bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Development of Human Megakaryocytes: III. Development of Mature Megakaryocytes From Highly Purified Committed Progenitors in Synthetic Culture Media and Inhibition of Thrombopoietin-Induced Polyploidization by Interleukin-3
Authors: Dolzhanskiy Alexandr , Basch Ross S. , Karpatkin Simon 
Published: October 2019

Abstract
Megakaryocyte (MK) progenitors, CD34+CD41+ cells, were isolated from human bone marrow with a purity greater than 98% and a viability of 95%, using affinity techniques with magnetic beads followed by fluorescence-activated cell sorting. These cells were incubated in synthetic media containing the cytokines thrombopoietin (TPO), interleukin-3 (IL-3), stem cell factor (SCF ), and IL-6, obviating the confounding effects of serum growth factors or cytokine secretions of non-MK cells on MK maturation. MK number, MK colony-forming units (CFU-MK), and MK ploidy and phenotype were examined during 7 days in culture. TPO in serum-free cultures without any other exogenously added cytokine supported MK growth and maturation. SCF synergized with TPO to augment MK production and maturation and could partially replace it under some conditions. Both TPO and IL-3 alone increased MK number (12- and 5-fold, respectively) and CFU-MK (∼15-fold each). SCF alone had no effect on MK proliferation in the absence of TPO, but increased both MK number and CFU-MK by 1.5- to 2.0-fold in the presence of TPO. When combined with IL-3, SCF increased both MK number and CFU-MK by 15- to 20-fold in the absence of TPO. In the presence of TPO, the combination of IL-3 and SCF produced only modest increases (1.5- to 2.0-fold) in both MK number and CFU-MK. The proportion of polyploid MK increased greater than fivefold in the presence of TPO. SCF had little effect on MK ploidy in the presence of TPO, but enhanced ploidy twofold to threefold in the absence of TPO. IL-3 alone never increased the level of polyploidization. Rather, it consistently inhibited TPO- and SCF-induced polyploidization of MK. This inhibition was observed in cultures with or without SCF or IL-6. Although IL-3 also supported the proliferation of CD41+ cells and CFU-MK production, the cells that developed under the influence of IL-3 were phenotypically unusual (CD41dim, CD42dim) and of relatively low ploidy. Mature MK were not produced. When added with TPO, IL-3 suppressed polyploidization. Therefore, TPO stimulates MK growth and maturation, whereas IL-3 stimulates growth without maturation and may serve to conserve the immature MK compartment.
The Unexpected G0/G1 Cell Cycle Status of Mobilized Hematopoietic Stem Cells From Peripheral Blood
Authors: Uchida Nobuko , He Dongping , Friera Annabelle M. , Reitsma Michael , Sasaki Dennis , Chen Ben , Tsukamoto Ann 
Published: October 2019

Abstract
Treatment with a combination of cytokines and chemotherapy can effectively stimulate the release of hematopoietic stem cells (HSC) into the peripheral blood (PB), which can then be harvested for transplantation. The cell cycle status of the harvested HSC from mobilized PB (MPB) is of interest because of the impact that cell cycling may have on optimizing the conditions for ex vivo expansion, retrovirus-mediated gene transfer, and the engraftment of transplanted tissues. Therefore, we characterized the cell cycling status of mobilized HSC from mice and humans. The murine HSC, which express the phenotype c-kit+ Thy-1.1lo Lin−/lo Sca-1+, were purified from PB, bone marrow (BM), and spleen after the mice were treated with the mobilizing regimen of granulocyte colony-stimulating factor (G-CSF ) or a combination of cyclophosphamide (CTX) and G-CSF. Human HSC (CD34+ Thy-1+ Lin−) and progenitor cells (CD34+ Thy-1− Lin−) were isolated from the BM of untreated healthy volunteers and from MPB of healthy volunteers and patients treated with G-CSF or a combination of CTX and GM-CSF. Cell cycle status was determined by quantitating the amount of DNA in the purified cells after staining with the dye Hoechst 33342. Fluorescence-activated cell sorting analysis of the progenitor cells from the murine and human samples showed an unexpected finding, ie, virtually none of the cells from the MPB was cycling. The G0/G1 status of HSC from MPB was surprising, because a significant proportion of HSC from BM are actively proliferating and, after mobilization, the HSC in the spleen and BM were also actively cycling.
Mapping of the Active Site of Recombinant Human Erythropoietin
Authors: Elliott Steve , Lorenzini Tony , Chang David , Barzilay Jack , Delorme Evelyne 
Published: October 2019

