bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Granulocyte Colony-Stimulating Factor Receptor Is Required for the Mobilization of Murine Hematopoietic Progenitors Into Peripheral Blood by Cyclophosphamide or Interleukin-8 But Not Flt-3 Ligand
Authors: Liu Fulu , Poursine-Laurent Jennifer , Link Daniel C. 
Published: October 2019

AbstractHematopoietic progenitor cells (HPC) can be mobilized from the bone marrow into the peripheral circulation in response to a number of stimuli including hematopoietic growth factors, cytotoxic agents, and certain chemokines. Despite significant differences in their biological activities, these stimuli result in the mobilization of HPC with a similar phenotype, suggesting that a common mechanism for mobilization may exist. In this study, the role of granulocyte colony-stimulating factor (G-CSF) in progenitor mobilization was examined using G-CSF receptor (G-CSFR)–deficient mice. In contrast to wild-type mice, no increase in circulating colony-forming cells (CFU-C), CD34+ lineage− progenitors, or day 12 colony-forming unit-spleen progenitors (CFU-S) was detected in G-CSFR–deficient mice after cyclophosphamide administration. This defect was not due to a failure to regenerate HPC following cyclophosphamide administration as the number of CFU-C in the bone marrow of G-CSFR–deficient mice was increased relative to wild-type mice. Likewise, no increase in circulating CFU-C was detected in G-CSFR–deficient mice following interleukin-8 (IL-8) administration. In contrast, mobilization of HPC in response to flt-3 ligand was nearly normal. These results show that the G-CSFR is required for mobilization in response to cyclophosphamide or IL-8 but not flt-3 ligand and suggest that the G-CSFR may play an important and previously unexpected role in HPC migration.
Bernard-Soulier Syndrome Caused by a Dinucleotide Deletion and Reading Frameshift in the Region Encoding the Glycoprotein Ibα Transmembrane Domain
Authors: Afshar-Kharghan Vahid , López José A. 
Published: October 2019

AbstractWe investigated the molecular genetic and biosynthetic basis of Bernard-Soulier syndrome in a severely affected white woman. Flow cytometric analysis showed a severe deficiency of glycoprotein (GP) Ib, GP IX, and GP V on the surface of her platelets. Similarly, GP Ibα was undetectable by immunoblot analysis of platelet lysates. Surprisingly, a large quantity of a 70-kD protein (which probably represents a GP Ibα degradation product) was found in the patient's plasma in much greater quantities than in the plasma of an unaffected individual. To analyze the molecular lesion responsible for the disorder, we amplified and sequenced gene segments corresponding to the entire coding regions of the GP Ibα, GP Ibβ, and GP IX genes. The patient was homozygous for a specific GP Ibα allele that contained two tandem VNTR repeats in the region encoding the macroglycopeptide (C variant) and three differences from the published GP Ibα gene sequence. Two mutations were unlikely to be involved in the disorder: the substitution of a single base (T → C) in the second nucleotide of exon 2, which is in the 5′ untranslated region of the GP Ibα transcript, and a silent mutation in the third base of the codon for Arg342 (A → G) that does not change the amino acid sequence. The third mutation was a deletion of the last two bases of the codon for Tyr492 (TAT). This mutation causes a frameshift that alters the GP Ibα amino acid sequence, beginning within its transmembrane region. The mutant polypeptide contains 81 novel amino acids and is 38 amino acids shorter than its wild-type counterpart. The new sequence changes the hydrophobic nature of the transmembrane domain and greatly decreases the net positive charge of what had been the cytoplasmic domain. The deletion mutation was introduced into the GP Ibα cDNA, alone and in combination with the 5′ mutation, and expressed in Chinese hamster ovary (CHO) cells. The deletion alone severely reduced GP Ibα expression on the cell surface. Expression was not decreased further by addition of the 5′ mutation, confirming that the deletion was the cause of the Bernard-Soulier phenotype. Stable cell lines expressing the mutant polypeptide secreted large amounts of the polypeptide into the medium, suggesting that the mutant anchors poorly in the plasma membrane. Nevertheless, a fraction of the mutant was able to associate with GP Ibβ, as demonstrated by their coimmunoprecipitation with a GP Ibβ antibody.
Thrombin Cleavage-Independent Deposition of Fibrinogen in Extracellular Matrices
Authors: Guadiz Gayle , Sporn Lee Ann , Simpson-Haidaris Patricia J. 
Published: October 2019

