bluuebunny/crossref_metadata_2025_split huggingface dataset:
Aspirin Down-Regulation of Platelet Reactivity in Patients with Acute Coronary Syndrome: Effects of Erythrocytes.
Authors: Santos Maria T. , Valles Juana , Fuset Maria P. , Perez Francisca , Moscardo Antonio , Aznar Justo , Ruano Miguel 
Published: October 2019

Abstract
Platelet-erythrocyte interactions enhance platelet reactivity (Santos et al.J Clin Invest1991;87:571;Valles et al. Blood2002; 99:3978) and reduce the effect of aspirin (ASA) as antithrombotic modality (Santos et al. Circulation1997;95:63; Valles et al.Circulation1998;97:350). These effects were studied using an experimental approach which independently evaluates platelet activation (TXA2, 5HT release) and recruitment (proaggregatory activity of cell-free releasates) during cell-cell interactions between erythrocytes (RBCs) and collagen-stimulated platelets (PTLs) or in whole blood (WB). In patients with chronic vascular disease treated with ASA, with blunted TXA2, three response groups were found: in Group A (39% of patients), recruitment was blocked in PTLs alone, PTL+RBCs and in WB; in Group B (45%), recruitment was abolished in PTLs alone but continued in the presence of RBCs and in WB; in Group C (16%), recruitment was detected in PTLs alone and was markedly enhanced by RBCs (Valles et al.Circulation1998; 97:350). Using the same experimental approach, 80 patients with acute coronary syndrome (ACS) treated with aspirin (100–300 mg/d) as the only antiplatelet therapy were studied. Other medications were administered according to guidelines. ASA-free (n=80) normal subjects were also evaluated as controls. Results: In ACS patients blockade of TXA2 was observed in only 76% of patients, while partial inhibition (44.7%) was achieved in 24%. In the latter patients platelet recruitment in WB was high, equal to that in ASA-free controls (ACS X=94.58 vs. controls 87.59). When the three response groups described above were studied in the 61 ACS patients with blocked TXA2 synthesis, the proportions differed markedly from chronic patients: Group A (3% vs. 30%), Group B (18% vs. 45%) and Group C (79% vs. 16%). Since TXA2 was blunted in these patients, the results indicate that ACS patients are highly susceptible to stimulation by COX-1 independent mechanisms. In 18% (Group B) this could be attributable to the ASA-insensitive prothrombotic effect of RBCs, while in 79% (Group C) platelet hyperactivity was intrinsic to PTLs and markedly enhanced by RBCs. Conclusions: Evaluation of TXA2 synthesis in ASA-treated ACS patients is advisable, since partial TXA2 inhibition results in full platelet recruitment. COX-1-independent mechanisms of platelet reactivity are prominent in ACS patients and increased by RBCs. Since platelet recruitment is essential in thrombus growth its inhibition by ASA alone or in combination with other antiplatelet drugs could reduce occlusive thrombus formation. (Grant FIS 03/0270).
AMD3100 Plus G-CSF Use in Proven Poor Mobilizers in a Compassionate Use Protocol (CUP).
Authors: McGuirk Joseph , McCarty John , Gibb Kaaren , Badel Karin , Bridger N Gary , Calandra Gary 
Published: October 2019

