal cells in vitro was investigated. Stromal cell coculture was shown to sustain the proliferation of B-lineage leukemic cells and to reduce leukemic cell apoptosis when exposed to Ara-C or VP-16. Direct contact with stromal cells was essential for the protection of leukemic cells during chemotherapy, whereas soluble factors had negligible effect. Specifically, signaling mediated through interaction with the stromal cell adhesion molecule VCAM-1 was required to maintain the maximum viability of leukemic cells during Ara-C and VP-16 exposure. In contrast, the interaction of leukemic cells with fibronectin did not confer significant resistance to either chemotherapeutic agent. These observations suggest a role for the bone marrow microenvironment in modulating the response of B-lineage leukemic cells to Ara-C or VP-16, and they indicate specific molecular interactions that may be important in determining the sensitivity of leukemic cells to treatment.
Red cells from glutathione peroxidase-1–deficient mice have nearly normal defenses against exogenous peroxides
Authors: Johnson Robert M. , Goyette Gerard , Ravindranath Yaddanapudi , Ho Ye-Shih
Published: October 2019

The role of glutathione peroxidase in red cell anti-oxidant defense was examined using erythrocytes from mice with a genetically engineered disruption of the glutathione peroxidase-1 (GSHPx-1) gene. Because GSHPx-1 is the sole glutathione peroxidase in the erythrocyte, all red cell GSH peroxidase activity was eliminated. Oxidation of hemoglobin and membrane lipids, using the cis-parinaric acid assay, was determined during oxidant challenge from cumene hydroperoxide and H2O2. No difference was detected between wild-type red cells and GSHPx-1–deficient cells, even at high H2O2 exposures. Thus, GSHPx-1 appears to play little or no role in the defense of the erythrocyte against exposure to peroxide. Simultaneous exposure to an H2O2 flux and the catalase inhibitor 3-amino-1,2,4-triazole supported this conclusion. Hemoglobin oxidation occurred only when catalase was depleted. Circulating erythrocytes from the GSHPx-1–deficient mice exhibited a slight reduction in membrane thiols, indicating that high exposure to peroxides might occur naturally in the circulation.
Bone morphogenetic protein-2 induces apoptosis in human myeloma cells with modulation of STAT3
Authors: Kawamura Chiharu , Kizaki Masahiro , Yamato Kenji , Uchida Hideo , Fukuchi Yumi , Hattori Yutaka , Koseki Takeyoshi , Nishihara Tatsuji , Ikeda Yasuo
Published: October 2019

Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)–β superfamily, are a group of related proteins that are capable of inducing the formation of cartilage and bone but are now regarded as multifunctional cytokines. We show in this report a novel function of BMPs in hematopoietic cells: BMP-2 induces apoptosis not only in human myeloma cell lines (U266, RPMI 8226, HS-Sultan, IM-9, OPM-2, and KMS-12 cells), but also in primary samples from patients with multiple myeloma. The mechanism of BMP-2–induced apoptosis was investigated with the use of U266 cells, which are dependent on the interleukin-6 autocrine loop. We showed that BMP-2 caused cell-cycle arrest in the G1 phase and the subsequent apoptosis of myeloma cells. BMP-2 up-regulated the expression of cyclin-dependent kinase inhibitors (p21CIP1/WAF1 and p27KIP1) and caused hypophosphorylation of retinoblastoma (Rb) protein. In studies of apoptosis-associated proteins, BMP-2 was seen to down-regulate the expression of Bcl-xL; however, BMP-2 had no effects on the expression of Bcl-2, Bax, or Bad. Therefore, BMP-2 induces apoptosis in various human myeloma cells by means of the down-regulation of Bcl-xL and by cell-cycle arrest through the up-regulation of p21CIP1/WAF1 and p27KIP1 and by the hypophosphorylation of Rb. Further analysis showed that the signal transducer and activator of transcription 3 (STAT3) was inactivated immediately after BMP-2 treatment. We conclude that BMP-2 would be useful as a novel therapeutic agent in the treatment of multiple myeloma both by means of its antitumor effect of inducing apoptotis and through its original bone-inducing activity, because bone lesions are frequently seen in myeloma patients.
Interferon-α directly represses megakaryopoiesis by inhibiting thrombopoietin-induced signaling through induction of SOCS-1
Authors: Wang Qin , Miyakawa Yoshitaka , Fox Norma , Kaushansky Kenneth
Published: October 2019

