bluuebunny/crossref_metadata_2025 huggingface dataset:
Bis-indols: a novel class of molecules enhancing the cytodifferentiating properties of retinoids in myeloid leukemia cells
Authors: Pisano Claudio , Kollar Peter , Giannı́ Maurizio , Kalac Yesim , Giordano Vincenzo , Ferrara Fabiana Fosca , Tancredi Richard , Devoto Antonio , Rinaldi Alessandra , Rambaldi Alessandro , Penco Sergio , Marzi Mauro , Moretti Giampiero , Vesci Loredana , Tinti Ornella , Carminati Paolo , Terao Mineko , Garattini Enrico
Published: October 2002

Enhancing the pharmacologic activity of all-transretinoic acid (ATRA) is potentially useful in the management of acute promyelocytic leukemia (APL) and other types of myeloid leukemia. In this report, we identify a novel class of experimental agents selectively potentiating the cytodifferentiating activity of ATRA and synthetic retinoic acid receptor α agonists in APL and other myeloid leukemia cell lines. These agents have a bis-indolic structure (BISINDS), and ST1346 is the prototypical compound of the series. Gene-profiling experiments and determination of the level of expression of myeloid-associated markers indicate that ST1346 stimulates many aspects of the granulocytic maturation process set in motion by ATRA. Stimulation of the cytodifferentiating activity of ATRA by ST1346 enhances the efficacy of the retinoid in vivo, as demonstrated in the APL model of the severe combined immunodeficiency (SCID) mouse receiving transplants of NB4 cells. Although the molecular mechanisms underlying the ATRA-potentiating action of ST1346 and congeners have not been completely clarified, bis-indols are not ligands and do not exert any direct effect on the ATRA-dependent transactivation of nuclear receptors. However, ST1346 inhibits the down-regulation of cyclic adenosine monophosphate (cAMP)–dependent CREB transcriptional complexes and enhances the level of expression of signal transducers and activators of transcription-1 (STAT1), 2 putative molecular determinants of the differentiation process activated by ATRA in APL cells. More importantly, ST1346 relieves the down-regulation of Jun N-terminal kinases (JNK) afforded by ATRA. In addition, a specific JNK inhibitor blocks the enhancing effect of ST1346 on ATRA-induced maturation of NB4 cells. This demonstrates an important role for the mitogen-activated protein kinase in the molecular mechanisms underlying the pharmacologic activity of the bis-indol.
B-cell development in the thymus is limited by inhibitory signals from the thymic microenvironment
Authors: Hashimoto Yoshiko , Montecino-Rodriguez Encarnacion , Leathers Hyosuk , Stephan Robert P. , Dorshkind Kenneth
Published: October 2002

B-cell precursors are present in the thymus, and the thymic microenvironment is the source of lymphopoietic factors that include interleukin-7 (IL-7). Despite the fact that intrathymic B-cell progenitors are bone marrow–derived cells, the data in this report demonstrate that these progenitors accumulate at an early pro–B-cell stage of development, cycle less than their bone marrow counterparts, and fail to differentiate efficiently. Additional studies presented herein indicate that these effects are mediated, at least in part, by soluble factors produced by the thymic microenvironment and suggest that they affect the ability of pro–B cells to respond optimally to IL-7. Taken together, these observations demonstrate a specific inhibition of intrathymic B lymphopoiesis, which in turn may explain why lymphoid cell production in the thymus is largely restricted to production of T-lineage cells despite the fact that B-cell precursors and B-lymphopoietic stimuli are present in that organ.
Lymphoproliferative defects in mice lacking the expression of neurofibromin: functional and biochemical consequences ofNf1 deficiency in T-cell development and function
Authors: Ingram David A. , Zhang Lei , McCarthy Jennifer , Wenning Mary Jo , Fisher Lucy , Yang Feng-Chun , Clapp D. Wade , Kapur Reuben
Published: October 2002

Ras plays an essential role in lymphocyte development and function. However, in vivo consequence(s) of regulation of Ras activity by guanosine triphosphatase (GTPase)–activating proteins (GAPs) on lymphocyte development and function are not known. In this study we demonstrate that neurofibromin, the protein encoded by theNF1 tumor suppressor gene functions as a GAP for Ras in T cells. Loss of Nf1 in T cells results in enhanced Ras activation, which is associated with thymic and splenic hyperplasia, and an increase in the absolute number of immature and mature T-cell subsets compared with control mice. Interestingly, in spite of a profound T-cell expansion and higher thymidine incorporation in unstimulated Nf1-deficient T cells, T-cell receptor and interleukin-2 receptor–mediated proliferation of thymocytes and mature T cells was substantially reduced compared with control mice. Collectively, these results identify neurofibromin as a GAP for Ras in T cells for maintaining immune homeostasis in vivo.
Urokinase receptor surface expression regulates monocyte adhesion in acute myocardial infarction
Authors: May Andreas E. , Schmidt Roland , Kanse Sandip M. , Chavakis Triantafyllos , Stephens Ross W. , Schömig Albert , Preissner Klaus T. , Neumann Franz-Josef
Published: October 2002

