bluuebunny/crossref_metadata_2025 huggingface dataset:
Defective antifungal T-helper 1 (TH1) immunity in a murine model of allogeneic T-cell–depleted bone marrow transplantation and its restoration by treatment with TH2 cytokine antagonists
Authors: Mencacci Antonella , Perruccio Katia , Bacci Angela , Cenci Elio , Benedetti Roberta , Martelli Massimo F. , Bistoni Francesco , Coffman Robert , Velardi Andrea , Romani Luigina
Published: July 2002

Patients undergoing full haplotype-mismatched hematopoietic transplantations may experience severe intractable invasive fungal infections. To verify whether an imbalanced production of T-helper 1 (TH1) and TH2 cytokines may be responsible for susceptibility to fungal infections, C3H/HeJ (H-2k) recipient mice were lethally irradiated, received transplantations with T-cell–depleted allogeneic bone marrow (BM) cells from mice ofH-2d haplotype, and were infected withCandida albicans. At different time-points after transplantation, mice were assessed for pattern of TH cytokine production and susceptibility to infection. The results show that a long-term, donor-type chimerism was achieved as early as 2 weeks after BM transplantation (BMT), at the time when high-level production of TH2 cytokines (interleukin-4 [IL-4] and IL-10) and impaired production of TH1 cytokines (interferon-γ [IFN-γ] and IL-12] were observed. At this time, mice were highly susceptible to both disseminated and mucosal infections, as indicated by decreased survival, uncontrolled fungal growth, and failure to develop protective TH1 immunity. However, a predominant production of TH1 cytokines was observed by week 5 after BMT, at the time when mice developed donor-type protective TH1 responses and were resistant to infections. Therapeutic ablation of IL-4 or IL-10 greatly increased resistance to candidiasis. These results indicate that a dysregulated production of TH cytokines occurs in mice undergoing T-cell–depleted allogeneic BMT. The transient predominant production of TH2 cytokines over that of IL-12 impaired the ability of mice to develop antifungal TH1 resistance, an activity that could be efficiently restored upon treatment with TH2 cytokine antagonists.
CD36 associates with CD9 and integrins on human blood platelets
Authors: Miao Wei-Min , Vasile Eliza , Lane William S. , Lawler Jack
Published: July 2002

The membrane glycoprotein CD36 is involved in platelet aggregation, inhibition of angiogenesis, atherosclerosis, and sequestration of malaria-parasitized erythrocytes. In this study, immunoprecipitations with anti-CD36 antibodies were performed to identify proteins that associate with CD36 in the platelet membrane. Platelets were solubilized in 1% Triton X-100, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), Brij 96, or Brij 99, and the proteins that coprecipitated with CD36 were identified by peptide mass spectrometry and Western blotting. The tetraspanin protein CD9 and the integrins αIIbβ3 and α6β1 specifically coprecipitated with CD36 from platelets that were solubilized in CHAPS and Brij 99 but not from platelets that were solubilized in Triton X-100. Only CD9 is coprecipitated with CD36 from platelets that were solubilized in Brij 96. Reciprocal immunoprecipitations with antibodies to CD9, α6, αIIb, or β3 from Brij 99–solubilized platelets coprecipitated CD36. Coprecipitation of CD36, CD9, and α6β1 was also observed on platelets from a patient with Glanzmann thrombasthenia, indicating that αIIbβ3 is not required for the other proteins to associate. Colocalization of α6 and CD36, of CD9 and CD36, and of α6 and CD9 was observed on intact platelets prior to solubilization, using double immunofluorescence microscopy. These data indicate that CD36 associates with CD9 and integrins on human blood platelets. These associated proteins may mediate or participate in some of the diverse biological functions of CD36.
Evidence for the presence of murine primitive megakarycytopoiesis in the early yolk sac
Authors: Xu Ming-jiang , Matsuoka Sahoko , Yang Feng-Chun , Ebihara Yasuhiro , Manabe Atsushi , Tanaka Ryuhei , Eguchi Mitsuoki , Asano Shigetaka , Nakahata Tatsutoshi , Tsuji Kohichiro
Published: July 2002

During mouse embryogenesis, primitive erythropoiesis occurs in blood islands of the yolk sac (YS) on the seventh day of gestation. This study demonstrated for the first time the presence of unique primitive megakaryocytic (Mk) progenitors in the early YS, which disappeared by 13.5 days postcoitum (dpc). When 7.5 dpc YS cells were incubated in the presence of stem cell factor (SCF), interleukin (IL)-3, IL-6, erythropoietin (EPO), thrombopoietin (TPO), and granulocyte colony-stimulating factor in methylcellulose clonal culture, not only erythroid bursts but also megakaryocyte colonies were observed. The megakaryocytes in the colonies matured to proplatelet stages and produced platelets as early as day 3 of culture, much earlier than those from adult bone marrow, although their ploidy class was lower. These megakaryocytes were stained with acetylcholine esterase, and expressed platelet glycoprotein (GP)Ibβ, GPIIIa, and platelet factor 4 by reverse transcription-polymerase chain reaction analysis. The analysis of hemoglobin types in erythrocytes obtained from hematopoietic multilineage colonies containing the megakaryocytes indicated that the Mk progenitors originated from primitive hematopoiesis. The primitive Mk progenitors formed colonies in the absence of any cytokines in fetal bovine serum (FBS)-containing culture, and SCF, IL-3, EPO, and TPO significantly enhanced the Mk colony formation. In FBS-free culture, however, no colony formation was induced without these cytokines. Because megakaryocytes were detected in 8.5-dpc YS, these unique primitive Mk progenitors may rapidly mature and give rise to platelets to prevent hemorrhage in the simultaneously developing blood vessels until definitive hematopoiesis begins to produce platelets.
Signal transduction pathways involved in soluble fractalkine–induced monocytic cell adhesion
Authors: Cambien Bèatrice , Pomeranz Manuel , Schmid-Antomarchi Heidy , Millet Marie-Ange , Breittmayer Violette , Rossi Bernard , Schmid-Alliana Annie
Published: July 2002

