bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential Effects of Anti-Fas Ligand and Anti-Tumor Necrosis Factor α Antibodies on Acute Graft-Versus-Host Disease Pathologies
Authors: Hattori Koichi , Hirano Takao , Miyajima Hiroaki , Yamakawa Norifumi , Tateno Masatoshi , Oshimi Kazuo , Kayagaki Nobuhiko , Yagita Hideo , Okumura Ko 
Published: October 2019

AbstractBoth tumor necrosis factor α (TNFα) and Fas ligand (FasL) have been implicated in the pathogenesis of graft-versus-host disease (GVHD). In this study, we examined the ameliorating effects of neutralizing anti-FasL and/or anti-TNFα monoclonal antibody (MoAb) in a lethal acute GVHD model in mice. Whereas the treatment with either anti-FasL or anti-TNFα MoAb alone significantly delayed the mortality and improved the body weight, a complete protection was achieved by the administration of both MoAbs. Pathological examination indicated differential effects of anti-FasL or anti-TNFα MoAb on GVHD-associated pathologies. Hepatic lesion was improved by anti-FasL but not anti-TNFα MoAb. In contrast, intestinal lesion was improved by anti-TNFα but not anti-FasL MoAb. Cutaneous and splenic lesions were improved by either MoAb. The combination of both MoAbs improved all these lesions. These results indicate that FasL and TNFα differentially contribute to the GVHD pathologies and a complete protection from mortality can be achieved by neutralization of both FasL and TNFα.
Quantitative Long-Term Culture-Initiating Cell Assays Require Accessory Cell Depletion That Can Be Achieved by CD34-Enrichment or 5-Fluorouracil Exposure
Authors: Koller Manfred R. , Manchel Ilana , Smith Alan K. 
Published: October 2019

Abstract
Characterization of hematopoietic cells and measurement of their proliferative potential is critical in many research and clinical applications. Because in vivo assay of human cells is not possible and xenogeneic assays are not yet routine, in vitro assays such as the long-term culture-initiating cell (LTC-IC) assay have been widely adopted. This study investigated LTC-IC assay linearity and reproducibility and resulting implications with respect to quantitation of primitive cell expansion. Measurement of secondary colony-forming cells (2° CFCs) from 5-week cultures of bone marrow (BM) mononuclear cells (MNCs) showed that 2° CFC frequency varied with assay plating density in a nonlinear fashion. The measured 2° CFC frequency increased from 4.6 to 63.8 (per 105 MNCs) as assay plating density was decreased from 5 × 105 to 2 × 104 MNCs per well (P &lt; 10−6, n = 37). In contrast, assay of CD34-enriched cells was linear within the range studied. Assays of cells obtained from expansion cultures initiated with either MNCs or CD34-enriched cells were also nonlinear. Consequently, calculated 2° CFC expansion ratios were ambiguous and dependent on the assay plating densities used. Limiting dilution analysis (LDA) results were also nonlinear, with LTC-IC frequency increasing from 8.2 to 22.4 per 105 MNCs (P &lt; 10−4, n = 100) as assay plating densities were decreased. Despite the nonlinearity, 2° CFC and LTC-IC assay results were consistent and reproducible over time with different samples and techniques and gave a semiquantitative indication of relative primitive cell frequency. Although CD34-enriched cells gave linear assay output, purification of cells for every assay is impractical. Therefore, exposure of cells to 5-fluorouracil (5-FU) was explored for improving assay linearity. Incubation of MNCs in 250 μg/mL 5-FU for 1 to 2 hours depleted accessory cells and resulted in a cell population that gave linear 2° CFC readout. The 5-FU–resistant LTC-ICs accounted for 49% of the total LTC-IC population, adding the potential benefit of restricting assay measurement to more primitive noncycling LTC-ICs. Consequently, similar linear assay results can be obtained with either the bulk 2° CFC or LDA LTC-IC methods after 5-FU, but multiple plating densities are nevertheless still required in both methods due to the greater than 100-fold range in primitive cell frequency present in normal human donor BM.
The Notch Ligand, Jagged-1, Influences the Development of Primitive Hematopoietic Precursor Cells
Authors: Varnum-Finney Barbara , Purton Louise E. , Yu Monica , Brashem-Stein Carolyn , Flowers David , Staats Steven , Moore Kateri A. , Le Roux Isabelle , Mann Robert , Gray Grace , Artavanis-Tsakonas Spyros , Bernstein Irwin D. 
Published: October 2019