Abstract
AMD3100 (A), an inhibitor of SDF-1∞ binding to CXCR4, has been used alone and with G-CSF (G) to mobilize CD34+ cells from healthy volunteers and donors and from cancer patients undergoing autologous transplantation. In several studies it enhanced mobilization of poor mobilizers. Therefore, a CUP was submitted to the FDA; more than 100 patients (most commonly NHL; N=42) have been entered. Results from the first 70 patients enrolled are reported here. Three patients underwent a second mobilization with A following initial failure (2) and disease relapse (1); data on the first mobilization is in this report. Entry requirements include documented inability to mobilize 2 x 106 CD34+ cells/kg. Patients must have acceptable cardiac and pulmonary status, no active infection, and to have WBC >3.0 x 109/L, ANC > 1.5 x109/L, PLT >100,000 x 109/L, LFT within 2 x ULN, and creatinine clearance >60 ml/min. as well as other acceptable laboratory values. After signing consent and undergoing history, physical, laboratory, CXR, and ECG evaluation, patients are given G (10 mg/kg) for 4 days. At about 10 p.m. on day 4 they receive 240 mg/kg of A subcutaneously. The next a.m. they receive G and undergo apheresis. The evening A, the a.m. G, and the apheresis are repeated until enough cells are collected or until it is clear insufficient cells could be collected. There were 38 males and 32 females. Five were 18 years or younger (10, 12, 13, 16 and 18 years). The rest ranged in age from 21 to 81 years. Most common diseases were NHL (31), MM (14), AML (8), and Hodgkin’s disease (HD) (10). The 70 patients had undergone 93 possible mobilizations and 16 patients failed multiple prior regimens. Nearly all (91/93) prior mobilization regimens contained G. The most common regimens were G alone (33), cyclophosphamide plus G (17), and cyclophosphamide plus G plus other agents (30). In a subset of patients (N=27) the median time between the prior failed mobilization and the CUP mobilization was 34 days. A was generally well tolerated with AEs being similar to that previously reported. Only one SAE was felt related - patient with worsening of GI symptoms. The percent of patients collecting >2 x 106 cells/kg for the more common cancers were 58 for NHL, 64 for MM, 50 for AML, and 70 for HD. For those collecting >2 x 106 cells/kg (N=40) the median collection was 4.72 106 cells/kg in a median of 3.5 aphereses. The number of these patients transplanted was 36 of 40 with 31 successful engraftments, 3 pending, and two deaths post successful PMN, but not PLT engraftment. Of all the patients transplanted 4 additional patients have died. Durability has been good with at least 24 patients at 6 months or beyond. The success of mobilization compares very favorably with prior published data. A offers a generally safe and effective way to enhance mobilization of patients who have previously failed mobilizations.
Adult Human Blood Leukocytes as an Efficient Source for Tissue-Committed Neural Progenitors.
Authors: Porat Yael , Fulga Valentin , Belkin Danny , Porozov Svetlana , Fisher Yehudit , Belkin Michael , Silverman Willam F. 
Published: October 2019

Abstract
In the last few years, significant progress has been made in the isolation and characterization of bone marrow stem cell populations and their potential to differentiate into a variety of cellular lineages. We hypothesized that peripheral blood can also be used as a source for precursor cells that can become committed progenitors for a variety of tissues. We report here the generation and characterization in vitro of neural progenitor cells from a newly discovered blood-derived multipotent cell population, named synergetic cell population (SCP). Human blood samples were obtained from the Israeli blood bank and SCP cells were purified based on cellular density. Neural progenitors were generated by culturing SCP cells in medium supplemented with autologous serum, followed by activation in a defined serum-free medium containing the specific differentiation-inducing factors F12, B27, bFGF, BDNF, and NGF.
An average of 13.5x106 neural progenitor cells was generated from 450 ml blood. These cells developed irregular perikarya, from which filamentous extensions spread contacting neighboring cells and forming net-like structures. Immunostaining revealed that some of the cells express the early neuronal progenitor markers nestin and b-tubulin and Neu-N, a nuclear protein present in mature neurons. Other cells expressed glial-specific antigens, such as O4 (a marker of oligodendrocytes) and GFAP (a marker of astrocytes). Flow cytometry analysis showed that 44.4% and 34% of the cells were positive for nestin and b-tubulin, respectively. In addition to exhibiting phenotypic evidence of markers specific for the neural lineage, these progenitor cells also responded to the neurotransmitters glutamate and GABA, as detected by calcium influx through voltage-gated calcium channels, demonstrating functional differentiation.
In this study we show that generation of neural progenitors from peripheral blood is feasible and efficient. Blood-derived angiogenic progenitors produced in our system are already safely and efficiently administrated to severe angina pectoris patients in a clinical trial we are conducting in Thailand (reported in a separate abstract by our group). The newly discovered source of these progenitors, the blood-derived multipotent population which we termed SCP, contains both hematopoietic stem cells as well as supportive cells that enable differentiation into various lineages. The therapeutic potential of these neural progenitors will be further characterized and evaluated in vivo using animal models.
Zebrafish KLF4 Is Essential for Primitive Haematopoiesis.
Authors: Perkins Andrew C. , Gardiner Melissa R. 
Published: October 2019

