bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites
Authors: Rance James B. , Follows George A. , Cockerill Peter N. , Bonifer Constanze , Lane David A. , Simmonds Rachel E. 
Published: May 2003

AbstractThe human endothelial cell protein C receptor (hEPCR) is normally expressed by the endothelium of large blood vessels, but the molecular basis for its in vivo specificity is uncertain. In this study, DNaseI hypersensitive site mapping demonstrated the presence of a hypersensitive site in the 5′ flanking region of the hEPCR gene in endothelial cells and certain transformed cells (HeLa and U937) known to express hEPCR in vitro. Conversely, this site was only weakly hypersensitive in HepG2 cells, cells which do not express hEPCR mRNA. Functional analysis of this 5′ flanking region by in vivo dimethylsulfate footprinting in cultured endothelial cells identified multiple regions, containing high and low homology consensus Sp1 binding sequences, that were protected from methylation in endothelial cells. These sequences were not protected in HepG2 cells. Reporter gene analysis of this region in endothelial cells demonstrated the presence of promoter activity conferred by the proximal 572 bp but failed to identify a functional TATA-box. This promoter was inactive in HepG2 cells. Electrophoresis mobility shift assays using endothelial cell nuclear extracts identified Sp1 family proteins binding to sites that were protected during footprinting. Sp1 sites were identified in regions at –368, –232, –226, –201, –146, and –102 bp relative to the translation start site. With the exception of the site at –102 bp, each identified Sp1 binding site made a positive contribution to reporter gene expression, although no individual site was critically important. We conclude that transcription factor binding to Sp1 binding sites in the 5′ flanking region is critical for normal hEPCR gene expression in endothelial cells.
Nonmyeloablative conditioning is sufficient to allow engraftment of EGFP-expressing bone marrow and subsequent acceptance of EGFP-transgenic skin grafts in mice
Authors: Andersson Goran , Illigens Ben M. W. , Johnson Kevin W. , Calderhead David , LeGuern Christian , Benichou Gilles , White-Scharf Mary E. , Down Julian D. 
Published: May 2003

AbstractImmunologic reactions against gene therapy products may prove to be a frequent problem in clinical gene therapy protocols. Enhanced green fluorescence protein (EGFP) is commonly used as a marker in gene transfer protocols, and immune responses against EGFP-expressing cells have been documented. The present study was designed to investigate the effect of a pharmacologic, nonmyeloablative, conditioning regimen on the development of EGFP+ donor/recipient mixed bone marrow chimerism and ensuing tolerance to EGFP-expressing transplants. To this end, C57BL/6J (B6) mice were treated with soluble formulations of either busulfan (Busulfex) or the closely related compound treosulfan, followed by transplantation of bone marrow cells from EGFP-transgenic (B6-EGFP.Tg) donor mice. Such conditioning regimens resulted in long-term persistence of donor EGFP+ cells among various hematopoietic lineages from blood, bone marrow, and thymus. Stable hematopoietic chimeras transplanted at 10 to 17 weeks after bone marrow transplantation (BMT) with B6-EGFP.Tg skin grafts all accepted their transplants, whereas non-EGFP chimeric B6 control animals were able to mount rejection of the EGFP+ B6 skin grafts. Control third-party grafts from major histocompatibility complex (MHC)–mismatched mice were rejected within 20 days, indicating that acceptance of EGFP-expressing skin grafts was the result of specific immune tolerance induction by the transplantation of EGFP-transgenic bone marrow. Long-term tolerance to EGFP in chimeric recipients was confirmed by the absence of anti-EGFP–reactive T cells and antibodies. These results broaden the therapeutic potential for using hematopoietic molecular chimerism in nonmyeloablated recipients as a means of preventing rejection of genetically modified cells.
The “stomatin” gene and protein in overhydrated hereditary stomatocytosis
Authors: Fricke Britta , Argent Annette C. , Chetty Margaret C. , Pizzey Arnold R. , Turner E. Jane , Ho Mei M. , Iolascon Achille , von Düring Monika , Stewart Gordon W. 
Published: May 2003

