bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stem cell factor amplifies newborn and sickle erythropoiesis in liquid cultures
Authors: Weinberg RS , Thomson JC , Lao R , Chen G , Alter BP 
Published: October 2019

Abstract
A two-phase liquid-culture system was used to substantially amplify and differentiate erythroblasts, starting with mononuclear cells from the blood of normal adults, newborn infants, and patients with sickle cell anemia. After the first 7 days (phase 1), in medium plus fetal bovine serum (FBS) alone, or in combination with stem cell factor (SCF) or conditioned medium (CM), the cell number was unchanged, and the cells all looked like lymphocytes. These cells were then diluted into medium with erythropoietin (Ep) alone, with Ep and either SCF or CM, or in methylcellulose with the same factors (phase 2). After 14 days in liquid phase 2 with SCF and Ep, the cell numbers increased an average of 30-fold in the sickle, 24-fold in the newborn, and 4-fold in the normal adult cultures; almost all the cells were erythroblasts and erythrocytes. SCF in phase 1 increased the number of late progenitors (CFU-E) assayed in methylcellulose, with the largest number in sickle, followed by newborn cultures and then adult cultures. We conclude that erythroid progenitor cells survive for at least 7 days without Ep (but with FBS). Progenitor cells are amplified, particularly with SCF. Later in culture, SCF with Ep increases the final number of differentiated erythroid cells. Both the early and the late effects of SCF are most effective in sickle, followed by newborn cultures and then adult cultures.
The roles of von Willebrand factor and factor VIII in arterial thrombosis: studies in canine von Willebrand disease and hemophilia A
Authors: Nichols TC , Bellinger DA , Reddick RL , Smith SV , Koch GG , Davis K , Sigman J , Brinkhous KM , Griggs TR , Read MS 
Published: October 2019

Abstract
We have studied the roles of von Willebrand factor (vWF) and factor VIII in arterial thrombosis in four canine phenotypes: normal (n = 6), hemophilia A (n = 11), von Willebrand disease (vWD) (n = 9), and hemophilia A/vWD (n = 1). vWF activity was determined by botrocetin- induced agglutination of fixed human platelets and vWF antigen (vWF:Ag) by Laurell electroimmunoassay and crossed immunoelectrophoresis. Plasma from normal dogs and those with hemophilia A had vWF activity, vWF:Ag, and a full range of vWF:Ag multimers on gel electrophoresis equivalent to normal canine plasma pool. Platelet cytosol contents were isolated by freezing and thawing, triton X-100 solubilization, or sonication of washed platelets with and without protease inhibitors and inhibitors of platelet activation. Washed platelets were also stimulated with calcium ionophore and MgCl2. There was no measurable vWF activity or vWF:Ag in platelet lysates or releasates in any dog regardless of phenotype. All dogs were studied using a standard arterial stenosis and injury procedure to induce arterial thrombosis. Thromboses were detected by cyclic reductions in Doppler blood flow velocity. Vessels were examined by light and scanning electron microscopy. Thrombosis developed in the arteries of normal (9 of 10) and hemophilia A dogs (16 of 16) but in none of the vWD dogs (0 of 10). Infusion of canine vWF cryoprecipitate into vWD dogs markedly shortened bleeding time but did not support thrombosis as seen in dogs with vWF in the plasma and subendothelium. Thrombosis, then, fails to occur when vWF is absent from the plasma and subendothelial compartments or present only in the plasma compartment. These data are consistent with the hypothesis that vWF in the plasma and subendothelium supports thrombosis. Neither plasma FVIII nor platelet vWF is essential for thrombosis in this model.
Effect of granulocyte-macrophage colony-stimulating factor on lymphokine-activated killer cell induction
Authors: Stewart-Akers AM , Cairns JS , Tweardy DJ , McCarthy SA 
Published: October 2019