AbstractWe examined the expression of two members of theNotch family, Notch-1 and Notch-2, and one Notch ligand, Jagged-1, in hematopoietic cells. Both Notch-1 and Notch-2 were detected in murine marrow precursors (Lin−Sca-1+c-kit+). The Notch ligand, Jagged-1, was not detected in whole marrow or in precursors. However, Jagged-1 was seen in cultured primary murine fetal liver stroma, cultured primary murine bone marrow stroma, and in stromal cell lines. These results indicate a potential role for Notch-Notch ligand interactions in hematopoiesis. To further test this possibility, the effect of Jagged-1 on murine marrow precursor cells was assessed by coculturing sorted precursor cells (Lin−Sca-1+c-kit+) with a 3T3 cell layer that expressed human Jagged-1 or by incubating sorted precursors with beads coated with the purified extracellular domain of human Jagged-1 (Jagged-1ext). We found that Jagged-1, presented both on the cell surface and on beads, promoted a twofold to threefold increase in the formation of primitive precursor cell populations. These results suggest a potential use for Notch ligands in expanding precursor cell populations in vitro.
Anti–LFA-1 Blocking Antibodies Prevent Mobilization of Hematopoietic Progenitor Cells Induced by Interleukin-8
Authors: Pruijt Johannes F.M. , van Kooyk Yvette , Figdor Carl G. , Lindley Ivan J.D. , Willemze Roel , Fibbe Willem E. 
Published: October 2019

AbstractPreviously, we have shown that interleukin (IL)-8 induces the rapid (15 to 30 minutes) mobilization of hematopoietic progenitor cells (HPC) in mice. Because integrins are essential for adhesion and transendothelial migration of HPC, we studied the involvement of the β2-integrin leukocyte function-associated antigen-1 (LFA-1) in IL-8–induced mobilization. After a single injection of blocking anti–LFA-1 antibodies, no mobilization of colony-forming cells was observed. In addition, when mice were pretreated with anti–LFA-1 or saline and subsequently injected with 30 μg of IL-8, mobilization of HPC was completely blocked. We showed that this was not due to anti–LFA-1 antibodies affecting colony formation, as addition of anti–LFA-1 antibodies to colony cultures in semisolid medium had no inhibitory activity. Also, anti-intercellular adhesion molecule (ICAM)-1 antibodies, directed to the main ligand of LFA-1 significantly inhibited the IL-8–induced mobilization. Furthermore, IL-1–induced mobilization was significantly inhibited by anti–LFA-1 antibodies. Because LFA-1 is reported to be expressed on more differentiated HPC, it was considered that the IL-8–induced mobilization of more primitive HPC would not be blocked by anti–LFA-1 antibodies. Transplantation of blood-derived mononuclear cells (MNC) from IL-8–mobilized animals pretreated with anti–LFA-1 antibodies protected only 25% of lethally irradiated recipient mice, whereas the radioprotection rate of control mice transplanted with MNC derived from IL-8-mobilized animals was 86% (P &lt; .01). Anti-LFA–1 antibodies did not interfere with stem cell homing, as transplantation of IL-8-mobilized blood MNC, incubated in vitro with these antibodies resulted in 100% radioprotection. We conclude that anti–LFA-1 antibodies completely prevent the rapid mobilization of colony-forming cells and of cells with radioprotective capacity induced by IL-8. These results indicate a major role for the β2-integrin LFA-1 in the IL-8–induced mobilization of hematopoietic stem cells.
NF-κB Transcription Factors Are Involved in Normal Erythropoiesis
Authors: Zhang Min-Ying , Sun Shao-Cong , Bell Laurie , Miller Barbara A. 
Published: October 2019

Abstract
NF-κB/Rel designates a widely distributed family of transcription factors involved in immune and acute phase responses. Here, the expression and function of NF-κB factors in erythroid proliferation and differentiation were explored. In an erythroleukemia cell line, TF-1, high levels of p105/p50, p100/p52, p65, and IκBα were detected 24 hours after growth factor deprivation. In response to granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation, significant induction of p52 expression was observed. GM-CSF also induced nuclear translocation of both p52 and p65. No induction of NF-κB factors was observed with erythropoietin stimulation of TF-1 cells. Overexpression of p52 and p65 in TF-1 cells by transient transfection resulted in significant induction of a κB-TATA-luciferase reporter plasmid, showing that these factors are functional in vivo in erythroid cells. To determine whether NF-κB factors may play a role in normal erythropoiesis, levels of these factors were determined in burst-forming unit-erythroid (BFU-E)–derived cells at different stages of differentiation. The NF-κB factors p105/p50, p100/p52, and p65 were highly expressed in early BFU-E–derived precursors, which are rapidly proliferating, and declined during maturation. Furthermore, nuclear levels of NF-κB factors p50, p52, and p65 were higher in less mature precursors (day 10 BFU-E–derived cells) compared with more differentiated (day 14) erythroblasts. In nuclear extracts from day 10 BFU-E–derived cells, p50, p52, and p65 were able to form complexes, which bound to κB sites in the promoters of both the c-myb and c-mycgenes, suggesting that c-myb and c-myc may be among the κB-containing genes regulated by NF-κB factors in normal erythroid cells. Taken together, these data show that NF-κB factors are modulated by GM-CSF and suggest they function to regulate specific κB containing genes involved in erythropoiesis.
A Directly Spliced Exon 10–Containing CD44 Variant Promotes the Metastasis and Homotypic Aggregation of Aggressive Non-Hodgkin's Lymphoma
Authors: Yakushijin Yoshihiro , Steckel Joshua , Kharbanda Samir , Hasserjian Robert , Neuberg Donna , Jiang Wei-meng , Anderson Ian , Shipp Margaret A. 
Published: October 2019