Abstract
Gene knockout studies of Krüppel-like factors (KLFs) in mice have demonstrated key roles in organogenesis. EKLF (Klf1) plays an essential role in definitive erythropoiesis and b-globin gene expression, but primitive (yolk sac) erythropoiesis and embryonic globin gene expression are essentially normal. Since expression of embryonic globin genes is dependent upon proximal CACC box elements, additional KLFs must function during the embryonic wave of erythropoiesis. A number of KLFs have been described in zebrafish. One of these, zKLF4, the homologue of neptune, a Xenopus Laevis KLF expressed in the ventral blood island, cement and hatching glands, is an early marker of lateral plate mesoderm (LPM). From 1 somite and prior to expression of scl and flk1, there is a continuous rim of expression of zKLF4 2–3 cells wide within the LPM extending from the postero-lateral boundary of the polster to the tail. Expression in the posterior lateral plate persists as it migrates medially in a zipper-like movement to generate the intermediate cell mass (ICM), the site of embryonic haematopoiesis. zKLF4 is also expressed in a patch of dorsal mesendodermal cells from 70% epiboly which are fated to become the polster. Morpholino knockdown of zKLF4 results in moderate anaemia which resolves as definitive erythropoiesis replaces primitive erythropoiesis at 48–72 hpf. zKLF4 morphants show a down-regulation in the expression of GATA-1 (>100 fold) and embryonic globin (>10 fold) by real time RT-PCR. Both are expressed during the primitive wave of haematopoiesis. In addition there is a dramatic up-regulation (>100 fold) in the level of cmyb expression in zKLF4 morphant compared to control embryos at 72 hpf, which is consistent with expansion of the secondary definitive wave of erythropoiesis, possibly in response to anemia. These results suggest mammalian KLF orthologs of zKLF4, either KLF4 or KLF2, may play roles in primitive erythropoiesis and embryonic globin gene expression in mice or man.
Development of a Novel Assay Using Bioluminescent Reporter Bacteria To Determine the Sensitivity of AML Cells to Cytosine Arabinoside.
Authors: Smith Graham , Angell Joanna E. , Alloush Habib M. , Smith Margaret A. , Hill Phil J. , Salisbury Vyv C. 
Published: October 2019

Abstract
The nucleoside analogue cytosine arabinoside(Ara-C) has been a cornerstone of remission induction therapy for patients with AML for three decades. Even today, approximately 70% of adults fail to achieve durable complete remission with regimens including Ara-C. This project involves the development of a simple, rapid, economical test to determine Ara-C sensitivity of patient cells to enable clinicians to consider the efficacy of this drug prior to patient treatment.
Bacteria expressing lux genes have been successfully used to study real time pharmacodynamics of antimicrobial agents. We have extended this elegant reporter gene technology to determine cellular penetration of Ara-C, into AML cells. AraC has no effect on E. coli since it does not express deoxycytidine kinase (dCK) and has the ability to deaminate AraC into AraU through the activity of cytidine deaminase (cdd). To render E. coli sensitive to AraC, a cdd-deficient strain was used as the host to express the human dCK gene carried on pTrc99-A plasmid under the control of the lac promoter. The expression of dCK, induced by the addition of IPTG, made E. coli sensitive to AraC. E. coli was then made self-bioluminescent by introducing the pAL2 plasmid carrying the lux operon (luxABCDE)
AML cell lines were incubated with therapeutic levels of Ara-C (100 μM),washed, and re-suspended in fresh media containing bioluminescent modified E. coli and 0.1% saponin, to lyse the AML cells. Light levels and optical density were monitored using a microtitre plate reader (Fluorstar Optima, BMG Labtec).
Cell lines minus drug were used so as to generate control lysates and supernantants.
It was possible to detect significant increases in light levels from the lysate and supernatant of an AML cell-line known to be Ara-C sensitive (KG-1a). The lysate from a cell-line (THP-1) known to deaminate Ara-C through the over production of cytidine deaminase showed no increase in light production in the presence of Ara-C. In addition no difference in light levels was observed from the supernatant of these resistant cells. The control supernatant from both cell lines showed no change in light output. These findings indicate that this assay could be used to evaluate the uptake and metabolism of Ara-C by leukaemia cells. With refinement, the assay could be used to specify at which point in Ara-C metabolism AML cells acquire resistance. Furthermore, this technology could be modified to examine the sensitivity of AML cells to other established chemotherapeutic agents such as anthracyclines thus giving a genuinely helpful chemosensitivity profile to clinicians involved in patient care and customising treatment for individual patients.
A Quick Method for Identifying the Molecular Targets of Potentially Diagnostic New Monoclonal Antibodies Using Protein Array Technology.
Authors: Barry Richard , Murphy Derek , Ip Cho Simon , Cordell Jacqueline , Jones Margaret , Roberton Helen , Cahill Dolores , Mason David 
Published: October 2019

