bluuebunny/crossref_metadata_2025 huggingface dataset:
Microchimerism in bone marrow–derived CD34+ cells of patients after liver transplantation
Authors: Nierhoff Dirk , Horvath Henrik Csaba , Mytilineos Joannis , Golling Markus , Bud Octavian , Klar Ernst , Opelz Gerhard , Voso Maria Teresa , Ho Anthony D. , Haas Rainer , Hohaus Stefan
Published: October 2019

Lymphoid and dendritic cells of donor origin can be detected in the recipient several years after a solid organ transplantation. This phenomenon is termed microchimerism and could play a role in the induction of tolerance. The fate of other hematopoietic cells transferred by liver transplantation, in particular of stem and progenitor cells, is unknown. For this reason, we studied peripheral blood and bone marrow samples of 12 patients who had received a liver transplant from an HLA-DR mismatched donor. Eight patients were long-term survivors between 2.8 and 10.1 years after allografting. CD34+ cells from bone marrow were highly enriched with the use of a 2-step method, and a nested polymerase chain reaction was applied to detect donor cells on the basis of allelic differences of the HLA-DRB1 gene. Rigorous controls with DRB1 specificities equal to the donor and host were included. In 5 of 8 long-term liver recipients, donor-specific CD34+ cells could be detected in bone marrow. Microchimerism in the CD34+ cell fraction did not correlate to the chimeric status in peripheral blood. In conclusion, our results demonstrate a frequent microchimerism among bone marrow–derived CD34+ cells after liver transplantation. The functional role of this phenomenon still needs to be defined.
Clinical relevance of intracellular vascular endothelial growth factor levels in B-cell chronic lymphocytic leukemia
Authors: Aguayo Alvaro , O'Brien Susan , Keating Michael , Manshouri Taghi , Gidel Cristi , Barlogie Bart , Beran Miloslav , Koller Charles , Kantarjian Hagop , Albitar Maher
Published: October 2019

Strong evidence exists for an association between high vascular endothelial growth factor (VEGF) levels and poor prognoses in patients with solid tumors and acute leukemia. Using Western blot analysis and solid-phase radioimmunoassay, we measured cellular VEGF levels in B-cell chronic lymphocytic leukemia (CLL) samples from 225 patients and correlated these levels with disease characteristics and prognoses. The median VEGF level in CLL samples was 7.26 times the median level detected in normal peripheral blood mononuclear cells. Patients with lower levels of VEGF protein showed a trend toward shorter survival (P = .07). However, in a subgroup of CLL patients with good prognoses or early-stage disease (Rai stages 0-II, Binet stages A,B; β2-M ≤ 2.8 mg/dL), lower levels of VEGF were associated with shorter survival times. For the entire group of patients, no correlation was found between VEGF levels and β2-M levels or Rai and Binet stage. Most samples from patients with CLL expressed the 43-kd VEGF isoform in addition to the commonly expressed 45-kd isoform. It remains to be seen whether the expression of the 43-kd isoform is responsible for this reversed correlation with outcome.
Lentiviral-mediated gene transfer into human lymphocytes: role of HIV-1 accessory proteins
Authors: Chinnasamy Dhanalakshmi , Chinnasamy Nachimuthu , Enriquez Melissa J. , Otsu Makoto , Morgan Richard A. , Candotti Fabio
Published: October 2019

Resting lymphocytes are refractory to gene transfer using Moloney murine leukemia virus (MMLV)-based retroviral vectors because of their quiescent status. Recently, it has been shown that lentiviral vectors are capable of transferring genes into nondividing and terminally differentiated cells. We used human immunodeficiency virus type-1 (HIV-1)–based vectors expressing enhanced green fluorescent protein (EGFP) driven by different promoters (CMV, MPSV, or PGK) and investigated their ability to transduce human T- and B-cell lines, as well as resting or activated primary peripheral and umbilical cord blood lymphocytes. The effects of the presence or the absence of HIV-1 accessory proteins (Vif, Vpr, Vpu, and Nef) in the vector system were also assessed. Flow cytometry analysis showed no differences in the ability of these vectors of transferring the reporter gene into lymphocytic lines and mitogen-stimulated primary lymphocytes in the presence or the absence of HIV-1 accessory proteins (APs). Similarly, viral supernatants generated in the presence of accessory genes could efficiently transduce various subsets of resting lymphocytes and provide long-term expression of the transgene. No significant transduction-induced changes in cell activation or cycling status were observed and Alu-HIV-1 long terminal repeat polymerase chain reaction (LTR PCR) analysis demonstrated integration of the vector sequences at the molecular level. In contrast, in the absence of HIV-1 APs, lentiviral vectors failed to integrate and express the transgene in resting lymphocytes. These results show that transduction of primary resting lymphocytes with HIV-1–based vectors requires the presence of viral accessory proteins.
Mild preconditioning and low-level engraftment confer methotrexate resistance in mice transplanted with marrow expressing drug-resistant dihydrofolate reductase activity
Authors: James Rohaizah I. , Warlick Christopher A. , Diers Miechaleen D. , Gunther Roland , McIvor R. Scott
Published: October 2019

