bluuebunny/crossref_metadata_2025 huggingface dataset:
Asynchronous Climate Changes in the North Atlantic and Japan During the Last Termination
Authors: Nakagawa Takeshi , Kitagawa Hiroyuki , Yasuda Yoshinori , Tarasov Pavel E. , Nishida Kotoba , Gotanda Katsuya , Sawai Yuki ,   Yangtze River Civilization Program Members
Published: January 2003

Pollen records from the annually laminated sediment sequence in Lake Suigetsu, Japan, suggest a sequence of climate changes during the Last Termination that resembles that of the North Atlantic region but with noticeable differences in timing. An interstadial interval commenced a few centuries earlier [∼15,000 years before the present (yr B.P.)] than the North Atlantic GI-1 (Bölling) event. Conversely, the onset of a Younger Dryas (YD)–like cold reversal (12,300 to 11,250 yr B.P.) postdated the North Atlantic GS-1 (YD) event by a few centuries. Climate in the Far East during the Last Termination reflected solar insolation changes as much as Atlantic influences.
A Gate-Controlled Bidirectional Spin Filter Using Quantum Coherence
Authors: Folk J. A. , Potok R. M. , Marcus C. M. , Umansky V.
Published: January 2003

We demonstrate a quantum coherent electron spin filter by directly measuring the spin polarization of emitted current. The spin filter consists of an open quantum dot in an in-plane magnetic field; the in-plane field gives the two spin directions different Fermi wavelengths resulting in spin-dependent quantum interference of transport through the device. The gate voltage is used to select the preferentially transmitted spin, thus setting the polarity of the filter. This provides a fully electrical method for the creation and detection of spin-polarized currents. Polarizations of emitted current as high as 70% for both spin directions (either aligned or anti-aligned with the external field) are observed.
Maintenance of Stable Heterochromatin Domains by Dynamic HP1 Binding
Authors: Cheutin Thierry , McNairn Adrian J. , Jenuwein Thomas , Gilbert David M. , Singh Prim B. , Misteli Tom
Published: January 2003

One function of heterochromatin is the epigenetic silencing by sequestration of genes into transcriptionally repressed nuclear neighborhoods. Heterochromatin protein 1 (HP1) is a major component of heterochromatin and thus is a candidate for establishing and maintaining the transcriptionally repressive heterochromatin structure. Here we demonstrate that maintenance of stable heterochromatin domains in living cells involves the transient binding and dynamic exchange of HP1 from chromatin. HP1 exchange kinetics correlate with the condensation level of chromatin and are dependent on the histone methyltransferase Suv39h. The chromodomain and the chromoshadow domain of HP1 are both required for binding to native chromatin in vivo, but they contribute differentially to binding in euchromatin and heterochromatin. These data argue against HP1 repression of transcription by formation of static, higher order oligomeric networks but support a dynamic competition model, and they demonstrate that heterochromatin is accessible to regulatory factors.
Modulation of Heterochromatin Protein 1 Dynamics in Primary Mammalian Cells
Authors: Festenstein Richard , Pagakis Stamatis N. , Hiragami Kyoko , Lyon Debbie , Verreault Alain , Sekkali Belaid , Kioussis Dimitris
Published: January 2003

Heterochromatin protein 1 (HP1β), a key component of condensed DNA, is strongly implicated in gene silencing and centromeric cohesion. Heterochromatin has been considered a static structure, stabilizing crucial aspects of nuclear organization and prohibiting access to transcription factors. We demonstrate here, by fluorescence recovery after photobleaching, that a green fluorescent protein–HP1β fusion protein is highly mobile within both the euchromatin and heterochromatin of ex vivo resting murine T cells. Moreover, T cell activation greatly increased this mobility, indicating that such a process may facilitate (hetero)chromatin remodeling and permit access of epigenetic modifiers and transcription factors to the many genes that are consequently derepressed.
Energetic Radiation Produced During Rocket-Triggered Lightning
Authors: Dwyer Joseph R. , Uman Martin A. , Rassoul Hamid K. , Al-Dayeh Maher , Caraway Lee , Jerauld Jason , Rakov Vladimir A. , Jordan Douglas M. , Rambo Keith J. , Corbin Vincent , Wright Brian
Published: January 2003

Using a NaI(Tl) scintillation detector designed to operate in electrically noisy environments, we observed intense bursts of energetic radiation (≫10 kiloelectron volts) during the dart leader phase of rocket-triggered lightning, just before and possibly at the very start of 31 out of the 37 return strokes measured. The bursts had typical durations of less than 100 microseconds and deposited many tens of megaelectron volts into the detector. These results provide strong evidence that the production of runaway electrons is an important process during lightning.
The Perfect Ocean for Drought
Authors: Hoerling Martin , Kumar Arun
Published: January 2003

The 1998–2002 droughts spanning the United States, southern Europe, and Southwest Asia were linked through a common oceanic influence. Cold sea surface temperatures (SSTs) in the eastern tropical Pacific and warm SSTs in the western tropical Pacific and Indian oceans were remarkably persistent during this period. Climate models show that the climate signals forced separately by these regions acted synergistically, each contributing to widespread mid-latitude drying: an ideal scenario for spatially expansive, synchronized drought. The warmth of the Indian and west Pacific oceans was unprecedented and consistent with greenhouse gas forcing. Some implications are drawn for future drought.
ARGONAUTE4
Control of Locus-Specific siRNA Accumulation and DNA and Histone Methylation
Authors: Zilberman Daniel , Cao Xiaofeng , Jacobsen Steven E.
Published: January 2003

