bluuebunny/crossref_metadata_2025_split huggingface dataset:
Donor γδ T Lymphocytes Promote Allogeneic Engraftment Across the Major Histocompatibility Barrier in Mice
Authors: Drobyski William R. , Majewski David 
Published: October 2019

AbstractT cells that express the αβ T-cell receptor are thought to be the T-cell population primarily responsible for facilitating alloengraftment. The role of γδ+ T cells that comprise only a minority of mature T cells in promoting allogeneic engraftment, however, has not been extensively studied. The purpose of this study was to determine whether γδ T cells were capable of facilitating alloengraftment in murine recipients of major histocompatibility complex-mismatched marrow grafts. We developed a model where engraftment of C57BL/6 × 129/F2 (H-2b) marrow in sublethally irradiated (800 cGy) recipients (AKR/J, H-2k) is dependent on the presence of mature donor T cells in the marrow graft. In this model, donor T-cell engraftment was significantly augmented by as few as 1 × 105 αβ T cells. The role of γδ T cells was then investigated using transgenic donors (C57BL/6 × 129 background) in which a portion of the T-cell receptor–β chain gene was deleted by gene targeting so that these mice lack αβ T cells. Addition of 10 × 106 naive γδ T cells to T-cell depleted marrow grafts was required to significantly increase alloengraftment, although donor T cells averaged <50% of total splenic T cells. To determine whether higher doses of γδ T cells would improve donor engraftment and eradicate residual host T cells, γδ T cells were ex vivo expanded with a γδ T-cell–specific monoclonal antibody and interleukin-2 and then transplanted into irradiated recipients. Transplantation of ≥ 160 × 106 activated γδ T cells was necessary to consistently and significantly augment donor cell chimerism and enhance hematopoietic reconstitution when compared to control mice, but host T cells persisted in these chimeras. Addition of 2.5 × 104 mature αβ T cells, which alone were incapable of facilitating engraftment, to T-cell depleted marrow grafts containing 160 × 106 activated γδ T cells resulted in long-term (<100 day) complete donor engraftment, indicating that limiting numbers of αβ T cells were required in the marrow graft for the eradication of residual host T cells. Using serial weight curves and B-cell reconstitution as end points, clinically significant graft-versus-host disease was not observed in these chimeras under these experimental conditions. These data show that, whereas less potent than αβ T cells, γδ T cells are able to promote engraftment and enhance hematopoietic reconstitution in allogeneic marrow transplant recipients.
A Single Injection of Pegylated Murine Megakaryocyte Growth and Development Factor (MGDF ) Into Mice Is Sufficient to Produce a Profound Stimulation of Megakaryocyte Frequency, Size, and Ploidization
Authors: Arnold Julie T. , Daw Najat C. , Stenberg Paula E. , Jayawardene Deepthi , Srivastava Deo Kumar , Jackson Carl W. 
Published: October 2019

Abstract
Despite numerous studies investigating the action of c-mpl ligand, no reports have defined the in vivo changes in megakaryocytopoiesis in response to a single injection of this cytokine. Here we compare the kinetics of the megakaryocytopoietic response in C57Bl/6J mice administered 25 μg/kg or 250 μg/kg of pegylated (PEG) murine megakaryocyte growth and development factor (MGDF ) as a single intravenous injection. Megakaryocytes of mice treated with MGDF had normal ultrastructure, showing a typical distribution of the demarcation membrane system, α-granules, and other cytoplasmic organelles. Megakaryocyte ploidy, size, and frequency were markedly increased with both MGDF doses. Megakaryocyte ploidy was maximally increased from a modal value of 16N to 64N on day 3, with both doses of MGDF. Similarly, a comparable increase in megakaryocyte size occurred in the two MGDF groups. Increased megakaryocyte size was coupled to the increase in megakaryocyte ploidy, and no evidence for independent regulation of megakaryocyte size within individual ploidy classes was apparent. In contrast to megakaryocyte ploidy and size, the increase in megakaryocyte frequency was markedly different with the two doses of MGDF. The proportion of 2N and 4N cells was increased from a baseline of 0.035% to 0.430% by day 4 in mice treated with the higher dose of MGDF, but only to 0.175% in mice administered 25 μg/kg of MGDF. The marked increase in the pool of these immature megakaryocytes translated to a sustained elevation in the frequency of polyploid megakaryocytes (8N cells and greater). In contrast to the sustained increase in the frequency of polyploid cells, the level of polyploidization was downregulated on days 6 to 10, but normalized by day 14. We conclude that a single injection of MGDF is able to expand the megakaryocytic pool in a dose-dependent manner, which, with subsequent maturation, should lead to an increased rate of platelet production.
Impaired Hematopoiesis in Paroxysmal Nocturnal Hemoglobinuria/Aplastic Anemia Is Not Associated With a Selective Proliferative Defect in the Glycosylphosphatidylinositol-Anchored Protein-Deficient Clone
Authors: Maciejewski Jaroslaw P. , Sloand Elaine M. , Sato Tadatsugu , Anderson Stacie , Young Neal S. 
Published: October 2019