Effective engraftment of hematopoietic cells targeted for gene transfer is facilitated by cytoreductive preconditioning such as high-dose total body irradiation (TBI). To minimize the adverse side effects associated with TBI, experiments were conducted to determine whether sublethal doses of TBI would allow sufficient engraftment of MTX-resistant hematopoietic cells to confer survival on recipient mice administered MTX. FVB/N animals were administered 1, 2, or 4 Gy TBI (lethal dose, 8.5 Gy), transplanted with 107 FVB/N transgenic marrow cells expressing an MTX-resistant dihydrofolate reductase (DHFR) transgene, and then administered MTX daily for 60 days. Control mice administered 1 Gy with or without subsequent transplantation of normal marrow cells succumbed to MTX toxicity by day 45. In contrast, nearly all animals transplanted with transgenic marrow survived MTX administration, regardless of the TBI dose used for preconditioning. The donor DHFR transgenic marrow engraftment level was proportional to the preconditioning dose of TBI but was surprisingly reduced in animals given 2 or 4 Gy TBI and subsequently administered MTX when compared with control animals administered phosphate-buffered saline. Animals preconditioned with 1 Gy were also protected from MTX toxicity when transplanted with reduced amounts (5 × 106 and 1 × 106 cells) of DHFR transgenic donor marrow, resulting in low-level (approximately 1%) engraftment. In conclusion, very mild preconditioning allows sufficient low-level engraftment of genetically modified stem cells for in vivo manifestation of the modified phenotype, suggesting the usefulness of mild preconditioning regimens in human gene therapy trials targeting hematopoietic stem cells.
Identification of a GATA-overlapping sequence within the enhancer of the murine GPIIb promoter that induces transcriptional deregulation in human K562 cells
Authors: Albanese Patricia , Leboeuf Marylène , Rosa Jean-Philippe , Uzan Georges
Published: October 2019

The human and the murine glycoprotein platelet IIb (GPIIb) promoters are megakaryocyte specific in human and murine cell systems, respectively. Here we show that the murine promoter is, however, highly active when transfected in K562 human cells in which the human promoter is almost inactive. A murine promoter, in which the enhancer element was replaced by the human, retrieves its megakaryocytic specificity in human cell lines. The human and murine GATA-binding sites located in the enhancer region display slight sequence divergence next to the consensus GATA core sequence. Gel shift experiments show that, although the murine and the human GATA sequences both bind GATA-1, the murine sequence alone forms an additional complex (B) not detected with the human sequence. When the murine GATA-containing region is replaced by the human in the context of the murine GPIIb promoter, megakaryocyte specificity is restored in the human cell lines. A G nucleotide 3′ to GATA appears crucial because its substitution abrogates B but not GATA-1 binding and restores megakaryocyte specificity to the murine promoter. Conversely, substitution of the human GATA-1 binding sequence by its murine homologue that binds both GATA-1 and complex B induces an abnormal activity for the human promoter in K562 cells. Altogether, our data suggest that limited changes in the GATA-containing enhancer of the GPIIb promoter can induce the recruitment of accessory proteins that could be involved in alteration of a megakaryocyte-restricted gene activation program.
Distinct functional properties of highly purified hematopoietic stem cells from mouse strains differing in stem cell numbers
Authors: de Haan Gerald , Szilvassy Stephen J. , Meyerrose Todd E. , Dontje Bert , Grimes Barry , Van Zant Gary
Published: October 2019

We have previously demonstrated that young adult DBA/2 (DBA) mice have more stem cells than C57BL/6 (B6) mice, as measured in a cobblestone area-forming cell (CAFC) assay using unfractionated marrow. To study the nature of this difference, we have now compared the proliferative fate of single, highly enriched Sca-1+c-kit+Lin−stem cells from these strains. Although equal in frequency, functional comparison revealed that Sca-1+c-kit+Lin−cells from DBA mice contained twice as many cells with CAFC activity. DBA clones persisted much longer in vitro, and developed later in time. To assess whether these differences were of any functional relevance in vivo, we compared engraftment of lethally irradiated mice transplanted with 1000 B6 or DBA Sca-1+c-kit+Lin−cells. Recipients of enriched DBA cells recovered much faster than animals transplanted with B6 cells. We also studied endogenous hematopoietic recovery after 5-fluorouracil (5-FU) treatment in vivo. Progenitors and peripheral blood cells recovered twice as fast in DBA mice. Thus, DBA stem cells have superior proliferative potential compared with phenotypically identical stem cells obtained from B6 mice. Such genetically determined quantitative and qualitative differences in stem cell behavior likely contribute to the dramatically different hematopoietic recovery rates observed in human transplant patients.
SHIP inhibits Akt activation in B cells through regulation of Akt membrane localization
Authors: Carver Deborah Jeannean , Aman Mohammad Javad , Ravichandran Kodimangalam S.
Published: October 2019

Activation of the serine/threonine kinase Akt and the regulation of its activation are recognized as critical in controlling proliferative/survival signals via many hematopoietic receptors. In B lymphocytes, the B-cell receptor (BCR)-mediated activation of Akt is attenuated by co–cross-linking of BCR with the inhibitory receptor FcγRIIB1, and the binding of the SH2 domain-containing inositol phosphatase, SHIP, to FcγRIIB1. Because SHIP dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PIP3) and activation of Akt requires PIP3, the destruction of this phospholipid has been proposed as the mechanism for Akt inhibition. However, upstream kinases that activate Akt, such as PDK1, also require PIP3 for activation. In this report, we addressed whether SHIP inhibits Akt directly at the level of Akt recruitment to the membrane, indirectly through PDK recruitment/phosphorylation of Akt, or both. We generated stable B-cell lines expressing a regulatable, but constitutively membrane-bound Akt that still required PDK-dependent phosphorylation for activation. Several lines of evidence suggested that activation of this membrane-targeted Akt is not inhibited by FcγRIIB1/SHIP and that PDK is not a target for SHIP-mediated inhibition. These data demonstrate that SHIP inhibits Akt primarily through regulation of Akt membrane localization. We also observed during these studies that FcγRIIB1/SHIP does not inhibit p70S6k activation, even though several other PIP3-dependent events were down-regulated. Because the enhanced activation of Akt in the absence of SHIP correlates with hyperproliferation in the myeloid lineage, our data have implications for SHIP and Akt-dependent regulation of proliferation in the hematopoietic lineage.
Gene expression networks underlying retinoic acid–induced differentiation of acute promyelocytic leukemia cells
Authors: Liu Ting-Xi , Zhang Ji-Wang , Tao Jiong , Zhang Ruo-Bo , Zhang Qing-Hua , Zhao Chun-Jun , Tong Jian-Hua , Lanotte Michel , Waxman Samuel , Chen Sai-Juan , Mao Mao , Hu Geng-Xi , Zhu Li , Chen Zhu
Published: October 2019