The urokinase receptor (urokinase plasminogen activator receptor; uPAR) regulates monocyte adhesion by direct binding to vitronectin and by forming complexes with integrins. Therefore, possible up-regulation of uPAR in acute myocardial infarction (AMI) may affect monocyte adhesion. In 20 patients with AMI, uPAR surface expression (measured by flow cytometry) was increased compared with that in patients with chronic stable angina (mean ± SD fluorescence, 179 ± 96 vs 80 ± 53; P = .002). Expression of uPAR correlated with activation of β2-integrins lymphocyte function–associated antigen 1 (LFA-1) and macrophage antigen 1 (Mac-1), measured by using monoclonal antibodies (mAbs) 24 and CBRM1/5. Isolated mononuclear cells (MNCs) from patients with AMI showed enhanced adhesiveness to human umbilical vein endothelial cells (HUVECs), to fibrinogen (Mac-1 ligand), and to vitronectin (uPAR ligand). Excessive adhesion of MNCs to HUVECs was inhibited by mAbs anti-CD18 (84%), anti-CD11a (51%), and anti-CD11b (57%), indicating a major contribution of LFA-1 and Mac-1. The mAb anti-uPAR R3 blocked adhesion of cells from patients with AMI to vitronectin (95%) but also β2-integrin–mediated adhesion to fibrinogen (79%) and HUVECs (66%). Incubation of monocytic MonoMac6 cells with plasma from patients with AMI enhanced uPAR messenger RNA expression and cell adhesion to HUVECs. Thus, released soluble factors may contribute to enhanced monocyte adhesion in AMI. Mouse pre-B lymphocytes (BAF3 cells) transfected with various amounts of uPAR complementary DNA showed a strong correlation of uPAR expression with β2-integrin–dependent adhesion to intercellular adhesion molecule 1, thus providing evidence for the functional relevance of uPAR up-regulation in an isolated in vitro system. In conclusion, we found that uPAR expression is elevated on monocytes in AMI and contributes to enhanced cell adhesion. Thus, uPAR may be a novel target for prevention of unwanted monocyte recruitment as part of inflammatory cardiovascular processes.
Interaction of human hematopoietic stem cells with bacterial pathogens
Authors: Kolb-Mäurer Annette , Wilhelm Martin , Weissinger Florian , Bröcker Eva-Bettina , Goebel Werner
Published: October 2002

Primitive hematopoietic stem cells (HSCs) in the bone marrow are rare pluripotent cells with the capacity to give rise to all lineages of blood cells. During commitment, progenitor cells are composed mainly of cells with the potential for differentiation into 1 or 2 lineages. This commitment involves the acquisition of specific growth factor receptors and the loss of others. Viral and bacterial infections may lead to profound disturbance of hematopoiesis, which is possibly due to different susceptibility of HSCs to infectious agents. Here, we show that quiescent human HSCs are fully resistant to infection by the intracellular bacteria, Listeria monocytogenes andSalmonella enterica serovariationtyphimurium, and the extracellular pathogen Yersinia enterocolitica. During myeloid/monocytic differentiation induced by incubation with stem cell factor, thrombopoietin, and flt-3 ligand, partially differentiated HSCs emerge, which readily take up these pathogens and also latex beads by macropinocytosis. After further monocytic differentiation, bacterial uptake by macropinocytosis still occurs but internalization of the pathogens is now mainly achieved by receptor-mediated phagocytosis. These results suggest that in the case of HSCs uptake mechanisms for bacteria develop sequentially.
Preclinical development of an adjuvant-free peptide vaccine with activity against CMV pp65 in HLA transgenic mice
Authors: La Rosa Corinna , Wang Zhongde , Brewer John C. , Lacey Simon F. , Villacres Maria C. , Sharan Rahul , Krishnan Radhika , Crooks Matthew , Markel Susan , Maas Rebecca , Diamond Don J.
Published: October 2002

Epitope vaccines have shown promise for inducing cellular immune responses in animal models of infectious disease. In cases where cellular immunity was augmented, peptide vaccines composed of covalently linked minimal cytotoxic T-lymphocyte (CTL) and T-helper (TH) epitopes generally showed the most efficacy. To address a clinical vaccine strategy for cytomegalovirus (CMV) in the context of HCT (hematopoietic cell transplantation), we observed that linking the synthetically derived pan-DR epitope peptide (PADRE) or one of several tetanus TH epitopes to the immunodominant human leukocyte antigen (HLA) A*0201–restricted CTL epitope from CMV-pp65 to create a fusion peptide caused robust cytotoxic cellular immune responses in HLA A*0201/Kbtransgenic mice. Significantly, the fusion peptides are immunogenic when administered in saline solution by either subcutaneous or intranasal routes. CpG-containing single-stranded DNA (ss-oligodeoxynucleotide [ODN]) added to the fusion peptides dramatically up-regulated immune recognition by either route. Notably, target cells that either expressed full-length pp65 protein from vaccinia viruses or were sensitized with the CTL epitope encoded in the vaccine were recognized by splenic effectors from immunized animals. Visualization of murine peptide–specific CTL by flow cytometry was accomplished using an HLA A*0201 tetramer complexed with the pp65495-503 CTL epitope. TH-CTL epitope fusion peptides in combination with CpG ss-ODN represent a new strategy for parenteral or mucosal delivery of vaccines in a safe and effective manner that has applicability for control or prophylaxis of infectious disease, especially in situations such as vaccination of donors or recipients of HCT, where highly inflammatory adjuvants are not desired.
Immortalization of yolk sac–derived precursor cells
Authors: Yu Wen-Mei , Hawley Teresa S. , Hawley Robert G. , Qu Cheng-Kui
Published: October 2002