Abstract
Paroxysmal nocturnal hemoglobinuria (PNH) results from somatic mutations in the PIG-A gene, leading to poor presentation of glycosylphosphatidylinositol (GPI)-anchored surface proteins. PNH frequently occurs in association with suppressed hematopoiesis, including frank aplastic anemia (AA). The relationship between GPI-anchored protein expression and bone marrow (BM) failure is unknown. To assess the hematopoietic defect in PNH, the numbers of CD34+ cells, committed progenitors (primary colony-forming cells [CFCs]), and long-term culture-initiating cells (LTC-ICs; a stem cell surrogate) were measured in BM and peripheral blood (PB) of patients with PNH/AA syndrome or patients with predominantly hemolytic PNH. LTC-IC numbers were extrapolated from secondary CFC numbers after 5 weeks of culture, and clonogenicity of LTC-ICs was determined by limiting dilution assays. When compared with normal volunteers (n = 13), PNH patients (n = 14) showed a 4.7-fold decrease in CD34+ cells and an 8.2-fold decrease in CFCs. LTC-ICs in BM and in PB were decreased 7.3-fold and 50-fold, respectively. Purified CD34+ cells from PNH patients had markedly lower clonogenicity in both primary colony cultures and in the LTC-IC assays. As expected, GPI-anchored proteins were decreased on PB cells of PNH patients. On average, 23% of monocytes were deficient in CD14, and 47% of granulocytes and 58% of platelets lacked CD16 and CD55, respectively. In PNH BM, 27% of CD34+ cells showed abnormal GPI-anchored protein expression when assessed by CD59 expression. To directly measure the colony-forming ability of GPI-anchored protein-deficient CD34+ cells, we separated CD34+ cells from PNH patients for the GPI+ and GPI− phenotype; CD59 expression was chosen as a marker of the PNH phenotype based on high and homogeneous expression on fluorescent staining. CD34+CD59+ and CD34+CD59− cells from PNH/AA patients showed similarly impaired primary and secondary clonogeneic efficiency. The progeny derived from CD34+CD59− cells were both CD59− and CD55−. A very small population of CD34+CD59− cells was also detected in some normal volunteers; after sorting, these CD34+CD59− cells formed normal numbers of colonies, but their progeny showed lower CD59 levels. Our results are consistent with the existence of PIG-A–deficient clones in some normal individuals. In PNH/AA, progenitor and stem cells are decreased in number and function, but the proliferation in vitro is affected similarly in GPI-protein–deficient clones and in phenotypically normal cells. As measured in the in vitro assays, expansion of PIG-A– clones appears not be caused by an intrinsic growth advantage of cells with the PNH phenotype.
Therapeutic Levels of Functional Human Factor X in Rats After Retroviral-Mediated Hepatic Gene Therapy
Authors: Le MaiThao , Okuyama Torayuki , Cai Shi-Rong , Kennedy Susan C. , Bowling William M. , Flye M. Wayne , Ponder Katherine Parker 
Published: October 2019

AbstractFactor X deficiency results in a rare but serious bleeding disorder that might be treated by expressing a normal factor X gene in patients. We generated an amphotropic retroviral vector with the human FX cDNA and delivered it to rat hepatocytes in vivo during liver regeneration. The human α1-antitrypsin promoter was chosen to direct expression because it was the most efficient of several tested in yielding expression of α1-antitrypsin protein from a retroviral vector in hepatocytes in vivo. We achieved expression of factor X in four rats at levels sufficient to maintain hemostasis in humans (10% to 43% of normal). The factor X was determined to be functional by using a chromogenic substrate assay after immunoprecipitation with human specific antibodies. Expression of factor X remained stable for more than 10 months in two rats. It is likely that expression will be maintained for the life of the animals, because retroviral vectors integrate into the chromosome and hepatocytes are long-lived. The high and stable levels of expression achieved using this liver-specific promoter overcomes one of the two major obstacles to successful human gene therapy for hemophilia.
Characterization of the Human Platelet/Endothelial Cell Adhesion Molecule-1 Promoter: Identification of a GATA-2 Binding Element Required for Optimal Transcriptional Activity
Authors: Gumina Richard J. , Kirschbaum Nancy E. , Piotrowski Kim , Newman Peter J. 
Published: October 2019

AbstractPlatelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the Ig gene superfamily that is expressed on platelets, endothelial cells, and certain leukocyte subsets. To examine the factors controlling vascular-specific expression of PECAM-1, we cloned the 5′-flanking region of the PECAM-1 gene and analyzed its transcriptional activity. 5′-Rapid amplification of cDNA ends (5′-RACE) analysis showed that transcription initiation occurred at several closely spaced nearby sites originating approximately 204 bp upstream from the translation start site. Analysis of the sequence immediately upstream from the transcription initiation site (TIS) showed no canonical TATA or CAAT elements, however an initiator element commonly found in TATA-less promoters encompassed the TIS. 5′-serially truncated PECAM-1 promoter segments cloned in front of a luciferase reporter drove transcription in both a lineage- and orientation-specific manner. Putative cis-acting control elements present within a 300-bp core promoter included two ets sites, an Sp1 site, tandem E-box domains, two GATA-associated sites (CACCC), an AP-2 binding site, and a GATA element at −24. Mutational analysis showed that optimal transcriptional activity required the GATA sequence at position −24, and gel-shift assays further showed that the GATA-2 transcription factor, but not GATA-1, bound to this region of the PECAM-1 promoter. Understanding the cis- and trans-acting factors that regulate the tissue-specific expression of PECAM-1 should increase our understanding of the mechanisms by which vascular-specific gene expression is achieved.
Evidence for a Large Compartment of IgM-Expressing Memory B Cells in Humans
Authors: Klein Ulf , Küppers Ralf , Rajewsky Klaus 
Published: October 2019

AbstractThe recent finding of somatically mutated μ heavy chain transcripts in human peripheral blood (PB) B lymphocytes suggests that T-dependent B-cell memory might not be restricted to class-switched cells. We provide here evidence that IgM-only PB B cells are likely to be the IgM-expressing counterpart of classical (IgM−IgD−) memory B cells in humans. As shown by molecular single cell analysis, most IgM-only cells carry mutated V region genes, like class-switched cells. Although both subsets represent populations of nonactivated, resting cells, they express higher levels of Ig mRNA than naive (IgM+IgD+) B cells. IgM-only and class-switched cells are CD38−CD77−, and mostly CD23−, thus neither resembling germinal center nor naive B cells. Because many IgM-expressing B cells located in secondary lymphoid tissues resemble IgM-only PB B cells in terms of cell phenotype, we propose that the human lymphoid system contains a large compartment of IgM-expressing memory cells. Moreover, these cells seem to represent the nonmalignant counterparts of IgM-expressing tumor cells in sporadic Burkitt's lymphoma, MALT lymphoma, monocytoid B-cell lymphoma, and diffuse large-cell lymphoma that were found to harbor somatically mutated V genes.
Telomerase Activity Is Induced in Human Peripheral B Lymphocytes by the Stimulation to Antigen Receptor
Authors: Igarashi Hideya , Sakaguchi Nobuo 
Published: October 2019

AbstractTo understand the molecular events for the proliferation of B cells, we studied the induction of telomerase activity in vitro after stimulation to B-cell antigen receptor (BCR) on human peripheral B cells. Although unstimulated purified B cells of tonsils and peripheral blood from healthy volunteers do not express detectable telomerase activity, anti-IgM beads induce telomerase activity in these B cells. Soluble anti-IgM antibody (Ab) alone does not induce telomerase activity, but the second signal, given by either one of the cytokines of interleukin-2 (IL-2), IL-4, and IL-13 or by anti-CD40 monoclonal Ab (MoAb), is effective as the costimulation for the induction of the activity. Stimulation with antiIgM Ab and anti-CD40 MoAb induces telomerase activity in most mature B cells of the tonsils and peripheral blood. The stimuli to both IgM and IgD receptors similarly induce the activity. Induction of telomerase activity is accompanied with the proliferation of B cells, but is not absolutely correlated with the extent of B-cell growth. Phorbol dibutylate (PDB) plus calcium (Ca) ionophore (PDB/Ca), which replace the activation through BCR and the costimulatory molecules, also induce telomerase activity. Moreover, it is suggested that phosphoinositide (PI) 3-kinase plays a role for the induction of telomerase activity in B cells stimulated with anti-IgM Ab and anti-CD40 MoAb. These results suggest that telomerase activity is induced in the B-cell activation of the antigen specific immune response.
Analysis of the Human Interleukin-6/Human Interleukin-6 Receptor Binding Interface at the Amino Acid Level: Proposed Mechanism of Interaction
Authors: Kalai Michael , Montero-Julian Fèlix A. , Grötzinger Joachim , Fontaine Véronique , Vandenbussche Paul , Deschuyteneer Rudi , Wollmer Axel , Brailly Hervé , Content Jean 
Published: October 2019

