bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of large numbers of enriched human megakaryocytes from liquid cultures of normal peripheral blood progenitor cells
Authors: Mazur EM , Basilico D , Newton JL , Cohen JL , Charland C , Sohl PA , Narendran A 
Published: October 2019

Abstract
Investigations linking human megakaryocyte development and cell biology have been hindered by an inability to obtain large, relatively pure megakaryocyte cell preparations from in vitro stem cell cultures. We report here that such preparations can be generated from liquid cultures of normal human peripheral blood mononuclear cells stimulated by a serum source of megakaryocyte colony stimulating activity (Meg- CSA, the 0% to 60% ammonium sulfate protein fraction of aplastic canine serum). Adherent-depleted peripheral blood mononuclear cells are suspended at 5 x 10(5) to 10(6) cells/mL in supplemented liquid culture medium, platelet-poor human plasma 20% (vol/vol) and 1 to 2 mg/mL serum Meg-CSA protein. After 12 to 14 days of incubation, megakaryocytes constitute 3.0 +/- 2.9% (mean +/- SD, n = 8) of the unseparated cultured cell population. Megakaryocytes can be enriched by counterflow centrifugal elutriation to a purity of 58 +/- 14% (+/- SD) with a recovery of 13 +/- 7% and a viability of 67 +/- 19%. This algorithm results in the average isolation of approximately 3 x 10(5) enriched megakaryocytes from a 100-mL starting volume of peripheral blood. Cultured megakaryocytes exhibit normal light and ultrastructural morphology by Wright-Giemsa staining and electron microscopic analysis. After a 12-day culture interval, enriched megakaryocyte preparations exhibit morphologic stage distributions that are similar to normal human marrow. Stage distributions move rightward with culture duration indicating partial synchrony of megakaryocyte maturation. On cytospin preparations, megakaryocyte diameter averages 30.2 +/- 1.5 microns and increases with maturation stage. Flow cytometric analyses demonstrate the expression of platelet glycoproteins (GP) Ib and IIb/IIIa by the cultured megakaryocytes. The modal ploidy of the enriched cells at day 12 of culture is 16N and most remaining megakaryocytes are 8N or 32N. Liquid culture of serum Meg-CSA-stimulated human peripheral blood mononuclear cells represents a valuable investigative tool that should permit studies of human megakaryocyte biology that have not been possible in the past.
Adenine and guanine nucleotide metabolism during platelet storage at 22 degrees C
Authors: Edenbrandt CM , Murphy S 
Published: October 2019

Abstract
Adenine and guanine nucleotide metabolism of platelet concentrates (PCs) was studied during storage for transfusion at 22 +/- 2 degrees C over a 7-day period using high-pressure liquid chromatography. There was a steady decrease in platelet adenosine triphosphate (ATP) and adenosine diphosphate (ADP), which was balanced quantitatively by an increase in plasma hypoxanthine. As expected, ammonia accumulated along with hypoxanthine but at a far greater rate. A fall in platelet guanosine triphosphate (GTP) and guanosine diphosphate (GDP) paralleled the fall in ATP + ADP. When adenine was present in the primary anticoagulant, it was carried over into the PC and metabolized. ATP, GTP, total adenine nucleotides, and total guanine nucleotides declined more slowly in the presence of adenine than in its absence. With adenine, the increase in hypoxanthine concentration was more rapid and quantitatively balanced the decrease in adenine and platelet ATP + ADP. Plasma xanthine rose during storage but at a rate that exceeded the decline in GTP + GDP. When platelet ATP + ADP was labeled with 14C- adenine at the initiation of storage, half of the radioactivity was transferred to hypoxanthine (45%) and GTP + GDP + xanthine (5%) by the time storage was completed. The isotopic data were consistent with the presence of a radioactive (metabolic) and a nonradioactive (storage) pool of ATP + ADP at the initiation of storage with each pool contributing approximately equally to the decline in ATP + ADP during storage. The results suggested a continuing synthesis of GTP + GDP from ATP + ADP, explaining the slower rate of fall of GTP + GDP relative to the rate of rise of plasma xanthine. Throughout storage, platelets were able to incorporate 14C-hypoxanthine into both adenine and guanine nucleotides but at a rate that was only one fourth the rate of hypoxanthine accumulation. All of these data should be helpful in improving the function and viability of PC as currently stored for 5 days, in devising methods for storage beyond 5 days, and in the development of synthetic media for PC storage.
Characterization of a novel self-associating Mr 40,000 platelet glycoprotein [see comments]
Authors: Hildreth JE , Derr D , Azorsa DO 
Published: October 2019