Hematopoiesis initiates in the extraembryonic yolk sac. To isolate various types of precursor cells from this blood cell–forming tissue, yolk sac cells were immortalized by retroviral-mediated expression of the HOX11 homeobox-containing gene. Among the cell lines derived, some were able to spontaneously generate adherent stromal-like cells capable of taking up acetylated low-density lipoprotein, and they could be induced to form tubelike structures when cultured on Matrigel. Although these cell lines were negative for hematopoietic cell surface markers, they gave rise to hematopoietic colonies—containing cells belonging to the monocytic, megakaryocytic, and definitive erythroid lineages—when plated in methylcellulose medium supplemented with hematopoietic growth factors. Low amounts of Flk-1 mRNA could be detected in these cells, and they showed significant responsiveness to vascular endothelial growth factor, stem cell factor, basic fibroblast growth factor, and interleukin 6. They also expressed the transcription factors SCL, GATA2, GATA1, PU.1, and c-myb. These yolk sac–derived cell lines may represent a transitional stage of early hematopoietic development.
How we manage venous thromboembolism during pregnancy
Authors: Bates Shannon M. , Ginsberg Jeffrey S.
Published: October 2002

During pregnancy, physiologic and anatomic changes can complicate the diagnosis of venous thromboembolism (VTE) as well as the management of patients with a high risk of or established VTE. As in nonpregnant subjects, clinical diagnosis of VTE by itself is unreliable and accurate objective testing is essential. Few diagnostic studies of VTE have been performed in pregnant women and, therefore, approaches are largely extrapolated from those used in nonpregnant subjects with modifications to limit the radiation exposure and overcome the limitations of diagnostic testing in pregnancy. Therapy of established VTE during pregnancy consists of therapeutic doses of unfractionated heparin (UFH) or low–molecular-weight heparin (LMWH), generally given throughout pregnancy subcutaneously and for 4 to 6 weeks after childbirth. A key unresolved issue includes the optimum dosing of LMWH therapy. Maternal warfarin can be safely used after childbirth because it is safe to use in the breast-fed infant of a mother receiving warfarin. Finally, pregnant women with prior VTE (with or without a hypercoagulable state) have an increased risk of recurrent venous thrombosis. A recent study has demonstrated that for women with a single episode of prior VTE, many can be managed without anticoagulants. However, for many, anticoagulant therapy with prophylactic UFH or LMWH is a reasonable option.
Interleukin-1 blockade does not prevent acute graft-versus-host disease: results of a randomized, double-blind, placebo-controlled trial of interleukin-1 receptor antagonist in allogeneic bone marrow transplantation
Authors: Antin Joseph H. , Weisdorf Daniel , Neuberg Donna , Nicklow Roberta , Clouthier Shawn , Lee Stephanie J. , Alyea Edwin , McGarigle Carol , Blazar Bruce R. , Sonis Stephen , Soiffer Robert J. , Ferrara James L.M.
Published: October 2002

Acute graft-versus-host disease (GVHD) is thought to derive from direct T-cell injury of target tissues through perforin/granzyme, Fas/FasL interactions, and the effects of inflammatory cytokines. Animal models and some clinical trials support the notion that inhibition of inflammatory mediators such as interleukin-1 (IL-1), tumor necrosis factor α, and interferon γ may ameliorate or prevent GVHD. We hypothesized that blockade of IL-1 during the period of initial T-cell activation would reduce the risk of severe GVHD. We tested this hypothesis in a double-blind, placebo-controlled randomized trial of recombinant human IL-1 receptor antagonist (IL-1Ra) in 186 patients undergoing allogeneic stem cell transplantation. Randomization was stratified by degree of histocompatibility and stem cell source. All patients were conditioned with cyclophosphamide and total body irradiation. GVHD prevention consisted of cyclosporine and methotrexate in all patients. Recombinant human IL-1Ra or saline placebo was given from day −4 to day +10. Randomization was stratified according to GVHD risk. The 2 groups were well-matched for pretreatment characteristics. Moderate to severe GVHD (grades B-D) developed in 57 (61%) of 94 patients receiving IL-1Ra and in 51 (59%) of 86 patients on placebo (P = .88). There was no difference in hematologic recovery, transplantation-related toxicity, event-free survival, or overall survival. We conclude that blockade of IL-1 using IL-1Ra during conditioning and 10 days immediately after transplantation is not sufficient to reduce GVHD or toxicity or to improve survival.
Sialyltransferase specificity in selectin ligand formation
Authors: Ellies Lesley G. , Sperandio Markus , Underhill Gregory H. , Yousif James , Smith Michael , Priatel John J. , Kansas Geoffrey S. , Ley Klaus , Marth Jamey D.
Published: October 2002

Selectin ligands are glycan structures that participate in leukocyte trafficking and inflammation. At least 6 ST3Gal sialyltransferases (I-VI) have been identified that may contribute to selectin ligand formation. However, it is not known which of these sialyltransferases are involved in vivo and whether they may differentially regulate selectin function. We have produced and characterized mice genetically deficient in ST3Gal-I, ST3Gal-II, ST3Gal-III, and ST3Gal-IV. Unlike mice bearing severe defects in selectin ligand formation, there was no finding of leukocytosis with these single ST3Gal deficiencies. Among neutrophils, only ST3Gal-IV was found to play a role in the synthesis of selectin ligands. In vitro rolling of marrow-derived neutrophils on E- or P-selectins presented by Chinese hamster ovary cells was reduced in the absence of ST3Gal-IV. However, in a tumor necrosis factor α (TNF-α)–induced inflammation model in vivo, no defect among P-selectin ligands was observed. Nevertheless, the number of leukocytes rolling on postcapillary venules in an E-selectin–dependent manner was decreased while E-selectin–dependent rolling velocity was increased. We propose that multiple ST3Gal sialyltransferases contribute to selectin ligand formation, as none of these ST3Gal deficiencies recapitulated the degree of E- and P-selectin ligand deficit observed on neuraminidase treatment of intact neutrophils. Our findings indicate a high degree of functional specificity among sialyltransferases and a substantial role for ST3Gal-IV in selectin ligand formation.
Leukemia proto-oncoprotein MLL is proteolytically processed into 2 fragments with opposite transcriptional properties
Authors: Yokoyama Akihiko , Kitabayashi Issay , Ayton Paul M. , Cleary Michael L. , Ohki Misao
Published: October 2002