Fractalkine displays features that distinguishes it from the other chemokines. In particular, besides its chemoattractant action it promotes, under physiologic flow, the rapid capture and the firm adhesion of a subset of leukocytes or intervenes in the neuron/microglia interaction. This study verified that indeed the human monocytic MonoMac6 cell line adheres to fibronectin-coated filters in response to soluble fractalkine (s-FKN). s-FKN stimulates, with distinct time courses, extracellular signal-related kinases (ERK1 and ERK2) and stress-activated protein kinases (SAPK1/JNK1 and SAPK2/p38). Both p60 Src and p72 Syk were activated under s-FKN stimulation with a rapid kinetic profile compatible with a downstream regulation on the mitogen-activated protein kinase (MAPK) congeners. The use of specific tyrosine kinase inhibitors revealed that the ERK pathway is strictly controlled by Syk, whereas c-Src up-regulated the downstream SAPK2/p38. In contrast, the SAPK1/JNK1 pathway was not regulated by any of these nonreceptor tyrosine kinases. The s-FKN–mediated increased adherence of MonoMac6 cells was partially inhibited by SB202190, a broad SAPKs inhibitor, PD98059, an MEK inhibitor, LY294002, a phosphatidyl inositol 3-kinase inhibitor, and a pertussis toxin-sensitive G protein. These data highlight that the integration of a complex array of signal transduction pathways is necessary to complete the full s-FNK–dependent adherence of human monocytic cells to fibronectin.
Identification of novel markers for monitoring minimal residual disease in acute lymphoblastic leukemia
Authors: Chen Jiann-Shiuh , Coustan-Smith Elaine , Suzuki Toshio , Neale Geoffrey A. , Mihara Keichiro , Pui Ching-Hon , Campana Dario
Published: July 2002

To identify new markers of minimal residual disease (MRD) in B-lineage acute lymphoblastic leukemia (ALL), gene expression of leukemic cells obtained from 4 patients with newly diagnosed ALL was compared with that of normal CD19+CD10+ B-cell progenitors obtained from 2 healthy donors. By cDNA array analysis, 334 of 4132 genes studied were expressed 1.5- to 5.8-fold higher in leukemic cells relative to both normal samples; 238 of these genes were also overexpressed in the leukemic cell line RS4;11. Nine genes were selected among the 274 overexpressed in at least 2 leukemic samples, and expression of the encoded proteins was measured by flow cytometry. Two proteins (caldesmon and myeloid nuclear differentiation antigen) were only weakly expressed in leukemic cells despite strong hybridization signals in the array. By contrast, 7 proteins (CD58, creatine kinase B, ninjurin1, Ref1, calpastatin, HDJ-2, and annexin VI) were expressed in B-lineage ALL cells at higher levels than in normal CD19+CD10+ B-cell progenitors (P < .05 in all comparisons). CD58 was chosen for further analysis because of its abundant and prevalent overexpression. An anti-CD58 antibody identified residual leukemic cells (0.01% to 1.13%; median, 0.03%) in 9 of 104 bone marrow samples from children with ALL in clinical remission. MRD estimates by CD58 staining correlated well with those of polymerase chain reaction amplification of immunoglobulin genes. These results indicate that studies of gene expression with cDNA arrays can aid the discovery of leukemia markers.
Ionizing radiation sensitizes erythroleukemic cells but not normal erythroblasts to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–mediated cytotoxicity by selective up-regulation of TRAIL-R1
Authors: Di Pietro Roberta , Secchiero Paola , Rana Rosalba , Gibellini Davide , Visani Giuseppe , Bemis Kristi , Zamai Loris , Miscia Sebastiano , Zauli Giorgio
Published: July 2002

Cytotoxic activity of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo-2 ligand), used alone or in different combinations with either a low (1.5 Gy) or a high (15 Gy) single dose of ionizing radiation (IR), was investigated on erythroleukemic cells (K562, HEL, Friend, primary leukemic erythroblasts) and on primary CD34+-derived normal erythroblasts. Human recombinant TRAIL alone variably affected the survival/growth of erythroleukemic cells; K562 cells were the most sensitive. Moreover, all erythroleukemic cells were radio-resistant, as demonstrated by the fact that cytotoxicity was evident only after treatment with high-dose (15 Gy) IR. Remarkably, when IR and TRAIL were used in combination, an additive effect was noticed in all erythroleukemic cells. Augmentation of TRAIL-induced cell death by IR was observed with both low and high IR doses and required the sequential treatment of IR 3 to 6 hours before the addition of TRAIL. Conversely, both TRAIL and IR showed a moderate cytotoxicity on primary CD34+-derived normal erythroblasts when used alone, but their combination did not show any additive effect. Moreover, the cytotoxicity of IR plus TRAIL observed in erythroleukemic cells was accompanied by the selective up-regulation of the surface expression of TRAIL-R1 (DR4), and it was completely blocked by the z-Val-Ala-Asp (OMe)-CH2 (z-VAD-fmk) caspase inhibitor. On the other hand, the surface expression of TRAIL-R1 in CD34+-derived normal erythroblasts was unaffected by IR, which induced the up-regulation of the decoy TRAIL-R3. These data demonstrate that treatment with IR provides an approach to selectively sensitize erythroleukemic cells, but not normal erythroblasts, to TRAIL-induced apoptosis through the functional up-regulation of TRAIL-R1.
The upstream stimulatory factor-2a inhibits plasminogen activator inhibitor-1 gene expression by binding to a promoter element adjacent to the hypoxia-inducible factor-1 binding site
Authors: Samoylenko Anatoly , Roth Ulrike , Jungermann Kurt , Kietzmann Thomas
Published: July 2002