AbstractRecombinant human erythropoietin (rHuEPO) variants have been constructed to identify amino acid residues important for biological activity. Immunoassays were used to determine the effect of each mutation on rHuEPO folding. With this strategy, we could distinguish between mutations that affected bioactivity directly and those that affected bioactivity because the mutation altered rHuEPO conformation. Four regions were found to be important for bioactivity: amino acids 11 to 15, 44 to 51, 100 to 108, and 147 to 151. EPO variants could be divided into two groups according to the differential effects on EPO receptor binding activity and in vitro biologic activity. This suggests that rHuEPO has two separate receptor binding sites. Mutations in basic residues reduced the biologic activity, whereas mutations in acidic residues did not. This suggests that electrostatic interactions between rHuEPO and the human EPO receptor may involve positive charges on rHuEPO.
Monocytes Upregulate Endothelial Cell Expression of Tissue Factor: A Role for Cell-Cell Contact and Cross-Talk
Authors: Napoleone Emanuela , Di Santo Angelomaria , Lorenzet Roberto 
Published: October 2019

AbstractMonocytes and endothelial cells interact at sites of vascular injury during inflammatory response, thrombosis, and development of atherosclerotic lesions. Such interactions result in modulation of several biological functions of the two cell types. Because both cells, on appropriate stimulation, synthesize tissue factor (TF), we examined the effect of human umbilical vein endothelial cell (HUVEC)/monocyte coculture on the expression of TF. We found that the coincubation resulted in TF generation, which was maximal at 4 hours, increased with increasing numbers of monocytes, and required mRNA and protein synthesis. Supernatant from HUVEC/monocyte coculture induced TF activity in HUVECs, but not in monocytes, indicating that HUVEC were the cells responsible for the activity, and that soluble mediators were involved. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), well-known inducers of TF in HUVECs, were found in the supernatant from the coculture, and specific antibodies directed against either cytokine inhibited TF generation. The need of IL-1β and TNF-α synthesis in order to elicit TF expression was also suggested by the delay observed in TF mRNA formation and TF activity generation when monocytes were incubated with HUVECs. IL-1β and TNF-α antigen levels in the coculture supernatant, and, consequently, HUVEC TF expression, were inhibited in the presence of anti-CD18 monoclonal antibody. These findings emphasize the role of cell-cell contact and cross-talk in the procoagulant activity, which could be responsible for the thromboembolic complications observed in those vascular disorders in which monocyte infiltration is a common feature.
Primary Non-Hodgkin's Lymphoma of the Intestine: High Prevalence of Epstein-Barr Virus in Mexican Lymphomas as Compared With European Cases
Authors: Quintanilla-Martı́nez Leticia , Lome-Maldonado Carmen , Ott German , Gschwendtner Andreas , Gredler Evelyn , Reyes Edgardo , Angeles-Angeles Arturo , Fend Falko 
Published: October 2019