Interferon (IFN)-α has proven useful for treating several clinical conditions, including chronic viral hepatitis and chronic myeloproliferative and lymphoproliferative disorders. In addition to its well-known antiviral effects, the cytokine exerts antiproliferative effects on many cell types, helping to explain its therapeutic usefulness in these latter conditions. However, this same property accounts for several undesirable effects, including thrombocytopenia, which can interfere with the successful clinical application of IFN-α. Unfortunately, the mechanisms responsible for the myelosuppressive effects of the cytokine are incompletely understood. The effects of IFN-α on megakaryocyte (MK) development were studied. Using several marrow cell purification techniques and quantitative culture methods, it was found that IFN-α directly inhibits thrombopoietin (TPO)-induced MK growth. Previous studies indicated that Janus kinase (JAK) and its substrates mediate the effects of TPO on cellular proliferation and survival. It was found that IFN-α directly suppresses TPO-induced phosphorylation of the JAK2 substrates c-Mpl and STAT 5 in a TPO-dependent hematopoietic cell line and of Mpl and STAT3 in primary murine MK. Moreover, IFN-α induces SOCS-1 production in these cells, which has been shown to inhibit TPO-induced cell growth. Because SOCS protein expression is induced by many cytokines and has been reported to extinguish signaling from several hematopoietic cytokine receptors, these results identify a molecular mechanism responsible for cytokine receptor cross-talk.
Soluble CD40 ligand induces selective proliferation of lymphoma cells in primary mantle cell lymphoma cell cultures
Authors: Andersen Niels S. , Larsen Jørgen K. , Christiansen Jette , Pedersen Lone B. , Christophersen Nicolaj S. , Geisler Christian H. , Jurlander Jesper
Published: October 2019

Interaction between CD40 and the CD40 ligand (CD40L) is critical for the survival and proliferation of B cells during immunopoiesis. However, the role of CD40L in the pathogenesis of malignant lymphomas is ambiguous. Primary mantle cell lymphoma (MCL) cells were cultured in the presence of recombinant human CD40L trimer (huCD40LT), and a significant time- and dose-dependent induction of DNA synthesis was observed in thymidine incorporation assays (n = 7,P < .04). The maximal rate of DNA synthesis was reached at huCD40LT doses of 100 ng/mL and above after 4 days of culture, but a significant increase of DNA synthesis was detected already at doses of 1 ng/mL (P = .03). HuCD40LT never inhibited the basal level of DNA synthesis. These findings established 400 ng/mL of huCD40LT for 4 days as standard conditions in the system. Under these conditions, huCD40LT significantly increased the proportion of cells in the S/G2/M phases of the cell cycle in 4 of 7 studied cases, while the fraction of apoptotic cells remained unchanged (n = 7). HuCD40LT also induced expression of CD80/B7-1, CD86/B7-2, and CD95/Fas and up-regulated the expression of HLA-DR (n = 6). With the use of bromodeoxyuridine incorporation in triple-color flow cytometric analysis, it was found that huCD40LT induced cell-cycle progression in light chain–restricted cells only, of which a median of 14% (range, 0.5% to 29%; n = 4) returned to G0/1 phase DNA content after bromodeoxyuridine incorporation, demonstrating completion of at least one cell cycle in the presence of huCD40LT. Thus, primary clonal MCL cells are activated and can proliferate in the presence of huCD40LT as a single agent.
Implantation of bone marrow beneath the kidney capsule results in transfer not only of functional stroma but also of hematopoietic repopulating cells
Authors: Varas Florencio , Grande Teresa , Ramı́rez Angel , Bueren Juan A.
Published: October 2019