AbstractVariants of the CD44 cell-surface adhesion molecule include additional sequences encoded by combinations of exons from the membrane proximal domain (exons 6–14). Preliminary studies suggest that these additional variable membrane proximal sequences may alter the ligand specificity, glycosylation, and biologic function of CD44. In earlier studies, we found that primary extranodal and widely disseminated aggressive non-Hodgkin's lymphomas (NHLs) and normal activated B cells expressed a directly spliced exon 10–containing variant (CD44ex10), whereas normal resting B cells expressed larger exon 10–containing variants (CD44ex10-14 and CD44ex7-14). To obtain additional information regarding the function of exon 10–containing CD44 variants in aggressive NHL, we generated aggressive NHL transfectants that expressed CD44ex10, CD44ex10-14, CD44ex7-14, the standard CD44 isoform (CD44H), or vector alone, and evaluated the local tumorogenicity, aggregation, and metastatic potential of these transfectants. CD44ex10 aggressive NHL transfectants were more likely to cause local tumor formation in nude mice than transfectants expressing the larger exon 10–containing variants, CD44H, or vector alone. In addition, cell suspensions derived from CD44ex10 local tumors exhibited far greater homotypic aggregation than those obtained from other CD44 or vector-only local tumors. In nude mice that received CD44ex10 transfectants, distant metastases were also significantly more likely to develop than in animals that were given either the CD44ex10-14, CD44ex7-14, CD44H, or vector-only transfectants. These data provide the first evidence that the directly spliced exon 10–containing CD44 variant (CD44ex10) has a unique biologic function in aggressive NHL.
Analysis of VH Genes in Follicular and Diffuse Lymphoma Shows Ongoing Somatic Mutation and Multiple Isotype Transcripts in Early Disease With Changes During Disease Progression
Authors: Ottensmeier Christian H. , Thompsett Andrew R. , Zhu Delin , Wilkins Bridget S. , Sweetenham John W. , Stevenson Freda K. 
Published: October 2019

Abstract
Investigations of VH gene mutational patterns in B-cell tumors are often performed at an arbitrary time point of disease. To assess the effects of disease progression, tumor-derived VHgenes have been monitored from presentation through treatment and relapse in one patient with follicle center lymphoma (FCL), and two patients with primary diffuse large B-cell lymphoma (DLCL). The patient with FCL and one patient with DLCL both achieved clinical remission, although this was only partial in the FCL. However, both subsequently relapsed, and the second patient with DLCL was refractory to radiotherapy and chemotherapy. In each case, the tumor-derived VH sequence was identified, and the CDR3 “clonal signature” was used to track tumor cell sequences in subsequent biopsies. All cases showed somatic mutations, with intraclonal heterogeneity evident at presentation, and some sequences were aberrant. The VH sequences of the DLCL which responded to treatment became homogeneous at relapse. The sequences of both the FCL and the refractory DLCL remained heterogeneous. In all cases, transcripts of multiple Ig isotypes could be identified, and there was immunophenotypic evidence for expression of several Ig isotypes. The case of refractory DLCL had identifiable transcripts from IgM, IgD, IgA, IgG, and IgE, but appeared to lose the ability to produce alternative isotype transcripts and protein at the late stage of disease. These cases indicate that VH gene analysis can be used to probe tumor cell behavior in cases of lymphoma and that perturbations caused by therapy and disease progression can occur.
p53 Expression in B-Cell Chronic Lymphocytic Leukemia: A Marker of Disease Progression and Poor Prognosis
Authors: Cordone Iole , Masi Serena , Mauro Francesca Romana , Soddu Silvia , Morsilli Ornella , Valentini Tiziana , Vegna Maria Luce , Guglielmi Cesare , Mancini Francesca , Giuliacci Sonia , Sacchi Ada , Mandelli Franco , Foa Robert 
Published: October 2019

