bluuebunny/crossref_metadata_2025_split huggingface dataset:
In vitro effects of hematopoietic growth factors on the proliferation, endoreplication, and maturation of human megakaryocytes
Authors: Debili N , Hegyi E , Navarro S , Katz A , Mouthon MA , Breton-Gorius J , Vainchenker W 
Published: October 2019

Abstract
A liquid culture technique was used to study regulation of human megakaryocytopoiesis in vitro. Low-density cells from adult bone marrow were cultured in the presence of normal plasma, plasma from patients with aplastic marrows (AP), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and interleukin-3 (IL-3). Megakaryocytes (MK) were studied at day 10 of culture by a two-color staining technique using a pool of monoclonal antibodies for their identification and propidium iodide to label DNA. Their ploidy distribution was analyzed by flow cytometry. In some experiments cytoplasmic maturation was also studied by ultrastructural techniques. Normal plasma provides a low number of MK with a ploidy distribution including 8 N and 16 N MK. AP promoted in a dose-dependent manner proliferation of MK and some batches favored endoreplication. This effect was clearly demonstrated when ploidy distribution was compared between normal plasma and AP on parallel marrow cultures. However, ploidy distribution was shifted toward low values compared with uncultured MK. rhGM-CSF had no significant effect on these two parameters. In contrast, rhIL-3 from 0.1 U/mL to 100 U/mL had a proliferative effect but was unable to induce endoreplication. Furthermore, when associated with AP it totally abrogated the effect of AP on endoreplication because in most experiments more than 90% of MK were 2 N and 4 N. This effect was also observed when rhIL-3 was added after 7 days of culture (when it has little proliferative effects). Studies of the maturation of MK grown with rhIL-3 indicate that the majority were small mature cells synthesizing alpha-granules and demarcation membranes. The effect of AP on MK proliferation and endoreplication was not related to IL-6 because its IL-6 content was identical to that of normal plasma and its neutralization did not modify these parameters. In conclusion, this study indicates that liquid culture technique in association with flow cytometry could be a powerful tool in identifying the humoral regulators of human megakaryocytopoiesis.
Developmental hematopoiesis in normal human fetal blood
Authors: Forestier F , Daffos F , Catherine N , Renard M , Andreux JP 
Published: October 2019

Abstract
Using an easy and safe procedure for fetal blood sampling in utero, we studied 3,415 fetuses for prenatal diagnosis. Retrospectively, 2,860 normal blood samples, performed from the 18th week of gestation to the end of pregnancy, were selected. Differentials were evaluated in 732 cases. Burst-forming unit erythroid (BFU-E) and erythropoietin (Epo) were measured in 27 and 163 cases, respectively. Total nucleated cell and platelet counts did not change from the 18th to the 30th week of gestation. The lymphocytes represented the main population and the decrease of normoblastic cells made up for the increase in neutrophils. The increase of red blood cells and hemoglobin was substantial during the studied period. At mid trimester threefold more BFU-E were obtained than at birth. Epo levels remained stable throughout the pregnancy and no correlation was found between Epo and gestational age. These normal values of fetal erythropoiesis will improve our knowledge of physiology and provide a better insight into developmental hematopoiesis.
Platelet activation by fMLP-stimulated polymorphonuclear leukocytes: the activity of cathepsin G is not prevented by antiproteinases
Authors: Evangelista V , Rajtar G , de Gaetano G , White JG , Cerletti C 
Published: October 2019

Abstract
Human polymorphonuclear leukocytes (PMN) activated by fMLP (in the presence of CaCl2, fibrinogen, and cytochalasin B) were able to induce aggregation, cytoplasmic Ca2+ increase, and thromboxane A2 production in coincubated autologousplatelets. Cell-free supernatants prepared from n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN were able also to induce platelet activation. Antibodies against cathepsin G and different serin protease inhibitors completely suppressed the activity of PMN-derived supernatants, indicating that cathepsin G is the major platelet activator released by PMN in our system. However, antiproteinases only partially affected platelet activation induced by PMN in mixed cell suspensions. Superoxide dismutase and catalase added to the cell suspension did not affect platelet activation nor potentiated serin protease inhibitors, making a role for short-lived oxygen radicals in our experimental system unlikely. Electron microscopic observation of stirred mixed cell suspensions preincubated for 2 minutes at 37 degrees C before stimulation showed a close PMN- platelets contact without any morphologic or biochemical event suggesting platelet activation. Preincubation of the cells without stirring to minimize PMN-platelet interaction before stimulation did not modify subsequent aggregation and platelet cytoplasmic Ca2+ increase in control samples. However, in this condition trypsin inhibitor from soybean completely prevented PMN-induced platelet activation. In samples preincubated without stirring in the presence of the antiproteinase, activated PMN stuck together but platelets preserved their discoid shape and did not appear significantly activated. We propose that membrane-to-membrane contact could create a microenvironment in which cathepsin G, discharged from stimulated PMN on adherent platelets, is protected from antiproteinases.
Tissue inhibitor of metalloproteinases-1 (TIMP-1) RNA is expressed at elevated levels in malignant non-Hodgkin's lymphomas
Authors: Kossakowska AE , Urbanski SJ , Edwards DR 
Published: October 2019