Abstract
A novel platelet glycoprotein has been purified and characterized. This glycoprotein, designated Pltgp40, is an acidic sialylated 40,000-dalton protein that bears both O-linked and N-linked oligosaccharides. Treatment of Pltgp40 with neuraminidase resulted in a 5,000-dalton reduction in its Mr and a 1.5 Unit alkaline shift in the isoelectric point, indicating the presence of a large number of sialic acid residues. A similar size reduction and change in pl were observed after treatment of Pltgp40 with O-glycanase showing that sialic acids are present on O-linked oligosaccharides. Digestion of Pltgp40 with N- glycanase reduced the Mr to approximately 20,000 daltons but did not affect the isoelectric point, suggesting that Pltgp40 contains six to seven nonsialylated N-linked carbohydrate chains. High Mr proteins were observed in affinity purified Pltgp40 and were identified as detergent- stable protein oligomers consisting of multiple 40,000-dalton monomers. Immunodepletion and direct binding studies indicated that Pltgp40 was not equivalent to Ig Fc receptor type II, another 40,000-dalton glycoprotein expressed on platelets. However, Pltgp40 copurified with Fc receptor type II when platelet extracts were loaded onto human IgG affinity columns, raising the possibility that Pltgp40 may associate with Fc receptors or Fc receptor-lg complexes. Amino acid sequence analysis of the N-terminus of Pltgp40 was performed and confirmed that Pltgp40 is a novel platelet glycoprotein. Epitopes on Pltgp40 appear to be widely expressed because monoclonal antibodies against Pltgp40 also reacted with a variety of myeloid, lymphoid, and epithelial cells. Pltgp40 was detected on activated but not resting platelets, indicating that Pltgp40 is a platelet activation marker.
T-cell receptor delta gene recombination in common acute lymphoblastic leukemia: preferential usage of V delta 2 and frequent involvement of the J alpha cluster
Authors: Yokota S , Hansen-Hagge TE , Bartram CR 
Published: October 2019

Abstract
A high frequency (greater than 80%) of acute lymphoblastic leukemias (ALL) exhibit a recombination of the T-cell receptor (TCR) delta chain locus. Interestingly, distinct TCR delta elements are preferentially used in immunologic subtypes. In a recent series of 201 children with common ALL (cALL) we observed a TCR delta rearrangement in 162 patients, 57% of the latter showing a hybridization pattern in Southern blots suggestive of a V delta 2 to D delta 3 recombination. To verify this interpretation and to elucidate in more detail the diversity of this common type of TCR delta recombination we amplified and sequenced the junctional region of nine cALL patients and cell line REH-6 by polymerase chain reaction (PCR). A V delta 2 D delta 3 recombination was confirmed in all cases; convincing evidence for the participation of D delta 1 or D delta 2 elements was not obtained. Eight of nine patients and REH-6 showed complete 5′ D delta 3 boundaries within V delta 2 D delta 3 segments, a limitation of junctional diversity also detected in 50% of peripheral blood cell clones derived from two healthy probands. Notably, sequence identity at the V delta 2 D delta 3 junction was demonstrated for a cALL and one of the control clones. Another group of 35 of 162 cALL patients was characterized by V delta 2 rearrangements and biallelic deletion of J delta and C delta sequences. Using a J alpha consensus primer, PCR-directed sequence analysis demonstrated V delta 2 D delta 3 J alpha recombinations in all four cases analyzed by this approach. The J alpha segments of these patients differed, but were identical or homologous to published J alpha elements. Our data suggest a recombination pathway of the TCR delta/alpha locus leading to chimeric TCR alpha molecules, containing V delta and, remarkably, also D delta sequences.
Human thymic epithelial cells produce interleukin-3
Authors: Dalloul AH , Arock M , Fourcade C , Hatzfeld A , Bertho JM , Debre P , Mossalayi MD 
Published: October 2019