To elucidate the molecular mechanism of all-trans-retinoic acid (ATRA)–induced differentiation of acute promyelocytic leukemia (APL) cells, the gene expression patterns in the APL cell line NB4 before and after ATRA treatment were analyzed using complementary DNA array, suppression-subtractive hybridization, and differential-display–polymerase chain reaction. A total of 169 genes, including 8 novel ones, were modulated by ATRA. The ATRA-induced gene expression profiles were in high accord with the differentiation and proliferation status of the NB4 cells. The time courses of their modulation were interesting. Among the 100 up-regulated genes, the induction of expression occurred most frequently 12-48 hours after ATRA treatment, while 59 of 69 down-regulated genes found their expression suppressed within 8 hours. The transcriptional regulation of 8 induced and 24 repressed genes was not blocked by cycloheximide, which suggests that these genes may be direct targets of the ATRA signaling pathway. A balanced functional network seemed to emerge, and it formed the foundation of decreased cellular proliferation, maintenance of cell viability, increased protein modulation, and promotion of granulocytic maturation. Several cytosolic signaling pathways, including JAKs/STAT and MAPK, may also be implicated in the symphony of differentiation.
Coagulation and fibrinolytic activities in 2 siblings with β2-glycoprotein I deficiency
Authors: Takeuchi Rie , Atsumi Tatsuya , Ieko Masahiro , Takeya Hiroyuki , Yasuda Shinsuke , Ichikawa Kenji , Tsutsumi Akito , Suzuki Koji , Koike Takao
Published: October 2019

β2-Glycoprotein I (β2GPI) is a major antigen for antiphospholipid antibodies, and its multiple in vitro functions have been reported. This glycoprotein not only down-regulates thrombin formation by inhibiting contact activation or prothrombinase activity, but also up-regulates coagulation by reducing protein C anticoagulant activity. However, the in vivo roles of β2GPI remain obscure. Coagulation and fibrinolytic characteristics were investigated in individuals with β2GPI deficiency. An apparently healthy woman and her brother are homozygotes for β2GPI deficiency. In these patients, Russell viper venom time was shortened (40.4 seconds; normal range, 47.8 ± 4.95 seconds), but all markers of thrombin generation and fibrin turnover were within normal ranges. Exogenous activated protein C adequately prolonged the clotting time of the β2GPI-deficient plasma, and euglobulin lysis time was also normal. Thus, elevated thrombin generation, enhancement of activated protein C response, and an altered fibrinolytic system were not found in congenitally β2GPI-deficient plasma.
Kinetics of CXCR4 and CCR5 up-regulation and human immunodeficiency virus expansion after antigenic stimulation of primary CD4+ T lymphocytes
Authors: Maier Reinhard , Bartolomé-Rodrı́guez Marı́a Matilde , Moulon Corinne , Weltzien Hans Ulrich , Meyerhans Andreas
Published: October 2019

The chemokine receptors CCR5 and CXCR4 are coreceptors for the human immunodeficiency virus (HIV) and determine the cell tropism of different HIV strains. Previous studies on their regulation were performed under conditions of unspecific T-lymphocyte stimulation and provided conflicting results. To mimick physiologic conditions, highly purified primary Staphylococcus enterotoxin B (SEB)-reactive CD4 T lymphocytes were stimulated in the presence of autologous antigen-presenting cells and the kinetics of CCR5 and CXCR4 surface expression and HIV replication were studied. Both chemokine receptors were transiently up-regulated with maximal expression at day 3 after stimulation. The stimulated T cells were equally susceptible to productive infection with R5-and X4-tropic virus strains. Thus, antigenic stimulation of T cells promotes efficient replication of both, T cell-tropic and macrophage-tropic HIV.
Interleukin-7 stimulates osteoclast formation by up-regulating the T-cell production of soluble osteoclastogenic cytokines
Authors: Weitzmann M. Neale , Cenci Simone , Rifas Leonard , Brown Christopher , Pacifici Roberto
Published: October 2019

In unstimulated conditions osteoclast renewal occurs as a result of the stromal cell production of the key osteoclastogenic factors, receptor activator of NFkB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Inflammation is known to cause increased osteoclastogenesis; however, the mechanisms responsible for this phenomenon are poorly understood. We now show that interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFα), cytokines typically produced in inflammatory conditions, increase the stromal cell production of IL-7. This factor, in turn, up-regulates production of osteoclastogenic cytokines by T cells leading to stimulation of osteoclast (OC) formation. Although T cells were found to produce soluble forms of both RANKL and M-CSF, saturating concentrations of osteoprotegerin failed to inhibit approximately 40% of the OC formation, suggesting that IL-7 acts via both RANKL-dependent and RANKL-independent pathways. Despite the identification of T-cell–secreted M-CSF, this cytokine was not essential for either RANKL-dependent or -independent OC formation, suggesting that T cells secrete other cytokines capable of substituting for M-CSF action. On the basis of our data, we propose a novel mechanism for inflammatory bone loss in which induction of IL-7 from stromal cells by IL-1 and TNFα leads to the production of soluble osteoclastogenic cytokines by T cells. Thus, the mechanism by which IL-7 causes bone resorption involves the activation of T cells and the T-cell–dependent augmentation of osteoclastogenesis.
Heparin and heparan sulfate bind interleukin-10 and modulate its activity
Authors: Salek-Ardakani Shahram , Arrand John R. , Shaw David , Mackett Mike
Published: October 2019