AbstractLung epithelial cells (A549) synthesize and secrete fibrinogen (FBG) in vitro when stimulated with interleukin-6 and dexamethasone. This FBG secretion is polarized in the basolateral direction, suggesting that FBG is a component of the extracellular matrix (ECM). Immunofluorescent staining of A549 cells showed a fibrillar pattern of FBG, similar to the staining detected using antibodies against the matrix constituents, collagen type IV and fibronectin (FN). The same pattern of staining was detected using antibodies against fibrinopeptides A and B, as well as with the T2G1 monoclonal antibody against the fibrin-specific epitope, β15-21. Matrix staining was unaltered in the presence of the thrombin inhibitor, hirudin, or the plasmin inhibitor, aprotinin, consistent with the interpretation that matrix deposition of FBG does not require such enzymatic action. Metabolic labeling studies confirmed that FBG secreted from A549 cells or deposited into the ECM showed no evidence of thrombin or plasmin proteolytic processing or of transglutaminase-mediated covalent cross-linking (γ-γ dimers or α-polymers). Incubation of either A549 cell-derived or purified plasma FBG with cultures of human foreskin fibroblasts resulted in FBG deposition in the ECM that colocalized with matrix fibrils containing endogenously produced FN and laminin (LN). Binding of FBG to this exogenously produced matrix was unaltered by inhibition of thrombin and plasmin action, yet also exhibited exposure of the fibrin-specific epitope, β15-21. The majority (∼70%) of newly synthesized and secreted FBG is bound to the cell surface as determined by its trypsin-sensitivity. Cell surface-bound FBG is initially deoxycholate-soluble, which, over time, becomes incorporated in the deoxycholate-insoluble ECM in a similar fashion to FN. These data suggest that matrix incorporation requires the binding of secreted FBG to cell-associated matrix assembly sites. However, unlike FN, FBG in the ECM is composed of the dimeric protamer (Aα/Bβ/γγ) and not high molecular weight polymers indicative of fibrin. This study provides evidence that deposition of FBG in both endogenous and exogenously produced matrices results in conformational changes that occur independently of thrombin cleavage. This matrix-bound FBG, on which unique cell-reactive domains are likely exposed, could augment cellular response mechanisms evoked during injury and inflammation.
Identification of T-Cell Epitopes on the Rhesus Polypeptides in Autoimmune Hemolytic Anemia
Authors: Barker Robert N. , Hall Andrew M. , Standen Graham R. , Jones Jeff , Elson Christopher J. 
Published: October 2019

Abstract
We have shown previously that the Rhesus (Rh) polypeptides are the commonest targets for pathogenic anti-red blood cell (RBC) autoantibodies in patients with autoimmune hemolytic anemia (AIHA). The aim of the current work was to determine whether activated T cells from such patients also mount recall responses to epitopes on these proteins. Two panels of overlapping 15-mer peptides, corresponding to the sequences of the 30-kD Rh proteins associated with expression of the D and Cc/Ee blood group antigens, were synthesized and screened for the ability to stimulate the in vitro proliferation of mononuclear cells from the peripheral blood or spleen of nine AIHA cases. Culture conditions were chosen that favor recall proliferation by previously activated T cells, rather than primary responses. In seven of the patients, including all four cases with autoantibody to the Rh proteins, two or more peptides elicited proliferation, but cells from eight of nine patients with other anemias and seven of nine healthy donors failed to respond to the panels. Multiple peptides were also stimulatory in two positive control donors who had been alloimmunized with Rh D-positive RBCs. Six different profiles of peptides elicited responses in the AIHA patients, and this variation may reflect the different HLA types in the group. Stimulatory peptides were identified throughout domains shared between, or specific to, each of the related 30-kD Rh proteins, but T cells that responded to nonconserved regions did not cross-react with the alternative sequences. Anti-major histocompatibility complex class II antibodies blocked the responses and depletion experiments confirmed that the proliferating mononuclear cells were T cells. Notably, splenic T cells that proliferated against multiple Rh peptides also responded to intact RBCs. We propose that pathogenic autoantibody production in many cases of AIHA is driven by the activation of T-helper cells specific for previously cryptic epitopes on the Rh proteins.
An Allelic Association Implicates Myeloperoxidase in the Etiology of Acute Promyelocytic Leukemia
Authors: Reynolds Wanda F. , Chang Eric , Douer Dan , Ball Edward D. , Kanda Vikas 
Published: October 2019