MLL (mixed lineage leukemia; alsoALL-1 or HRX) is a proto-oncogene that is mutated in a variety of acute leukemias. Its product is normally required for the maintenance of Hox gene expression during embryogenesis and hematopoiesis through molecular mechanisms that remain poorly defined. Here we demonstrate that MLL (mixed lineage leukemia) is proteolytically processed into 2 fragments (MLLN and MLLC) that display opposite transcriptional properties and form an intramolecular MLL complex in vivo. Proteolytic cleavage occurs at 2 amino acids (D2666 and D2718) within a consensus processing sequence (QXD/GZDD, where X is a hydrophobic amino acid and Z is an alanine or a valine) that is conserved in TRX, the Drosophila homolog of MLL, and in the MLL-related protein MLL2, suggesting that processing is important for MLL function. Processed MLLN and MLLC associate with each other via N-terminal (1253-2254 amino acids) and C-terminal (3602-3742 amino acids) intramolecular interaction domains. MLL processing occurs rapidly within a few hours after translation and is followed by the phosphorylation of MLLC. MLLNdisplays transcriptional repression activity, whereas MLLChas strong transcriptional activation properties. Leukemia-associated MLL fusion proteins lack the MLL processing sites, do not undergo cleavage, and are unable to interact with MLLC. These observations suggest that posttranslational modifications of MLL may participate in regulating its activity as a transcription factor and that this aspect of its function is perturbed by leukemogenic fusions.
Prominent role of TGF-β1 in thrombopoietin-induced myelofibrosis in mice
Authors: Chagraoui Hédia , Komura Emiko , Tulliez Micheline , Giraudier Stéphane , Vainchenker William , Wendling Françoise
Published: October 2002

Several studies suggest an implication of transforming growth factor-β1 (TGF-β1) in the promotion of myelofibrosis associated with hematopoietic malignancies, but the involvement of this cytokine is not fully investigated. To test directly the impact of TGF-β1 in the pathogenesis of myelofibrosis, bone marrow stem cells from homozygous TGF-β1 null (TGF-β1−/−) and wild-type (WT) littermates were infected with a retrovirus encoding the murine thrombopoietin (TPO) protein and engrafted into lethally irradiated wild-type hosts for long-term reconstitution. Over the 4 months of follow-up, TPO levels in plasma were markedly elevated in both groups of mice, and animals typically developed a myeloproliferative syndrome characterized by thrombocytosis, leukocytosis, splenomegaly, increased numbers of progenitors in blood, and extramedullary hematopoiesis. Severe fibrosis was observed in spleen and marrow from all the mice engrafted with WT cells. In contrast, none of the mice repopulated with TGF-β1−/− cells (chimerism > 70%) showed deposition of reticulin fibers at any time during the follow-up. In accordance with the development of fibrosis, latent TGF-β1 levels in plasma and extracellular fluid of the spleen from mice engrafted with WT cells were increased 6-fold and 4-fold, respectively, over levels found in normal hosts, whereas no increase over baseline levels could be demonstrated in animals undergoing transplantation with TGF-β1−/− cells. These data provide evidence that TGF-β1 produced by hematopoietic cells is pivotal for the pathogenesis of myelofibrosis that develops in mice with TPO overexpression.
The genotype distribution of the XRCC1gene indicates a role for base excision repair in the development of therapy-related acute myeloblastic leukemia
Authors: Seedhouse Claire , Bainton Rowena , Lewis Michael , Harding Alexander , Russell Nigel , Das-Gupta Emma
Published: October 2002

Polymorphisms in several DNA repair genes have been described. These polymorphisms may affect DNA repair capacity and modulate cancer susceptibility by means of gene-environment interactions. We investigated DNA repair capacity and its association with acute myeloblastic leukemia (AML). We studied polymorphisms in 3 DNA repair genes: XRCC1, XRCC3, and XPD. We also assessed the incidence of a functional polymorphism in theNQO1 gene, which is involved in protection of cells from oxidative damage. We genotyped the polymorphisms by using polymerase chain reaction–restriction fragment-length polymorphism analysis in 134 patients with de novo AML, 34 with therapy-related AML (t-AML), and 178 controls. The distributions of theXRCC3 Thr241Met and NQO1 Pro187Ser genotypes were not significantly different in patients and controls. However, the distribution of the XRCC1 Arg399Gln genotypes was significantly different when comparing the t-AML and control groups (χ2, P = .03). The presence of at least oneXRCC1 399Gln allele indicated a protective effect for the allele in controls compared with patients with t-AML (odds ratio 0.44; 95% confidence interval, 0.20-0.93). We found no interactions between the XRCC1 or XRCC3 and NQO1genotypes. We also found no differences in the distribution of the XPD Lys751Gln or XRCC1 Arg194Trp genotypes. Our data provide evidence of a protective effect against AML in individuals with at least one copy of the variant XRCC1 399Gln allele compared with those homozygous for the common allele.
Evaluation of KIR ligand incompatibility in mismatched unrelated donor hematopoietic transplants
Authors: Davies Stella M. , Ruggieri Loredana , DeFor Todd , Wagner John E. , Weisdorf Daniel J. , Miller Jeffrey S. , Velardi Andrea , Blazar Bruce R.
Published: October 2002