Proteins of the ARGONAUTE family are important in diverse posttranscriptional RNA-mediated gene-silencing systems as well as in transcriptional gene silencing in
Drosophila
and fission yeast and in programmed DNA elimination in
Tetrahymena
. We cloned
ARGONAUTE4
(
AGO4
) from a screen for mutants that suppress silencing of the
Arabidopsis SUPERMAN
(
SUP
) gene. The
ago4-1
mutant reactivated silent
SUP
alleles and decreased CpNpG and asymmetric DNA methylation as well as histone H3 lysine-9 methylation. In addition,
ago4-1
blocked histone and DNA methylation and the accumulation of 25-nucleotide small interfering RNAs (siRNAs) that correspond to the retroelement
AtSN1
. These results suggest that AGO4 and long siRNAs direct chromatin modifications, including histone methylation and non-CpG DNA methylation.
Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations
Authors: Levene M. J. , Korlach J. , Turner S. W. , Foquet M. , Craighead H. G. , Webb W. W.
Published: January 2003

Optical approaches for observing the dynamics of single molecules have required pico- to nanomolar concentrations of fluorophore in order to isolate individual molecules. However, many biologically relevant processes occur at micromolar ligand concentrations, necessitating a reduction in the conventional observation volume by three orders of magnitude. We show that arrays of zero-mode waveguides consisting of subwavelength holes in a metal film provide a simple and highly parallel means for studying single-molecule dynamics at micromolar concentrations with microsecond temporal resolution. We present observations of DNA polymerase activity as an example of the effectiveness of zero-mode waveguides for performing single-molecule experiments at high concentrations.
The Emperor's New Design
Authors: Miller Kenneth R.
Published: January 2003

Where Darwin Meets the Bible
. Creationists and Evolutionists in America. Larry A. Witham. Oxford University Press, New York, 2002. 338 pp. $30, £25. ISBN 0-19-515045-7.

A supporter of intelligent design offers an even-handed account of the creation-evolution debates that is focused on the current protagonists rather than the arguments per se.
Platelet activation induced by human antibodies to interleukin-8
Authors: Regnault Véronique , de Maistre Emmanuel , Carteaux Jean-Pierre , Gruel Yves , Nguyen Philippe , Tardy Brigitte , Lecompte Thomas
Published: January 2003

Some cases of heparin-induced thrombocytopenia (HIT) have been reported to be associated with antibodies against interleukin-8 (IL-8), a chemokine related to platelet factor 4. We found that sera from 5 HIT patients containing immunoglobulin G (IgG) or IgM antibodies to IL-8, as evidenced using surface plasmon resonance spectroscopy, were able to trigger IL-8–dependent activation of washed platelets, leading to procoagulant activity. This activation occurred at IL-8 concentrations achievable in vivo and was facilitated by heparin (0.1 U/mL). Activation was also induced by affinity-purified anti–IL-8 IgG and involved FcγRIIa. In the 2 patients who could be followed up, antibodies were no longer detectable 4 months after heparin withdrawal. One additional patient with paraneoplastic recurrent thrombosis without thrombocytopenia was found to have platelet-activating anti–IL-8 IgM, but in this case heparin was inhibitory. This is another example of potentially pathogenic platelet activation by antibodies.
The protein tyrosine kinase inhibitor SU5614 inhibits FLT3 and induces growth arrest and apoptosis in AML-derived cell lines expressing a constitutively activated FLT3
Authors: Spiekermann Karsten , Dirschinger Ralf J. , Schwab Ruth , Bagrintseva Ksenia , Faber Florian , Buske Christian , Schnittger Susanne , Kelly Louise M. , Gilliland D. Gary , Hiddemann Wolfgang
Published: January 2003

Activating mutations of the protein tyrosine kinase (PTK) FLT3 can be found in approximately 30% of patients with acute myeloid leukemia (AML), thereby representing the most frequent single genetic alteration in AML. These mutations occur in the juxtamembrane (FLT3 length mutations; FLT3-LMs) and the second tyrosine kinase domain of FLT3-TKD and confer interleukin 3 (IL-3)–independent growth to Ba/F3 cells. In the mouse bone marrow transplantation model, FLT3-LMs induce a myeloproliferative syndrome stressing their transforming activity in vivo. In this study, we analyzed the pro-proliferative and antiapoptotic potential of FLT3 in FLT3-LM/TKD-mutation–transformed Ba/F3 cells and AML-derived cell lines. The PTK inhibitor SU5614 has inhibitory activity for FLT3 and selectively induces growth arrest, apoptosis, and cell cycle arrest in Ba/F3 and AML cell lines expressing a constitutively activated FLT3. In addition, the compound reverts the antiapoptotic and pro-proliferative activity of FLT3 ligand (FL) in FL-dependent cells. No cytotoxic activity of SU5614 was found in leukemic cell lines that express a nonactivated FLT3 or no FLT3 protein. At the biochemical level, SU5614 down-regulated the activity of the hyperphosphorylated FLT3 receptor and its downstream targets, signal transducer and activator of (STAT) 3, STAT5, and mitogen-activated protein kinase (MAPK), and the STAT5 target genes BCL-XL and p21. Our results show that SU5614 is a PTK inhibitor of FLT3 and has antiproliferative and proapoptotic activity in AML-derived cell lines that endogenously express an activated FLT3 receptor. The selective and potent cytotoxicity of FLT3 PTK inhibitors support a clinical strategy of targeting FLT3 as a new molecular treatment option for patients with FLT3-LM/TKD-mutation+ AML.
Antithymocyte globulin with or without cyclosporin A: 11-year follow-up of a randomized trial comparing treatments of aplastic anemia
Authors: Frickhofen Norbert , Heimpel Hermann , Kaltwasser Joachim P. , Schrezenmeier Hubert
Published: January 2003