Renal ossicles are ossified structures developed after the implantation of a bone marrow (BM) plug beneath the kidney capsule. The authors have investigated the origin of the hematopoietic cells in murine renal ossicles by conducting sex-mismatched implants into Ly-5 congenic mice. BM plugs from transgenic mice provided additional genotypic tracers. Flow cytometry analyses on nonadherent cells from long-term cultures established with ossicles excised at 17 to 40 weeks postimplantation evidenced the presence of 5% to 70% of donor-derived myeloid cells. The genetic analysis of the day 12 colony-forming unit (CFU-S12) population in ossicles excised at 10 to 40 weeks postimplantation revealed that 16% to 93% of the colonies were of donor origin. Moreover, we describe for the first time the presence of long-term repopulating cells of donor origin in ossicles excised at 10 to 19 weeks postimplantation.
Evidence that juvenile myelomonocytic leukemia can arise from a pluripotential stem cell
Authors: Cooper Laurence J. N. , Shannon Kevin M. , Loken Michael R. , Weaver Molly , Stephens Karen , Sievers Eric L.
Published: October 2019

Children with neurofibromatosis type 1 (NF1) carry germline mutations in one allele of the NF1 gene and are predisposed to myeloid malignancies, particularly juvenile myelomonocytic leukemia (JMML). Disruption of the remaining NF1 allele can be found in malignant cells. Flow cytometric cell sorting techniques to isolate the malignant cell populations and molecular genetic methods to assay for somatic loss of the normal NF1 allele were used to study an unusual child with NF1 and JMML who subsequently had T-cell lymphoma. The data show that malignant JMML and lymphoma cells share a common loss of genetic material involving the normal NF1gene and approximately 50 Mb of flanking sequence, suggesting that the abnormal T-lymphoid and myeloid populations were derived from a common precursor cell. These data support the hypothesis that JMML can arise in a pluripotent hematopoietic cell.
Probability in transcriptional regulation and its implications for leukocyte differentiation and inducible gene expression
Authors: Hume David A.
Published: October 2019

The phenotype of individual hematopoietic cells, like all other differentiated mammalian cells, is determined by selective transcription of a subset of the genes encoded within the genome. This overview summarizes the recent evidence that transcriptional regulation at the level of individual cells is best described in terms of the regulation of the probability of transcription rather than the rate. In this model, heterogeneous gene expression among populations of cells arises by chance, and the degree of heterogeneity is a function of the stability of the mRNA and protein products of individual genes. The probabilistic nature of transcriptional regulation provides one explanation for stochastic phenomena, such as stem cell lineage commitment, and monoallelic expression of inducible genes, such as lymphokines and cytokines.
Analysis of the CC chemokine receptor 3 gene reveals a complex 5′ exon organization, a functional role for untranslated exon 1, and a broadly active promoter with eosinophil-selective elements
Authors: Zimmermann Nives , Daugherty Bruce L. , Kavanaugh Jessica L. , El-Awar Faisal Y. , Moulton Elizabeth A. , Rothenberg Marc E.
Published: October 2019

To understand the regulation of CC chemokine receptor 3 (CCR3) expression, its gene structure and promoter have been characterized. The CCR3 gene contains 4 exons that give rise to multiple messenger RNA (mRNA) species by alternative splicing. Exon 1 is present in all transcripts, whereas exon 2 or 3 is present at low frequency (< 10%). Exon 4 contains the open reading frame and 11 bp of the 5′ untranslated region. Northern analysis revealed 4 species of CCR3 mRNA. Direct sequencing revealed that the first 1 kb of the promoter and exon 1 contained only one mutation in 19 individuals, indicating that the CCR3 promoter and exon 1 are conserved between individuals. The first 1.6 kb of the 5′ flanking region of exon 1 contained promoter elements including a TATA box and motifs for myeloid transcription factors and had strong promoter activity in eosinophilic, lymphoid, myeloid, and respiratory epithelial cell lines. Deletion analysis revealed differential regulation of the CCR3 promoter in eosinophilic and epithelial cells suggesting the presence of lineage-specific elements. Interestingly, exon 1 enhanced the activity of the promoter and this effect was especially prominent in eosinophilic cells. Thus, the humanCCR3 gene has a complex 5′ exon structure, a conserved promoter with strong activity in multiple cell types, and a functional 5′ untranslated exon.
Sulfated glycans induce rapid hematopoietic progenitor cell mobilization: evidence for selectin-dependent and independent mechanisms
Authors: Frenette Paul S. , Weiss Linnea
Published: October 2019