One of the functions of HLA class I alleles is interaction with natural killer (NK) cells. Receptors termed killer immunoglobulinlike receptors (KIRs) on NK cells recognize groups of HLA class I alleles, and interaction between receptor and class I allele inhibits reactivity of the NK cell. Failure to recognize the appropriate KIR ligand on a mismatched cell can trigger NK cell elimination of that target cell. Recent analysis of haploidentical hematopoietic transplantations has shown a reduction of graft failure, graft-versus-host disease, and relapse in those with KIR ligand incompatibility in the graft-versus-host direction. In this study we analyzed the effect of KIR ligand incompatibility on outcomes of unrelated donor bone marrow transplantations. The data show no advantage for KIR ligand incompatibility in this clinical setting as assessed by HLA-Bw4 and HLA-C alleles. It is possible that there will be a benefit of NK cell alloreactivity if strategies of haploidentical transplantation are used: high stem cell doses, extensive T-cell depletion, and no postgrafting immune suppression.
Murine hematopoietic stem cell distribution and proliferation in ablated and nonablated bone marrow transplantation
Authors: Zhong Jiang F. , Zhan Yuxia , Anderson W. French , Zhao Yi
Published: October 2002

The engraftment of donor bone marrow (BM) cells in nonablated mice is inefficient. Niche availability has been thought to be the reason, and cytoablation with irradiation or cytotoxic agents is routinely used with the belief that this frees the preoccupied niches in recipients. In this study, donor cell redistribution and proliferation in ablated and nonablated mice were compared by implanting donor cells directly into the femur cavity of sedated mice. The redistribution of Lin− donor cells into BM was similar between ablated and nonablated mice. Poor engraftment in nonablated mice was shown to be the result of inefficient donor cell proliferation rather than because of a lack of space. Competitive repopulation assays demonstrated that the donor hematopoietic stem cells (HSCs) were present in nonirradiated recipients for at least 6 months after transplantation, but that they did not expand as did their counterparts in lethally irradiated mice. This study suggests that efficient bone marrow transplantation in nonablated recipients may be possible as a result of better understanding of HSC proliferative regulation and appropriate in vitro manipulation.
Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease
Authors: Weinstein David A. , Roy Cindy N. , Fleming Mark D. , Loda Massimo F. , Wolfsdorf Joseph I. , Andrews Nancy C.
Published: October 2002

The anemia of chronic disease is a prevalent, poorly understood condition that afflicts patients with a wide variety of diseases, including infections, malignancies, and rheumatologic disorders. It is characterized by a blunted erythropoietin response by erythroid precursors, decreased red blood cell survival, and a defect in iron absorption and macrophage iron retention, which interrupts iron delivery to erythroid precursor cells. We noted that patients with large hepatic adenomas had severe iron refractory anemia similar to that observed in anemia of chronic disease. This anemia resolved spontaneously after adenoma resection or liver transplantation. We investigated the role of the adenomas in the pathogenesis of the anemia and found that they produce inappropriately high levels of hepcidin mRNA. Hepcidin is a peptide hormone that has been implicated in controlling the release of iron from cells. We conclude that hepcidin plays a major, causative role in the anemia observed in our subgroup of patients with hepatic adenomas, and we speculate that it is important in the pathogenesis of the anemia of chronic disease in general.
Identification of distal regulatory regions in the human αIIb gene locus necessary for consistent, high-level megakaryocyte expression
Authors: Thornton Michael A. , Zhang Chunyan , Kowalska Maria A. , Poncz Mortimer
Published: October 2002

The αIIb/β3-integrin receptor is present at high levels only in megakaryocytes and platelets. Its presence on platelets is critical for hemostasis. The tissue-specific nature of this receptor's expression is secondary to the restricted expression of αIIb, and studies of the αIIb proximal promoter have served as a model of a megakaryocyte-specific promoter. We have examined the αIIb gene locus for distal regulatory elements. Sequence comparison between the human (h) and murine (m) αIIb loci revealed high levels of conservation at intergenic regions both 5′ and 3′ to the αIIb gene. Additionally, deoxyribonuclease (DNase) I sensitivity mapping defined tissue-specific hypersensitive (HS) sites that coincide, in part, with these conserved regions. Transgenic mice containing various lengths of the hαIIb gene locus, which included or excluded the various conserved/HS regions, demonstrated that the proximal promoter was sufficient for tissue specificity, but that a region 2.5 to 7.1 kb upstream of the hαIIb gene was necessary for consistent expression. Another region 2.2 to 7.4 kb downstream of the gene enhanced expression 1000-fold and led to levels of hαIIb mRNA that were about 30% of the native mαIIb mRNA level. These constructs also resulted in detectable hαIIb/mβ3 on the platelet surface. This work not only confirms the importance of the proximal promoter of the αIIb gene for tissue specificity, but also characterizes the distal organization of the αIIb gene locus and provides an initial localization of 2 important regulatory regions needed for the expression of the αIIb gene at high levels during megakaryopoiesis.
Primitive interleukin 3 null hematopoietic cells transduced with BCR-ABL show accelerated loss after culture of factor-independence in vitro and leukemogenic activity in vivo
Authors: Jiang Xiaoyan , Ng Eddy , Yip Calvin , Eisterer Wolfgang , Chalandon Yves , Stuible Matthew , Eaves Allen , Eaves Connie J.
Published: October 2002