AbstractIn overhydrated hereditary stomatocytosis (OHSt), Coomassie- and silver-stained polyacrylamide gels show an apparently complete deficit of the 32-kDa membrane protein, stomatin. We have used an antistomatin antibody to examine peripheral blood films, bone marrow, splenic tissue, and hepatic tissue from these patients by immunocytochemistry. This technique revealed that, in fact, some red cells did show positive stomatin immunoreactivity; and consistent with this result, Western blot analysis of the red cell membranes confirmed that about one twentieth to one fiftieth of the normal amount of stomatin was in fact present. Flow cytometry, combining immunoreactive quantitation of stomatin expression with thiazole orange staining for reticulocytes, showed that in OHSt, it was the young cells that had more stomatin. Magnetic-activated cell separation studies, using beads to which an anti–transferrin receptor antibody was conjugated, confirmed that in OHSt there was a correspondence between expression of stomatin and the transferrin receptor. Immunocytochemistry and Western blotting revealed that in OHSt patients, the protein was present in spleen, liver, neutrophils, platelets, monocytes, and about 50% of the peripheral lymphocytes, with the same distribution as in healthy controls. Neither Southern blots, nor direct sequencing of multiple subclones of the cDNA, nor sequencing of amplicons from genomic DNA revealed any significant abnormality in stomatin gene sequence in these patients. The deficiency of stomatin from red cells appears to be due to a loss of stomatin from these red cells on maturation in the bone marrow and in the circulation.
L-selectin stimulation enhances functional expression of surface CXCR4 in lymphocytes: implications for cellular activation during adhesion and migration
Authors: Ding Ziqiang , Issekutz Thomas B. , Downey Gregory P. , Waddell Thomas K. 
Published: May 2003

AbstractL-selectin mediates leukocyte tethering and rolling, the first step in a sequential process of leukocyte adhesion and migration. Additionally, L-selectin has important signaling roles perhaps contributing to leukocyte activation and integrin-mediated adhesion. Because chemokines are critically involved in leukocyte activation, we questioned whether L-selectin signaling affects chemokine receptor expression and function. We observed that whereas only 5% to 15% of freshly isolated lymphocytes expressed CXCR4 on the cell surface, intracellular CXCR4 was detectable in all cells. Engagement of L-selectin by antibody cross-linking or the L-selectin ligands fucoidan or sulfatide mobilized intracellular CXCR4 to significantly increase surface CXCR4 expression but did not affect CCR5, CCR7, or β2-integrin expression. L-selectin stimulation also inhibited stromal-derived factor 1 (SDF-1)–induced CXCR4 internalization. The combined effects of L-selectin on CXCR4 trafficking are likely important in markedly enhancing cell activation by SDF-1. Blockade of SDF-1–induced CXCR4 internalization resulted in enhanced actin polymerization on subsequent exposure to SDF-1. Physiologically more important, L-selectin stimulation increased SDF-1–induced lymphocyte adhesion and transendothelial migration, which were inhibited by anti–leukocyte function-associated antigen 1 antibodies, tyrosine kinase inhibitors, and pertussis toxin. To further corroborate the additive stimulating effects, L-selectin signaling and SDF-1 increased β2-integrin activation. Taken together, L-selectin–mediated signals specifically enhance CXCR4 expression and function, suggesting a novel mechanism for the modulation of lymphocyte activation during cell adhesion and transmigration.
Activation of pp125FAK by type 2B recombinant von Willebrand factor binding to platelet GPIb at a high shear rate occurs independently of αIIbβ3 engagement
Authors: Mekrache Médina , Bachelot-Loza Christilla , Ajzenberg Nadine , Saci Abdelhafid , Legendre Paulette , Baruch Dominique 
Published: May 2003