Immunosuppression with antithymocyte globulin, (methyl)prednisolone, and cyclosporin A is considered the treatment of choice for the patient with aplastic anemia without a donor for standard-risk stem cell transplantation. This consensus is supported by the results of several series, including a randomized German trial. Here we report 11-year results of the latter trial. With stringent response criteria and 4 months as the time to evaluate responses, this analysis confirms the superiority of the cyclosporine regimen regarding the response rate in all patients treated (70% vs 41%, with or without cyclosporine; P = .015) and in patients with severe aplastic anemia (65% vs 31%; P = .011). Patients responded more rapidly after treatment with cyclosporine (median, 60 vs 82 days; P = .019). Most patients treated with cyclosporine needed only one course of immunosuppression, whereas many patients treated without cyclosporine required repeated immunosuppressive treatment. Because of the efficacy of salvage treatment, overall survival was not different between the 2 treatment groups. However, failure-free survival favored the cyclosporine regimen (39% vs 24%; P = .04). The relapse rate, projected at 38% after 11.3 years, was similar between the 2 treatment groups. Remissions were cyclosporine dependent in 26% of the patients responding to a regimen that included cyclosporine. Clonal or malignant diseases developed in 25% of the patients. These data demonstrate that antithymocyte globulin, methylprednisolone, and cyclosporin A are an effective regimen for the treatment of aplastic anemia. However, remissions are unstable, and secondary diseases are common. In contrast to the results of stem cell transplantation, most patients are not cured.
Modulation of VEGFR-2–mediated endothelial-cell activity by VEGF-C/VEGFR-3
Authors: Matsumura Kazuyoshi , Hirashima Masanori , Ogawa Minetaro , Kubo Hajime , Hisatsune Hiroshi , Kondo Nobuyuki , Nishikawa Satomi , Chiba Tsutomu , Nishikawa Shin-Ichi
Published: January 2003

Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3), a receptor for VEGF-C, was shown to be essential for angiogenesis as well as for lymphangiogenesis. Targeted disruption of theVEGFR-3 gene in mice and our previous study using an antagonistic monoclonal antibody (MoAb) for VEGFR-3 suggested that VEGF-C/VEGFR-3 signals might be involved in the maintenance of vascular integrity. In this study we used an in vitro embryonic stem (ES) cell culture system to maintain the VEGFR-3+ endothelial cell (EC) and investigated the role of VEGFR-3 signals at the cellular level. In this system packed clusters of ECs were formed. Whereas addition of exogenous VEGF-A induced EC dispersion, VEGF-C, which can also stimulate VEGFR-2, promoted EC growth without disturbing the EC clusters. Moreover, addition of AFL4, an antagonistic MoAb for VEGFR-3, resulted in EC dispersion. Cytological analysis showed that VEGF-A– and AFL4-treated ECs were indistinguishable in many aspects but were distinct from the cytological profile induced by antagonistic MoAb for VE-cadherin (VECD-1). As AFL4- induced EC dispersion requires VEGF-A stimulation, it is likely that VEGFR-3 signals negatively modulate VEGFR-2. This result provides new insights into the involvement of VEGFR-3 signals in the maintenance of vascular integrity through modulation of VEGFR-2 signals. Moreover, our findings suggest that the mechanisms underlying AFL4-induced EC dispersion are distinct from those underlying VECD-1–induced dispersion for maintenance of EC integrity.
CD4+ T-cell clones specific for wild-type factor VIII: a molecular mechanism responsible for a higher incidence of inhibitor formation in mild/moderate hemophilia A
Authors: Jacquemin Marc , Vantomme Valérie , Buhot Cécile , Lavend'homme Renaud , Burny Wivine , Demotte Nathalie , Chaux Pascal , Peerlinck Kathelijne , Vermylen Jos , Maillere Bernard , van der Bruggen Pierre , Saint-Remy Jean-Marie
Published: January 2003

Mild/moderate hemophilia A patients carrying certain mutations in the C1 domain of factor VIII (FVIII) have a higher risk of inhibitor occurrence. To analyze the mechanisms responsible for inhibitor development in such patients, we characterized FVIII-specific CD4+ T-cell clones derived from a mild hemophilia A patient carrying an Arg2150His substitution in the C1 domain and who presented with a high titer inhibitor toward normal but not self-FVIII. All T-cell clones recognized synthetic peptides encompassing Arg2150. The peptides were presented to the T-cell clones by DRB1*0401/DRB4*01 or DRB1*1501/DRB5*01. Interestingly, the latter haplotype was previously reported as being associated with an increased incidence of inhibitor formation. Peptide I2144-T2161 also bound to other DR molecules such as DRB1*0101 and DRB1*0701, indicating that the peptide binds to major histocompatibility complex (MHC) class II molecules expressed in more than 60% of the population. None of the T-cell clones recognized recombinant FVIII carrying the substitution Arg2150His, even when FVIII was presented by an FVIII-specific B-cell line. The mutation likely alters T-cell recognition of the mutated peptide associated to MHC molecules, because the mutated peptide bound to immunopurified DR molecules nearly as effectively as the native peptide. These observations demonstrate that T cells of this patient with mutation Arg2150His distinguish between self- and wild-type FVIII and provide a plausible mechanism for the frequent occurrence of an inhibitor in patients carrying this substitution. A similar phenomenon may occur with other mutations associated to an increased incidence of inhibitor formation.
Thrombopoietin promotes mixed lineage and megakaryocytic colony-forming cell growth but inhibits primitive and definitive erythropoiesis in cells isolated from early murine yolk sacs
Authors: Xie Xiaodong , Chan Rebecca J. , Johnson Scott A. , Starr Mark , McCarthy Jennifer , Kapur Reuben , Yoder Mervin C.
Published: January 2003