Plasminogen activator inhibitor-1 (PAI-1) expression is induced by hypoxia (8% O2) via the PAI-1 promoter region −175/−159 containing a hypoxia response element (HRE-2) binding the hypoxia-inducible factor-1 (HIF-1) and an adjacent response element (HRE-1) binding a so far unknown factor. The aim of the present study was to identify this factor and to investigate its role in the regulation of PAI-1 expression. It was found by supershift assays that the upstream stimulatory factor-2a (USF-2a) bound mainly to the HRE-1 of the PAI-1 promoter and to a lesser extent to HRE-2. Overexpression of USF-2a inhibited PAI-1 messenger RNA and protein expression and activated L-type pyruvate kinase expression in primary rat hepatocytes under normoxia and hypoxia. Luciferase (Luc) gene constructs driven by 766 and 276 base pairs of the 5′-flanking region of the PAI-1 gene were transfected into primary hepatocytes together with expression vectors encoding wild-type USF-2a and a USF-2a mutant lacking DNA binding and dimerization activity (ΔHU2a). Cotransfection of the wild-type USF-2a vector reduced Luc activity by about 8-fold, whereas cotransfection of ΔHU2a did not influence Luc activity. Mutation of the HRE-1 (−175/−168) in the PAI-1 promoter Luc constructs decreased USF-dependent inhibition of Luc activity. Mutation of the HRE-2 (−165/−158) was less effective. Cotransfection of a HIF-1α vector could compete for the binding of USF at HRE-2. These results indicated that the balance between 2 transcriptional factors, HIF-1 and USF-2a, which can bind adjacent HRE sites, appears to be involved in the regulation of PAI-1 expression in many clinical conditions.
Surgical and physical stress increases circulating blood dendritic cell counts independently of monocyte counts
Authors: Ho Christopher S. K. , López Jose Alejandro , Vuckovic Slavica , Pyke Christopher M. , Hockey Richard L. , Hart Derek N. J.
Published: July 2002

Dendritic cells (DCs) are specialized antigen-presenting cells that have the unique ability to initiate a primary immune response. The effect of physiologic stress on circulating blood DCs has thus far not been studied. In this study, we applied a recently developed method of counting blood DCs to test the hypothesis that significant stress to the body such as surgery and exercise might induce measurable changes in the DC numbers, subsets, phenotype, and function. Twenty-six patients scheduled for elective laparoscopic cholecystectomy, 4 for elective hysterectomy, 56 controls, and 5 volunteers who underwent a stress exercise test were enrolled in the study. Absolute DC counts increased acutely (71.7% ± 11% [SEM],P = .0001) in response to the stress of surgery and dropped below preoperative levels (−25% ± 14% [SEM],P = .05) on days 2-3. The perioperative DC subset balance remained constant. Interestingly, DC counts changed independently of monocyte counts. Exercise also induced a rise in DC counts but coincidentally with monocyte counts. Surprisingly, no phenotypic or functional activation of DCs was seen in either stress situations in vivo. DCs are rapidly mobilized into the circulation in response to surgical and exercise stress, which may serve to prepare the host's immune defenses against trauma. The independent regulation of the DC and monocyte counts reinforces the distinction between these 2 cell populations.
Platelet characteristics in patients with X-linked macrothrombocytopenia because of a novel GATA1mutation
Authors: Freson Kathleen , Devriendt Koen , Matthijs Gert , Van Hoof Achiel , De Vos Rita , Thys Chantal , Minner Kristien , Hoylaerts Marc F. , Vermylen Jos , Van Geet Chris
Published: July 2002

A new mutation is described in the X-linked geneGATA1, resulting in macrothrombocytopenia and mild dyserythropoietic features but no marked anemia in a 4-generation family. The molecular basis for the observed phenotype is a substitution of glycine for aspartate in the strictly conserved codon 218 (D218G) of the amino-terminal zinc finger loop of the transcription factor GATA1. Zinc finger interaction studies demonstrated that this mutation results in a weak loss of affinity of GATA1 for its essential cofactor FOG1, whereas direct D218G-GATA1 binding to DNA was normal. The phenotypic effects of this mutation in the patients' platelets have been studied. Semiquantitative RNA analysis, normalized for β-actin messenger RNA, showed extremely low transcription of the GATA1 target genes GPIbβ and GPIXbut also a significantly lower expression of the nondirectly GATA1-regulated Gsα gene, suggestive of incomplete megakaryocyte maturation. In contrast, GPIIIa expression was close to normal in agreement with its early appearance during megakaryocyte differentiation. Flow cytometric analysis of patient platelets confirmed the existence of a platelet population with abnormal size distribution and reduced GPIb complex levels but with normal GPIIIa expression. It also showed the presence of very immature platelets lacking almost all membrane glycoproteins studied (GPIbα, GPIbβ, GPIIIa, GPIX, and GPV). Patients' platelets showed weak ristocetin-induced agglutination, compatible with the disturbed GPIb complex. Accordingly, electron microscopy of the patients' platelets revealed giant platelets with cytoplasmic clusters consisting of smooth endoplasmic reticulum and abnormal membrane complexes. In conclusion,GATA1 mutations can lead to isolated X-linked macrothrombocytopenia without anemia.
The acute promyelocytic leukemia–associated protein, promyelocytic leukemia zinc finger, regulates 1,25-dihydroxyvitamin D3–induced monocytic differentiation of U937 cells through a physical interaction with vitamin D3receptor
Authors: Ward Jeremy O. , McConnell Melanie J. , Carlile Graeme W. , Pandolfi Pier Paolo , Licht Jonathan D. , Freedman Leonard P.
Published: July 2002