Glycosaminoglycans (GAG) are a group of negatively charged molecules that have been shown to bind and directly regulate the bioactivity of growth factors and cytokines such as basic fibroblast growth factor, transforming growth factor-β, IL-7, and interferon-γ. The ability of GAG to interact with human IL-10 (hIL-10) and the effect of these interactions on its biologic activity were analyzed. It was demonstrated by affinity chromatography that hIL-10 binds strongly to heparin–agarose at physiological pH. Biosensor-based binding kinetic analysis indicated an equilibrium dissociation constant, Kd, of 54 nmol/L for this interaction. Human IL-10 stimulated CD16 and CD64 expression on the monocyte/macrophage population within peripheral blood mononuclear cells, with optimal concentrations between 1 and 10 ng/mL. Soluble heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate were shown to inhibit the hIL-10–induced expression of CD16 and CD64 in a concentration-dependent manner. Heparin and heparan sulfate were most effective with IC50 values of 100 to 500 μg/mL. Considerably higher concentrations of dermatan sulfate and chondroitin 4-sulfate were required with an IC50 of 2000 to 5000 μg/mL, whereas chondroitin 6-sulfate was essentially inactive. The antagonistic effect of heparin on hIL-10 activity was shown to be dependent on N-sulfation, inasmuch as de-N-sulfated heparin had little or no inhibitory effect on the IL-10– induced expression of CD16, whereas the effect of de-O-sulfated heparin was comparable to that of unmodified heparin. Furthermore, the inhibition of cell-bound proteoglycan sulfation reduced the hIL-10–mediated expression of CD16 molecules on monocytes/macrophages. Taken together, these findings support the hypothesis that soluble and cell-surface GAG and, in particular, their sulfate groups are important in binding and modulation of hIL-10 activity.
Abnormal rearrangement within the α/δ T-cell receptor locus in lymphomas from Atm-deficient mice
Authors: Liyanage Marek , Weaver Zoë , Barlow Carrolee , Coleman Allen , Pankratz Daniel G. , Anderson Stacie , Wynshaw-Boris Anthony , Ried Thomas
Published: October 2019

Atm-deficient mice (Atm−/−) recapitulate many aspects of the ataxia telangiectasia (AT) syndrome, including the susceptibility to tumors of lymphoid origin. To investigate the mechanism of tumorigenesis, we have examined a panel of 8 thymic lymphomas from Atm−/− mice. AllAtm−/− tumors are of thymic lymphoblastoid origin, display an immature CD3− and CD4+/CD8+ phenotype, and arise coincident with V(D)J recombination. Cytogenetically, all tumors are diploid or near diploid but exhibit multiple chromosome aberrations with an average of 4 abnormal chromosomes per tumor. All the tumors revealed chromosome 14 rearrangements precisely at the T-cell receptorα/δ(Tcrα/δ) locus, suggesting the involvement of V(D)J recombination in these translocations. In addition, 11.5% ofAtm−/− peripheral T cells showed chromosome 14 translocations, suggesting that rearrangements at theTcrα/δ locus occur early during tumor development in the absence of ATM. However, additional genetic aberrations are required for tumorigenesis. For example, translocations involving chromosome 12, often with chromosome 14 (more than 60%), and partial or complete trisomy of chromosome 15, with copy number increases of the c-myc oncogene were frequently observed. These observations suggest that ATM is required for normal rearrangement of the Tcrα/δ locus but not for V(D)J recombination at other loci. The mechanisms that lead to tumorigenesis may be due to the involvement of ATM in monitoring double-stranded DNA breaks.
CD34 expression by murine hematopoietic stem cells mobilized by granulocyte colony-stimulating factor
Authors: Tajima Fumihito , Sato Takashi , Laver Joseph H. , Ogawa Makio
Published: October 2019

Controversy has existed about CD34 expression by hematopoietic stem cells. We recently reported that CD34 expression reflects the activation state of stem cells by using a murine transplantation model. It has been generally held that mobilized blood stem cells express CD34.However, it has also been reported that mobilized stem cells and progenitors are in G0/G1 phases of the cell cycle. To address the state of CD34 expression by the mobilized stem cells, we again used the mouse transplantation model. We prepared CD34− and CD34+ populations of nucleated blood cells from granulocyte colony-stimulating factor–treated Ly-5.1 mice and assayed each population for long-term engrafting cells in lethally irradiated Ly-5.2 mice. The majority of the stem cells were in the CD34+population. The CD34 expression by mobilized stem cells was reversible because re-transplantation of Ly-5.1 CD34− marrow cells harvested from the Ly-5.2 recipients of CD34+-mobilized stem cells 8 months posttransplantation revealed long-term engraftment. These results may support the use of total CD34+ cells in mobilized blood as a predictor for engraftment and CD34 selection for enrichment of human stem cells.
Solitary plasmacytoma of bone and asymptomatic multiple myeloma
Authors: Dimopoulos Meletios A. , Moulopoulos Lia A. , Maniatis Alice , Alexanian Raymond
Published: October 2019