Abstract
The treatment of cancer with lymphokine-activated killer (LAK) cells in conjunction with high-dose interleukin-2 (IL-2) has been limited by the toxicity of IL-2 and the narrow range of tumors that respond to therapy. Cytokines that are capable of augmenting lower doses of IL-2 are, therefore, a major focus of research. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF) can augment low-dose IL-2 LAK induction from murine splenocytes. Anti-tumor necrosis factor alpha (anti-TNF alpha) or anti-interferon gamma (anti- IFN gamma) monoclonal antibodies did not inhibit (IL-2 + GM-CSF)- induced LAK generation, indicating that GM-CSF augmentation does not require TNF alpha or IFN gamma activity. Depletion of natural killer cells before culture did not inhibit low-dose IL-2-induced LAK generation or the ability of GM-CSF to augment LAK generation. In contrast, depletion of both CD4+ and CD8+ T cells before culture inhibited the generation of LAK activity. However, depletion of only CD4+ T cells, or only CD8+ T cells, did not inhibit the generation of IL-2 or (IL-2 + GM-CSF) LAK activity. These results suggest that LAK precursors are present in both the CD4+ and CD8+ T-cell populations and suggest that the addition of GM-CSF to low-dose IL-2 may result in the generation of T-derived LAK cells.
Adhesion molecule expression on B-cell chronic lymphocytic leukemia cells: malignant cell phenotypes define distinct disease subsets
Authors: De Rossi G , Zarcone D , Mauro F , Cerruti G , Tenca C , Puccetti A , Mandelli F , Grossi CE 
Published: October 2019

Abstract
Expression of surface adhesion molecules of the Ig superfamily (CD54 and CD58), of the integrin family (beta 1, beta 2, and beta 3 chains), of the selectin family (L-selectin), and of the lymphocyte homing receptor (CD44) was analyzed on B-cell chronic lymphocytic leukemia (B- CLL) cells from 74 patients. The aim of the study was the definition of phenotypically distinct disease subsets and the correlation of adhesion molecule phenotypes with clinical parameters. Expression of CD58 on B- CLL cells defined more advanced disease stages. In comparison with beta chain-positive cases, patients whose cells did not express beta 1, beta 2, and beta 3 integrin chains fell into the most favorable prognostic group, with lower lymphocytosis and the absence of splenomegaly, diffuse bone marrow infiltration, and therapy requirement. A novel finding was the expression of beta 3 chains on cells from a minority (12 of 74) of B-CLL cases. beta 3 chains were always coexpressed with beta 1 and beta 2 chains. Two-color immunofluorescence analyses of adhesion molecules such as alpha x beta 2 integrin (LeuM5) and L- selectin (Leu8) showed that these markers were detectable on variable proportions of leukemic cells, thus confirming the intraclonal phenotypic heterogeneity of B-CLL. Differences in the intensity of CD44 expression were also shown among the various B-CLL clones. Finally, no major variations were shown by comparison of adhesion molecule phenotypes of leukemic cells simultaneously obtained from blood and bone marrow, and of CD5+ versus CD5- clones.
A novel syndrome of severe neutrophil dysfunction: unresponsiveness confined to chemotaxin-induced functions
Authors: Roos D , Kuijpers TW , Mascart-Lemone F , Koenderman L , de Boer M , van Zwieten R , Verhoeven AJ 
Published: October 2019

Abstract
We have identified a patient with a number of neutrophil dysfunctions. The patient was a female baby who lived for 8 months. During her life, she developed severe bacterial infections and showed omphalitis, impaired wound healing, and a pronounced leukocytosis. She was not a patient with leukocyte adhesion deficiency, because all leukocyte CD18 complex proteins were expressed at normal levels. Yet, neutrophil polarization and chemotaxis to platelet-activating factor, leukotriene B4, or formyl-methionyl-leucyl-phenylalanine (FMLP) were completely absent. We found a strong defect in actin polymerization in response to chemotactic stimuli, but only a retarded or even normal reaction with other stimuli. This indicates that the cellular dysfunctions were not due to an intrinsic defect in actin metabolism. Instead, the regulation of actin polymerization with chemotactic stimuli seemed to be defective. We concentrated on FMLP-induced responses in the patient<s neutrophils. Functions dependent on activation of complement receptor type 3, such as aggregation or adherence to endothelial cells, were normally induced. Binding to serum-coated coverslips was normal in cell number; however, spreading was not observed. Exocytosis from the specific granules was readily induced. In contrast, FMLP failed to induce a respiratory burst activity or degranulation of the azurophil granules. FMLP induced a normal increase in free intracellular Ca2+, but a decreased formation of diglycerides (especially the 1-O-alkyl,2- acyl compounds). Thus, we have described a patient whose neutrophils show a severe defect in functional activation via chemotaxin receptors, resulting in a selective absence of NADPH oxidase activity, exocytosis from the azurophil granules, and actin polymerization. Our findings show that actin polymerization for neutrophil spreading and locomotion is regulated differently from that for phagocytosis. Also, the release of azurophil and specific granule contents is clearly shown to be regulated in a different way.
A splice site mutation of alpha-spectrin gene causing skipping of exon 18 in hereditary elliptocytosis
Authors: Alloisio N , Wilmotte R , Marechal J , Texier P , Denoroy L , Feo C , Benhadji- Zouaoui Z , Delaunay J 
Published: October 2019