AbstractMyeloperoxidase (MPO) catalyzes a reaction between chloride and hydrogen peroxide to generate hypochlorous acid and other reactive compounds that have been linked to DNA damage. The MPO gene is expressed at high levels in normal myeloid precursors and in acute myeloid leukemias (AMLs) which are clonal derivatives of myeloid precursors that have lost the ability to differentiate into mature blood cells. Two MPO alleles differ at -463 G/A within a cluster of nuclear receptor binding sites in an Alu element. The -463 G creates a stronger SP1 binding site and retinoic acid (RA) response element (RARE) in the allele termed Sp. In this study, we investigate potential links between MPO genotype, MPO expression level, and myeloid leukemia. The SpSp MPO genotype is shown to correlate with increased MPO mRNA levels in primary myeloid leukemia cells. This higher-expressing SpSp genotype is further shown to be overrepresented in acute promyelocytic leukemia-M3 (APL-M3) and AML-M4, suggesting that higher levels of MPO are associated with an increased risk for this subset of leukemias.
Inhibition of Metastasis of Intraocular Melanomas by Adenovirus-Mediated Gene Transfer of Plasminogen Activator Inhibitor Type 1 (PAI-1) in an Athymic Mouse Model
Authors: Ma Ding , Gerard Robert D. , Li Xiao-Yan , Alizadeh Hassan , Niederkorn Jerry Y. 
Published: October 2019

AbstractUveal melanoma is the most common intraocular malignancy in adults and results in the death of 50% of the patients. Plasminogen activators (PA) are believed to facilitate tumor metastasis by promoting invasion of tissue barriers. The present study explored the possibility of preventing the metastasis of intraocular melanomas by disrupting plasminogen activator function through gene transfer. A replication-deficient adenovirus vector was used for the in vivo transfer of plasminogen activator inhibitor type 1 (PAI-1) cDNA. Intraocular injection of an adenovirus vector (AdCMV-PAI-1) expressing plasminogen activator inhibitor-1 resulted in: (1) the transduction of more than 95% of human and murine uveal melanoma cells in the eyes of nude mice; (2) a 50% reduction in the number of animals developing liver metastases; and (3) a 78% reduction in the metastatic tumor burden in animals that eventually developed metastases. In other experiments intravenous injections of AdCMV-PAI-1 resulted in transduction of normal liver cells and culminated in a sharp reduction in the incidence of metastases and a significant prolongation of host survival. The results support the feasibility of disruption of PA function through gene transfer as a therapeutic strategy for preventing metastases and prolonging host survival.
Molecular Interactions Between Hb α-G Philadelphia, HbC, and HbS: Phenotypic Implications for SC α-G Philadelphia Disease
Authors: Lawrence Christine , Hirsch Rhoda Elison , Fataliev Nazim A. , Patel Snehal , Fabry Mary E. , Nagel Ronald L. 
Published: October 2019

AbstractWe show here that αG-Phila.2βC2 has an increased rate of crystal nucleation compared to α2 βC2 (HbC). We conclude from this finding that position α68, the mutation site of αG-Phila.2 β2 (HbGPhiladelphia), is a contact site in the crystal of HbC. In addition, that HbS enhances HbC crystallization (additive to the effect of αG-Phila, as shown here) and that αG-Phila. inhibits polymerization of HbS are pathogenically relevant previously known facts. All of these findings help explain the phenotype of an individual simultaneously heterozygous for the βS, βC, and the αG-Phila. genes (SCα-G Philadelphia disease). This disease is characterized by a mild clinical course, abundant circulating intraerythrocytic crystals, and increased folded red cells. This phenotype seems to be the result of increased crystallization and decreased polymerization brought about by the opposite effects of the gene product of the αG-Phila. gene on the βC and βS gene products. Some of the intraerythrocytic crystals in this syndrome are unusually long and thin, resembling sugar canes, unlike those seen in SC disease. The mild clinical course associated with increased crystallization implies that, in SC disease, polymerization of HbS is pathogenically more important than the crystallization induced by βC chains. The SCα-G Philadelphia disease is an example of multiple hemoglobin chain interactions (epistatic effect among globin genes) creating a unique phenotype.
Stroma-Contact Prevents Loss of Hematopoietic Stem Cell Quality During Ex Vivo Expansion of CD34+ Mobilized Peripheral Blood Stem Cells
Authors: Breems Dimitri A. , Blokland Ellen A.W. , Siebel Karen E. , Mayen Angelique E.M. , Engels Lilian J.A. , Ploemacher Rob E. 
Published: October 2019