Most patients with multiple myeloma (MM) present with symptoms, have evidence of generalized disease, and require chemotherapy promptly to reduce the malignant clone. Some patients present with a local symptom from a single plasmacytoma but no myeloma elsewhere. Such patients usually become free of symptoms after local radiotherapy. In patients with MM without symptoms, the diagnosis is made on the basis of screening laboratory tests. In patients with either solitary plasmacytoma of bone or asymptomatic MM, systemic treatment should be deferred until there is evidence of disease progression.
Ligand-inducible interaction of the DRIP/TRAP coactivator complex with retinoid receptors in retinoic acid–sensitive and –resistant acute promyelocytic leukemia cells
Authors: Shao Wenlin , Rosenauer Angelika , Mann Koren , Chang Chao-Pei Betty , Rachez Christophe , Freedman Leonard P. , Miller Wilson H.
Published: October 2019

Retinoic acid (RA) signaling is mediated by its nuclear receptors RXR and RAR, which bind to their cognate response elements as a heterodimer, RXR/RAR, and act in concert with coregulatory factors to regulate gene transcription on ligand binding. To identify specific cofactors that interact with the RXR/RAR heterodimer in acute promyelocytic leukemia (APL) cells, a double cistronic construct was used that allowed coexpression of the RXR LBD (ligand binding domain) with the RAR LBD as an affinity matrix to pull down interacting proteins from nuclear extracts prepared from a human APL cell line, NB4. A group of proteins was detected whose interaction with RXR/RAR is ligand inducible. The molecular weight pattern of these proteins is similar to that of a complex of proteins previously identified as DRIP or TRAP, which are ligand-dependent transcription activators of VDR and TR, respectively. The RXR/RAR-interacting proteins from NB4 were confirmed to be identical to the DRIP subunits by comparative electrophoresis, Western blot analysis, and in vitro protein interaction assay. In addition to RXR/RAR, the DRIP component can interact directly with the APL-specific PML-RARα fusion protein. The same DRIP complex is present in RA-resistant APL cells and in a variety of cancer cell lines, supporting its global role in transcriptional regulation.
Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes
Authors: Aguayo Alvaro , Kantarjian Hagop , Manshouri Taghi , Gidel Cristi , Estey Elihu , Thomas Deborah , Koller Charles , Estrov Zeev , O'Brien Susan , Keating Michael , Freireich Emil , Albitar Maher
Published: October 2019

Angiogenesis has been associated with the growth, dissemination, and metastasis of solid tumors. The aims of this study were to evaluate the vascularity and the levels of angiogenic factors in patients with acute and chronic leukemias and myelodysplastic syndromes (MDS). The numbers of blood vessels were measured in 145 bone marrow biopsies and the levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor necrosis growth factor-α (TNF-α), tumor growth factor-α (TGF-α), and hepatocyte growth factor (HGF) were determined in 417 plasma samples. Except for chronic lymphocytic leukemia (CLL), vascularity was significantly higher in all leukemias and MDS compared with control bone marrows. The highest number of blood vessels and largest vascular area were found in chronic myeloid leukemia (CML). VEGF, bFGF, and HGF plasma levels were significantly increased in acute myeloid leukemia (AML), CML, CLL, chronic myelomonocytic leukemia (CMML), and MDS. HGF, TNF-α, and bFGF but not VEGF were significantly increased in acute lymphoblastic leukemia (ALL). TNF-α levels were significantly increased in all diseases except for AML and MDS. No significant increase was found in TGF-α in any leukemia or MDS. The highest plasma levels of VEGF were in CML, and the highest plasma levels of bFGF were in CLL. The levels of HGF were highest in CMML. These data suggest that vascularity and angiogenic factors are increased in leukemias and MDS and may play a role in the leukemogenic process.
CGP57148B (STI-571) induces differentiation and apoptosis and sensitizes Bcr-Abl–positive human leukemia cells to apoptosis due to antileukemic drugs
Authors: Fang Guofu , Kim Caryn Naekyung , Perkins Charles L. , Ramadevi Nimmanapalli , Winton Elliott , Wittmann Sylvie , Bhalla Kapil N.
Published: October 2019

The differentiation and apoptosis-sensitizing effects of the Bcr-Abl–specific tyrosine kinase inhibitor CGP57148B, also known as STI-571, were determined in human Bcr-Abl–positive HL-60/Bcr-Abl and K562 cells. First, the results demonstrate that the ectopic expression of the p185 Bcr-Abl fusion protein induced hemoglobin in the acute myeloid leukemia (AML) HL-60 cells. Exposure to low-dose cytosine arabinoside (Ara-C; 10 nmol/L) increased hemoglobin levels in HL-60/Bcr-Abl and in the chronic myeloid leukemia (CML) blast crisis K562 cells, which express the p210 Bcr-Abl protein. As compared with HL-60/neo, HL-60/Bcr-Abl and K562 cells were resistant to apoptosis induced by Ara-C, doxorubicin, or tumor necrosis factor-α (TNF-α), which was associated with reduced processing of caspase-8 and Bid protein and decreased cytosolic accumulation of cytochrome c (cyt c). Exposure to CGP57148B alone increased hemoglobin levels and CD11b expression and induced apoptosis of HL-60/Bcr-Abl and K562 cells. CGP57148B treatment down-regulated antiapoptotic XIAP, cIAP1, and Bcl-xL, without affecting Bcl-2, Bax, Apaf-1, Fas (CD95), Fas ligand, Abl, and Bcr-Abl levels. CGP57148B also inhibited constitutively active Akt kinase and NFκB in Bcr-Abl–positive cells. Attenuation of NFκB activity by ectopic expression of transdominant repressor of IκB sensitized HL-60/Bcr-Abl and K562 cells to TNF-α but not to apoptosis induced by Ara-C or doxorubicin. Importantly, cotreatment with CGP57148B significantly increased Ara-C– or doxorubicin-induced apoptosis of HL-60/Bcr-Abl and K562 cells. This was associated with greater cytosolic accumulation of cyt c and PARP cleavage activity of caspase-3. These in vitro data indicate that combinations of CGP57148B and antileukemic drugs such as Ara-C may have improved in vivo efficacy against Bcr-Abl–positive acute leukemia.
Bcr/Abl activates transcription of theBcl-X gene through STAT5
Authors: Gesbert Franck , Griffin James D.
Published: October 2019