Monocyte differentiation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is interrupted during the course of acute promyelocytic leukemia (APL). One form of APL is associated with the translocation t(11;17), which joins the promyelocytic leukemia zinc finger (PLZF) and retinoic acid receptor α (RARα) genes. Because PLZF is coexpressed in the myeloid lineage with the vitamin D3 receptor (VDR), the interplay between PLZF and VDR was examined. It was found that PLZF interacts directly with VDR. This occurred at least partly through contacts in the DNA-binding domain of VDR and the broad complex, tram-trak, bric-a-brac/pox virus zinc finger (BTB/POZ) domain of PLZF. Moreover, PLZF altered the mobility of VDR derived from nuclear extracts when bound to its cognate binding site, forming a slowly migrating DNA-protein complex. Overexpression of PLZF in a monocytic cell line abrogated 1,25(OH)2D3 activation from both a minimal VDR responsive reporter and the promoter of p21WAF1/CIP1, a target gene of VDR. Deletion of the BTB/POZ domain significantly relieved PLZF-mediated repression of 1,25(OH)2D3-dependent activation. In addition, stable, inducible expression of PLZF in U937 cells inhibited the ability of 1,25(OH)2D3 to induce surface expression of the monocytic marker CD14 and morphologic changes associated with differentiation. These results suggest that PLZF may play an important role in regulating the process by which 1,25(OH)2D3 induces monocytic differentiation in hematopoietic cells.
Combined genomic and antisense analysis reveals that the transcription factor Erg is implicated in endothelial cell differentiation
Authors: McLaughlin Fiona , Ludbrook Valerie J. , Cox Joanne , von Carlowitz Ira , Brown Suzy , Randi Anna M.
Published: July 2002

It has recently been shown that the transcription factor Erg, an Ets family member, drives constitutive expression of the intercellular adhesion molecule 2 (ICAM-2) in human umbilical vein endothelial cells (HUVECs) and that its expression is down-regulated by the pleiotropic cytokine tumor necrosis factor α (TNF-α). To identify other Erg target genes and to define its function in the endothelium, a combined approach of antisense oligonucleotides (GeneBloc) and differential gene expression was used. Treatment of HUVECs with Erg-specific GeneBloc for 24, 48, and 72 hours suppressed Erg mRNA and protein levels at all time points. Total RNA extracted from HUVECs treated withErg-specific or control GeneBloc was analyzed for differences in gene expression using high-density, sequence-verified cDNA arrays containing 482 relevant genes. Inhibition ofErg expression resulted in decreased expression ofICAM-2, as predicted. Four more genes decreased in Erg-deficient HUVECs were the extracellular matrix proteinsSPARC and thrombospondin, the adhesive glycoprotein von Willebrand factor, and the small GTPaseRhoA. Each of these molecules has been directly or indirectly linked to angiogenesis because of its role in vascular remodeling, adhesion, or shape change. Therefore, the role of Erg in vascular remodeling was tested in an in vitro model, and the results showed that HUVECs treated with Erg GeneBloc had a decreased ability to form tubulelike structures when grown on Matrigel. These results suggest that Erg may be a mediator of the TNF-α effects on angiogenesis in vivo.
Clinical and virologic characteristics of chronic active Epstein-Barr virus infection
Authors: Kimura Hiroshi , Hoshino Yo , Kanegane Hirokazu , Tsuge Ikuya , Okamura Takayuki , Kawa Keisei , Morishima Tsuneo
Published: July 2002

Thirty patients with chronic active Epstein-Barr virus (CAEBV) infection were analyzed. The study group included 18 male and 12 female patients, ranging in age from 5 to 31 years with a mean age of 14.2 years. Not all patients had high titers of EBV-specific antibodies, but all patients had high viral loads in their peripheral blood (more than 102.5 copies/μg DNA). Fifty percent of the patients displayed chromosomal aberrations, and 79% had monoclonality of EBV. Patients were divided into 2 clinically distinct groups, based on whether the predominantly infected cells in their peripheral blood were T cells or natural killer (NK) cells. Over a 68-month period of observation, 10 patients died from hepatic failure, malignant lymphoma, or other causes. Patients with T-cell CAEBV had a shorter survival time than those with NK-cell type of disease.
The functional interactions between CD98, β1-integrins, and CD147 in the induction of U937 homotypic aggregation
Authors: Cho Jae Youl , Fox David A. , Horejsi Vaclav , Sagawa Kimitaka , Skubitz Keith M. , Katz David R. , Chain Benjamin
Published: July 2002

CD98 is expressed on both hematopoietic and nonhematopoietic cells and has been implicated in a variety of different aspects of cell physiology and immunobiology. In this study, the functional interactions between CD98 and other adhesion molecules on the surface of the promonocyte line U937 are examined by means of a quantitative assay of cell aggregation. Several of the CD98 antibodies induced homotypic aggregation of these cells without affecting cellular viability or growth. Aggregation induced by CD98 antibodies could be distinguished from that induced by β1-integrin (CD29) ligation by lack of sensitivity to EDTA and by increased sensitivity to deoxyglucose. Aggregation induced via CD98 and CD29 could also be distinguished by the pattern of protein tyrosine phosphorylation induced. Some CD29 antibodies partially inhibited CD98-induced aggregation, and these antibodies were neither agonistic for aggregation nor inhibitors of β1-integrin binding to substrates. Conversely, some CD98 antibodies were potent inhibitors of CD29-induced aggregation. Antibodies to β2 integrins also partially inhibited CD98-induced aggregation. Unexpectedly, 2 antibodies to CD147, an immunoglobulin superfamily member whose function has remained unclear, were also potent inhibitors of both the aggregation and the protein tyrosine phosphorylation induced via CD98 ligation. The results of this study support a central role for CD98 within a multimolecular unit that regulates cell aggregation.
Genomic and proteomic analysis of the myeloid differentiation program
Authors: Lian Zheng , Wang Le , Yamaga Shigeru , Bonds Wesley , Beazer-Barclay Y. , Kluger Yuval , Gerstein Mark , Newburger Peter E. , Berliner Nancy , Weissman Sherman M.
Published: July 2002