Abstract
Spectrin Oran (alpha II/21) has been reported previously as a variant of the alpha II domain. Its expression level is low (10% of total spectrin) in heterozygotes denoting a major disadvantage of the mutated alpha-chain dimer or tetramer with respect to their normal counterparts. Spectrin Oran is associated with symptomatic elliptocytosis in the homozygous state. A 1-minute digestion time allowed to perceive a fast trypsin cleavage (not existing normally) after Arg 890 (helix 3 of repeating segment alpha 9). The responsible change was the lack of amino acids 822 to 862 (helix 2 of repeating segment alpha 8). Such a situation fits with the phasing of spectrin according to which mutated helix 2 and distorted helix 3 are adjacent to one another. The internal position of the structural change accounts for the slight self-association defect. The ultimate genetic lesion was a G to A substitution (intronic position-1) in the acceptor splice site of intron 17 resulting in skipping of exon 18. The substitution also created an acceptor splice site 1 base downstream, but the latter was used at a low grade.
The human Kell blood group gene maps to chromosome 7q33 and its expression is restricted to erythroid cells
Authors: Lee S , Zambas ED , Marsh WL , Redman CM 
Published: October 2019

Abstract
The Kell blood group is one of the major antigenic systems in human red blood cells. To determine the location of the Kell gene on human chromosomes, panels containing genomic DNA of human-hamster somatic cell hybrids were hybridized with radiolabeled cDNA probe specific for the Kell locus. Only the samples containing DNA from chromosome 7 gave positive hybridization signals. In situ hybridization analysis, using genomic clones isolated with the cDNA, localized the KEL gene to 7q33. Northern blot analysis of poly(A)+ RNA from human brain, kidney, lung, fetal and adult liver, and bone marrow showed that Kell transcripts were only present in fetal liver and bone marrow. This indicates that the Kell protein, which carries the Kell antigens, may only be expressed in erythroid tissues.
A murine stromal cell line allows the proliferation of very primitive human CD34++/CD38- progenitor cells in long-term cultures and semisolid assays
Authors: Issaad C , Croisille L , Katz A , Vainchenker W , Coulombel L 
Published: October 2019

Abstract
Analysis of molecular mechanisms associated with stem cell commitment and differentiation requires an in vitro assay that identifies the most primitive hematopoietic stem cells in human bone marrow. Such primitive stem cells usually do not form colonies in short-term semisolid assays and are best identified by their ability to initiate sustained hematopoiesis when they are cocultured with competent stromal cells. In this study, we investigated whether a murine marrow stromal cell line (MS-5) that supports colony-forming unit-spleen (CFU-S) maintenance would permit, both in short-term colony assays and long-term cultures, the development of primitive human stem cells sorted on the basis of their high expression of CD34 and lack of expression of CD38 antigen. In short-term colony assays, this population included almost exclusively primitive progenitor cells. MS-5 cells synergized with any combination of interleukin-3, Steel factor, granulocyte colony- stimulating factor, agar-leukocyte conditioned medium, and erythropoietin and increased at least twofold both the cloning efficiency of CD34++/CD38- cells and the size of the colonies. Furthermore, MS-5 cells triggered the development of multipotent blast cell progenitors with a high proliferative potential, which in these conditions represented 1% to 2% of CD34++/CD38- cells. When MS-5 cells were substituted by human stromal cells or when growth factor combinations were used in the absence of stromal cells, much lower numbers of CFU-blast were detected. This selective action of MS-5 on early progenitors was also observed when MS-5 cells were used as feeders in long-term cultures of CD34++/CD38- cells. Murine cells promoted the expansion of high proliferative potential primitive progenitor cells up to 3 months, although they did not support their differentiation in mature clonogenic progenitors or terminally differentiated cells. Sustained hematopoiesis in these longterm cultures was accounted for by 2% to 5% of initial CD34++/CD38- cells as estimated by limiting dilution experiments. Mechanisms by which murine stromal cells act specifically on human primitive stem cells are unclear, but from our data this effect is unlikely to be explained solely by known species cross-reactive growth factors. Further manipulation of this long-term coculture system should prove useful in identifying stromal molecules regulating commitment and differentiation of early human progenitor cells.
Identification of a distinct low-affinity receptor for human interleukin-4 on pre-B cells
Authors: Fanslow WC , Spriggs MK , Rauch CT , Clifford KN , Macduff BM , Ziegler SF , Schooley KA , Mohler KM , March CJ , Armitage RJ 
Published: October 2019