Abstract
We have analyzed by immunocytochemistry (ICC) the frequency of p53 protein expression in 181 cases of B-cell chronic lymphocytic leukemia (CLL) followed at a single institution to assess the relationship between p53 and the clinical and morphological features of the disease, as well as the possible involvement of this protein in the pathogenesis of the more aggressive forms of CLL. The overall frequency of p53 protein positivity in CLL was 15% (27 of 181 cases). There were no significant differences in age, sex, absolute lymphocyte count, or lymphocyte doubling time between p53-positive and -negative patients. By contrast, p53-positive patients had a significantly higher percentage of prolymphocytes (P = .002) and a significantly lower percentage of residual CD3-positive T lymphocytes (P = .0001). No correlation was found between the percentage of p53-positive cells and the percentage of cells in cycle assessed by the monoclonal antibody Ki-67. When the percentage of p53 positivity was correlated with the clinical stage of the disease, the proportion of p53-positive cases increased significantly from Binet's stage A (8 of 108; 7.4%), to stage B (12 of 49; 24.4%) and C (7 of 24; 29.2%) (P = .002). p53 positivity correlated also with the phase of the disease, showing a low expression at diagnosis (8 of 112; 7.1%) and a significantly higher expression in patients studied during the course of the disease (7 of 35; 20%) and, to a further extent, with disease progression (12 of 34; 35.3%) (P = .0001). The association of p53 protein expression with mutations in the gene was confirmed by direct sequence of the entire cDNA in 15 of the 17 ICC positive cases tested (88%). A significantly shorter treatment-free interval from diagnosis (P = .003) and a poorer response to therapy (P = .007) was observed in p53-positive compared with p53-negative patients. Overall survival from the time of diagnosis, as well as from the time of p53 protein analysis, was significantly shorter in patients with p53 protein expression (P = .03 and .0001, respectively). Moreover, in multivariate analysis, p53 expression and stage C were independently associated with a short survival. The results of this study indicate that in CLL the expression of the p53 protein, analyzed by a simple and reliable immunocytochemical method, is strongly associated with p53 gene mutations, a morphological variant (CLL with &gt;10% prolymphocytes), advanced clinical stage, progressive disease, poor response to therapy, and short survival.
A Large Deletion Within the Protein 4.1 Gene Associated With a Stable Truncated mRNA and an Unaltered Tissue-Specific Alternative Splicing
Authors: Venezia N. Dalla , Maillet P. , Morlé L. , Roda L. , Delaunay J. , Baklouti F. 
Published: October 2019

AbstractProtein 4.1 is a major protein of the red blood cell skeleton. It binds to the membrane through its 30-kD N-terminal domain and to the spectrin-actin lattice through its 10-kD domain. We describe here the molecular basis of a heterozygous hereditary elliptocytosis (HE) associated with protein 4.1 partial deficiency. The responsible allele displayed a greater than 70-kb genomic deletion, beginning within intron 1 and ending within a 1.3-kb region upstream from exon 13. This deletion encompassed both erythroid and nonerythroid translation initiation sites. It accounts for the largest deletion known in genes encoding proteins of the red blood cell membrane. The corresponding mRNA was shortened by 1727 bases, due to the absence of exons 2 to 12. Nevertheless, this mRNA was stable. It showed a similar pattern in lymphoblastoid cells as in reticulocytes. Differential splicing of exons within the undeleted region remained regulated in a tissue-specific manner. Exons 14, 15, and 17a were absent from both reticulocyte and lymphocyte mRNAs, whereas exon 16 was present in reticulocytes but absent from lymphocytes. Thus, differential splicing on a local scale was not dependent on the overall structure of protein 4.1 mRNA in this particular instance.
The Efficacy of Reducing Agents or Antioxidants in Blocking the Formation of Dense Cells and Irreversibly Sickled Cells In Vitro
Authors: Gibson Xunda A. , Shartava Archil , McIntyre Jonah , Monteiro Carlos A. , Zhang Yalin , Shah Arvind , Campbell Naomi F. , Goodman Steven R. 
Published: October 2019