AbstractThe interaction between interleukin-6 (IL-6) and IL-6 receptor (IL-6R) is the initial and most specific step in the IL-6 signaling pathway. Understanding its mechanism at the amino acid level is the basis for developing small IL-6–inhibiting molecules. We studied the human IL-6 (hIL-6)/hIL-6R binding interface by a combination of molecular modelling and site-directed mutagenesis. Our model suggests that the center of the interface between the two molecules consists of hydrophobic contacts predicted to account for most of the binding-free energy. These contacts can be regarded as a hydrophobic core shielded by hydrophilic residues that are also needed for recognition. Following this hypothesis, we altered in hIL-6 and hIL-6R residues predicted to reside in the contact region and to interact with each other. We studied the capacity of these mutants to form an IL-6/IL-6R complex and their ability to transduce the signal. This combined approach has led to the identification of certain residue-clusters in the binding interface and to a rational explanation of their specific interactions, suggesting therein a likely mechanism of complex formation. The results confirm the predictive model and strongly support our hypothesis. Comparison with other cytokines and their α-subunit receptors suggests that the structural location of certain binding sites are conserved.
Activated Cytotoxic T Cells as Prognostic Marker in Hodgkin's Disease
Authors: Oudejans J.J. , Jiwa N.M. , Kummer J.A. , Ossenkoppele G.J. , van Heerde P. , Baars J.W. , Kluin Ph.M. , Kluin-Nelemans J.C. , van Diest P.J. , Middeldorp J.M. , Meijer C.J.L.M. 
Published: October 2019

AbstractAlthough the results of treatment of Hodgkin's disease (HD) have improved considerably in the last decades, the disease remains fatal in a minority of patients. We have recently shown that numbers of activated cytotoxic T cells (CTLs), present in tumor biopsy specimens, differ considerably among individual HD patients. Because CTLs are the major effector cells in elimination of neoplastic cells, we investigated whether the number of activated CTLs is related to the clinical outcome of the individual patient with HD. Activated CTLs present in tumor biopsy specimens of patients with nodular sclerosis or mixed cellularity HD were identified by immunohistochemistry using an antibody directed against granzyme B (GrB), a major constituent of the cytotoxic granules of activated CTLs and natural killer cells, and an antibody directed against CD8. The presence of a high percentage of GrB+ lymphocytes was found to be an unfavorable prognostic marker. The large majority of GrB+ cells were also CD8+, indicating that these cells are activated CTLs. Prognosis was found to decrease with increasing percentages of GrB+ lymphocytes. Optimal discrimination between patients with good and poor prognosis was obtained when the threshold was set at 15% GrB+ cells; 6 of 10 patients with ≥15% GrB+ lymphocytes died as a result of the disease, as compared with 6 of 70 patients with less than 15% GrB+ lymphocytes (P < .0001). In stage-2 patients, the percentage of GrB+ lymphocytes retained its predictive value in a multivariate analysis including histology, sex, age, erythrocyte sedimentation rate, and the presence of B symptoms as covariables. In addition, patients with ≥15% GrB+ lymphocytes had a shortened progression-free survival time (P = .002). We conclude that a high percentage of activated CTLs present in biopsy material of HD patients is a strong indicator for an unfavorable clinical outcome.
A Tyrosine-Phosphorylated Protein of 140 kD Is Constitutively Associated With the Phosphotyrosine Binding Domain of Shc and the SH3 Domains of Grb2 in Acute Myeloid Leukemia Cells
Authors: Jücker Manfred , Schiffer Charles A. , Feldman Ricardo A. 
Published: October 2019