Primitive chronic myeloid leukemia cells display a unique autocrine interleukin 3 (IL-3)/granulocyte–colony-stimluating factor (G-CSF) mechanism that may explain their abnormal proliferation and differentiation control. Here we show that BCR-ABL transduction of primitive Sca-1+ lin− mouse bone marrow (BM) cells causes immediate activation of IL-3, G-CSF, and granulocyte macrophage–colony-stimulating factor (GM-CSF) expression in these cells. Their autocrine IL-3–mediated growth dependence is thus demonstrable only in clonal cultures where paracrine effects are reduced. Interestingly, upon continued culture, these cells produce large populations of rapidly proliferating mast cells in which only the IL-3 autocrine mechanism is consistently maintained, together with evidence of hyperphosphorylation of p210BCR-ABL and STAT5 and retention of a multilineage but attenuated in vivo leukemogenic potential characterized by a prolonged latency. BCR-ABL transduction of IL-3−/− Sca-1+ lin− BM cells initially activates GM-CSF and G-CSF production, factor independence, and the ability to generate phenotypically indistinguishable populations of mast cells. However, maintenance of factor independence, and p210BCR-ABL and STAT 5 activation beyond 4 to 6 weeks, requires rescue with an IL-3 transgene. The cultured BCR-ABL–transduced IL-3−/− cells also lack leukemogenic activity in vivo. These findings provide new evidence that IL-3 production is a rapid, sustained, and biologically relevant consequence of BCR-ABL expression in primitive hematopoietic cells with multilineage leukemogenic activity.
Cytomegalovirus reactivation following allogeneic stem cell transplantation is associated with the presence of dysfunctional antigen-specific CD8+ T cells
Authors: Özdemir Evren , St. John Lisa S. , Gillespie Geraldine , Rowland-Jones Sarah , Champlin Richard E. , Molldrem Jeffrey J. , Komanduri Krishna V.
Published: October 2002

Cytomegalovirus (CMV) infection causes significant morbidity and mortality in the setting of immunodeficiency, including the immune reconstitution phase following allogeneic stem cell transplantation (SCT). We assessed CMV-specific CD4+ and CD8+T-cell responses in 87 HLA-A*0201–positive (A2+) and/or B*0702-positive (B7+) allogeneic stem cell transplant recipients using HLA-peptide tetramer staining and cytokine flow cytometry (CFC) to examine the association of CMV-specific immune reconstitution and CMV antigenemia following SCT. Strong CMV-specific T-cell responses recovered in most subjects (77 of 87, 88%) after SCT. Frequencies of CMV-specific CD8+ T cells were significantly higher in those subjects who experienced early antigenemia relative to those who did not (2.2% vs 0.33%, P = .0002), as were frequencies of CMV-specific CD4+ T cells (1.71% vs 0.75%,P = .002). Frequencies of CMV-specific CD8+ T cells were also higher in subjects experiencing late antigenemia (2.4% vs 0.57%). When we combined tetramer staining and an assessment of cytokine production in a single assay, we found that individuals who experienced CMV antigenemia had lower tumor necrosis factor-α (TNF-α)–producing fractions of tetramer-staining CMV-specific CD8+ T cells than subjects who did not (25% vs 65%,P = .015). Furthermore, individuals at high risk for CMV reactivation, including patients with acute graft-versus-host disease and those receiving steroids, had low fractions of cytokine-producing CMV-specific CD8+ T cells (25% and 27%, respectively). These data suggest that the inability to control CMV reactivation following allogeneic SCT is due to the impaired function of antigen-specific CD8+ T cells rather than an inability to recover sufficient numbers of CMV-specific T cells.
Cloning, expression, and functional characterization of the von Willebrand factor–cleaving protease (ADAMTS13)
Authors: Plaimauer Barbara , Zimmermann Klaus , Völkel Dirk , Antoine Gerhard , Kerschbaumer Randolf , Jenab Pegah , Furlan Miha , Gerritsen Helen , Lämmle Bernhard , Schwarz Hans Peter , Scheiflinger Friedrich
Published: October 2002

Deficient von Willebrand factor (VWF) degradation has been associated with thrombotic thrombocytopenic purpura (TTP). In hereditary TTP, the specific VWF-cleaving protease (VWF-cp) is absent or functionally defective, whereas in the nonfamilial, acquired form of TTP, an autoantibody inhibiting VWF-cp activity is found transiently in most patients. The gene encoding for VWF-cp has recently been identified as a member of the metalloprotease family and designatedADAMTS13, but the functional activity of the ADAMTS13 gene product has not been verified. To establish the functional activity of recombinant VWF-cp, we cloned the complete cDNA sequence in a eukaryotic expression vector and transiently expressed the encoded recombinant ADAMTS13 in HEK 293 cells. The expressed protein degraded VWF multimers and proteolytically cleaved VWF to the same fragments as those generated by plasma VWF-cp. Furthermore, recombinant ADAMTS13-mediated degradation of VWF multimers was entirely inhibited in the presence of plasma from a patient with acquired TTP. These data show that ADAMTS13 is responsible for the physiologic proteolytic degradation of VWF multimers.
Familial idiopathic methemoglobinemia revisited: original cases reveal 2 novel mutations in NADH-cytochrome b5 reductase
Authors: Percy Melanie J. , Gillespie Matthew J. S. , Savage Geraldine , Hughes Anne E. , McMullin Mary Frances , Lappin Terry R. J.
Published: October 2002