Abstract
Secreted metalloproteinases (MPs) and their specific inhibitors (TIMPs, tissue inhibitors of MPs) are important mediators of extracellular matrix metabolism. Previous studies have linked either excessive MP release or reduced TIMP-1 production to the invasive and metastatic phenotypes of cancer cells. In the present study we investigated the relationship between the expression of TIMP-1 and the clinical behavior of 28 non-Hodgkin's lymphomas. Northern blot analysis showed that levels of TIMP-1 mRNAs correlated directly with clinical aggressiveness: tumors in the high-grade category contained the highest levels of TIMP-1 transcripts approaching those found in maximally growth factor-stimulated fibroblasts in vitro. In situ hybridization localized the TIMP-1 expression to stromal cells of endothelial and fibroblastic origin. In contrast, transcripts hybridizing with metalloproteinase gene probes (interstitial collagenase and 72-Kd type IV collagenase) were expressed at very low levels in malignant lymphomas and their expression was not coordinately regulated with that of TIMP-1. The majority of tumors expressed either interstitial collagenase or 72-Kd type IV collagenase, and only a small number expressed both. Interstitial collagenase transcripts were only detected in high-grade tumors. The relative levels of TIMP-1 expression did not correlate with the degree of fibrosis of the tumors. Our data suggest the importance of tumor-stromal interactions in non-Hodgkin's lymphomas, and moreover, our results indicate a possible relationship between high-level, localized expression of TIMP-1 and the malignant phenotype of high-grade advanced-stage lymphomas.
Autologous bone marrow transplantation in 69 patients with a history of low-grade B-cell non-Hodgkin's lymphoma
Authors: Freedman AS , Ritz J , Neuberg D , Anderson KC , Rabinowe SN , Mauch P , Takvorian T , Soiffer R , Blake K , Yeap B 
Published: October 2019

Abstract
Sixty-nine patients with a history of low-grade B-cell non-Hodgkin's lymphoma (NHL) in sensitive relapse or incomplete first remission underwent high-dose chemoradiotherapy and anti-B-cell monoclonal antibody (MoAb)-treated autologous bone marrow transplantation (ABMT). At ABMT, 51 patients had low-grade histology and 18 patients had a history of low-grade NHL that had undergone histologic transformation to a higher-grade NHL. Before ABMT, only 20 of the 51 low-grade patients and 10 of the 18 patients with transformed histologies were in complete remission. Moreover, at the time of marrow harvest, 24 of the low-grade and eight of the transformed histology patients had histologic evidence of lymphoma cells infiltrating the marrow. Following high-dose therapy, only one acute, in-hospital death was observed. There was no significant difference in the disease-free survival (DFS) between patients with low-grade and patients with transformed histologies. Among patients with low-grade NHL, the patients in complete remission before ABMT experienced significantly longer DFS than those in partial remission (P less than .05). This preliminary study suggests that some patients with relapsed low-grade NHL may experience prolonged DFS following high-dose ablative therapy.
Toxicity and immunomodulatory effects of interleukin-2 after autologous bone marrow transplantation for hematologic malignancies
Authors: Higuchi CM , Thompson JA , Petersen FB , Buckner CD , Fefer A 
Published: October 2019