Abstract
During the course of raising monoclonal antibodies, reagents are often produced that are not directed against the immunising antigen. These may pass unnoticed unless a screening step based on immunostaining of human tissue is included. Many of these reagents are auto-antibodies, often directed against intracellular targets (e.g. nuclear components), and the process of hybridoma production serves to “rescue” these self-reactive B cell clones. Such unexpected antibodies of this sort may prove of interest if their distribution is analysed on normal tissues and within the spectrum of leukaemia/lymphoma samples showing specific patterns of staining. However, before they can be used diagnostically or therapeutically, their target molecule needs to be identified, and this can be technically demanding. We describe a novel approach that can be used to define the targets of new monoclonal antibodies to intracellular molecules. The technique involves screening against protein arrays comprising thousands of recombinant human proteins expressed in bacteria. In our initial experiments we showed that a control monoclonal antibody against nucleophosmin identified 22 clones encoding this ubiquitous protein in a human foetal brain cDNA library. Monoclonal antibody JC1 (first published in 1992) was known to recognise an unidentified proliferation-associated nuclear molecule, and screening on the recombinant protein array defined nuclear factor I/X (NFIX) as its probable target. A second antibody (JJ166), reactive with cell nuclei, was shown by the same technique to recognise the mitotic spindle-associated molecule NUMA1 (the gene for which is involved in rare chromosomal translocations). To confirm these putative specificities, the antibodies were shown to react with Cos-1 cells that had been transiently transfected with the corresponding clones from the array (after transfer into a mammalian vector pcDNA4HisMaxC, incorporating an Xpress tag). This new method represents an effective and quick strategy for defining the protein targets of new monoclonal antibodies identified as having diagnostic or other potential value on the basis of their immunostaining patterns.
Proplatelet Formation Is Regulated by the Rho/Rock Pathway.
Authors: Chang Yunhua , Aurade Frederic , Larbret Frederic , Le Couedic Jean Pierre , Momeux Laurence , Zhang Yan Yan , Larghero Jerome , Louache Fawzia , Bertoglio Jacques , Cramer Elisabeth , Vainchenker William , Debili Najet 
Published: October 2019

Abstract
Megakaryopoiesis is a highly specialized cellular process which sustains platelet production. At the end of megakaryopoiesis, megakaryocyte (MKs) fragments into platelets via long and thin cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) is associated essentially with cytoskeleton changes, including actin dynamics. The Rho/Rock pathway is a well characterized regulator of the actin reorganization. In the present study, we have tried to understand the precise role of the Rho/Rock pathway in PPF from human CD34+ derived MKs. Our results show that Rho is expressed in MKs and that its expression and activity remain stable during megakaryopoiesis. Overexpression of a RhoA dominant negatif (RhoA N19) in MKs leads to an increase in PPF. Conversely overexpression of a RhoA spontaneous active (RhoA V14) in MKs leads to a decrease in PPF. These results indicate that Rho activation could inhibit PPF in vitro. It is known that Rho/ROCK promotes actin cytoskeleton dynamics by regulating myosin light chain 2 (MLC2) phosphorylation. To demonstrate that Rho/Rock inhibits PPF through MLC2 phosphorylation, we added MLC kinase inhibitor (P18), Rho inhibitor (TatC3) and ROCK inhibitor (Y27362) in MKs culture just before PPF. Western blot analysis shows that MLC2 phosphorylation was inhibited by these 3 compounds, in contrast, PPF was significantly increased. Moreover, the platelet produced have an identical size and ultrastructure as control platelets and could be normally activated. These results suggest that Rho/ROCK could inhibit PPF through MLC phosphorylation during megakaryopoiesis.
Differential Mapping of the Amino Acids Mediating Agonist and Antagonist Coordination with the Human Thromboxane A2 Receptor Protein.
Authors: Khasawneh Fadi T. , Huang Jin-Sheng , Turek Joseph W. , Le Breton Guy C. 
Published: October 2019