Several tyrosine kinase oncogenes have been associated with myeloproliferative diseases, including Bcr/Abl, Tel/Abl, Tel/Jak2, and Tel/PDGFR. One target molecule shared by these oncogenes is known to be STAT5. We generated sublines of Ba/F3 cells in which either wild-type STAT5 or a constitutively active mutant of STAT5 (STAT5-1*6) were expressed under the control of a tetracycline-inducible promoter. These cell lines were compared with a Ba/F3 cell line in which the expression of p210Bcr/Abl was made inducible by a similar promoter. Before induction, all cells were dependent on interleukin 3 (IL-3) for growth and survival. Both STAT5-1*6 and Bcr/Abl enhanced viability and induced proliferation in the absence of IL-3. We found that the proviability protein Bcl-XL, but not Bcl-2, was induced by both p210Bcr/Abl and STAT5-1*6. Using a Bcl-X gene promoter construct fused to a luciferase complementary DNA (cDNA), both p210Bcr/Abl and STAT5-1*6 were shown to induce transcription of Bcl-X. The increase in transcription of the Bcl-X promoter and the increase in Bcl-X protein, due to p210Bcr/Abl, were blocked by expression of a dominant negative STAT5 mutant. Interestingly, however, STAT5-1*6 required the continued presence of IL-3 to cause a significant increase in Bcl-XL protein, whereas p210Bcr/Abl did not need IL-3. Studies with enzyme inhibitors suggest that the extra signal supplied by IL-3 may be supplied by the PI3K pathway. Overall, these data suggest that constitutively activated STAT5 can increase viability and proliferation of Ba/F3 cells. This may contribute to, but is not likely sufficient for, the enhanced viability associated with Bcr/Abl transformation.
Stat5a/b contribute to interleukin 7–induced B-cell precursor expansion, but abl- andbcr/abl-induced transformation are independent of Stat5
Authors: Sexl Veronika , Piekorz Roland , Moriggl Richard , Rohrer Juerg , Brown Michael P. , Bunting Kevin D. , Rothammer Kristen , Roussel Martine F. , Ihle James N.
Published: October 2019

The cytokines interleukin 7 (IL-7) and interleukin 4 (IL-4) regulate lymphoid differentiation and function and activate the transcription factor Stat5. Using mice deficient for the 2 highly related transcription factors, Stat5a and Stat5b (Stat5a/b−/−), we investigated the role of Stat5 for B-cell differentiation, expansion, and function. Peripheral blood B cells of Stat5-deficient mice are significantly reduced, but no proliferation defects in response to various mitogenic stimuli are found. Also, IgM and IgG1 antibody production and immunoglobulin class switching are not affected. Pre- and pro-B cells of Stat5-deficient animals were found to have reduced responses to IL-7. Pro- and pre-B cells are the target cells of the abloncogene and numerous studies have suggested that Stat5a/b is essential for transformation by derivatives of the Abelson(abl) gene. To assess the role of Stat5a/b in transformation, we have evaluated the ability of variousabl derivatives to transform cells from Stat5a/b-deficient mice in vitro or in vivo. We demonstrate that the absence of Stat5a/b is not essential for the induction of lymphoid or myeloid tumors in vivo or on the ability to transform bone marrow cells in vitro.
High-dose chemoradiotherapy and autologous stem cell transplantation for patients with primary refractory aggressive non-Hodgkin lymphoma: an intention-to-treat analysis
Authors: Kewalramani Tarun , Zelenetz Andrew D. , Hedrick Eric E. , Donnelly Gerard B. , Hunte Sonia , Priovolos Anna C. , Qin Jing , Lyons Nancy Coady , Yahalom Joachim , Nimer Stephen D. , Moskowitz Craig H.
Published: October 2019

High-dose chemoradiotherapy (HDT) with autologous stem cell transplantation (ASCT) is the treatment of choice for patients with relapsed aggressive non-Hodgkin lymphoma (NHL). However, its role in the treatment of patients with primary refractory disease is not well defined. The outcomes of 85 patients with primary refractory aggressive NHL who underwent second-line chemotherapy with ICE with the intent of administering HDT/ASCT to those patients with chemosensitive disease were reviewed. Patients were retrospectively classified as induction partial responders (IPR) if they attained a partial response to doxorubicin-based front-line therapy or as induction failures (IF) if they had less than partial response. Forty-three patients (50.6%) had ICE-chemosensitive disease; there was no difference in the response rate between the IPR and the IF groups. Intention-to-treat analysis revealed that 25% of the patients were alive and 21.9% were event-free at a median follow-up of 35 months. Among 42 patients who underwent transplantation, the 3-year overall and event-free survival rates were 52.5% and 44.2%, respectively, similar to the outcomes for patients with chemosensitive relapsed disease. No differences were observed between the IPR and IF groups, and there were no transplantation-related deaths. More than one extranodal site of disease and a second-line age-adjusted International Prognostic Index of 3 or 4 before ICE chemotherapy were predictive of poor survival. These results suggest that patients with primary refractory aggressive NHL should receive second-line chemotherapy, with the intent of administering HDT/ASCT to those with chemosensitive disease. Newer therapies are needed to improve the outcomes of patients with poor-risk primary refractory disease.
Biologic significance of GATA-1 activities in Ras-mediated megakaryocytic differentiation of hematopoietic cell lines
Authors: Matsumura Itaru , Kawasaki Akira , Tanaka Hirokazu , Sonoyama Junko , Ezoe Sachiko , Minegishi Naoko , Nakajima Koichi , Yamamoto Masayuki , Kanakura Yuzuru
Published: October 2019