AbstractShear-induced platelet aggregation (SIPA) involves the sequential interaction of von Willebrand factor (VWF) with both glycoprotein Ib (GPIb) and αIIbβ3 receptors. Type 2B recombinant VWF (2B-rVWF), characterized by an increased affinity for GPIb, induces strong SIPA at a high shear rate (4000 s–1). Despite the increased affinity of 2B-rVWF for GPIb, patients with type 2B von Willebrand disease have a paradoxical bleeding disorder, which is not well understood. The purpose of this study was to determine if SIPA induced by 2B-rVWF was associated with αIIbβ3-dependent platelet activation. To this end, we have addressed the influence of 2B-rVWF (Val553Met substitution) on SIPA-dependent variations of tyrosine protein phosphorylation (P-Tyr) and the effect of αIIbβ3 blockers. At a high shear rate, 2B-rVWF induced a strong SIPA, as shown by a 92.7% ± 0.4% disappearance of single platelets (DSP) after 4.5 minutes. In these conditions, increased P-Tyr of proteins migrating at positions 64 kd, 72 kd, and 125 kd were observed. The band at 125 kd was identified as pp125FAK using anti–phospho-FAK antibody. This effect, which required a high level of SIPA (&gt; 70% DSP), was observed at 4000 s–1 but not at 200 s–1. Monoclonal antibodies (MoAbs) 6D1 (anti-GPIb) and 328 (anti-VWF A1 domain), completely abolished SIPA and p125FAK phosphorylation mediated by 2B-rVWF. In contrast, neither RGDS peptide nor MoAb 7E3, both known to block αIIbβ3 engagement, had any effect on SIPA and pp125FAK. The size of aggregates formed at a high shear rate in the presence of 2B-rVWF was decreased by genistein, demonstrating the biologic relevance of pp125FAK. These findings provide a unique mechanism whereby the enhanced interaction of 2B-rVWF with GPIb, without engagement of αIIbβ3, is sufficient to induce SIPA but does not lead to stable thrombus formation.
Host stem cells can selectively reconstitute missing lymphoid lineages in irradiation bone marrow chimeras
Authors: Rivera Amariliz , Chen Chiann-Chyi , Dougherty Joseph P. , Ben-Nun Avraham , Ron Yacov 
Published: May 2003

AbstractThe regulatory elements governing the process of lymphopoiesis from pluripotential stem cells to mature lymphocytes are not well understood. In this study we found that in bone marrow chimeras made by reconstituting lethally irradiated normal mice with bone marrow taken from genetically B-cell–deficient animals (μMT.B6 → F1) the B-cell compartment is reconstituted with host-derived B cells. Similarly, in animals reconstituted with bone marrow taken from mice with genetic deficiencies in the development of T cells (TCR–/– → F1) or both B and T cells (RAG–/– → F1), the missing lymphocyte lineage(s) was specifically reconstituted from host-derived cells. In all chimeras, all other blood lineages were generated from donor-derived stem cells. Control chimeras (B6 → F1) had only donor-derived hematopoietic cells as expected. The reconstituted, host-derived lymphoid compartments contained normal functional cell populations as determined by the presence of T cells expressing all 16 common TCR Vβ families, and the presence of all antibody isotypes in the serum. Reconstituted TCR–/– → F1 chimeras were also able to mount T-cell proliferative responses to foreign antigens equal to those of control animals. This observation would seem to suggest that during lymphopoietic reconstitution, missing lymphoid lineages can dictate their own reconstitution.
Peripheral T-cell lymphomas unspecified presenting in the skin: analysis of prognostic factors in a group of 82 patients
Authors: Bekkenk Marcel W. , Vermeer Maarten H. , Jansen Patty M. , van Marion Ariënne M. W. , Canninga-van Dijk Marijke R. , Kluin Philip M. , Geerts Marie-Louise , Meijer Chris J. L. M. , Willemze Rein 
Published: May 2003

Abstract
In the present study the clinicopathologic and immunophenotypic features of 82 patients with a CD30– peripheral T-cell lymphoma, unspecified, presenting in the skin were evaluated. The purpose of this study was to find out whether subdivision of these lymphomas on the basis of cell size, phenotype, or presentation with only skin lesions is clinically relevant. The study group included 46 primary cutaneous CD30– large cell lymphomas and 17 small/medium-sized T-cell lymphomas as well as 17 peripheral T-cell lymphomas with both skin and extracutaneous disease at the time of diagnosis. Patients with primary cutaneous small- or medium-sized T-cell lymphomas had a significantly better prognosis (5-year-overall survival, 45%) than patients with primary cutaneous CD30– large T-cell lymphomas (12%) and patients presenting with concurrent extracutaneous disease (12%). The favorable prognosis in this group with primary cutaneous small- or medium-sized T-cell lymphomas was particularly found in patients presenting with localized skin lesions expressing a CD3+CD4+CD8– phenotype. In the primary cutaneous T-cell lymphoma (CTCL) group and in the concurrent group, neither extent of skin lesions nor phenotype had any effect on survival. Our results indicate that peripheral T-cell lymphomas, unspecified, presenting in the skin have an unfavorable prognosis, irrespective of the presence or absence of extracutaneous disease at the time of diagnosis, cell size, and expression of a CD4+ or CD8+ phenotype. The only exception was a group of primary cutaneous small- or medium-sized pleomorphic CTCLs with a CD3+CD4+CD8– phenotype and presenting with localized skin lesions.
Ectopic hTERT expression extends the life span of human CD4+ helper and regulatory T-cell clones and confers resistance to oxidative stress–induced apoptosis
Authors: Luiten Rosalie M. , Pène Jérôme , Yssel Hans , Spits Hergen 
Published: May 2003