Abstract
Stroma-supported long-term cultures (LTC) allow estimation of stem cell quality by simultaneous enumeration of hematopoietic stem cell (HSC) frequencies in a graft using the cobblestone area forming cell (CAFC) assay, and the ability of the graft to generate progenitors in flask LTC (LTC-CFC). We have recently observed that the number and quality of mobilized peripheral blood stem cells (PBSC) was low in patients having received multiple rounds of chemotherapy. Moreover, grafts with low numbers of HSC and poor HSC quality had a high probability to cause graft failure upon their autologous infusion. Because ex vivo culture of stem cells has been suggested to present an attractive tool to improve hematological recovery or reduce graft size, we have studied the possibility that such propagation may affect stem cell quality. In order to do so, we have assessed the recovery of different stem cell subsets in CD34+ PBSC after a 7-day serum-free liquid culture using CAFC and LTC-CFC assays. A numerical expansion of stem cell subsets was observed in the presence of interleukin-3 (IL-3), stem cell factor, and IL-6, while stroma-contact, stromal soluble factors, or combined addition of FLT3-ligand and thrombopoietin improved this parameter. In contrast, ex vivo culture severely reduced the ability of the graft to produce progenitors in LTC while stromal soluble factors partly abrogated this quality loss. The best conservation of graft quality was observed when the PBSC were cultured in stroma-contact. These data suggest that ex vivo propagation of PBSC may allow numerical expansion of various stem cell subsets, however, at the expense of their quality. In addition, cytokine-driven PBSC cultures require stroma for optimal maintenance of graft quality.
Unbalanced Expression of Bcl-2 Family Proteins in Follicular Lymphoma: Contribution of CD40 Signaling in Promoting Survival
Authors: Ghia Paolo , Boussiotis Vassiliki A. , Schultze Joachim L. , Cardoso Angelo A. , Dorfman David M. , Gribben John G. , Freedman Arnold S. , Nadler Lee M. 
Published: October 2019

AbstractAlthough highly responsive, advanced stage follicular lymphoma (FL) is not curable with conventional treatment. This relative resistance is thought to be due to the t(14;18) that results in the constitutive overexpression of the death-inhibiting protein bcl-2. However, the observation that FL cells are sensitive to treatment in vivo and prone to apoptosis on in vitro culture questions whether bcl-2 alone is responsible for the pathogenesis and clinical behavior of this disease. Therefore, multiple genes are likely to be involved in both the lymphomagenesis and the clinical course of FL. We examined whether expression of other bcl-2 family genes might also be operative. Here, we show that FL cells display a different pattern of expression of bcl-2 family proteins from normal germinal center (GC) B cells that are thought to be their normal counterpart. FL cells express the death-suppressor proteins bcl-2, bcl-xL, and mcl-1; whereas GC B cells express bcl-xL and mcl-1 but also the proapoptotic proteins bax-α and bad. Although maintaining constitutive levels of bcl-2 and mcl-1, FL cells are not protected from apoptosis when cultured in vitro. Their propensity to undergo apoptosis is temporally associated with downregulation of bcl-xL. More importantly, activation of FL cells via CD40 not only prevents downregulation but increases the level of bcl-xL expression and results in promotion of survival. These results support the hypothesis that the overexpression of bcl-2 is not the only antiapoptotic mechanism responsible for the pathogenesis of FL. Survival of FL cells is determined by a number of death-inhibiting proteins, among which bcl-xL appears to have the most critical role. Moreover, these findings are consistent with the hypothesis that, although FL cells are malignant, they respond to microenvironmental signals such as CD40L that appear to contribute to their survival through the upregulation of death-inhibiting proteins.
Myeloid Development Is Selectively Disrupted in PU.1 Null Mice
Authors: Anderson Karen L. , Smith Kent A. , Conners Kris , McKercher Scott R. , Maki Richard A. , Torbett Bruce E. 
Published: October 2019