The role of thrombopoietin (Tpo) in promoting hematopoiesis has been extensively studied in late fetal, neonatal, and adult mice. However, the effects of Tpo on early yolk sac hematopoiesis have been largely unexplored. We examined whole embryos or the cells isolated from embryo proper and yolk sacs and identified both Tpo and c-mpl (Tpo receptor) mRNA transcripts in tissues as early as embryonic day 6.5 (E6.5). Presomite whole embryos and somite-staged yolk sac and embryo proper cells were plated in methylcellulose cultures and treated with selected hematopoietic growth factors in the presence or absence of Tpo. Tpo alone failed to promote colony-forming unit (CFU) formation. However, in the presence of other growth factors, Tpo caused a substantial dose-dependent reduction in primitive and definitive erythroid CFU growth in cultures containing E7.5 and E8.0 whole embryos and E8.25 to 9.5 yolk sac–derived cells. Meanwhile, Tpo treatment resulted in a substantial dose-dependent increase in CFU-mixed lineage (CFU-Mix) and CFU-megakaryocyte (CFU-Meg) formation in cultures containing cells from similar staged tissues. Addition of Tpo to cultures of sorted E9.5 yolk sac c-Kit+CD34+ hematopoietic progenitors also inhibited erythroid CFU growth but augmented CFU-Mix and CFU-Meg activity. Effects of Tpo on CFU growth were blocked in the presence of a monoclonal antibody with Tpo-neutralizing activity but not with control antibody. Thus, under certain growth factor conditions, Tpo directly inhibits early yolk sac erythroid CFU growth but facilitates megakaryocyte and mixed lineage colony formation.
Identification of P2Y12-dependent and -independent mechanisms of glycoprotein VI–mediated Rap1 activation in platelets
Authors: Larson Mark K. , Chen Hong , Kahn Mark L. , Taylor Anne M. , Fabre Jean-Etienne , Mortensen Richard M. , Conley Pamela B. , Parise Leslie V.
Published: January 2003

Glycoprotein (GP) VI is a critical platelet collagen receptor, yet the steps involved in GPVI-mediated platelet activation remain incompletely understood. Because activation of Rap1, an abundant small guanosine triphosphatase (GTPase) in platelets, contributes to integrin αIIbβ3 activation, we asked whether and how GPVI signaling activates Rap1 in platelets. Here we show that platelet Rap1 is robustly activated upon addition of convulxin, a GPVI-specific agonist. Using a reconstituted system in RBL-2H3 cells, we found that GPVI-mediated Rap1 activation is dependent on FcRγ but independent of another platelet collagen receptor, α2β1. Interestingly, GPVI-mediated Rap1 activation in human platelets is largely dependent on adenosine diphosphate (ADP) signaling through the P2Y12 and not the P2Y1 receptor. However, experiments with specific ADP receptor antagonists and platelets from knockout mice deficient in P2Y1 or the P2Y12-associated G-protein, Gαi2, indicate that human and murine platelets also have a significant P2Y12-independent component of GPVI-mediated Rap1 activation. The P2Y12-independent component is dependent on phosphatidylinositol 3-kinase and is augmented by epinephrine-mediated signaling. P2Y12-dependent and -independent components are also observed in GPVI-mediated platelet aggregation, further supporting a role for Rap1 in aggregation. These results define mechanisms of GPVI-mediated platelet activation and implicate Rap1 as a key signaling protein in GPVI-induced platelet signaling.
Impact of simian immunodeficiency virus (SIV) infection on lymphocyte numbers and T-cell turnover in different organs of rhesus monkeys
Authors: Sopper Sieghart , Nierwetberg Dagmar , Halbach Astrid , Sauer Ursula , Scheller Carsten , Stahl-Hennig Christiane , Mätz-Rensing Kerstin , Schäfer Frank , Schneider Thomas , ter Meulen Volker , Müller Justus G.
Published: January 2003

HIV infection leads to reduced numbers and increased turnover of CD4+ T cells in blood. However, blood represents only 2% of the total lymphocyte pool, and information about other organs is lacking, leading to controversy about the effects of HIV infection on T-cell homeostasis. Therefore, we have determined phenotype and turnover of lymphocyte subsets in various tissues of macaques. Infection with simian immunodeficiency virus (SIV) resulted in increased proliferation rates of T cells in all organs. Despite reduced CD4 counts in blood, absolute numbers of CD4+ T cells were increased in spleen and lymph nodes and remained stable in nonlymphoid organs such as liver, lung, bone marrow, and brain during the asymptomatic phase, indicative for an altered tissue distribution. In animals killed with first signs of AIDS, total body CD4 counts and proliferation rates had returned to control levels, whereas thymocytes were almost completely absent. Our data show that a drastically increased turnover in the early stages of HIV infection, driven by a generalized immune activation rather than a homeostatic response to CD4+ T-cell destruction, is followed by exhaustion of the regenerative capacity of the immune system.
Shielding the front-strand β3 of the von Willebrand factor A1 domain inhibits its binding to platelet glycoprotein Ibα
Authors: Bonnefoy Arnaud , Yamamoto Hiroshi , Thys Chantal , Kito Morikazu , Vermylen Jos , Hoylaerts Marc F.
Published: January 2003

Platelet adhesion to damaged vessel wall and shear-induced platelet aggregation necessitate binding of the von Willebrand factor (VWF) A1 domain to platelet GPIbα. Blocking this interaction represents a promising approach to the treatment of arterial thrombosis. Comparison of amino acid sequences of the VWF A1 domain in several species, expressing VWF recognized by the blocking monoclonal antibody AJvW-2, suggested 9 residues (His563, Ile566, Asp570, Ala581, Val584, Ala587, Arg616, Ala618, and Met622) to contribute to the epitope for AJvW-2 or to be part of the GPIbα-binding site. Glutathione-S-transferase (GST)–human VWF A1 fusion proteins, in which these amino acids were mutated to their murine counterparts, were tested for their capacity to bind AJvW-2 or heparin, to interfere with botrocetin- or ristocetin-mediated VWF binding to GPIb, or to induce flow-dependent platelet tethering in a perfusion chamber. Thus, mutations His563Arg, Ile566Leu, Asp570Ala, and Ala587Thr, clustered on the outer surface of the A1 domain, dramatically impaired binding of AJvW-2 to A1. The His563Arg, Ile566Leu, and Asp570Ala mutations also impaired the binding of heparin, which competes with AJvW-2 for binding to A1. Perfusion studies revealed that His563, Ile566, Asp570, Arg616, and Ala618 take part in GPIbα binding, their mutation-impairing platelet recruitment. In agreement with the surface distribution of VWF type 2M mutations, this study demonstrates overlapping of the epitope for AJvW-2 and the GPIbα-binding site, located around the front pocket of the A1 domain and defined by strands β3, β4, and helix α3, and it provides a mechanistic basis for VWF neutralization by this antibody.
Convergence of the adhesive and fibrinolytic systems: recognition of urokinase by integrin αMβ2 as well as by the urokinase receptor regulates cell adhesion and migration
Authors: Pluskota Elzbieta , Soloviev Dmitry A. , Plow Edward F.
Published: January 2003