Lineage-specific transcription factors play crucial roles in the development of hematopoietic cells. In a previous study, it was demonstrated that Ras activation was involved in thrombopoietin-induced megakaryocytic differentiation. In this study, constitutive Ras activation by H-rasG12V evoked megakaryocytic maturation of erythroleukemia cell lines F-36P and K562, but not of myeloid cell line 32D cl3 that lacks GATA-1. However, the introduction of GATA-1 led to reprogramming of 32D cl3 toward erythrocytic/megakaryocytic lineage and enabled it to undergo megakaryocytic differentiation in response to H-rasG12V. In contrast, the overexpression of PU.1 and c-Myb changed the phenotype of K562 from erythroid to myeloid/monocytic lineage and rendered K562 to differentiate into granulocytes and macrophages in response to H-rasG12V, respectively. In GATA-1–transfected 32D cl3, the endogenous expression of PU.1 and c-Myb was easily detectable, but their activities were reduced severely. Endogenous GATA-1 activities were markedly suppressed in PU.1-transfected and c-myb–transfected K562. As for the mechanisms of these reciprocal inhibitions, GATA-1 and PU.1 were found to associate through their DNA-binding domains and to inhibit the respective DNA-binding activities of each other. In addition, c-Myb bound to GATA-1 and inhibited its DNA-binding activities. Mutant GATA-1 and PU.1 that retained their own transcriptional activities but could not inhibit the reciprocal partner were less effective in changing the lineage phenotype of 32D cl3 and K562. These results suggested that GATA-1 activities may be crucial for Ras-mediated megakaryocytic differentiation and that its activities may be regulated by the direct interaction with other lineage-specific transcription factors such as PU.1 and c-Myb.
Syk-deficient eosinophils show normal interleukin-5–mediated differentiation, maturation, and survival but no longer respond to FcγR activation
Authors: Lach-Trifilieff Estelle , Menear Keith , Schweighoffer Edina , Tybulewicz Victor L. J. , Walker Christoph
Published: October 2019

The tyrosine kinase Syk has been proposed to play a critical role in the antiapoptotic effect of interleukin (IL)-5 in human eosinophils. However, little is known about the involvement of Syk in other IL-5–mediated activation events. To further address these questions, the role of Syk in IL-5–induced eosinophil differentiation, activation, and survival was analyzed using cells obtained from Syk-deficient mice. We could demonstrate that Syk-deficient fetal liver cells differentiate into mature eosinophils in response to IL-5 at the same rate as wild-type fetal liver cells and generate the same total number of eosinophils. Moreover, no difference in IL-5–induced survival of mature eosinophils between Syk−/− and wild-type eosinophils could be demonstrated, suggesting that the antiapoptotic effect of IL-5 does not require Syk despite the activation of this tyrosine kinase upon IL-5 receptor ligation. In contrast, eosinophils derived from Syk-deficient but not wild-type mice were incapable of generating reactive oxygen intermediates in response to Fcγ receptor (FcγR) engagement. Taken together, these data clearly demonstrate no critical role for Syk in IL-5–mediated eosinophil differentiation or survival but underline the importance of this tyrosine kinase in activation events induced by FcγR stimulation.
New recurring cytogenetic abnormalities and association of blast cell karyotypes with prognosis in childhood T-cell acute lymphoblastic leukemia: a Pediatric Oncology Group report of 343 cases
Authors: Schneider Nancy R. , Carroll Andrew J. , Shuster Jonathan J. , Pullen D. Jeanette , Link Michael P. , Borowitz Michael J. , Camitta Bruce M. , Katz Julie A. , Amylon Michael D.
Published: October 2019

To further define the cytogenetic differences between B-cell lineage (B-lineage) acute lymphoblastic leukemia (ALL) and T-cell lineage ALL (T-ALL) and to determine the prognostic value of cytogenetics in childhood T-ALL, the blast cell karyotypes of 343 cases of pediatric T-ALL, the largest series reported to date, were evaluated. Cytogenetics were performed in a single central laboratory, and the children were treated using a single Pediatric Oncology Group protocol. Clear differences between the karyotypic characteristics of B-lineage ALL and T-ALL were confirmed. This study suggests that there may be survival differences associated with some T-ALL blast cell karyotypes. Better survival is associated with only normal karyotypes and with t(10;14) (translocation of chromosomes 10 and 14); worse survival is associated with the presence of any derivative chromosome. Two new recurring chromosome aberrations previously not reported in T-ALL were found: del(1)(p22) and t(8;12)(q13;p13). Ten aberrations found in this series, which were reported only once previously in T-ALL, can now be considered recurring abnormalities in T-ALL. All 12 of these new recurring aberrations are targets for discovery and characterization of new genes that are important in T-cell development and leukemogenesis.
An IL6 promoter polymorphism is associated with a lifetime risk of development of Kaposi sarcoma in men infected with human immunodeficiency virus
Authors: Foster Charles B. , Lehrnbecher Thomas , Samuels Susan , Stein Steven , Mol Femke , Metcalf Julie A. , Wyvill Kathleen , Steinberg Seth M. , Kovacs Joseph , Blauvelt Andrew , Yarchoan Robert , Chanock Stephen J.
Published: October 2019