Abstract
Autologous bone marrow transplantation (ABMT) for advanced hematologic malignancies is associated with high relapse rates. Interleukin-2 (IL- 2) and lymphokine-activated killer (LAK) cells represent a potentially non-cross-resistant therapeutic modality that might prevent or delay relapses if used early after ABMT at a time when the tumor burden is minimal. However, high-dose chemoradiotherapy and ABMT might increase patients' susceptibility to IL-2 toxicity, and might interfere with immunologic responses to IL-2 in vivo. Therefore, to determine safety, tolerance, and immunomodulatory effects of IL-2 therapy early after ABMT, IL-2 was administered by continuous intravenous infusion to 16 patients 14 to 91 days (median, 33) after ABMT for acute leukemia, lymphoma, or multiple myeloma. Patients were sequentially assigned to escalating IL-2 “induction” doses (0.3 to 4.5 x 10(6) U/m2/d, days 1 to 5), and all patients received a nonescalating IL-2 “maintenance” dose (0.3 x 10(6) U/m2/d, days 12 to 21). Most patients exhibited mild to moderate fever, nausea, diarrhea, and/or skin rash with IL-2 infusions. The maximum tolerated “induction” dose was 3.0 x 10(6) U/m2/d; dose- limiting toxicities were hypotension and thrombocytopenia. All toxicities reversed on stopping the IL-2 infusions, and all patients completed “maintenance.” Postinfusion lymphocytosis was exhibited by patients at all IL-2 dose levels. With the higher IL-2 doses, increased percentages of patients' PBMC expressed CD16 and CD56, with augmented lysis of K562 and Daudi, reflecting the induction of natural killer and circulating LAK effector activities. Increased LAK precursor activity was exhibited by patients at all IL-2 dose levels. Thus, the IL-2 therapy regimen was safely tolerated after ABMT, and pronounced immunomodulatory effects were observed with the higher IL-2 doses. These studies support the planned use of IL-2 and LAK cells after ABMT in an attempt to reduce relapses of advanced hematologic malignancies.
Erythropoietin receptor characteristics on primary human erythroid cells
Authors: Broudy VC , Lin N , Brice M , Nakamoto B , Papayannopoulou T 
Published: October 2019

Abstract
Erythropoietin (EP) exerts its effects on erythropoiesis by binding to a cell surface receptor. We examined EP receptor expression during normal human erythroid differentiation and maturation from the burst- forming unit-erythroid (BFU-E) to the reticulocyte level. In contrast to previous studies, we assessed EP receptor number and affinity in erythroid precursors immunologically purified from fresh bone marrow aspirates or fetal liver samples and in reticulocytes purified from peripheral blood. EP receptors were quantitated by equilibrium binding experiments with 125I EP. We found that purified primary erythroblasts from both adult and fetal sources exhibited a single high-affinity (kd 100 pmol/L) binding site for EP under our experimental conditions, and 135 or 250 receptors per cell, respectively. Reticulocytes were devoid of EP receptors. We compared these data to in vitro-derived BFU-E progeny at both early and late stages of maturation. Cultured BFU-E progeny also displayed a single class of receptors of slightly lower affinity (210 to 220 pmol/L). Preparations enriched in colony-forming units-erythroid (CFU-E) and proerythroblasts (day 9 BFU-E progeny) displayed approximately 1,100 receptors per cell, whereas populations containing mature erythroblasts (day 14 BFU-E progeny) exhibited approximately 300 receptors per cell. Furthermore, information from binding experiments was complemented by autoradiography in both enriched BFU-E preparations, cultured BFU-E progeny (days 9 and 14), and marrow mononuclear cells. These studies are consistent with a peak in EP receptor expression at the CFU-E/proerythroblast stage and a decrease with further maturation to undetectable levels at the reticulocyte stage. These data examining EP receptor characteristics on freshly isolated erythroid precursor cells complement previous data on EP receptor biology using culture-derived erythroblasts.
Identification and comparison of CD34-positive cells and their subpopulations from normal peripheral blood and bone marrow using multicolor flow cytometry
Authors: Bender JG , Unverzagt KL , Walker DE , Lee W , Van Epps DE , Smith DH , Stewart CC , To LB 
Published: October 2019