In 1943, the first description of familial idiopathic methemoglobinemia in the United Kingdom was reported in 2 members of one family. Five years later, Quentin Gibson (then of Queen's University, Belfast, Ireland) correctly identified the pathway involved in the reduction of methemoglobin in the family, thereby describing the first hereditary trait involving a specific enzyme deficiency. Recessive congenital methemoglobinemia (RCM) is caused by a deficiency of reduced nicotinamide adenine dinucleotide (NADH)–cytochrome b5 reductase. One of the original propositi with the type 1 disorder has now been traced. He was found to be a compound heterozygote harboring 2 previously undescribed mutations in exon 9, a point mutation Gly873Ala predicting a Gly291Asp substitution, and a 3-bp in-frame deletion of codon 255 (GAG), predicting loss of glutamic acid. A brother and a surviving sister are heterozygous; each bears one of the mutations. Thirty-three different mutations have now been recorded for RCM. The original authors' optimism that RCM would provide material for future genetic studies has been amply justified.
Deletion of the mouse α-globin regulatory element (HS −26) has an unexpectedly mild phenotype
Authors: Anguita Eduardo , Sharpe Jacqueline A. , Sloane-Stanley Jacqueline A. , Tufarelli Cristina , Higgs Douglas R. , Wood William G.
Published: October 2002

Natural deletions of the region upstream of the human α-globin gene cluster, together with expression studies in cell lines and transgenic mice, identified a single element (HS −40) as necessary and perhaps sufficient for high-level expression of the α-globin genes. A similar element occupies the corresponding position upstream of the mouse (m) α-globin genes (mHS −26) and was thought to have similar functional properties. We knocked out mHS −26 by homologous recombination and observed the surprising result that instead of the expected severe α-thalassemia phenotype, the mice had a mild disease. Transcription levels of the mouse genes were reduced by about 50%, but homozygotes were healthy, with normal hemoglobin levels and only mild decreases in mean corpuscular volume and mean corpuscular hemoglobin. These results may indicate differences in the regulation of the α-globin clusters in mice and humans or that additionalcis-acting elements remain to be characterized in one or both clusters.
Increased frequency of mannose-binding lectin insufficiency among children with acute lymphoblastic leukemia
Authors: Schmiegelow Kjeld , Garred Peter , Lausen Birgitte , Andreassen Bente , Petersen Bodil Laub , Madsen Hans Ole
Published: October 2002

Epidemiological data indicate that acute lymphoblastic leukemia (ALL) could be induced by interactions between the immune system and early childhood infections. Mannose-binding lectin (MBL) plays a critical role in the immune response in early childhood before specific immune protection develops. We investigated whether there may be an association between childhood ALL and low-producing MBLgenotypes. Serum MBL levels depend on normal (A)or defective (O) alleles, and on normal (Y) or reduced (X) promoter activities. For this study, 137 noninfants with ALL and 250 controls were classified into 3 MBL genotype groups according to their influence on the serum level of functional MBL: group I, YA/YA andYA/XA (higher levels); group II, XA/XA andYA/O (intermediate levels); and group III, MBLinsufficiency with XA/O or O/O(MBL-deficient) genotypes. Compared with controls, cases more often had low-level genotypes (I/II/III: 63 [46%]/44 [32%]/30 [22%] vs 145 [58%]/65 [26%]/40 [16%];P = .02) and MBL deficiency (8.8% vs 2.8%;P = .009). Thus, the ALL odds ratio forMBL-deficient versus nondeficient individuals was 3.3 (95% CI, 1.3-8.7), whereas the ALL odds ratio for group I versus group II/III genotypes was 0.62 (95% CI, 0.41-0.94). MBL group III patients were significantly younger at diagnosis than patients in group I/II (median, 3.9 vs 5.2 years; P = .04). The study shows that the presence of low-level MBL genotypes is associated with an increased risk of childhood ALL, particularly with early age at onset.
Recombinant human thrombopoietin: basic biology and evaluation of clinical studies
Authors: Kuter David J. , Begley C. Glenn
Published: October 2002

Thrombocytopenia is a common medical problem for which the main treatment is platelet transfusion. Given the increasing use of platelets and the declining donor population, identification of a safe and effective platelet growth factor could improve the management of thrombocytopenia. Thrombopoietin (TPO), the c-Mpl ligand, is the primary physiologic regulator of megakaryocyte and platelet development. Since the purification of TPO in 1994, 2 recombinant forms of the c-Mpl ligand—recombinant human thrombopoietin (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF)—have undergone extensive clinical investigation. Both have been shown to be potent stimulators of megakaryocyte growth and platelet production and are biologically active in reducing the thrombocytopenia of nonmyeloablative chemotherapy. However, neither TPO has demonstrated benefit in stem cell transplantation or leukemia chemotherapy. Other clinical studies have investigated the use of TPO in treating chronic nonchemotherapy-induced thrombocytopenia associated with myelodysplastic syndromes, idiopathic thrombocytopenic purpura, thrombocytopenia due to human immunodeficiency virus, and liver disease. Based solely on animal studies, TPO may be effective in reducing surgical thrombocytopenia and bleeding, ex vivo expansion of pluripotent stem cells, and as a radioprotectant. Ongoing and future studies will help define the clinical role of recombinant TPO and TPO mimetics in the treatment of chemotherapy- and nonchemotherapy-induced thrombocytopenia.
hnRNP-K and Purα act together to repress the transcriptional activity of the CD43 gene promoter
Authors: Da Silva Nicolas , Bharti Ajit , Shelley Carl S.
Published: October 2002