The adhesive mechanisms leading to the mobilization of hematopoietic progenitor cells (HPCs) from the bone marrow into the blood are poorly understood. We report on a role for selectins and fucoidan in progenitor mobilization. Baseline levels of circulating HPCs are increased in endothelial selectin-deficient (P/E−/−) mice. Similar levels are observed when E-selectin null (E−/−) mice are treated with anti-P-selectin antibody or with fucoidan (which inhibits P- and L-selectin function). In particular, administration of 2 doses of fucoidan (25 mg/kg) over 6 hours produces profound mobilization of progenitors in wild-type mice and the response is greatly enhanced in E−/− and P/E−/− mice. Competitive reconstitution experiments reveal that fucoidan also elicits long-term (more than 6 months) repopulating stem cells. Mobilization assays using chimeric mice harboring L-selectin–deficient progenitors and wild-type progenitors expressing the green fluorescence protein suggest that L-selectin expression is not required but confers an advantage for fucoidan-induced mobilization. Sulfation is critical as desulfated fucoidan is ineffective. In addition, sulphogalactosylceramide (sulfatide) but not heparin can induce HPC mobilization. Our results indicate that administration of sulfated glycans, especially with concurrent inhibition of E-selectin function, represents a powerful novel method for rapid mobilization of long-term–repopulating stem cells. These findings may help elucidate the mechanisms of HPC trafficking during development and adult life.
A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing
Authors: Margaglione Maurizio , Santacroce Rosa , Colaizzo Donatella , Seripa Davide , Vecchione Gennaro , Lupone Maria Rosaria , De Lucia Domenico , Fortina Paolo , Grandone Elvira , Perricone Corrado , Di Minno Giovanni
Published: October 2019

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.
Identification of critical antigen-specific mechanisms in the development of immune thrombocytopenic purpura in mice
Authors: Nieswandt Bernhard , Bergmeier Wolfgang , Rackebrandt Kirsten , Gessner J. Engelbert , Zirngibl Hubert
Published: October 2019

The pathogenic effects of antiplatelet antibodies were investigated in mice. Monoclonal antibodies (mAbs) of different immunoglobulin G subclass directed against mouse GPIIbIIIa, GPIIIa, GPIbα, GPIb-IX, GPV, and CD31 were generated and characterized biochemically. MAbs against GPIb-IX, GPV, CD31, and linear epitopes on GPIIIa had mild and transient effects on platelet counts and induced no spontaneous bleeding. Anti-GPIbα mAbs induced profound irreversible thrombocytopenia (< 3% of normal) by Fc-independent mechanisms but only had minor effects on hematocrits. In contrast, injection of intact mAbs, but not F(ab)2 fragments, against conformational epitopes on GPIIbIIIa, induced irreversible thrombocytopenia, acute systemic reactions, hypothermia, decreased hematocrits, and a paradoxical loss of surface GPIIbIIIa on platelets in vivo, the latter suggesting the formation of platelet-derived microparticles. Blockage of platelet-activating factor receptors inhibited the acute reactions, but not thrombocytopenia, loss of GPIIbIIIa, and decreases in hematocrits. Repeated injections of low doses of anti-GPIIbIIIa antibodies resulted in profound thrombocytopenia and bleeding, whereas no acute systemic reactions were observed. These data strongly suggest that the identity of the target antigen recognized by antiplatelet antibodies determines the mechanisms of platelet destruction and the severity of bleeding in mice, the latter depending on previously unrecognized anti-GPIIbIIIa-specific inflammatory mechanisms.