Abstract
We show that N-acetylcysteine (NAC) has the ability to cause statistically significant diminishment in the in vitro formation of irreversibly sickled cells (ISCs) at concentrations greater than 250 μmol/L. Other antioxidants, approved for human use (cysteamine, succimer, dimercaprol), were not efficacious. NAC had the ability to cause statistically significant conversion of ISCs formed in vivo back to the biconcave shape. NAC was also shown to reduce the formation of dense cells and increase the available thiols in β-actin. We showed that diminishing reduced glutathione (GSH), by treatment with 1-chloro-2,4-dinitrobenzene, resulted in increased dense cells. We conclude the NAC blocks dense cell formation and ISC formation by targeting channels involved in cellular dehydration and β-actin, respectively. The efficacy of NAC is probably due to its combined antioxidant activity and ability to increase intracellular GSH.
Fetal Hemoglobin and F-Cell Responses to Long-Term Hydroxyurea Treatment in Young Sickle Cell Patients
Authors: Maier-Redelsperger Micheline , de Montalembert Mariane , Flahault Antoine , Neonato Maria Grazia , Ducrocq Rolande , Masson Marie-Pierre , Girot Robert , Elion Jacques , French Study Group on Sickle Cell Disease the 
Published: October 2019

Abstract
We have studied the cellular and molecular responses to long-term hydroxyurea (HU) treatment in 29 severely affected young patients with sickle cell disease (mean age, 10.9 ± 4.1 years). Patients received HU at 20 mg/kg/d on 4 consecutive days per week initially, with a monthly escalated dose avoiding marrow-toxicity (mean steady-state dose, 34.2 ± 4.6 mg/kg/d) for 12 to 36 months (mean duration, 22 months). The studied parameters were hemoglobin F (HbF), F reticulocytes (F retics), F cells, the amount of HbF per F cell (F/F cell), polymer tendency at 40% and 70% oxygen saturation, and hemolysis. Initial HbF (Fi) was dispersed (from 0.85% to 13.9%). HbF increased in all patients but 1. HbF at maximal response (Fmax) reached a sustained level varying from a 1.5-fold to a 16-fold Fi after a variable delay (6 to 24 months). Fmax was not related to HU dosage, but ▵F (Fmax − Fi) was strongly correlated to ▵MCV (MCVmax − MCVi). HbF increase resulted from the increase of both F cells and F/F cell. In this rather short series, Fi and Fmax were not significantly associated with age, gender, or β-globin haplotype. Neither Fmax nor ▵F was related to bone marrow reserve, as measured by baseline reticulocyte or neutrophil counts. However, Fmax was highly dependent on Fi. When patients are individualized into three groups according to Fmax (group 1, Fmax &gt;20% [12 patients]; group 2, 10% &lt; Fmax &lt; 20% [11 patients]; group 3, Fmax &lt;10% [5 patients]), Fi is significantly different between groups, being the highest in group 1. In addition, the best responders (group 1) were significantly different from patients in the two other groups with higher levels of total hemoglobin, decreased bilirubin, and decreased polymer tendency.
Pgp and MRP Activities Using Calcein-AM Are Prognostic Factors in Adult Acute Myeloid Leukemia Patients
Authors: Legrand Ollivier , Simonin Ghislaine , Perrot Jean-Yves , Zittoun Robert , Marie Jean-Pierre 
Published: October 2019

AbstractThirteen cell lines with different levels of Pgp and MRP expression were used to assess the ability of calcein acetoxymethyl ester (calcein-AM) uptake and calcein efflux to measure Pgp and MRP functions, respectively. There was a good correlation between MRP expression and the modulatory effect of probenecid (a specific modulator of MRP) on the calcein efflux (r = .91, P= .0003) and between Pgp expression and the modulatory effect of CsA on calcein-AM uptake (r = .96, P &lt; .0001). In light of the high correlations for both proteins, we tested calcein-AM uptake and efflux in fresh myeloid leukemic cells. In 53 acute myeloid leukemia (AML) patients, there was also a good correlation between MRP expression (measured by reverse transcription-polymerase chain reaction and by MRPm6 expression by flow cytometry) and the modulatory effect of probenecid on the calcein fluorescence (r = .92, P &lt; .0001) and between Pgp expression as measured by UIC2 antibody binding on flow cytometry and the modulatory effect of cyclosporin A on calcein-AM uptake (r = .83,P &lt; .0001). Pgp activity was higher in CD34+leukemia than in CD34− leukemia (2.26 ± 1.50 v1.46 ± 1.21, respectively; P = .003), and MRP activity was higher in CD34− leukemia than in CD34+leukemia (1.77 ± 0.40 v 1.4 ± 0.29, respectively; P= .004). Pgp expression and activity (P = .004 andP = .01, respectively) and MRP activity (P = .03) but not MRP expression were prognostic factors for achievement of complete remission. These results suggest that functional testing (with calcein-AM ± modulators) for the presence of both MRP and Pgp activities is of prognostic value and that MRP contributes to drug resistance in AML.