AbstractHuman somatic cells have a limited life span in vitro. Upon aging and with each cell division, shortening of telomeres occurs, which eventually will lead to cell cycle arrest. Ectopic hTERT expression has been shown to extend the life span of human T cells by preventing this telomere erosion. In the present study, we have shown that ectopic hTERT expression extends the life span of CD4+ T helper type 1 or 2 and regulatory T-cell clones and affected neither the in vitro cytokine production profile nor their specificity for antigen. In mixed cell cultures, ectopic hTERT-expressing clones were found to expand in greater numbers than untransduced cells of the same replicative age. This ectopic hTERT-induced growth advantage was not due to an enhanced cell division rate or number of divisions following T-cell receptor–mediated activation, as determined in carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeling experiments. Moreover, the susceptibility to activation-induced cell death of both cell types was similar. However, cultures of resting hTERT-transduced T cells contained higher frequencies of Bcl-2–expressing cells and lower active caspase-3–expressing cells, compared with wild-type cells. Furthermore, hTERT-transduced cells were more resistant to oxidative stress, which causes preferential DNA damage in telomeres. Taken together, these results show that ectopic hTERT expression not only protects proliferating T cells from replicative senescence but also confers resistance to apoptosis induced by oxidative stress.
In vitro identification of human pro-B cells that give rise to macrophages, natural killer cells, and T cells
Authors: Reynaud Damien , Lefort Nathalie , Manie Elodie , Coulombel Laure , Levy Yves 
Published: May 2003

AbstractIn this study we report the molecular and functional characterization of very early interleukin 7 receptor α (IL-7Rα)+-CD79a+CD19– B-cell progenitors, produced by human CD34+CD19–CD10– cord blood cells grown in the presence of stromal cells and cytokines. Purified IL-7Rα+CD79a+CD19– cells transcribed the B-lymphoid specific genes E2A, EBF, TdT, Rag-1, had initiated DJH rearrangements, but almost lacked Pax-5 mRNA. When exposed to appropriate environmental conditions, these cells repressed B-cell genes and completely differentiated into CD14+ macrophages, CD56+ natural killer cells, and CD4high T cells. Retention of the DJH rearranged genes in both CD14+ and CD56+ cells unambiguously demonstrates that early B-cell genes, expressed prior to Pax-5, can be activated in a multipotent human progenitor cell whose final fate, including in non-B lineages, is determined by external signals.
The carboxy-terminal region of the granulocyte colony-stimulating factor receptor transduces a phagocytic signal
Authors: Santini Valeria , Scappini Barbara , Indik Zena K. , Gozzini Antonella , Rossi Ferrini Pierluigi , Schreiber Alan D. 
Published: May 2003

AbstractGranulocyte colony-stimulating factor (G-CSF) induces proliferation, maturation, and functional activities of myeloid progenitors and mature neutrophils through a specific receptor, the G-CSF-R. Different signals are mediated by distinct regions of the cytoplasmic domain of G-CSF-R, but the precise role of each region has not yet been fully clarified. We evaluated the involvement of Syk kinase, essential in mediating phagocytic signals by Fcγ receptors, in G-CSF–induced phagocytosis, using murine myeloid 32D cells transfected with wild-type (WT) human G-CSF-R (hG-CSF-R) or with a G-CSF-R mutant truncated at cytoplasmic amino acid 715. The G-CSF-R mutant lacks the immunoreceptor tyrosine-based activation motif (ITAM), putative binding site for Syk. Following treatment of WT hG-CSF-R transfectants with IgG-coated particles, there was a significant increase in phagocytosis in G-CSF–stimulated cells, in which Syk tyrosine phosphorylation occurred, paralleled by enhancement of its tyrosine kinase activity. In the mutant transfectants, no significant increase in phagocytosis or Syk tyrosine phosphorylation occurred after stimulation with G-CSF. We also demonstrated that tyrosine phosphorylation of the Src kinases Hck and Lyn occurs following G-CSF stimulation of cells expressing WT G-CSF-R, but that Hck is not phosphorylated in mutant G-CSF-R transfectants. The increase in phagocytosis following G-CSF stimulation cannot be attributed to a rapid de novo increase in expression of Fcγ receptors. G-CSF induced expression of Fcγ receptors only after prolonged stimulation. Our data provide evidence that the carboxy-terminal region of G-CSF-R plays a role in the phagocytosis of IgG-coated particles and that Syk and Hck kinase tyrosine phosphorylation is involved.
Differential response of human acute myeloid leukemia cells to gemtuzumab ozogamicin in vitro: role of Chk1 and Chk2 phosphorylation and caspase 3
Authors: Amico Donatella , Barbui Anna Maria , Erba Eugenio , Rambaldi Alessandro , Introna Martino , Golay Josée 
Published: May 2003

AbstractGemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated to the anticancer agent calicheamicin, approved for the treatment of CD33+-relapsed acute myeloid leukemia. We have investigated the effects of GO on 4 human myeloid leukemia lines of different French-American-British (FAB) types (KG-1, THP-1, HL-60, and NB-4), observing 3 different types of response. Exposure to GO (10-1000 ng/mL) induced G2 arrest (up to 80% of the cells) followed by apoptosis (45% of the cells) in HL-60 and NB-4 cells. By contrast, in THP-1 cells we observed a strong G2 arrest (up to 75% of the cells) with little apoptosis. Finally, the KG-1 line was completely resistant to the same concentrations of GO. These different responses did not correlate with the levels of expression of either CD33 or multiple-drug resistance proteins, although the higher cyclosporin A (CsA)–inhibitable efflux activity of KG-1 cells may play a role in the resistance of this line to the drug. We could show that Chk1 and Chk2 phosphorylation, but not p53 or p21 expression, correlated with G2 arrest, implicating the ataxia-telangiectasia mutated/ataxia-telangiectasia related (ATM/ATR)–Chk1/Chk2 pathway in the cell cycle response to GO. However, apoptosis was associated with caspase 3 activation. Freshly isolated acute myeloid leukemia (AML) cells showed patterns of response to GO in vitro similar to those observed with the cell lines, including phosphorylation of Chk2 and caspase 3 activation. Our results suggest that the different molecular pathways induced by the drug in vitro may reflect, at least in part, the variable response to GO obtained in vivo.
Estimating the rate of thrombin and fibrin generation in vivo during cardiopulmonary bypass
Authors: Chandler Wayne L. , Velan Tomas 
Published: May 2003

AbstractOur objective was to estimate the in vivo rates of thrombin and fibrin generation to better understand how coagulation is regulated. Studied were 9 males undergoing cardiopulmonary bypass (CPB). The rates of thrombin, total fibrin, and soluble fibrin generation in vivo were based on measured levels of prothrombin activation peptide F1.2, thrombin-antithrombin complex, fibrinopeptide A, and soluble fibrin, combined with a computer model of the patient's vascular system that accounted for marker clearance, hemodilution, blood loss, and transfusion. Prior to surgery, the average thrombin generation rate was 0.24 ± 0.11 pmol/s. Each thrombin molecule in turn generated about 100 fibrin molecules, of which 1% was soluble fibrin. The thrombin generation rate did not change after sternotomy or administration of heparin, then rapidly increased 20-fold to 5.60 ± 6.65 pmol/s after 5 minutes of CPB (P = .000 05). Early in CPB each new thrombin generated only 4 fibrin molecules, of which 35% was soluble fibrin. The thrombin generation rate was 2.14 ± 1.88 pmol/s during the remainder of CPB, increasing again to 5.47 ± 4.08 pmol/s after reperfusion of the ischemic heart (P = .000 08). After heparin neutralization with protamine, thrombin generation remained high (5.34 ± 4.01 pmol/s, P = .0002) and total fibrin generation increased, while soluble fibrin generation decreased. By 2 hours after surgery, thrombin and fibrin generation rates were returning to baseline levels. We conclude that cardiopulmonary bypass and reperfusion of the ischemic heart results in bursts of nonhemostatic thrombin generation and dysregulated fibrin formation, not just a steady increase in thrombin generation as suggested by previous studies.