Although the mature neutrophil is one of the better characterized mammalian cell types, the mechanisms of myeloid differentiation are incompletely understood at the molecular level. A mouse promyelocytic cell line (MPRO), derived from murine bone marrow cells and arrested developmentally by a dominant-negative retinoic acid receptor, morphologically differentiates to mature neutrophils in the presence of 10 μM retinoic acid. An extensive catalog was prepared of the gene expression changes that occur during morphologic maturation. To do this, 3′-end differential display, oligonucleotide chip array hybridization, and 2-dimensional protein electrophoresis were used. A large number of genes whose mRNA levels are modulated during differentiation of MPRO cells were identified. The results suggest the involvement of several transcription regulatory factors not previously implicated in this process, but they also emphasize the importance of events other than the production of new transcription factors. Furthermore, gene expression patterns were compared at the level of mRNA and protein, and the correlation between 2 parameters was studied.
The Hox cofactor and proto-oncogene Pbx1 is required for maintenance of definitive hematopoiesis in the fetal liver
Authors: DiMartino Jorge F. , Selleri Licia , Traver David , Firpo Meri T. , Rhee Joon , Warnke Roger , O'Gorman Stephen , Weissman Irving L. , Cleary Michael L.
Published: July 2002

Pbx1 is the product of a proto-oncogene originally discovered at the site of chromosomal translocations in acute leukemias. It binds DNA as a complex with a broad subset of homeodomain proteins, but its contributions to hematopoiesis have not been established. This paper reports that Pbx1 is expressed in hematopoietic progenitors during murine embryonic development and that its absence results in severe anemia and embryonic lethality at embryonic day 15 (E15) or E16. Definitive myeloerythroid lineages are present inPbx1−/−fetal livers, but the total numbers of colony-forming cells are substantially reduced. Fetal liver hypoplasia reflects quantitative as well as qualitative defects in the most primitive multilineage progenitors and their lineage-restricted progeny. Hematopoietic stem cells from Pbx1−/−embryos have reduced colony-forming activity and are unable to establish multilineage hematopoiesis in competitive reconstitution experiments. Common myeloid progenitors (CMPs), the earliest known myeloerythroid-restricted progenitors, are markedly depleted inPbx1−/−embryos at E14 and display clonogenic defects in erythroid colony formation. Comparative cell-cycle indexes suggest that these defects result largely from insufficient proliferation. Megakaryocyte- and erythrocyte-committed progenitors are also reduced in number and show decreased erythroid colony-forming potential. Taken together, these data indicate that Pbx1 is essential for the function of hematopoietic progenitors with erythropoietic potential and that its loss creates a proliferative constriction at the level of the CMP. Thus, Pbx1 is required for the maintenance, but not the initiation, of definitive hematopoiesis and contributes to the mitotic amplifications of progenitor subsets through which mature erythrocytes are generated.
Myelopoiesis in the zebrafish, Danio rerio
Authors: Bennett Carolyn M. , Kanki John P. , Rhodes Jennifer , Liu Ting X. , Paw Barry H. , Kieran Mark W. , Langenau David M. , Delahaye-Brown Anne , Zon Leonard I. , Fleming Mark D. , Look A. Thomas
Published: July 2002

Genome-wide chemical mutagenesis screens in the zebrafish(Danio rerio) have led to the identification of novel genes affecting vertebrate erythropoiesis. In determining if this approach could also be used to clarify the molecular genetics of myelopoiesis, it was found that the developmental hierarchy of myeloid precursors in the zebrafish kidney is similar to that in human bone marrow. Zebrafish neutrophils resembled human neutrophils, possessing segmented nuclei and myeloperoxidase-positive cytoplasmic granules. The zebrafish homologue of the human myeloperoxidase (MPO) gene, which is specific to cells of the neutrophil lineage, was cloned and used to synthesize antisense RNA probes for in situ hybridization analyses of zebrafish embryos. Granulocytic cells expressing zebrafishmpo were first evident at 18 hours after fertilization (hpf) in the posterior intermediate cell mass (ICM) and on the anterior yolk sac by 20 hpf. By 24 hpf, mpo-expressing cells were observed along the ICM and within the developing vascular system. Thus, the mpo gene should provide a useful molecular probe for identifying zebrafish mutants with defects in granulopoiesis. The expression of zebrafish homologues was also examined in 2 other mammalian hematopoietic genes, Pu.1, which appears to initiate a commitment step in normal mammalian myeloid development, andL-Plastin, a gene expressed by human monocytes and macrophages. The results demonstrate a high level of conservation of the spatio-temporal expression patterns of these genes between zebrafish and mammals. The morphologic and molecular genetic evidence presented here supports the zebrafish as an informative model system for the study of normal and aberrant human myelopoiesis.
Ascorbic acid enhances arsenic trioxide–induced cytotoxicity in multiple myeloma cells
Authors: Grad Jennifer M. , Bahlis Nizar J. , Reis Isildinha , Oshiro Marc M. , Dalton William S. , Boise Lawrence H.
Published: July 2002