Abstract
Recent studies in Western European populations have shown that peripheral T-cell non-Hodgkin's lymphomas (T-NHLs) are associated with Epstein-Barr virus (EBV) in a higher percentage than sporadic B-cell NHL (B-NHLs), and that the frequency of EBV-positivity might be influenced by the primary site of the tumor. Because of the geographic differences in EBV expression in Burkitt's lymphoma (BL) and Hodgkin's disease (HD), and the lack of studies of sporadic NHL from developing countries, we decided to survey the presence of EBV in a series of primary intestinal lymphomas from patients in Mexico and in Western Europe, and to analyze whether EBV status is influenced by tumor phenotype, and geographic or ethnic determinants. Paraffin-embedded tissue from 43 primary intestinal NHLs (19 cases from Mexico and 24 from Western Europe) were examined, including 17 high grade B-NHLs, 9 low grade B-NHLs, and 17 T-NHLs; 6 of which were enteropathy associated T-cell lymphomas. The distribution of histologic subtypes was similar in both groups. The presence of EBV was investigated with a combined approach using a nested polymerase chain reaction technique as well as immunohistochemistry for latent membrane protein-1 and in situ hybridization for EBV early RNA transcripts (EBER 1/2) RNAs. The median age of the Mexican patients was significantly lower than the median age of the European patients (32 v 62 years). This difference was most pronounced in patients with T-cell lymphoma (24 v 63 years). EBER-positive tumor cells were detected in 13 of the 43 (30%) cases of primary intestinal lymphoma, including 5 of 26 sporadic B-NHL (3 high grade and 2 low grade), and 8 of 17 T-NHL, all of which were classified as pleomorphic, medium and large cell. The rates of EBV-positivity were markedly different for European and Mexican cases. Whereas 7 of 7 (100%) T-NHL and 5 of 12 (42%) sporadic B-NHL of Mexican origin were EBER-positive, only 1 of 10 T-NHL and 0 of 14 sporadic B-NHL from Europe showed EBER expression in tumor cells. Latent membrane protein was positive in only 2 of 43 cases, 1 of which was an EBER-negative high grade B-NHL from Mexico that showed intact total mRNA in control hybridization. CD30 expression was found in 4 of 8 EBV-positive T-NHL and in none of the EBV-positive B-NHL. In contrast to European cases, intestinal NHLs from Mexico show a very high frequency of EBV-positivity, which is not limited to T-NHL, but includes a significant proportion of B-NHL. This study strongly suggests that similar to HD and probably BL, there are important epidemiologic differences in EBV association in intestinal T-cell NHL between European and Mexican populations. These differences might be the result of environmental factors, for example, earlier contact with childhood viruses on intestinal lymphomagenesis.
Molecular Monitoring of Minimal Residual Disease in Follicular and Mantle Cell Non-Hodgkin's Lymphomas Treated With High-Dose Chemotherapy and Peripheral Blood Progenitor Cell Autografting
Authors: Corradini Paolo , Astolfi Monica , Cherasco Cristina , Ladetto Marco , Voena Claudia , Caracciolo Daniele , Pileri Alessandro , Tarella Corrado 
Published: October 2019

Abstract
Minimal residual disease (MRD) was evaluated in 30 patients with follicular or mantle cell non-Hodgkin's lymphoma (NHL) undergoing an intensive treatment with high-dose sequential (HDS) chemotherapy and peripheral blood progenitor cell (PBPC) autografting. To minimize the potential tumor cell contamination, PBPC harvests were scheduled at the end of HDS pretransplant phase. All patients had advanced-stage disease and most of them presented with bone marrow (BM) involvement. A tumor marker could be generated in 90% of patients using bcl-2 or Ig heavy-chain genes. MRD was analyzed on PBPC, BM harvests, and after autografting by polymerase chain reaction (PCR). All evaluable follicular and 6 of 9 mantle cell patients achieved clinical complete remission. PCR negativity of PBPC and/or BM harvests was documented in 68% of follicular and 12% of mantle cell lymphomas. Molecular remission of PBPC and/or BM harvests was achieved in 9 of 15 patients with overt marrow involvement and in all patients with only molecular marrow infiltration at onset. Molecular follow-up was conducted on 14 patients: all 7 evaluable patients who received at least one PCR-negative graft maintained the negative status at a median follow-up of 24 months and none of them relapsed so far. Thus, the results show that (1) a molecular marker to monitor MRD can be obtained in most follicular and mantle cell NHL patients, (2) the HDS regimen may provide PCR-negative PBPC and/or BM harvests even from patients with BM disease, and (3) autograft with at least one PCR-negative harvest is associated with a durable clinical and molecular remission.
Characterization of a New Alloantigen (SH) on the Human Neutrophil Fcγreceptor IIIb
Authors: Bux Juergen , Stein Ernst-Ludwig , Bierling Philippe , Fromont Patricia , Clay Mary , Stroncek David , Santoso Sentot 
Published: October 2019