Previous studies demonstrated that integrin αMβ2 (CD11b/18, Mac-1) forms a physical complex with the urokinase-type plasminogen activator receptor (uPAR/CD87) on leukocytes. In this study, we used human peripheral blood neutrophils and transfected cells expressing αMβ2, uPAR, or both receptors to show that the integrin can directly interact with urokinase (uPA). We demonstrate that αMβ2 supported adhesion and migration of these cells to uPA, and, in each case, blockade of αMβ2 suppressed the response. Within uPA, both the kringle and proteolytic domains are recognized by αMβ2, which are distinct from the growth factor domain that binds to uPAR. Within the αM subunit of the integrin, the I domain interacts with uPA, which is distinct from the region that interacts with uPAR. On cells expressing uPAR and αMβ2, both receptors mediated adhesion and migration. This cooperation was particularly apparent in the responses of neutrophils to uPA, where blockade of αMβ2 reduced uPAR-mediated responses and engagement of uPAR enhanced recognition of uPA by αMβ2. Thus, recognition of uPA by αMβ2 allows for formation of a multicontact trimolecular complex, in which a single uPA ligand may bind simultaneously to both uPAR and αMβ2. This complex may play an important role in the control of inflammatory cell migration and vascular homeostasis.
T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-B–lineage leukemia effect
Authors: Cooper Laurence J. N. , Topp Max S. , Serrano Lisa Marie , Gonzalez Sergio , Chang Wen-Chung , Naranjo Araceli , Wright Christine , Popplewell Leslie , Raubitschek Andrew , Forman Stephen J. , Jensen Michael C.
Published: January 2003

Relapse of B-lineage acute lymphoblastic leukemia (B-ALL) after allogeneic hematopoietic stem cell transplantation (HSCT) commonly results from the failure of a graft-versus-leukemia (GVL) effect to eradicate minimal residual disease. Augmenting the GVL effect by the adoptive transfer of donor-derived B-ALL–specific T-cell clones is a conceptually attractive strategy to decrease relapse rates without exacerbating graft-versus-host disease (GVHD). Toward this end, we investigated whether a genetic engineering approach could render CD8+ cytotoxic T lymphocytes (CTLs) specific for tumor cells that express the B-cell lineage cell surface molecule CD19. This was accomplished by the genetic modification of CTLs to express a chimeric immunoreceptor composed of a CD19-specific single-chain immunoglobulin extracellular targeting domain fused to a CD3-ζ intracellular signaling domain. CD19-redirected CTL clones display potent CD19-specific lytic activity and chimeric immunoreceptor-regulated cytokine production and proliferation. Because B-ALL cells can evade T-cell/natural killer- cell recognition by down-regulation of cell surface accessory molecules that participate in the formation of a functional immunologic synapse, we compared the CD19-specific effector function of genetically modified CD8+ CTLs toward CD19+ cells with disparate levels of intercellular adhesion molecule 1 (ICAM-1), leukocyte function-associated antigen 1 (LFA-1), and LFA-3. We observed that recognition of B-lineage tumor lines by CD19-specific CTLs was not impaired by low levels of ICAM-1, LFA-1, and LFA-3 cell surface expression, a functional attribute that is likely a consequence of our high-affinity CD19-specific chimeric immunoreceptor. Furthermore, the CD19-specific CTLs could lyse primary B-ALL blasts. These preclinical observations form the basis for implementing clinical trials using donor-derived CD19-specific T-cell clones to treat or prevent relapse of B-ALL after allogeneic HSCT.
LCR-regulated transgene expression levels depend on the Oct-1 site in the AT-rich region of β-globin intron-2
Authors: Bharadwaj Rikki R. , Trainor Cecelia D. , Pasceri Peter , Ellis James
Published: January 2003

Human β-globin transgenes regulated by the locus control region (LCR) express at all integration sites in transgenic mice. For such LCR activity at ectopic sites, the 5′HS3 element requires the presence of the AT-rich region (ATR) in β-globin intron-2. Here, we examine the dependence of 5′HS3 LCR activity on transcription factor binding sites in the ATR. In vitro DNaseI footprint analysis and electrophoretic mobility shift assays of the ATR identified an inverted double Gata-1 site composed of 2 noncanonical sequences (GATT and GATG) and an Oct-1 consensus site. Mutant Oct-1, Gata-1, or double mutant sites were created in the ATR of the BGT50 construct composed of a 5′HS3 β/γ-globin hybrid transgene. Transgenes with double mutant sites expressed at all sites of integration, but mean expression levels in transgenic mice were reduced from 64% per copy (BGT50) to 37% (P < .05). Mutation of the inverted double Gata-1 site had no effect at 61% per copy expression levels. In contrast, mutation of the Oct-1 site alone reduced per-copy expression levels to 31% (P < .05). We conclude that the ability of 5′HS3 to activate expression from all transgene integration sites is dependent on sequences in the ATR that are not bound at high affinity by transcription factors. In addition, the Oct-1 site in the ATR is required for high-level 5′HS3 β/γ-globin transgene expression and should be retained in LCRβ-globin expression cassettes designed for gene therapy.
Disruption of a novel regulatory element in the erythroid-specific promoter of the human PKLR gene causes severe pyruvate kinase deficiency
Authors: van Wijk Richard , van Solinge Wouter W. , Nerlov Claus , Beutler Ernest , Gelbart Terri , Rijksen Gert , Nielsen Finn C.
Published: January 2003