AbstractThe Shc gene encodes three proteins that have been implicated as mediators of signal transduction from growth factor receptors and nonreceptor tyrosine kinases to Ras. Overexpression of Shc in established murine fibroblasts results in oncogenic transformation, indicating that Shc has oncogenic potential. Shc proteins contain a carboxy terminal SH2 domain and a novel non-SH2 phosphotyrosine-binding (PTB) domain that specifically recognizes a phosphorylated NPXpY motif in target proteins such as the epidermal growth factor receptor. We show here that Shc is constitutively tyrosine-phosphorylated in all primary acute myeloid leukemias analyzed and that, in some of these leukemias, Shc is associated through its PTB domain with a tyrosinephosphorylated protein of 140 kD (p140) in vivo. In factor-dependent cells, this 140-kD protein can be tyrosine-phosphorylated in vitro in response to cytokines involved in myeloid proliferation and differentiation, ie, granulocyte-macrophage colony-stimulating factor and colony-stimulating factor-1. A similar or identical protein of 140 kD is constitutively bound to the C-terminal SH3 domain of Grb2 in the same acute myeloid leukemias. In addition to p140, other tyrosine-phosphorylated proteins of 61 and 200 kD are constitutively associated with Shc in some of the leukemias analyzed. Our results implicate Shc, Grb2, p140, and additional tyrosine-phosphorylated proteins of 61 and 200 kD in signalling of acute myeloid leukemia cells.
International Scoring System for Evaluating Prognosis in Myelodysplastic Syndromes
Authors: Greenberg Peter , Cox Christopher , LeBeau Michelle M. , Fenaux Pierre , Morel Pierre , Sanz Guillermo , Sanz Miguel , Vallespi Teresa , Hamblin Terry , Oscier David , Ohyashiki Kazuma , Toyama Keisuke , Aul Carlo , Mufti Ghulam , Bennett John 
Published: October 2019

Abstract
Despite multiple disparate prognostic risk analysis systems for evaluating clinical outcome for patients with myelodysplastic syndrome (MDS), imprecision persists with such analyses. To attempt to improve on these systems, an International MDS Risk Analysis Workshop combined cytogenetic, morphological, and clinical data from seven large previously reported risk-based studies that had generated prognostic systems. A global analysis was performed on these patients, and critical prognostic variables were re-evaluated to generate a consensus prognostic system, particularly using a more refined bone marrow (BM) cytogenetic classification. Univariate analysis indicated that the major variables having an impact on disease outcome for evolution to acute myeloid leukemia were cytogenetic abnormalities, percentage of BM myeloblasts, and number of cytopenias; for survival, in addition to the above, variables also included age and gender. Cytogenetic subgroups of outcome were as follows: “good” outcomes were normal, −Y alone, del(5q) alone, del(20q) alone; “poor” outcomes were complex (ie, ≥3 abnormalities) or chromosome 7 anomalies; and “intermediate” outcomes were other abnormalities. Multivariate analysis combined these cytogenetic subgroups with percentage of BM blasts and number of cytopenias to generate a prognostic model. Weighting these variables by their statistical power separated patients into distinctive subgroups of risk for 25% of patients to undergo evolution to acute myeloid leukemia, with: low (31% of patients), 9.4 years; intermediate-1 (INT-1; 39%), 3.3 years; INT-2 (22%), 1.1 years; and high (8%), 0.2 year. These features also separated patients into similar distinctive risk groups for median survival: low, 5.7 years; INT-1, 3.5 years; INT-2, 1.2 years; and high, 0.4 year. Stratification for age further improved analysis of survival. Compared with prior risk-based classifications, this International Prognostic Scoring System provides an improved method for evaluating prognosis in MDS. This classification system should prove useful for more precise design and analysis of therapeutic trials in this disease.
Hemoglobin-Specific Antibody in a Multiply Transfused Patient With Sickle Cell Disease
Authors: Noronha Peter A. , Vida Loyda N. , Park C. Lucy , Honig George R. 
Published: October 2019

AbstractHuman hemoglobins (Hbs) are known to be immunogenic, and both normal and variant forms of Hb have been shown to stimulate antibody formation in a variety of animal species. In patients who are homozygous for the sickle Hb (HbS) mutation, transfusion of normal, HbA-containing erythrocytes provides a potential stimulus for HbA alloimmunization. We tested serum samples for the presence of anti-Hb antibody by a solid-phase enzyme-linked immunosorbent assay (ELISA) using Hb-coated polystyrene microtiter plates. Hb-bound antibody was identified using an antihuman IgG antibody. Serum samples from 89 patients with sickle cell disease were initially tested for evidence of Hb antibody. The serum from three individuals exhibited antibody activity against HbA with little or no activity against HbS. Only one of them, a multiply transfused adult with HbSS, was available for further study. When this patient's antibody was tested against a variety of normal and mutant Hbs using antibody either to human IgG or to κ chains, the anti-Hb antibody demonstrated specificity for the region of the Hb β chain corresponding to the site of the amino acid substitution of HbS. The level of activity of the patient's anti-HbA showed no significant change over 1.5 years of observation. The transfusion of erythrocytes containing Hb structurally different from that of the recipient appeared to be capable of stimulating the production of Hb-specific alloimmune antibody.