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by slow-growing plasma cells in the bone marrow (BM). Patients with MM typically respond to initial chemotherapies; however, essentially all progress to a chemoresistant state. Factors that contribute to the chemorefractory phenotype include modulation of free radical scavenging, increased expression of drug efflux pumps, and changes in gene expression that allow escape from apoptotic signaling. Recent data indicate that arsenic trioxide (As2O3) induces remission of refractory acute promyelocytic leukemia and apoptosis of cell lines overexpressing Bcl-2 family members; therefore, it was hypothesized that chemorefractory MM cells would be sensitive to As2O3. As2O3 induced apoptosis in 4 human MM cell lines: 8226/S, 8226/Dox40, U266, and U266/Bcl-xL. The addition of interleukin-6 had no effect on cell death. Glutathione (GSH) has been implicated as an inhibitor of As2O3-induced cell death either through conjugating As2O3 or by sequestering reactive oxygen induced by As2O3. Consistent with this possibility, increasing GSH levels with N-acetylcysteine attenuated As2O3 cytotoxicity. Decreases in GSH have been associated with ascorbic acid (AA) metabolism. Clinically relevant doses of AA decreased GSH levels and potentiated As2O3-mediated cell death of all 4 MM cell lines. Similar results were obtained in freshly isolated human MM cells. In contrast, normal BM cells displayed little sensitivity to As2O3 alone or in combination with AA. Together, these data suggest that As2O3 and AA may be effective antineoplastic agents in refractory MM and that AA might be a useful adjuvant in GSH-sensitive therapies.
Antibodies from patients with heparin-induced thrombocytopenia stimulate monocytic cells to express tissue factor and secrete interleukin-8
Authors: Arepally Gowthami M. , Mayer Irene M.
Published: July 2002

Thrombosis is a life-threatening complication that occurs in a subset of patients with heparin-induced thrombocytopenia (HITT). The pathogenic mechanisms underlying the variable occurrence of thrombosis in HITT is poorly understood. It was hypothesized that monocyte activation leading to tissue factor expression may play a role in promoting a thrombogenic state in HITT. This study demonstrates that a human platelet factor 4 (PF4)/heparin-specific murine monoclonal antibody (KKO) binds to peripheral blood-derived human monocytes in a PF4-dependent manner. KKO and antibodies from patients with HITT induce monocytes to synthesize and secrete interleukin-8 and induce cell-surface procoagulant activity, which is abrogated following treatment with antihuman tissue factor antibody. The findings suggest a novel mechanism by which PF4/heparin antibodies may promote a hypercoagulable state in patients with HITT.
Specific von Willebrand factor–cleaving protease in thrombotic microangiopathies: a study of 111 cases
Authors: Veyradier Agnès , Obert Bernadette , Houllier Anne , Meyer Dominique , Girma Jean-Pierre
Published: July 2002

Retrospective studies of patients with thrombotic microangiopathies (TMAs) have shown that a deficient activity of von Willebrand factor (vWF)–cleaving protease is involved in thrombotic thrombocytopenic purpura (TTP) but not in the hemolytic-uremic syndrome (HUS). To further analyze the relevance of this enzymatic activity in TMA diagnosis, a 20-month multicenter study of vWF-cleaving protease activity was conducted in adult patients prospectively enrolled in the acute phase of TMA. Patients with sporadic (n = 85), intermittent (n = 21), or familial recurrent (n = 5) forms of TMA (66 manifesting as TTP and 45 as HUS) were included. TMA was either idiopathic (n = 42) or secondary to an identified clinical context (n = 69). vWF-cleaving protease activity was normal in 46 cases (7 TTP and 39 HUS) and decreased in 65 cases (59 TTP and 6 HUS). A protease inhibitor was detected in 31 cases and was observed only in patients manifesting TTP with a total absence of protease activity. Among the 111 patients, mean vWF antigen levels were increased and the multimeric distribution of vWF was very heterogeneous, showing either a defect of the high-molecular-weight forms (n = 40), a normal pattern (n = 21), or the presence of unusually large multimers (n = 50). Statistical analysis showed that vWF-protease deficiency was associated with the severity of thrombocytopenia (P < .01). This study emphasizes that vWF-cleaving protease deficiency specifically concerns a subgroup of TMA corresponding to the TTP entity.
Bifurcation of osteoclasts and dendritic cells from common progenitors
Authors: Miyamoto Takeshi , Ohneda Osamu , Arai Fumio , Iwamoto Katsuya , Okada Seiji , Takagi Katsumasa , Anderson Dirk M. , Suda Toshio
Published: July 2002

Osteoclasts and dendritic cells are derived from monocyte/macrophage precursor cells; however, how their lineage commitment is regulated is unknown. This study investigated the differentiation pathways of osteoclasts and dendritic cells from common precursor cells at the single-cell level. Osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor–κB ligand (RANKL) or tumor necrosis factor-α (TNF-α) is completely inhibited by addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 at early stages of differentiation. GM-CSF–treated cells express both c-Fms and RANK and also low levels of CD11c and DEC205, which are detected on dendritic cells. Addition of GM-CSF also reduces expression of both c-Fos and Fra-1, which is an important event for inhibition of osteoclastogenesis. Overexpression of c-Fos by retroviral infection or induction in transgenic mice can rescue a failure in osteoclast differentiation even in the presence of GM-CSF. By contrast, differentiation into dendritic cells is inhibited by M-CSF, indicating that M-CSF and GM-CSF reciprocally regulate the differentiation of both lineages. Dendritic cell maturation is also inhibited when c-Fos is expressed at an early stage of differentiation. Taken together, these findings suggest that c-Fos is a key mediator of the lineage commitment between osteoclasts and dendritic cells. The lineage determination of osteoclast progenitors seen following GM-CSF treatment functions through the regulation of c-Fos expression.
The MLL fusion partner AF10 binds GAS41, a protein that interacts with the human SWI/SNF complex
Authors: Debernardi Silvana , Bassini Alessandra , Jones Louise K. , Chaplin Tracy , Linder Britta , de Bruijn Diederik R. H. , Meese Eckart , Young Bryan D.
Published: July 2002