Abstract
Biotinylated interleukin-4 (IL-4) was used to examine IL-4 receptor (IL- 4R) expression on a range of human B-cell lines by flow cytometry. Using high concentrations of biotinylated IL-4, we have identified a novel low-affinity IL-4 receptor expressed at high levels on pre-B lines. Expression of this low-affinity receptor did not correlate with detected mRNA levels for the previously cloned receptor or with reactivity of two anti-human IL-4R monoclonal antibodies (MoAb). Radiolabeled IL-4 cross-linking studies using pre-B lines showed a doublet of 65 to 75 Kd in contrast to the 110- to 130-Kd molecule detected on cells expressing the cloned IL-4R. A soluble IL-4 binding protein (IL-4bp) was purified from the supernatants of three pre-B lines expressing the low-affinity receptor on their surface. IL-4bp could block both IL-4-mediated CD23 induction on tonsil B cells and IL- 4-induced inhibition of proliferation of the pre-B line JM1. Partial N- terminal amino acid sequence was obtained from purified IL-4bp that confirmed this protein to be novel. A 12 amino acid peptide based on the IL-4bp sequence was used to produce a polyclonal antiserum that was reactive with purified IL-4bp, and also bound to the surface of pre-B cells but not to murine CTLL cells transfected with the human IL-4R. Blocking MoAb against the previously characterized high-affinity receptor inhibited IL-4-mediated proliferation of hIL-4R+ CTLL cells but had no effect on IL-4-induced inhibition of JM1 cell proliferation, and only partially inhibited IL-4-mediated CD23 and sIgM induction and proliferation of tonsil B cells. The data presented here provide evidence for a novel cell-surface expressed low-affinity IL-4R that also exists as a biologically active soluble IL-4 binding protein.
Human t(1;19)(q23;p13) pre-B acute lymphoblastic leukemia in mice with severe combined immunodeficiency
Authors: Uckun FM , Downing JR , Gunther R , Chelstrom LM , Finnegan D , Land VJ , Borowitz MJ , Carroll AJ , Crist WM 
Published: October 2019

Abstract
Severe combined immunodeficient (SCID) mice were injected intravenously with 5 x 10(6) primary bone marrow (BM) blasts from newly diagnosed patients with E2A-PBX1 fusion transcript positive t(1;19)(q23;p13) pre- B acute lymphoblastic leukemia (ALL). A marked variation existed in the pattern and extent of leukemic cell engraftment in SCID mice challenged with t(1;19) pre-B ALL blasts. Blasts from some patients caused disseminated leukemia that was detected by histopathology and/or flow cytometry, whereas blasts from other patients produced occult leukemia that was only detected by flow cytometry and/or polymerase-chain reaction. Notably, the ability of primary t(1;19) pre-B ALL blasts to cause disseminated leukemia in SCID mice was associated with poor prognosis. Six of six patients whose blasts caused disseminated leukemia in SCID mice relapsed at a median of 7.8 months (range: 5.7 to 25.2 months). In contrast, the remaining four patients whose blasts did not engraft or only partially engrafted remain in complete remission at 28 to 47 months. A new E2A-PBX-1 fusion transcript positive t(1;19) pre- B ALL cell line (designated LC1;19) with the composite immunophenotype CD7-CD10+CD19+CD45-HLA-DR+C mu+ was established by expanding BM blasts from a SCID mouse, which died of human t(1;19) ALL at 7 weeks after inoculation of primary leukemic blasts from a t(1;19) ALL patient. This cell line caused disseminated and invariably fatal leukemia when greater than 10(4) cells were injected intravenously into SCID mice. Total body irradiation followed by syngeneic BM transplantation (BMT) showed limited efficacy against LC1;19 leukemia in SCID mice. To our knowledge, this study is the first to (1) examine the in vivo growth of primary t(1;19) pre-B ALL blasts in SCID mice and (2) show that leukemic blasts from a majority of newly diagnosed t(1;19) pre-B ALL patients cause disseminated human leukemia in SCID mice. Our results indicate that t(1;19) pre-B ALL is biologically heterogeneous with regard to its in vivo growth pattern in SCID mice, a feature that may be predictive of prognosis. The described LC1;19 SCID mouse model may prove particularly useful for designing more effective treatment strategies against poor-prognosis t(1;19) ALL.
Levels of soluble stem cell factor in serum of patients with aplastic anemia
Authors: Wodnar-Filipowicz A , Yancik S , Moser Y , dalle Carbonare V , Gratwohl A , Tichelli A , Speck B , Nissen C 
Published: October 2019