Abstract
Four-color flow cytometry was used with a cocktail of antibodies to identify and isolate CD34+ hematopoietic progenitors from normal human peripheral blood (PB) and bone marrow (BM). Mature cells that did not contain colony forming cells were resolved from immature cells using antibodies for T lymphocytes (CD3), B lymphocytes (CD20), monocytes (CD14), and granulocytes (CD11b). Immature cells were subdivided based on the expression of antigens found on hematopoietic progenitors (CD34, HLA-DR, CD33, CD19, CD45, CD71, CD10, and CD7). CD34+ cells were present in the circulation in about one-tenth the concentration of BM (0.2% v 1.8%) and had a different spectrum of antigen expression. A higher proportion of PB-CD34+ cells expressed the CD33 myeloid antigen (84% v 43%) and expressed higher levels of the pan leukocyte antigen CD45 than BM-CD34+ cells. Only a small fraction of PB-CD34+ cells expressed CD71 (transferrin receptors) (17%) while 94% of BM-CD34+ expressed CD71+. The proportion of PB-CD34+ cells expressing the B-cell antigens CD19 (10%) and CD10 (3%) was not significantly different from BM-CD34+ cells (14% and 17%, respectively). Few CD34+ cells in BM (2.7%) or PB (7%) expressed the T-cell antigen CD7. CD34+ cells were found to be predominantly HLA-DR+, with a wide range of intensity. These studies show that CD34+ cells and their subsets can be identified in normal PB and that the relative frequency of these cells and their subpopulations differs in PB versus BM.
Stimulatory effects of granulocyte colony-stimulating factor on colony- forming units-spleen (CFU-S) differentiation and pre-CFU-S proliferation in mice
Authors: Tanaka T , Suda T , Suda J , Inoue T , Hirabayashi Y , Hirai H , Takaku F , Miura Y 
Published: October 2019

Abstract
Granulocyte colony-stimulating factor (G-CSF) was reported to increase the number of colony-forming units-spleen (CFU-S) and multilineage colonies as well as myeloid-committed cells. We investigated the effects of G-CSF on myeloid progenitors and primitive stem cells in a mouse bone marrow transplantation (BMT) system. Lethally irradiated mice received BM cells from untreated or 5-fluorouracil-treated mice, and then were administered G-CSF or carrier buffer (control) for 5 days from immediately after BMT. A pre-CFU-S assay was performed by the repeated transplantation of BM cells from the first BMT recipients to other mice. By the method of polymerase chain reaction, most of the spleen colonies in the secondary recipients were confirmed to be derived from the first donors. G-CSF did not increase the peripheral white blood cell count significantly, but did increase the number of immature myeloid cells and granulocyte-macrophage colony-forming cells in the BM. The number of erythroid cells in the BM was initially suppressed and then increased by G-CSF treatment. In addition, the pre- CFU-S assay showed an increase in pre-CFU-S cells due to G-CSF administration. The number of spleen colonies of first BMT recipients did not increase, but a higher percentage of them were committed to a certain lineage by G-CSF treatment. These findings suggest that G-CSF has important roles in the early stages of hematopoiesis.
DNA synthesis in human bone marrow is circadian stage dependent
Authors: Smaaland R , Laerum OD , Lote K , Sletvold O , Sothern RB , Bjerknes R 
Published: October 2019

Abstract
Fraction of human bone marrow (BM) cells in DNA synthesis has been studied by sampling BM from the sternum or the iliac crests every 4 hours during one 24-hour period in 16 healthy male volunteers. Three of the subjects underwent the sampling procedure twice, resulting in 19 24- hour profiles. The percentage of cells in DNA synthesis measured by flow cytometry demonstrated a large variation along the circadian time scale for each 24-hour profile, with a range of variation from 29% to 339% from lowest to highest value. Seventeen profiles (89.5%) had the highest DNA synthesis during waking hours between 08:00 hours and 20:00 hours, and the lowest percentage of cells in DNA synthesis between 00:00 hours and 04:00 hours. The mean value of the lowest DNA synthesis for each 19 24-hour period was 8.7% +/- 0.6%, while the mean value of the highest DNA synthesis was 17.6% +/- 0.6%, ie, a twofold difference. There was no difference in DNA synthesis between winter and summer. A significantly higher DNA synthesis was demonstrated for samples obtained from sternum as compared with the iliac crests, but the same circadian pattern was demonstrated for both localizations. By taking circadian stage-dependent variations in DNA synthesis into account it may be possible to reduce BM sensitivity to cytotoxic chemotherapy, to increase the effect of hematopoietic growth factors as well as increase the fraction of proliferating cells with careful selection of time of day for harvesting BM cells for auto- or allografting.
The importance of N- and O-linked oligosaccharides for the biosynthesis and in vitro and in vivo biologic activities of erythropoietin
Authors: Wasley LC , Timony G , Murtha P , Stoudemire J , Dorner AJ , Caro J , Krieger M , Kaufman RJ 
Published: October 2019