Abstract
Interleukin-3 (IL-3) is a hematopoietic growth factor suggested to be produced by activated T lymphocytes. Meanwhile, supernatants from human thymic stroma could promote the proliferation of myeloid stem cells. Thus, we investigated whether IL-3 accounts for this activity. Therefore, human thymic epithelial cells (TEC), fibroblasts, and adherent cells were isolated, and their culture supernatants assayed for myeloid colony promotion. Only supernatants from thymic epithelial cells supported colony-forming unit growth in semisolid media. This effect decreased following anti-IL-3 monoclonal antibody addition to these cultures. Furthermore, in situ hybridization showed the presence of IL-3 mRNA in epithelial cells. Effect of TEC culture conditions on IL-3 production by these cells was also studied. Together, these data show that IL-3 production is not the exclusive property of human activated T lymphocytes.
Effect of murine mast cell growth factor (c-kit proto-oncogene ligand) on colony formation by human marrow hematopoietic progenitor cells
Authors: Broxmeyer HE , Cooper S , Lu L , Hangoc G , Anderson D , Cosman D , Lyman SD , Williams DE 
Published: October 2019

Abstract
Purified natural (n) and recombinant (r) murine (mu) mast cell growth factor (MGF, a c-kit ligand) were evaluated alone and in combination with r human (hu) erythropoietin (Epo), rhu granulocyte-macrophage colony-stimulating factor (rhuGM-CSF), rhuG-CSF, and/or rhuM-CSF for effects in vitro on colony formation by multipotential (colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit erythroid [BFU-E]) and granulocyte- macrophage (CFU-GM) progenitor cells from normal human bone marrow. MGF was a potent enhancing cytokine for Epo-dependent CFU-GEMM and BFU-E colony formation, stimulating more colonies and of a larger size than either rhu interleukin-3 (rhuIL-3) or rhuGM-CSF. MGF, especially at lower concentrations, also acted with rhuIL-3 or rhuGM-CSF to enhance Epo-dependent CFU-GEMM and BFU-E colony formation. MGF had little stimulating activity for CFU-GM colonies by itself, but in combination with suboptimal to optimal amounts of rhuGM-CSF enhanced the numbers and the size of CFU-GM colonies in an additive to greater than additive manner. While we did not detect an effect of MGF on CFU-G colony numbers stimulated by maximal concentrations of rhuG-CSF, MGF did enhance the size of CFU-G-derived colonies. MGF did not enhance the activity of rhuM-CSF. In a comparative assay, maximal concentrations of rmu and rhuMGF were equally effective in the enhancement of human bone marrow colony formation, but rhuMGF, in contrast to rmuMGF, did not at the concentrations tested enhance colony formation by mouse bone marrow cells. MGF effects on BFU-E, CFU-GM, and CFU-GEMM may be direct acting ones as MGF-enhanced colony formation by these cells in highly enriched progenitor cell populations of CD34 HLA-DR+ and CD34 HLA-DR+CD33- sorted cells in which greater than or equal to 1 of 2 cells was a BFU-E plus CFU-GM plus CFU-GEMM. MGF appears to be an early acting cytokine that preferentially stimulates the growth of immature hematopoietic progenitor cells.
Granulocyte colony-stimulating factor in cerebrospinal fluid from patients with meningitis
Authors: Shimoda K , Okamura S , Omori F , Mizuno Y , Hara T , Aoki T , Ueda K , Niho Y 
Published: October 2019

Abstract
Granulocyte colony-stimulating factor (G-CSF) in the cerebrospinal fluid from patients with meningitis was measured by our modified enzyme- linked immunosorbent assay for G-CSF. The minimal detection level was 20 pg/mL G-CSF. In patients with bacterial meningitis, the G-CSF levels in the cerebrospinal fluid were extremely elevated, showing a mean value of approximately 1,500 pg/mL. On the other hand, G-CSF levels in the cerebrospinal fluid from 67% patients with aseptic meningitis were moderately increased, showing a mean value of about 80 pg/mL, whereas G- CSF levels in 33% samples remained undetectable. The G-CSF levels and neutrophil counts in the cerebrospinal fluid were proven to be related by Spearman's rank correlation coefficient analysis (r = .724). These elevations of G-CSF levels at inflammation sites associated with bacterial meningitis may indicate that G-CSF plays an important role in the combat of bacterial infections.
Mycobacterium avium-intracellulare induces interleukin-6 from human monocytes and large granular lymphocytes
Authors: Blanchard DK , Michelini-Norris MB , Pearson CA , Freitag CS , Djeu JY 
Published: October 2019