Abstract
Aplastic anemia (AA) is a rare bone marrow (BM) disorder characterized by an unexplained failure of hematopoietic precursors to proliferate. In vitro growth of AA BM cells can be improved by the addition of the hematopoietic growth factor SCF (stem cell factor), which suggests that deficiency of SCF may be one of the underlying causes of the disease. In this study, we measured the concentration of SCF in sera of patients with severe AA. One hundred twenty-eight serum samples from 32 patients, at diagnosis and following therapy, were analyzed. Before treatment, SCF levels varied between 0.33 and 6.1 ng/mL; no correlation between hematopoietic function and SCF serum levels was apparent. Therapy with antilymphocyte globulin (ALG) or bone marrow transplantation (BMT) did not result in a recognizable pattern of changes in SCF levels. However, serum concentration of SCF in many patients with AA was at the low range of control serum levels determined in healthy blood donors. Of 128 AA serum samples tested before and after therapy, 107 were below the mean normal value of 3.3 ng/mL, including 26 samples below the minimum normal value of 1.3 ng/mL, as estimated in 267 controls. We also found that SCF levels in peripheral blood serum correlate well with factor concentrations in the BM plasma. Clinical observations suggest that higher SCF serum levels are often associated with a better clinical status of the patients in terms of survival and transfusion requirements. The data indicate that a deficient production of soluble SCF may contribute to AA in some patients; thus, suggesting a potential therapeutic benefit of SCF in this disorder.
In vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits
Authors: Griffin LC , Tidmarsh GF , Bock LC , Toole JJ , Leung LL 
Published: October 2019

Abstract
Using a novel in vitro selection/amplification technique, we have recently identified a new class of thrombin inhibitors based on single- stranded DNA oligonucleotides. One oligonucleotide, GGTTGGTGTGGTTGG (thrombin, aptamer), showed potent anticoagulant activity in vitro. We have initiated pharmacologic studies in cynomolgus monkeys to study the thrombin aptamer's in vivo anticoagulant properties. Upon infusion of the thrombin aptamer, anticoagulation was rapidly achieved, with a plateau reached within 10 minutes. There was a linear dose-response relationship between thrombin aptamer infusion rate and prolongation of plasma prothrombin time. Ten minutes after the infusion was stopped, no prolongation of prothrombin time was observed, indicating that the thrombin aptamer has an extremely short in vivo half-life, estimated to be 108 +/- 14 seconds. In addition, inhibition of thrombin-induced platelet aggregation in platelet-rich plasma was observed ex vivo without an effect on collagen-induced aggregation, indicating that the inhibition was specific for thrombin and not due to a nonspecific inhibitory effect on platelets. To exploit the short in vivo half-life of the thrombin aptamer, its ability to achieve regional anticoagulation in an extracorporeal hemofiltration circuit in sheep was tested. Doubling of the prothrombin time in the circuit was observed, whereas the systemic prothrombin time was minimally prolonged. We conclude that the thrombin aptamer is a potent anticoagulant in vivo, and specifically inhibits thrombin-induced platelet aggregation ex vivo. The rapid onset of action and short half- life in vivo suggest that the thrombin aptamer may be useful in anticoagulation with extracorporeal circuits and may have distinct advantages in certain acute clinical settings.
Neonatal alloimmune thrombocytopenia due to a new platelet-specific alloantibody
Authors: McFarland JG , Blanchette V , Collins J , Newman PJ , Wang R , Aster RH 
Published: October 2019

Abstract
An infant with severe neonatal alloimmune thrombocytopenia is described in whom an antibody directed at a new platelet-specific alloantigen, Ca (HPA-6b), is implicated. The new alloantigen is of low frequency in the population and was localized to platelet glycoprotein (GP) IIIa. Immunoprecipitation studies using murine monoclonal antibodies specific for the GP complex IIb-IIIa and GPIIIa alone (AP2 and AP3) suggest that the location of the Ca epitope on GPIIIa may be near the binding site for AP3. Neonatal alloimmune thrombocytopenia associated with Ca is likely to be as severe as that seen in cases due to incompatibilities for the HPA-1 (PIA) and HPA-4 (Pen) platelet alloantigen systems, because each is located on GPIIIa, a densely represented molecule on the platelet surface.