Abstract
Erythropoietin (EPO) plays a critical role in stimulating the proliferation and differentiation of erythroid precursor cells. EPO is heavily glycosylated with three asparagine (N)-linked tetraantennary oligosaccharides that may contain N-acetyl-lactosamine repeats and a single serine (O)-linked oligosaccharide. EPO expressed in Chinese hamster ovary cells exhibits biologic properties and amino acid and carbohydrate composition similar to natural urinary EPO. The importance of the complex N-linked and the O-linked carbohydrate was studied by expressing EPO in cells that are deficient in UDP-galactose/UDP-N- acetylgalactosamine 4-epimerase activity. In these cells, the ability to add galactose and N-acetylgalactosamine to glycoproteins can be controlled by the addition of these sugars to the culture medium. The results demonstrate that a block in O-linked glycosylation and/or the ability to process N-linked carbohydrate to completion does not alter EPO secretion. EPO produced without O-linked carbohydrate exhibits normal in vitro and in vivo biologic activity and in vivo clearance. However, EPO produced with incompletely processed N-linked oligosaccharides exhibits normal in vitro activity but is at least 500- fold less effective in stimulating erythropoiesis in vivo. Studies on the survival of bioactive EPO remaining in the circulation demonstrated that EPO with incomplete N-linked oligosaccharides exhibits a sevenfold increased rate of clearance. However, this increased clearance may not fully account for the 500-fold loss of in vivo activity. These results suggest a potentially important unique requirement for appropriate complex N-linked oligosaccharides for the intrinsic biologic activity of EPO in vivo.
Genotype establishments for protein C deficiency by use of a DNA polymorphism in the gene
Authors: Yamamoto K , Tanimoto M , Matsushita T , Kagami K , Sugiura I , Hamaguchi M , Takamatsu J , Saito H 
Published: October 2019

Abstract
During the course of structural gene analyses for protein C deficiency, we have confirmed that a T or G nucleotide variation is present at exon 6 of the protein C gene. This single-base substitution was located at the third nucleotide coding for Ser (TCT) at 99 residue, and neither produces an amino acid substitution nor creates a new restriction enzyme site. By using mutagenic primers that could introduce A instead of G at the third nucleotide 3′ to the de novo polymorphic site, we have created the polymorphic Xba I site (T/CTAGA) in a more-frequent allele. Polymerase chain reaction using these mutagenic primers and subsequent Xba I digestion of 20 normal Japanese genomic samples showed that the frequency of this new sequence polymorphism designated as PC- 493 was 0.18 and that the estimated heterozygosity rate was 28.9%. In Caucasians, the frequency of this polymorphism was 0.25, and a significant difference did not exist between Japanese and Caucasian populations. The examination of the haplotype inter-relationships with PC-493 and the Msp I polymorphism 5′ to the protein C gene established that PC-493 gave a 16.7% chance of new information per individual for people who were previously homozygous for the Msp I polymorphism. We have performed a family study of the protein C-deficient pedigree using this sequence polymorphism, and found that the PC-493 DNA polymorphism was a useful marker for tracing the affected gene in protein C- deficient family members.
Inhibitor of the thrombin time in systemic amyloidosis: a common coagulation abnormality
Authors: Gastineau DA , Gertz MA , Daniels TM , Kyle RA , Bowie EJ 
Published: October 2019

Abstract
Patients with primary systemic amyloidosis (AL) often experience bleeding, and we report a newly recognized coagulation abnormality in AL. Of 103 patients with primary systemic AL studied over 2 years, 41 had prolongation of the thrombin time (range, 25 to 46 seconds; normal, less than 22 seconds) and reptilase time (range, 17 to 39 seconds; normal, 14 to 16 seconds). The fibrinogen from the plasma of 36 patients was precipitated by beta-alanine and diluted to a concentration of approximately 200 mg/dL. The thrombin times of the precipitated fibrinogens were normal in 34 patients, implying that an inhibitor was responsible for the abnormal tests. The addition of patient fibrinogen-free plasma to normal plasma prolonged the thrombin times, and this result confirmed the presence of an inhibitor. The inhibitor is more likely to be present in patients with nephrotic syndrome (20 of our patients) and congestive heart failure (six). A circulating monoclonal protein (24 patients), the presence of amyloid liver involvement (eight), and the presence of amyloid neuropathy (nine) were not predisposing factors. Only one patient had deficiency of factor X. We conclude that inhibition of fibrinogen conversion to a fibrin clot rather than dysfibrinogenemia is the cause of the prolonged thrombin time in primary systemic AL.