Abstract
Mycobacterium avium-intracellulare (MAI) is an opportunistic pathogen commonly found in acquired immunodeficiency syndrome patients, whose immune systems are severely compromised. However, normal responses to this bacterium are apparently sufficient to prevent disseminated infection because disease is rarely found unless an immunocompromised state is present. Because interleukin-6 (IL-6) is an inflammatory cytokine with a multitude of activities, we investigated the potential of MAI to induce IL-6 from normal human leukocytes. Peripheral blood mononuclear cells were fractionated into monocytes (Mo), large granular lymphocytes (LGL), and T cells and stimulated with bacteria. Culture supernatants were collected and assayed for IL-6 activity by bioassay. Mo and LGL, but not T cells, were found to release IL-6 within 12 hours of stimulation, with optimal production occurring by 2 days of culture. Production of IL-6 from human leukocyte subsets was confirmed by Northern blot analysis and by neutralization of biologic function of the culture supernatants with specific antisera. Taken together, these results indicate that production of IL-6 is a key response of Mo and LGL to MAI. The role of IL-6 in MAI infection, therefore, needs to be further investigated.
Correlation of response of aplastic anemia patients to antilymphocyte globulin with in vitro lymphocyte stimulatory effect: predictive value of in vitro test for clinical response
Authors: Abe T , Matsuoka H , Kojima S , Kamachi Y , Tsuge I , Kodera Y , Matsuyama T 
Published: October 2019

Abstract
Therapy with antilymphocyte globulin (ALG) has been shown to be effective in restoring hematopoiesis to some patients with aplastic anemia. It would be useful to have a method for predicting those likely to be responders versus nonresponders. The mode of immunostimulatory action of ALG is of interest in addition to its immunosuppressive action. We examined in vitro the distribution of the proliferative responses of ALG-stimulated peripheral blood mononuclear cells (PBMCs) obtained from 18 patients with aplastic anemia, eight of whom responded to ALG and 10 who did not. We found a significant difference in the proliferative response of PBMCs obtained from the eight responders versus the 10 nonresponders (P less than .01). Two-color flow cytometry analysis of the patients' PBMCs stimulated by ALG in vitro showed that the CD4-positive subsets were activated to a greater extent by ALG than the CD8-positive subsets. Moreover, a positive correlation with the clinical response of patients to ALG with granulocyte-macrophage colony- stimulating factor produced by their PBMCs stimulated by ALG suggests that the immunostimulatory property of ALG has an important role in the treatment of aplastic anemia. Our results suggest that the clinical response to ALG therapy is correlated with its lymphocyte proliferative effect in vitro, and indicates that the assessment of the proliferative response of PBMCs in vitro would be useful in predicting the clinical response to ALG therapy.
Human herpesvirus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction
Authors: Torelli G , Marasca R , Luppi M , Selleri L , Ferrari S , Narni F , Mariano MT , Federico M , Ceccherini-Nelli L , Bendinelli M 
Published: October 2019