Kaposi sarcoma (KS) is an angioproliferative inflammatory condition that occurs commonly in patients infected with human immunodeficiency virus (HIV). Inflammatory cytokines and growth factors promote the development of KS. Because physiologically important cytokine polymorphisms modulate host inflammatory responses, we investigated the association between KS and common regulatory polymorphisms in 5 proinflammatory cytokine genes encoding interleukin (IL) IL-1α, IL-1β, tumor necrosis factor (TNF) α, TNF-β, and IL-6 and in the IL-1 receptor antagonist (IL1RN). We also examined the contribution of stromal-derived factor 1 and chemokine receptor 5 (Δ32) polymorphisms to KS development. The population consisted of 115 HIV-infected men with KS and 126 deceased HIV-infected men without KS. The only strong association was observed between an IL6promoter polymorphism (G-174C) and susceptibility to KS in HIV-infected men (P = .0035). Homozygotes for IL6 allele G, associated with increased IL6 production, were overrepresented among patients with KS (P = .0046), whereas allele C homozygotes were underrepresented (P = .0062). Substantial in vitro evidence indicates that IL-6 contributes to the pathogenesis of KS. Our results show thatIL6 promoter genotypes associated with altered gene expression are risk factors for development of KS. Identification of a genetic risk factor for development of KS has important clinical implications for prevention and therapy.
Involvement of SNAP-23 and syntaxin 6 in human neutrophil exocytosis
Authors: Martı́n-Martı́n Belén , Nabokina Svetlana M. , Blasi Juan , Lazo Pedro A. , Mollinedo Faustino
Published: October 2019

To understand the molecular basis of exocytosis in human neutrophils, the role of syntaxin 6 and SNAP-23 in neutrophil degranulation was examined. Human syntaxin 6 was cloned and identified as a 255-amino acid protein with a carboxy-terminal transmembrane region and two coiled-coil domains. Syntaxin 6 was localized mainly in the plasma membrane of human resting neutrophils, whereas SNAP-23 was located primarily in the mobilizable tertiary and specific granules. SNAP-23 was translocated to the cell surface, colocalizing with syntaxin 6, on neutrophil activation. In vitro binding studies established that SNAP-23 binds to syntaxin 6. Coimmunoprecipitation assays indicated that SNAP-23 interacts with syntaxin 6 in vivo, and this interaction was dramatically increased on neutrophil activation. Antibodies against SNAP-23 inhibited Ca++ and GTP-γ-S–induced exocytosis of CD67-enriched specific granules, but they hardly affected exocytosis of the CD63-enriched azurophilic granules, when introduced into electropermeabilized neutrophils. Anti–syntaxin 6 antibodies prevented exocytosis of both CD67- and CD63-enriched granules in electropermeabilized neutrophils. These data show that syntaxin 6 and SNAP-23 are involved in human neutrophil exocytosis, demonstrating that vesicle SNAP receptor-target SNAP receptor (v-SNARE– t-SNARE) interactions modulate neutrophil secretion. Syntaxin 6 acts as a target for secretion of specific and azurophilic granules, whereas SNAP-23 mediates specific granule secretion.
Comprehensive gene expression profile of LPS-stimulated human monocytes by SAGE
Authors: Suzuki Takuji , Hashimoto Shin-ichi , Toyoda Nobuaki , Nagai Shigenori , Yamazaki Nobuyuki , Dong Hong-Yan , Sakai Jun , Yamashita Taro , Nukiwa Toshihiro , Matsushima Kouji
Published: October 2019

Monocytes play a pivotal role in various human infectious and inflammatory diseases. To reveal a whole picture of pathophysiologic function of activated human monocytes, this study used the serial analysis of gene expression (SAGE) procedure in lipopolysaccharide (LPS)-stimulated human monocytes. A total of 35 874 tags corresponding to more than 12 000 different transcripts were sequenced. Comparison of gene expression profile with that of resting monocytes revealed the LPS-inducible gene expression profile. Many cytokines and chemokines, including interleukin (IL)-6, IL-1α, IL-1β, tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1β, MIP-2β, MIP-2α, liver and activation-regulated chemokine (LARC), MIP-1α, thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), regulated on activation, normal T cell expressed and secreted (RANTES), growth-regulated oncogene (GRO) α, and IL-8, were observed in the highest inducible transcripts. Other genes encoding plasminogen activator inhibitor type 2 (PAI-2), Hc-gp39, apolipoproteins, malate dehydrogenase, matrix metalloproteinase-9 (MMP-9), and cyclooxygenase (COX2) were also highly elevated in LPS-stimulated monocytes. Moreover, up-regulation of Naf1β, IL-7 receptor, adenosine receptor A2a, and many novel genes was newly identified. These results suggest that the LPS-inducible gene products may be involved in cell activation and migration, angiogenesis, tissue remodeling, and metabolism, and thus may orchestrate the inflammatory reactions. On the other hand, the expression of numerous sets of novel genes was discovered to be down-regulated on LPS stimulation. This study represents the first comprehensive analysis of LPS-inducible gene expression in human monocytes and provides tremendous novel information for the function of LPS-activated monocytes and targets for diagnosing, monitoring, and treating sepsis and various human infectious and inflammatory diseases.