Abstract
Despite the well-documented involvement of thromboxane A2 receptor (TPR) signaling in the pathogenesis of thrombotic diseases, there are currently no rationally-designed antagonists available for clinical use. To a large extent this derives from a lack of knowledge regarding the topography of the TPR ligand binding pocket. On this basis, the purpose of the current study was to identify the specific amino acid residues in the TPR protein which regulate ligand coordination and binding. The sites selected for mutation reside within or in close proximity to a region we previously defined as a TPR ligand binding site, i.e., the C-terminus of the second extracellular loop and the leading edge of the fifth transmembrane domain. Mutation of these residues caused varying effects on the TPR-ligand coordination process. Specifically, the D193A mutant lacked both SQ29,548 (antagonist) binding and U46619 (agonist)-induced calcium mobilization. Three other mutants, F184Y, T186A and S191T, discriminated between SQ29,548 binding and the U46619-mediated functional response. Furthermore, these mutants also revealed a divergence in the binding of two structurally different antagonists, SQ29,548 and BM13.505. Conversely, two separate mutants which exhibited SQ29,548 binding activity yielded either a normal (F196Y) or reduced (S201T) U46619 response. Finally, mutation of other residues directly adjacent to those described above, e.g., E190A and F200A, produced no detectable effects on either SQ29,548 binding or the U46619-induced functional response. In summary, these results identify key amino acids involved in TPR ligand coordination and demonstrate that TPR-specific ligands do not necessarily interact with the same residues in the ligand-binding pocket.
“Maturational” Globin Switching in Primary Primitive Erythroid Cells.
Authors: Palis James , Malik Jeff , Emerson Rachael L. , Bushnell Tim P. , McGrath Kathleen E. , Bulger Michael , Kingsley Paul D. 
Published: October 2019

Abstract
Mammals have two distinct erythroid lineages. The “definitive” erythroid lineage generates small, enucleated erythrocytes that constitute the predominant cell type in the fetal and postnatal circulation. It is preceded by the “primitive” erythroid lineage, which originates in the yolk sac and generates a semi-synchronous wave of large erythroblasts that terminally differentiate in the bloodstream. This feature provides a unique opportunity to investigate changes in gene expression during erythroid maturation. Here, we have examined expression of the various α- and β-globin genes in purified populations of primary primitive erythroid cells isolated from progressive developmental time-points of mouse embryogenesis. Our studies, using both in situ hybridization and quantitative PCR, indicate that βH1 is the predominant β-globin transcript in the early yolk sac. Thus, unlike the human, the murine β-globin genes are not up-regulated in the order of their chromosomal arrangement. As primitive erythroblasts mature from proerythroblasts to reticulocytes, they undergo a βH1- to εy-globin switch, up-regulate low levels of the adult β1- and β2-globins, and down-regulate ζ-globin. These changes in transcript levels correlate with changes in RNA polymerase II density at their promoters and transcribed regions as assayed by ChIP assays. Furthermore, we found that the εy- and βH1-globin genes in primitive erythroblasts reside within a single large hyperacetylated domain. Taken together, these results are consistent with the notion that this βH1- to εy-globin “maturational” switch is dynamically regulated at the transcriptional level. We conclude that the embryonic to adult globin switch that occurs during murine ontogeny is due not only to the sequential appearance of primitive and definitive erythroid lineages but also to striking changes in globin gene expression that occur as primitive erythroblasts mature in the bloodstream.