AbstractPolymorphic structures of the neutrophil Fcγreceptor IIIb (FcγRIIIb) result in alloantibody formation that causes alloimmune neonatal neutropenia and transfusion reactions. Alloantigens located on FcγRIIIb include the antigens NA1 and NA2. In four cases of alloimmune neonatal neutropenia, granulocyte-specific alloantibodies directed against a thus far unknown antigen were detected by granulocyte agglutination and immunofluorescence tests in the maternal sera. By the use of the monoclonal antibody–specific immobilization of granulocyte antigens (MAIGA) assay, the new antigen, termed SH, was located on the FcγRIIIb. Nucleotide sequence analysis of the FcγRIIIb coding region from a SH(+) individual showed a single-base C→A mutation at position 266, which results in an Ala78Asp amino acid substitution. A family study confirmed that this nucleotide difference is inherited, and corresponds to the SH phenotype. Serologic typing of 309 randomly selected individuals showed an antigen frequency of 5% in the white population. The same frequency was found by genotyping, for which a technique based on polymerase chain reaction (PCR) using sequence-specific primers (PCR-SSP) was developed. Typing of all SH(+) individuals for NA1 and NA2, and PCR-restriction fragment length polymorphism analysis of the NA-specific PCR products from five SH(+) individuals using the SH-specific endonuclease SfaN I showed that SH antigen is very probably the result of an additional mutational event in the NA2 form of the FcγRIIIB gene. Immunochemical studies also demonstrated that the SH determinants reside on the 65- to 80-kD NA2 isoform of the FcγRIIIb. Our findings show the existence of an additional polymorphism of the FcγRIIIb, which can result in alloantibody formation causing alloimmune neonatal neutropenia.
Identification of the cDNA for Human Red Blood Cell–Specific Hexokinase Isozyme
Authors: Murakami Koko , Piomelli Sergio 
Published: October 2019

AbstractA unique cDNA for hexokinase (HK) was identified from poly(A)+ RNA of human reticulocytes by anchored polymerase chain reaction. This appeared to represent the cDNA for the red blood cell (RBC)–specific HK isozyme (HKR ) described in our previous study (Murakami et al: Blood 75:770, 1990). Its nucleotide sequence was identical to HKI cDNA except for the 5′ extreme end. It lacked the first 62 nucleotides of the HKI coding region: instead, it contained a unique sequence of 60 nucleotides at the beginning of the coding sequence as well as another unique sequence upstream of the putative translation initiation site. It lacked the porin-binding domain which facilitates binding to the mitochondria, thus explaining the exclusive cytoplasmic localization of HKR . It was the major cDNA derived from reticulocytes, consistent with the observation that HKR activity is predominant in reticulocytes. Northern blot analysis showed that it was expressed in the reticulocytes and in the K562 erythroleukemic cell line, but not in a lymphocytic cell line. In the extract of K562 cells, HKR activity co-eluted with the HKR of human RBCs on a MonoQ column (Pharmacia, Piscataway, NJ) chromatography, using a salt gradient elution. The separate genetic control of the RBC-specific HK isozyme explains the clinical reports of two types of HK deficiency, one in which the HK activity was reduced exclusively in the RBC (HKR defect) and another with general decrease of HK activity in several tissues (HKI defect).
Granulocyte Colony-Stimulating Factor (Filgrastim) Accelerates Granulocyte Recovery After Intensive Postremission Chemotherapy for Acute Myeloid Leukemia With Aziridinyl Benzoquinone and Mitoxantrone: Cancer and Leukemia Group B Study 9022
Authors: Moore Joseph O. , Dodge Richard K. , Amrein Philip C. , Kolitz Jonathan , Lee Edward J. , Powell Bayard , Godfrey Scott , Robert Francisco , Schiffer Charles A. 
Published: October 2019

AbstractThis study evaluated the effect of filgrastim (granulocyte colony-stimulating factor [G-CSF]) on the duration of granulocytopenia and thrombocytopenia after intensive consolidation therapy with diaziquone (AZQ) and mitroxantrone for patients less than 60 years of age with acute myeloid leukemia (AML) in complete remission. Patients less than 60 years of age with AML who achieved complete remission (CR) with daunorubicin and cytarabine induction therapy, were scheduled to receive three sequential courses of high-dose cytarabine, cyclophosphamide/etoposide, AZQ, and mitroxantrone in a pilot study to determine their tolerance of these three sequential consolidation regimens. The initial patients treated with AZQ and mitoxantrone experienced prolonged bone marrow suppression and, therefore, subsequent cohorts were treated with G-CSF, 5 μg/kg, beginning the day after completion of the third cycle of chemotherapy. There was a marked decrease in the duration of granulocytopenia less than 500/μL in two groups of patients receiving two different dose levels of AZQ and the same dose of mitoxantrone compared with patients not receiving the G-CSF. There was also a decrease in the need for hospitalization, as well as the duration of hospitalization. There was a trend towards shortening of the duration of thrombocytopenia, as well. The duration of complete remission and overall survival was similar in patients who received or did not receive G-CSF. G-CSF markedly shortened the duration of granulocytopenia in patients with AML receiving intensive postremission consolidation with AZQ and mitoxantrone. There was no adverse effect on CR duration or survival.
Frequency and Severity of Central Nervous System Lesions in Hemophagocytic Lymphohistiocytosis
Authors: Haddad Elie , Sulis Maria-Luisa , Jabado Nada , Blanche Stephane , Fischer Alain , Tardieu Marc 
Published: October 2019