AbstractThe ets family transcription factor PU.1 is expressed in monocytes/macrophages, neutrophils, mast cells, B cells, and early erythroblasts, but not in T cells. We have recently shown that PU.1 gene disruption results in mice with no detectable monocytes/macrophages and B cells but T-cell development is retained. Although neutrophil development occurred in these mice, it was delayed and markedly reduced. We now proceed to demonstrate that PU.1 null hematopoietic cells fail to proliferate or form colonies in response to macrophage colony-stimulating factor (M-CSF), granulocyte CSF (G-CSF), and granulocyte/macrophage CSF (GM-CSF). In contrast, PU.1 null cells did proliferate and form colonies in response to interleukin-3 (IL-3), although the response was reduced as compared with control littermates. Compared with control cells, PU.1 null cells had minimal expression of G- and GM-CSF receptors and no detectable M-CSF receptors. The size of individual myeloid colonies produced from PU.1 null primitive and committed myeloid progenitors in the presence of IL-3, IL-6, and stem cell factor (SCF) were reduced compared with controls. Under these conditions, PU.1 null progenitors produced neutrophils but not monocytes/macrophages. These observations suggest that PU.1 gene disruption induces additional cell-autonomous effects that are independent of the alterations in myeloid growth factor receptor expression. Our results demonstrate that PU.1 gene disruption affects a number of developmentally regulated hematopoietic processes that can, at least in part, explain the changes in myeloid development and reduction in myeloid and neutrophil expansion observed in PU.1 null mice.
Effect of L-Carnitine on Human Immunodeficiency Virus-1 Infection-Associated Apoptosis: A Pilot Study
Authors: Moretti Sonia , Alesse Edoardo , Di Marzio Luisa , Zazzeroni Francesca , Ruggeri Barbara , Marcellini Sonia , Famularo Giuseppe , Steinberg Seth M. , Boschini Antonio , Cifone M. Grazia , De Simone Claudio 
Published: October 2019

AbstractThe Fas/Fas ligand system is involved in uncontrolled apoptosis, which ultimately leads to the loss of T lymphocytes in human immunodeficiency virus (HIV)-infected individuals. The signal transduced by Fas receptor involves the activation of an acidic sphingomyelinase, sphingomyelin breakdown, and ceramide production. Our recent reports have shown that L-carnitine inhibits Fas-induced apoptosis and ceramide production both in vitro and in vivo. The aim of this study was to study, in a preliminary fashion, the impact of long-term L-carnitine administration on CD4 and CD8 absolute counts, rate, and apoptosis in HIV-1–infected subjects. The generation of cell-associated ceramide and HIV-1 viremia was also investigated. Eleven, asymptomatic, HIV-1–infected subjects, who refused any antiretroviral treatment despite experiencing a progressive decline of CD4 counts, were treated with daily infusions of L-carnitine (6 g) for 4 months. Immunologic and virologic measures and safety were monitored at the start of the treatment and then on days 15, 30, 90, and 150. L-carnitine therapy resulted in an increase of absolute CD4 counts, which was statistically significant on day 90 and 150 (P = .010 and P = .019, respectively). A positive, not significant trend was also observed even in the change in absolute counts of CD8 lymphocytes. L-carnitine therapy also led to a drop in the frequency of apoptotic CD4 and CD8 lymphocytes. This reduction occurred gradually, but changes in actual values between each time point and baseline were strongly significant (P = .001 at the end of the study compared with the baseline). A strong reduction (P = .001) in cell-associated ceramide levels was found at the end of the study. In general, HIV-1 viremia increased slightly. No toxicity related to L-carnitine therapy was observed and dose reductions were not necessary. In HIV-1–infected subjects, long-term infusions of L-carnitine produced substantial increases in the rate and absolute counts of CD4 and, to a lesser degree, of CD8 lymphocytes. This was paralleled by a reduced frequency of apoptotic cells of both subgroups and a decline in the levels of ceramide. No clinically relevant change of HIV-1 viremia was observed.