The AF10 gene encodes a putative transcription factor containing an N-terminal LAP/PHD zinc finger motif, a functional nuclear localization signal, an AT-hook domain, and a leucine zipper toward the C-terminus. AF10 is involved in 2 distinct chromosomal translocations associated with hematologic malignancy. The chimeric fusion proteins MLL/AF10 and CALM/AF10, resulting from the t(10;11)(p12;q23) and the t(10;11)(p12;q14), respectively, consistently retain the leucine zipper motif of AF10. This part of the C-terminal region was used as bait in a yeast 2 hybrid screening of a testis complementary DNA library. The leucine zipper interacted with GAS41, a protein previously identified as the product of an amplified gene in a glioblastoma. GAS41 shows significant homology to theSaccharomyces cerevisiae protein ANC1 and to the human MLL fusion partners AF9 and ENL. The interaction was confirmed in vivo. Furthermore, the study showed by coimmunoprecipitation that GAS41 interacts with INI1 (Integrase Interactor 1) and that INI1 was present in the AF10 immunoprecipitate. INI1 is the human homologue of the yeast SNF5 protein, a component of the SWI/SNF complex, which acts to remodel chromatin and to modulate transcription. The retention of the leucine zipper in the MLL and CALM fusions suggests that a key feature of these chimeric proteins may be their ability to interfere in normal gene regulation through interaction with the adenosine triphosphate–dependent chromatinremodeling complexes.
Correlation of human T-cell lymphotropic virus type 1 (HTLV-1) mRNA with proviral DNA load, virus-specific CD8+ T cells, and disease severity in HTLV-1–associated myelopathy (HAM/TSP)
Authors: Yamano Yoshihisa , Nagai Masahiro , Brennan Meghan , Mora Carlos A. , Soldan Samantha S , Tomaru Utano , Takenouchi Norihiro , Izumo Shuji , Osame Mitsuhiro , Jacobson Steven
Published: July 2002

To investigate the role of viral expression in individuals infected with human T-cell lymphotropic virus type 1 (HTLV-1), a real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) of HTLV-1 tax messenger RNA (mRNA) using ABI Prism 7700 Sequence Detection System was developed. Using this system, the HTLV-1tax mRNA load was compared with HTLV-1 proviral DNA load, HTLV-1 Tax protein expression, HTLV-1 Tax-specific CD8+T-cell frequency, and disease severity of HTLV-1–associated myelopathy/tropical spastic paraparesis (HAM/TSP). This approach was a sensitive and specific technique for the precise quantification of HTLV-1 tax mRNA. The total amount of HTLV-1 taxmRNA and mRNA expression level in HTLV-1–infected cells (mRNA/DNA ratio) were higher in HAM/TSP patients than in asymptomatic HTLV-1 carriers. The HTLV-1 tax mRNA load correlated with the HTLV-1 proviral DNA load ex vivo, the Tax protein expression in vitro, and the Tax-specific CD8+ T-cell frequency ex vivo. The HTLV-1 tax mRNA load also correlated with disease severity in HAM/TSP patients. These data suggest that increased HTLV-1 expression plays an important role in the pathogenesis of HAM/TSP, and the HTLV-1 tax mRNA level could be a useful predictor of disease progression in patients with HAM/TSP.
Increased and prolonged inflammation and angiogenesis in delayed-type hypersensitivity reactions elicited in the skin of thrombospondin-2–deficient mice
Authors: Lange-Asschenfeldt Bernhard , Weninger Wolfgang , Velasco Paula , Kyriakides Themis R. , von Andrian Ulrich H. , Bornstein Paul , Detmar Michael
Published: July 2002

Angiogenesis and enhanced microvascular permeability are hallmarks of a large number of inflammatory diseases. Although up-regulation of proangiogenic factors such as vascular endothelial growth factor and interleukin-8 have been previously reported in inflamed tissue, the biologic role of endogenous inhibitors of angiogenesis in inflammation has remained unclear. To investigate the biologic role of the potent angiogenesis inhibitor thrombospondin-2 (TSP-2) in the control of cutaneous inflammation, delayed-type hypersensitivity reactions were elicited in the ear skin of wild-type and TSP-2–deficient mice by topical sensitization and challenge with oxazolone. Cutaneous TSP-2 expression was up-regulated in the inflamed skin of wild-type mice, predominantly in dermal fibroblasts and microvessels. Lack of TSP-2 resulted in a significantly enhanced inflammatory response with increased angiogenesis, edema formation, and inflammatory infiltration. Ear swelling and inflammation persisted for more than 2 weeks in TSP-2–deficient mice, as compared with 1 week in wild-type mice. Although baseline vascular permeability was unchanged, significantly enhanced microvascular leakage was found in the inflamed skin of TSP-2–deficient mice. Moreover, the fraction of rolling leukocytes was significantly increased in the untreated skin of TSP-2–deficient mice. These results reveal an important role of TSP-2 in limiting the extent and the duration of edema formation, angiogenesis, and inflammatory cell infiltration during acute and chronic inflammation.
The mechanism of tumor cell clearance by rituximab in vivo in patients with B-cell chronic lymphocytic leukemia: evidence of caspase activation and apoptosis induction
Authors: Byrd John C. , Kitada Shinichi , Flinn Ian W. , Aron Jennifer L. , Pearson Michael , Lucas David , Reed John C.
Published: July 2002