AbstractWe have retrospectively assessed the neurological manifestations in 34 patients with hemophagocytic lymphohistiocytosis (HLH) in a single center. Clinical, radiological, and cerebrospinal fluid (CSF ) cytology data were analyzed according to treatment modalities. Twenty-five patients (73%) had evidence of central nervous system (CNS) disease at time of diagnosis, stressing the frequency of CNS involvement early in the time course of HLH. Four additional patients who did not have initial CNS disease, who did not die early from HLH complications, and who were not transplanted, also developed a specific CNS disease. Therefore, all surviving and nontransplanted patients had CNS involvement. Initially, CNS manifestations consisted of isolated lymphocytic meningitis in 20 patients and meningitis with clinical and radiological neurological symptoms in nine patients. For these nine patients, neurological symptoms consisted of seizures, coma, brain stem symptoms, or ataxia. The outcome of patients treated by systemic and intrathecal chemotherapy and/or immunosuppression exclusively (n = 16) was poor, as all died following occurrence of multiple relapses or CNS disease progression in most cases. Bone marrow transplantation (BMT) from either an HLA identical sibling (n = 6) or haplo identical parent (n = 3) was performed in nine patients, once first remission of CNS and systemic disease was achieved. Seven are long-term survivors including three who received an HLA partially identical marrow. All seven are off treatment with normal neurological function and cognitive development. In four other patients, BMT performed following CNS relapses was unsuccessful. Given the frequency and the poor outcome of CNS disease in HLH, BMT appears, therefore, to be the only available treatment procedure that is capable of preventing HLH CNS disease progression and that can result in cure when performed early enough after remission induction.
Induction of Fibrinogen Biosynthesis and Secretion From Cultured Pulmonary Epithelial Cells
Authors: Simpson Haidaris Patricia J. 
Published: October 2019

AbstractAlthough the liver is the primary site of fibrinogen (FBG) synthesis, epithelial cells from diverse tissues have been shown to express one or more of the FBG Aα, Bβ, and γ chain genes. In contrast, marrow megakaryocytes, which store FBG in the α-granules, are thought not to express the FBG genes. Our earlier studies have shown that epithelial cells in a variety of extrahepatic tissues express the γ chain gene ubiquitously, while the mRNAs for the Aα and Bβ chain genes are essentially undetectable. During systemic inflammation, the liver secretes increased levels of FBG into the circulation, and lung epithelium responds to local inflammation during pulmonary infection by increased transcription of the γ-FBG gene. Therefore, to determine whether extrahepatic epithelial cells express the Aα, Bβ, and γ chain genes in response to proinflammatory mediators, cultured lung epithelial cells were treated with interleukin-6 (IL-6) and dexamethasone (DEX). Northern blot analysis demonstrated that the levels of γ-FBG mRNA in cultured lung (A549) and liver (HepG2) epithelial cells increased 2- to 10-fold in response to treatment. Reverse-transcriptase-polymerase chain reaction amplification demonstrated increased accumulation of steady state levels of FBG Aα, Bβ, and γ chain mRNAs in lung epithelial cells after treatment. The basal level of lung cell γ-FBG gene transcription was not accompanied by appreciable levels of Aα and Bβ chain gene transcription; however, nuclear run-on analysis suggested that the increase in lung cell FBG mRNAs in response to DEX ± IL-6 was due to new transcription. Furthermore, stimulation of lung epithelial cells with IL-6 + DEX resulted in maximal secretion of intact FBG (340 kD) composed of the characteristic Aα, Bβ, and γ chain polypeptides. The data suggest that basal expression of the γ-FBG gene in extrahepatic tissue occurs ubiquitously in the absence of detectable levels of Aα- and Bβ-FBG gene expression, which are then upregulated on induction of an inflammatory response. This would result in local synthesis and secretion of FBG in the injured tissue, supporting the hypothesis that production of FBG by extrahepatic epithelial cells in response to inflammation plays a role in wound repair.