We established the molecular basis for pyruvate kinase (PK) deficiency in a white male patient with severe nonspherocytic hemolytic anemia. The paternal allele exhibited the commonPKLR cDNA sequence (c.) 1529G>A mutation, known to be associated with PK deficiency. On the maternal allele, 3 in cis mutations were identified in the erythroid-specific promoter region of the gene: one deletion of thymine −248 and 2 single nucleotide substitutions, nucleotide (nt) −324T>A and nt −83G>C. Analysis of the patient's RNA demonstrated the presence of only the 1529A allele, indicating severely reduced transcription from the allele linked to the mutated promoter region. Transfection of promoter constructs into erythroleukemic K562 cells showed that the most upstream −324T>A and −248delT mutations were nonfunctional polymorphisms. In contrast, the −83G>C mutation strongly reduced promoter activity. Site-directed mutagenesis of the promoter region revealed the presence of a putative regulatory element (PKR-RE1) whose core binding motif, CTCTG, is located between nt −87 and nt −83. Electrophoretic mobility shift assay using K562 nuclear extracts indicated binding of an as-yet-unidentified trans-acting factor. This novel element mediates the effects of factors necessary for regulation of pyruvate kinase gene expression during red cell differentiation and maturation.
Retroviral-mediated expression of recombinant Fancc enhances the repopulating ability of Fancc−/−hematopoietic stem cells and decreases the risk of clonal evolution
Authors: Haneline Laura S. , Li Xiaxin , Ciccone Samantha L. M. , Hong Ping , Yang Yanzhu , Broxmeyer Hal E. , Lee Suk-Hee , Orazi Attilio , Srour Edward F. , Clapp D. Wade
Published: January 2003

Fanconi anemia (FA) is a chromosomal instability disorder characterized by a progressive bone marrow (BM) failure and an increased incidence of myeloid leukemias. Children with FA are currently being enrolled in clinical trials to evaluate the safety of retroviral-mediated gene transfer. Previously, we usedFancc−/− mice to show thatFancc−/− hematopoietic stem cells (HSCs) have a profound defect in repopulating ability. Here, we examined whether retroviral-mediated gene transfer of recombinantFancc (rFancc) would restore the repopulating ability of Fancc−/− HSC to wild-type levels. Fancc−/− HSCs transduced with a retrovirus encoding rFancc exhibited a repopulating ability that approached wild-type levels. Interestingly, ∼30% of primary recipients (7 of 22) transplanted with uncorrectedFancc−/− cells developed a range of hematopoietic abnormalities including pancytopenia and BM hypoplasia similar to individuals with FA. Hematopoietic abnormalities were detected in only 1 of 22 mice transplanted withFancc−/− cells transduced with a retrovirus encoding rFancc. Moreover, several mice with hematopoietic defects had progenitors that displayed a marked resistance to IFN-γ, TNF-α, and MIP-1α compared to both Fancc−/−progenitors, which are uniquely hypersensitive to these cytokines, and wild-type progenitors. These data are analogous to studies using progenitors from patients with myelodysplasia and provide functional support for clonal evolution in these mice. Collectively, these data show that gene transfer can enhance HSC repopulating ability and suppresses the tendency for clonal evolution. These studies also reveal potential detrimental effects of ex vivo manipulation for untransducedFancc−/− HSCs.
Amplification and overexpression of JUNB is associated with primary cutaneous T-cell lymphomas
Authors: Mao Xin , Orchard Guy , Lillington Debra M. , Russell-Jones Robin , Young Bryan D. , Whittaker Sean J.
Published: January 2003

Primary cutaneous lymphomas (PCLs) represent a heterogeneous group of extranodal T- and B-cell malignancies. The underlying molecular pathogenesis of this malignancy remains unclear. This study aimed to characterize oncogene abnormalities in PCLs. Using genomic microarray, we detected oncogene copy number gains of RAF1(3p25), CTSB (8p22), PAK1 (11q13), and JUNB (19p13) in 5 of 7 cases of mycosis fungoides (MF)/Sezary syndrome (SS) (71%), gains of FGFR1 (8p11), PTPN (20q13), andBCR (22q11) in 4 cases (57%), and gains ofMYCL1 (1p34), PIK3CA (3q26), HRAS(11p15), MYBL2 (20q13), and ZNF217 (20q13) in 3 cases (43%). Amplification of JUNB was studied in 104 DNA samples from 78 PCL cases using real-time polymerase chain reaction. Twenty-four percent of cases, including 7 of 10 cases of primary cutaneous CD30+ anaplastic large-cell lymphoma (C-ALCL), 4 of 14 MF, 4 of 22 SS, and 2 of 23 primary cutaneous B-cell lymphoma (PCBCL) showed amplification ofJUNB, and high-level amplification of this oncogene was present in 3 C-ALCL and 2 MF cases. JUNB protein expression was analyzed in tissue sections from 69 PCL cases, and 44% of cases, consisting of 21 of 23 SS, 6 of 8 C-ALCL, 5 of 10 MF, and 9 of 21 PCBCL, demonstrated nuclear expression of JUNB by tumor cells. Overexpression of JUNB also was detected in 5 C-ALCL and 2 SS cases. These results have revealed, for the first time, amplification and expression patterns of JUNB in PCL, suggesting thatJUNB may be critical in the pathogenesis of primary cutaneous T-cell lymphomas.
Translocation t(11;14)(q13;q32) is the hallmark of IgM, IgE, and nonsecretory multiple myeloma variants
Authors: Avet-Loiseau Hervé , Garand Richard , Lodé Laurence , Harousseau Jean-Luc , Bataille Régis
Published: January 2003