Rituximab is a chimeric monoclonal antibody directed at CD20 with significant activity in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). A variety of pathways of tumor cytotoxicity different from cytotoxic chemotherapy have been proposed for this therapeutic antibody including antibody-dependent cellular cytotoxicity and complement-mediated cell lysis. This report describes that a proportion of patients with CLL receiving rituximab treatment have in vivo activation of caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP) cleavage in blood leukemia cells immediately following infusion of rituximab. This suggests that apoptosis using a pathway similar to fludarabine and other chemotherapeutic agents is intricately involved in the blood elimination of tumor cells after rituximab treatment. Patients having caspase-3 activation and PARP cleavage in vivo had a significantly lower blood leukemia cell count after treatment as compared to those without caspase activation. Significant down-modulation of the antiapoptotic proteins XIAP and Mcl-1 was also noted, possibly explaining in part how rituximab sensitizes CLL cells to the cytotoxic effect of chemotherapy in vivo. These findings suggest that the therapeutic benefit of antibody-based therapy in vivo for patients with CLL depends in part on induction of apoptosis and provides another area of focus for studying mechanisms of antibody-resistance in neoplastic cells.
The cytokines IL-3 and GM-CSF regulate the transcriptional activity of retinoic acid receptors in different in vitro models of myeloid differentiation
Authors: Johnson Barton S. , Mueller LeMoyne , Si Jutong , Collins Steven J.
Published: July 2002

The disruption of retinoic acid receptor (RAR) activity that characterizes human acute promyelocytic leukemia (APL) is associated with a block to granulocytic differentiation indicating that RARs are critical regulators of normal myeloid differentiation. Nevertheless, how RAR activity might be regulated in the presumably homogenous concentration of retinoids in blood and bone marrow and how these receptors might interact with specific hematopoietic cytokines to regulate normal myeloid differentiation remain unclear. Here, using several cytokine-dependent in vitro models of myeloid development, it was observed that specific hematopoietic cytokines that normally regulate myeloid lineage commitment and differentiation (interleukin-3 and granulocyte-macrophage colony-stimulating factor) trigger the enhancement of both ligand-dependent and ligand-independent transcriptional activity of both endogenous and exogenous (transiently transfected) RARs. This cytokine-mediated enhancement of RAR activity is not associated with any observed changes in expression of the RARs or their respective coactivators/corepressors. These studies define a previously unknown cytokine-RAR interaction during myelopoiesis and suggest that RAR activation might be a critical downstream event following interleukin-3 and granulocyte-macrophage colony-stimulating factor signaling during myeloid differentiation. This observation of ligand-independent activation of RARs that is mediated by certain cytokines represents a new paradigm with respect to how RAR activity might be modulated during hematopoiesis and also suggests a molecular basis for the differential sensitivity of human acute myelogenous leukemia cells to retinoids.
Unique patterns of surface receptors, cytokine secretion, and immune functions distinguish T cells in the bone marrow from those in the periphery: impact on allogeneic bone marrow transplantation
Authors: Zeng Defu , Hoffmann Petra , Lan Fengshuo , Huie Philip , Higgins John , Strober Samuel
Published: July 2002

The “conventional” NK1.1− T cells from mouse blood and marrow were compared with regard to surface receptors, cytokine secretion, and function. Most blood NK1.1−CD4+ and CD8+ T cells expressed the naive CD44int/loCD62LhiCD45RBhiT-cell phenotype typical of those in the peripheral lymphoid tissues. In contrast, most marrow NK1.1−CD4+ and CD8+ T cells expressed an unusual CD44hiCD62LhiCD45RBhiphenotype. The blood NK1.1− CD4+ T cells had a naive T-helper cytokine profile and a potent capacity to induce lethal graft versus host (GVH) disease in a C57BL/6 donor to a BALB/c host bone marrow transplantation model. In contrast, the marrow NK1.1− CD4+ T cells had a Th0 cytokine profile and failed to induce lethal GVH disease, even at 20-fold higher numbers than those from the blood. NK1.1− CD8+ T cells from the blood but not the marrow induced lethal GVH disease. Nevertheless, the marrow NK1.1− CD8+ T cells induced potent antitumor activity that was augmented by marrow NK1.1− CD4+ T cells and facilitated hematopoietic progenitor engraftment. The inability of marrow CD4+ and CD8+ T cells to induce GVH disease was associated with their inability to expand in the blood and gut of allogeneic recipients. Because neither the purified marrow CD4+ or CD8+ T cells induced GVH disease, their unique features are desirable for inclusion in allogeneic bone marrow or hematopoietic progenitor transplants.
Residual factor VII activity and different hemorrhagic phenotypes in CRM+ factor VII deficiencies (Gly331Ser and Gly283Ser)
Authors: Pinotti Mirko , Etro Daniela , Bindini Debora , Papa Maria Luisa , Rodorigo Giuseppina , Rocino Angiola , Mariani Guglielmo , Ciavarella Nicola , Bernardi Francesco
Published: July 2002

Two cross-reacting material–positive (CRM+) factor VII (FVII) mutations, associated with similar reductions in coagulant activity (2.5%) but with mild to asymptomatic (Gly331Ser, c184 [in chymotrypsin numbering]) or severe (Gly283Ser, c140) hemorrhagic phenotypes, were investigated. The affected glycines belong to structurally conserved regions in the c184 through c193 and c140s activation domain loops, respectively. The natural mutants 331Ser-FVII and 283Ser-FVII were expressed, and in addition 331Ala-FVII and 283Ala-FVII were expressed because 3 functional serine-proteases bear alanine at these positions. The 331Ser-FVII, present in several asymptomatic subjects, showed detectable factor Xa generation activity in patient plasma (0.7% ± 0.2%) and in reconstituted system with the recombinant molecules (2.7% ± 1.1%). The reduced activity of recombinant 283Ala-FVII (7.2% ± 2.2%) indicates that the full function of FVII requires glycine at this position, and the undetectable activity of 283Ser-FVII suggests that the oxydrile group of Ser283 participates in causing severe CRM+ deficiency. Furthermore, in a plasma system with limiting thromboplastin concentration, 283Ser-FVII inhibited wild-type FVIIa activity in a dose-dependent manner.