Abstract
In search of a possible involvement of the human herpesvirus type 6 (HHV-6) in human Hodgkin's and non-Hodgkin's lymphomas, we studied the levels of anti-HHV-6 antibodies in the sera of 94 cases by an immunofluorescence assay, as well as the presence of HHV-6 sequences in the affected tissues of 66 cases by polymerase chain reaction, using one set of primer oligonucleotides. Our results showed higher anti-HHV- 6 antibody titers in human lymphomas than in normal blood donors, but the difference is statistically significant only when normal donors are compared with Hodgkin's lymphoma cases. HHV-6 sequences were detected in 3 of 25 Hodgkin's lymphomas and 0 of the 41 cases of non-Hodgkin's lymphomas studied. The three cases positive for HHV-6 sequences belong to the nodular sclerosis-lymphocyte depletion histologic subtype and share remarkable similarities in their clinical features. Furthermore, Southern blot analysis of total genomic DNA obtained from the neoplastic tissues of two of the three patients showed the same restriction fragment length polymorphism. Our results suggest that: (1) the high level of anti-HHV-6 antibodies in Hodgkin's disease is due to an activation of the immune system not related to the presence of HHV-6 sequences in affected lymph nodes; (2) the presence of HHV-6 sequences in human lymphoid tissues is not a frequent event, rather it is in fact a very rare event in non-Hodgkin's lymphomas, while in Hodgkin's cases it is more frequent than previously reported on the basis of Southern blot analysis; and (3) the presence of HHV-6 sequences in Hodgkin's lymphomas may have a relation with the clinical presentation of the disease.
Functional properties of the beta-globin locus control region in K562 erythroleukemia cells
Authors: Moon AM , Ley TJ 
Published: October 2019

Abstract
In this report, we compare the function of the human beta-globin locus control region (LCR) in three K562 erythroleukemia cell assays, including (1) a transient transfection assay for “classical' enhancer activity, (2) a colony assay that detects “productive integration events,” and (3) an assay that detects the ability of LCR fragments to confer hemin inducibility on linked, stably integrated gamma-globin promoters. Various LCR fragments were inserted into an expression vector consisting of an A gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo). Using these vectors, we determined that a 2.5-kb DNA fragment containing LCR sites I through IV (previously named mu locus activation region [mu LAR]) had activity in all three assays; of the individual LCR sites, only site II was highly active in all three assays. One region within site II, consisting of tandem AP-1/NF-E2 consensus elements, had approximately 10% as much colony assay activity as the entire mu LAR. However, this region did not have detectable activity in a transient enhancer assay in uninduced K562 cells, nor was it capable of conferring hemin inducibility on linked gamma-globin promoters in stably transfected cells. Finally, we tested the ability of the mu LAR to activate promoters (beta-globin and cathepsin G) that are not normally expressed in K562 cells. beta-neo was minimally activated by the mu LAR in transient transfection experiments. The mu LAR increased the number of stably transfected colonies produced by beta-neo, but the absolute number of beta-neo colonies, with or without the mu LAR, was approximately 10% to 20% that of gamma-neo. In contrast, a minimal cathepsin G promoter was activated by the mu LAR in K562 cells. Our results suggest that LCR functions are dependent in part on the environments and the promoters with which the LCR is tested.
Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor
Authors: Bernstein ID , Andrews RG , Zsebo KM 
Published: October 2019

Abstract
We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.
Iron uptake by human reticulocytes at physiologic and sub-physiologic concentrations of iron transferrin: the effect of interaction with aluminum transferrin [see comments]
Authors: Cochran M , Chawtur V , Jones ME , Marshall EA 
Published: October 2019

Abstract
We have studied the interaction, in vitro, between diferric transferrin (FeTr), aluminum transferrin (AlTr), and human reticulocytes harvested from human placental blood. In particular, we aimed to determine the extent to which the kinetics of AlTr and FeTr differed. Using transferrin labeled with either 59Fe or 125I, the association of radiotracer with reticulocytes, as a function both of time and of transferrin concentration, was examined. Under the conditions of the experiments, the data are consistent with a mechanism involving at least three processes. Two early processes acting in parallel behave as a high-affinity saturable receptor and a low-affinity non-saturable receptor, neither of which distinguish between AlTr and FeTr. In a subsequent process, AlTr and FeTr exhibit different kinetics. This third process may be saturated by FeTr but not by AlTr. Interpreted in terms of a current conventional view of metallo-transferrin uptake, we conjecture that the early parallel processes involve cell surface phenomena including classical transferrin-receptor binding, and that the subsequent process represents events, possibly intracellular, involved in metallo-transferrin dissociation or further iron transport. The extent to which AlTr influences the interaction of FeTr with reticulocytes offers insight into both the normal physiology of iron uptake and the potential for toxicity by aluminum.