In an attempt to address the issue of cytogenetic features of multiple myeloma (MM) variants, we have analyzed a series of 8 IgM, 9 IgD, 2 IgE, and 14 nonsecretory (NS) MM cases using fluorescence in situ hybridization. A very high incidence (83%) of t(11;14)(q13;q32) was detected in the IgM (7 of 8), IgE (2 of 2), and NS (11 of 14) MM cases, but not in the IgD cases (2 of 9). Of note, no t(4;14) was observed in this cohort of patients. This increased incidence of t(11;14) was associated with 2 dominant features in these variants, namely, a “lymphoplasmacytic” presentation mainly in IgM MM and a lower secreting capacity in the others, 2 features previously associated with t(11;14). Of major interest, t(11;14) was never observed in Waldenström macroglobulinemia or in IgG/IgA “lymphoplasmacytic” lymphomas. Thus, for unknown reasons, t(11;14) is the hallmark of IgM, IgE, and NS MM, (but not IgD MM), with a 5-fold increase of its incidence compared to that of IgG and IgA MM.
New designs for phase 2 clinical trials
Authors: Estey Elihu H. , Thall Peter F.
Published: January 2003

Conventional phase 2 clinical trials are typically single-arm experiments, with outcome characterized by one binary “response” variable. Clinical investigators are poorly served by such conventional methodology. We contend that phase 2 trials are inherently comparative, with the results of the comparison determining whether to conduct a subsequent phase 3 trial. When different treatments are studied in separate single-arm trials, actual differences between response rates associated with the treatments, “treatment effects,” are confounded with differences between the trials, “trial effects.” Thus, it is impossible to estimate either effect separately. Consequently, when the results of separate single-arm trials of different treatments are compared, an apparent treatment difference may be due to a trial effect. Conversely, the apparent absence of a treatment effect may be due to an actual treatment effect being cancelled out by a trial effect. Because selection involves comparison, single-arm phase 2 trials thus fail to provide a reliable means for selecting which therapies to investigate in phase 3. Moreover, reducing complex clinical phenomena, including both adverse and desirable events, to a single outcome wastes important information. Consequently, conventional phase 2 designs are inefficient and unreliable. Given the limited number of patients available for phase 2 trials and the increasing number of new therapies that must be evaluated, it is critically important to conduct these trials efficiently. These concerns motivated the development of a general paradigm for randomized selection trials evaluating several therapies based on multiple outcomes. Three illustrative applications of trials using this approach are presented.
Global down-regulation of HOX gene expression in PML-RARα+ acute promyelocytic leukemia identified by small-array real-time PCR
Authors: Thompson Alexander , Quinn Michael F. , Grimwade David , O'Neill Claire M. , Ahmed Momin R. , Grimes Sean , McMullin Mary Frances , Cotter Finbarr , Lappin Terence R. J.
Published: January 2003

Acute promyelocytic leukemia (APL) is associated with a reciprocal and balanced translocation involving the retinoic acid receptor-α(RARα). All-trans retinoic acid (ATRA) is used to treat APL and is a potent morphogen that regulatesHOX gene expression in embryogenesis and organogenesis.HOX genes are also involved in hematopoiesis and leukemogenesis. Thirty-nine mammalian HOX genes have been identified and classified into 13 paralogous groups clustered on 4 chromosomes. They encode a complex network of transcription regulatory proteins whose precise targets remain poorly understood. The overall function of the network appears to be dictated by gene dosage. To investigate the mechanisms involved in HOX gene regulation in hematopoiesis and leukemogenesis by precise measurement of individual HOX genes, a small-array real-timeHOX (SMART-HOX) quantitative polymerase chain reaction (PCR) platform was designed and validated. Application of SMART-HOX to 16 APL bone marrow samples revealed a global down-regulation of 26 HOX genes compared with normal controls. HOX gene expression was also altered during differentiation induced by ATRA in thePML-RARα+ NB4 cell line. PML-RARα fusion proteins have been reported to act as part of a repressor complex during myeloid cell differentiation, and a model linkingHOX gene expression to this PML-RARα repressor complex is now proposed.
Prospective Evaluation of a Follow-Up Schedule in Cutaneous Melanoma Patients: Recommendations for an Effective Follow-Up Strategy
Authors: Garbe Claus , Paul Andrena , Kohler-Späth Hanna , Ellwanger Ulf , Stroebel Waltraud , Schwarz Monika , Schlagenhauff Bettina , Meier Friedegund , Schittek Birgit , Blaheta Hans-Juergen , Blum Andreas , Rassner Gernot
Published: January 2003

Purpose: To prospectively examine and evaluate the results of follow-up procedures in a large cohort of cutaneous melanoma patients.

Patients and Methods: This was a prospective study in 2,008 consecutive patients with stage I to IV cutaneous melanoma from 1996 to 1998 on the yield of stage-appropriate follow-up examinations according to the German guidelines. Documentation of patient and follow-up data comprised patient demography, primary tumor specifics, and any clinical and technical examinations performed. The detection of metastasis was classified as early or late, and the means of their detection and the resulting overall survival probabilities were examined.

Results: A total of 3,800 clinical examinations and 12,398 imaging techniques were documented. Sixty-two second primary melanomas in 46 patients and 233 disease recurrences in 112 patients were detected during this time. In stage I to III disease, physical examination was responsible for the discovery of 50% of all recurrences. In the primary tumor stages, 21% of all recurrences were discovered by lymph node sonography, with the majority being classified as early detection. Forty-eight percent of the recurrences were classified as early detection, and these patients had a significant benefit of overall survival probability.