CD43 is an abundant, heavily glycosylated molecule expressed specifically on the surface of leukocytes and platelets. When leukocytes are at rest, CD43 acts to prevent both homotypic and heterotypic interactions. However, during leukocyte activation CD43 expression is repressed, facilitating the intercellular contact required for chemotaxis, phagocytosis, aggregation, adhesion to endothelium, and transendothelial migration. Consequently, CD43 repression plays a vital role both in innate and acquired immunity. Here we report that a dramatic down-regulation of CD43 mRNA levels occurs during activation of the leukocytic cell line K562. This repression coincides with repression of the transcriptional activity of the CD43 gene promoter. We have determined that heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and Purα act together to mediate repression of the CD43 promoter during K562 activation. The hnRNP-K molecule and Purα bind single-stranded DNA. Therefore, exposure of single-stranded structures within theCD43 promoter probably plays a major role in effectingCD43 repression.
In vivo trafficking, cell cycle activity, and engraftment potential of phenotypically defined primitive hematopoietic cells after transplantation into irradiated or nonirradiated recipients
Authors: Plett P. Artur , Frankovitz Stacy M. , Orschell-Traycoff Christie M.
Published: October 2002

Recent interest in bone marrow (BM) transplantation in nonconditioned or minimally conditioned recipients warrants investigation of homing patterns of transplanted hematopoietic progenitor cells (HPCs) in irradiated and nonirradiated recipients. To this end, phenotypically defined populations of BM cells were tracked in lethally irradiated or nonirradiated mice at 1, 3, 6, and 24 hours after transplantation. Recovery of transplanted cells at all time points was higher in BM of nonirradiated mice, similar to earlier suggestions. The percentage of lineage-negative Sca-1+cells and Sca-1+ cells expressing CD43, CD49e, and CD49d steadily increased in BM of nonirradiated mice up to 24 hours, while fluctuating in irradiated mice. Cell cycle status and BrdU incorporation revealed that less than 20% of Sca-1+ cells and fewer Sca-1+lin− cells had cycled by 24 hours after transplantation. To more directly examine trafficking of primitive HPCs, purified grafts of CD62L− or CD49e+ subfractions of Sca-1+lin−cells, previously shown to be enriched for long-term repopulating cells, also were tracked in vivo. Recovery of purified cells was similarly increased in BM of nonirradiated mice. When 50 to 100 of these BM-homed cells were examined in serial transplantation studies, BM-homed cells from initially nonirradiated mice were enriched 5- to 30-fold for cells capable of long-term hematopoiesis in secondary recipients. Collectively, these data suggest that homing or survival of transplanted cells in irradiated recipients is less efficient than that in nonirradiated recipients, implicating an active role of radiation-sensitive microenvironmental cues in the homing process. These results may have important clinical implications in the design of BM transplantation protocols.
Characterization of gene expression of CD34+ cells from normal and myelodysplastic bone marrow
Authors: Hofmann Wolf-K. , de Vos Sven , Komor Martina , Hoelzer Dieter , Wachsman William , Koeffler H. Phillip
Published: October 2002

Gene patterns of expression in purified CD34+ bone marrow cells from 7 patients with low-risk myelodysplastic syndrome (MDS) and 4 patients with high-risk MDS were compared with expression data from CD34+ bone marrow cells from 4 healthy control subjects. CD34+ cells were isolated by magnetic cell separation, and high-density oligonucleotide microarray analysis was performed. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction. Class membership prediction analysis selected 11 genes. Using the expression profile of these genes, we were able to discriminate patients with low-risk from patients with high-risk MDS and both patient groups from the control group by hierarchical clustering (Spearman confidence). The power of these 11 genes was verified by applying the algorithm to an unknown test set containing expression data from 8 additional patients with MDS (3 at low risk, 5 at high risk). Patients at low risk could be distinguished from those at high risk by clustering analysis. In low-risk MDS, we found that the retinoic-acid–induced gene (RAI3), the radiation-inducible, immediate-early response gene (IEX1), and the stress-induced phosphoprotein 1 (STIP1) were down-regulated. These data suggest that CD34+cells from patients with low-risk MDS lack defensive proteins, resulting in their susceptibility to cell damage. In summary, we propose that gene expression profiling may have clinical relevance for risk evaluation in MDS at the time of initial diagnosis. Furthermore, this study provides evidence that in MDS, hematopoietic stem cells accumulate defects that prevent normal hematopoiesis.
Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C
Authors: Pearce Andrew C. , Wilde Jonathan I. , Doody Gina M. , Best Denise , Inoue Osamu , Vigorito Elena , Tybulewicz Victor L. J. , Turner Martin , Watson Steve P.
Published: October 2002

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)–coupled collagen receptor GPVI and by the G protein–coupled receptor agonist thrombin. The glycoprotein VI (GPVI)–specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.