Conclusion: The results of our study suggest that an elaborated follow-up schedule in cutaneous melanoma is suitable for the early detection of second primary melanomas and early recurrences. The intensity of clinical and technical examinations can be reduced during follow-up of patients in the primary tumor stages and may be intensified in locoregional disease. Recommendations for an effective follow-up strategy are outlined.
Effect of Upper Extremity Exercise on Secondary Lymphedema in Breast Cancer Patients: A Pilot Study
Authors: McKenzie Donald C. , Kalda Andrea L.
Published: January 2003

Purpose: To examine the effect of a progressive upper-body exercise program on lymphedema secondary to breast cancer treatment.

Methods: Fourteen breast cancer survivors with unilateral upper extremity lymphedema were randomly assigned to an exercise (n = 7) or control group (n = 7). The exercise group followed a progressive, 8-week upper-body exercise program consisting of resistance training plus aerobic exercise using a Monark Rehab Trainer arm ergometer. Lymphedema was assessed by arm circumference and measurement of arm volume by water displacement. Patients were evaluated on five occasions over the experimental period. The Medical Outcomes Trust Short-Form 36 Survey was used to measure quality of life before and after the intervention. Significance was set at alpha ≤ 0.01.

Results: No changes were found in arm circumference or arm volume as a result of the exercise program. Three of the quality-of-life domains showed trends toward increases in the exercise group: physical functioning (P = .050), general health (P = .048), and vitality (P = .023). Mental health increased, although not significantly, for all subjects (P = .019). Arm volume measured by water displacement was correlated with calculated arm volume (r = .973, P < .001), although the exercise and control group means were significantly different (P < .001).

Conclusions: Participation in an upper-body exercise program caused no changes in arm circumference or arm volume in women with lymphedema after breast cancer, and they may have experienced an increase in quality of life. Additional studies should be done in this area to determine the optimum training program.
Thymidylate Synthase Expression in Hepatic Tumors Is a Predictor of Survival and Progression in Patients With Resectable Metastatic Colorectal Cancer
Authors: Gonen Mithat , Hummer Amanda , Zervoudakis Alice , Sullivan Deidre , Fong Yuman , Banerjee Debabrata , Klimstra David , Cordon-Cardo Carlos , Bertino Joseph , Kemeny Nancy
Published: January 2003

Purpose: To investigate the role of thymidylate synthase (TS),p53, and epidermal growth factor receptor (EGF-R) expressions in hepatic tumors in predicting overall survival (OS), progression-free survival (PFS), and hepatic progression-free survival (HPFS) in patients with resectable metastatic colorectal cancer who were randomly assigned to receive either systemic chemotherapy (SYS) alone or systemic and hepatic arterial infusion (HAI+SYS) chemotherapy following liver surgery.

Patients and Methods: Tissues from metastatic tumors were collected during liver resection from 156 patients, and marker expressions were determined using immunohistochemistry on frozen samples. Univariate associations between marker expressions and baseline variables with OS, PFS, and HPFS were examined. Independent predictors of outcome were determined using a multivariate Cox model.

Results: In multivariate analyses, TSoverexpression was found to be an independent factor of poor prognosis in OS (P < .01), PFS (P = .06), and HPFS (P < .01). In addition, resection margin was a significant independent factor for all three outcomes. Patients who received HAI+SYS experienced delayed progression in general, and in the liver, specifically. Increased levels of serum alkaline phosphatase correlated with hepatic progression. We also found a significant TS-treatment interaction for OS (P = .01) in multivariate analysis. In particular, TS+ patients receiving HAI+SYS had significantly higher survival than those receiving SYS (64 monthsv 21 months; P = .01).

Conclusion: TSlevels in hepatic tumors and resection margin are independent predictors of survival and progression in patients with metastatic colorectal cancer, whereasp53and EGFRare not independent predictors. Treatment with HAI+SYS significantly improved the survival profile of TS+ patients.
Elevated Expression of Valosin-Containing Protein (p97) in Hepatocellular Carcinoma Is Correlated With Increased Incidence of Tumor Recurrence
Authors: Yamamoto Shinji , Tomita Yasuhiko , Nakamori Shoji , Hoshida Yoshihiko , Nagano Hiroaki , Dono Keizo , Umeshita Koji , Sakon Masato , Monden Morito , Aozasa Katsuyuki
Published: January 2003

Purpose: Valosin-containing protein (VCP; also known as p97) has been shown to be associated with antiapoptotic function and metastasis via activation of the nuclear factor-κB signaling pathway. In this study, association of VCP expression with recurrence of hepatocellular carcinoma (HCC) and patient survival was examined.

Patients and Methods: VCP expression in 170 patients (139 male and 31 female) with ages ranging from 31 to 81 years (median, 61 years) was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, in which staining intensity in tumor cells was categorized as weaker (level 1) or equal to or stronger (level 2) than that in endothelial cells.

Results: Immunohistochemically, 57 patients (35.2%) showed level 1, and 105 patients (64.8%) showed level 2, VCP expression. Quantitative RT-PCR analysis revealed higher VCP mRNA expression in level 2 patients (n = 7) than level 1 (n = 4) (P < .05). Patients with VCP-level 2 HCC showed higher rate of portal vein invasion in the tumor (P < .01) and poorer disease-free and overall survival (P < .0001 and P < .05, respectively) compared with level 1 patients. Multivariate analysis revealed VCP expression level, tumor multiplicity, and degree of fibrosis in the noncancerous liver tissue to be independent prognosticators for disease-free and overall survival. VCP level was an indicator for disease-free survival in each early- (I and II) and advanced- (III and IV) stage group of pathologic tumor-node-metastasis classification (P < .001 and P < .01, respectively).

Conclusion: VCP expression level has prognostic significance for disease-free and overall survival of patients with HCC.