bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential Requirement for Cell Division in the Development of Terminally Differentiated Neutrophils and Macrophages from Their Common Progenitors.
Authors: Sugimoto Yuka , Katayama Naoyuki , Masuya Masahiro , Shiku Hiroshi 
Published: October 2019

Abstract
Neutrophils and monocytes are the main population of peripheral blood nucleated cells and progenies of common progenitor cells, CFU-GM, that are derived from hematopoietic stem cells. Monocytes are non-dividing immediate precursors for macrophages. Common clinical observation that monocytes more rapidly recover than do neutrophils in myelosuppressive patients indicates the difference in regulatory mechanism of terminal differentiation between neutrophils and macrophages. In this study, we investigated the cell division profiles in the differentiation pathway from common progenitors for neutrophils and macrophages to respective post-mitotic cells in culture. We prepared lineage-negative (Lin−) bone marrow cells from normal C57BL/6 mice by using immunomagnetic beads. The Lin− cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), a cytoplasmic dye that is equally diluted between daughter cells, and stained with anti-Sca-1 antibody and lineage antibodies. CFSEhighSca-1+Lin− cells were sorted on a FACSVantage or FACSAria. When the CFSEhighSca-1+Lin− cells were cultured in the presence of steel factor (SF) and IL-11 for 5 days, the resulting cells could be separated into four populations based on staining with anti-fms and anti-Gr-1 antibodies: fms-Gr-1−, fms+Gr-1−, fms−Gr-1+, and fms+Gr-1+ cells. The fms+Gr-1+ cells showed morphological features typical of macrophages. The commitment of fms−Gr-1+ cells into neutrophilic lineage was evident, although they exhibited an immature morphology. CFSE labeling pattern in fms+Gr-1+ macrophages was equal to that of fms−Gr-1+ neutrophilic cells, indicating that fms+Gr-1+ macrophages and fms−Gr-1+ neutrophilic cells passed through similar successive rounds of cell division in culture of CFSEhighSca-1+Lin− cells. These fms+Gr-1+ macrophages and fms−Gr-1+ neutrophilic cells were subsequently cultured for additional 3 days in the presence of SF, IL-11, IL-3, GM-CSF, G-CSF, M-CSF, erythropoietin, and thrombopoietin. While a decline in the number of fms+Gr-1+ macrophages was observed, the number of fms−Gr-1+ neutrophilic cells increased approximately 5-fold during subsequent culture. The proliferation of fms−Gr-1+ neutrophilic cells and no cell division of fms+Gr-1+ macrophages were confirmed by Ki-67 antibody staining, BrdU incorporation, and DNA staining. The cultured fms−Gr-1+ neutrophilic cells also predominantly acquired a mature morphology. These results suggest distinct cell division histories between terminally differentiated neutrophils and macrophages, possibly explaining the early appearance of monocytes in the blood after myeloablative therapy, as compared to neutrophils.
Truncated and Microparticle Free Soluble Tissue Factor Bound to Peripheral Monocytes Preferentially Activate Factor VII.
Authors: Khan Mohammad , Hattori Takashi , Niewiarowski Stefan , Edmunds L. Henry , Colman Robert W. 
Published: October 2019

Abstract
Soluble plasma tissue factor (TF) circulates in picomolar concentrations in healthy individuals and increases in a wide spectrum of diseases. This study tests the hypothesis that both truncated or long length TF antigens in low concentrations combine with monocytes or platelets to convert factor VII (fVII) to fVIIa. Both recombinant truncated TF (rsTF, kDa 33) and long length (47 kDa) plasma TF (pTF), obtained from pericardial wounds of patients having cardiac surgery using cardiopulmonary bypass, were studied in association with blood cells and TF bearing microparticles. Tissue factor was measured by ELISA. RsTF binds to erythrocytes, platelets, mononuclear cells and neutrophils. The rate of fVII conversion with rsTF (1.0 – 103nmoles/L) is fastest with mononuclear cells, less with platelets, minimal with neutrophils and undetectable with erythrocytes. Both unstimulated and stimulated mononuclear cells or platelets in the presence of 3.5 pmoles/L rsTF or pTF convert fVII (10 nmoles/L) to fVIIa, but the amounts of fVIIa produced is greater in mononuclear cells. When leukocytes or platelets are absent, microparticles associated with 3.5 pmoles/L TF antigen, derived from pericardial wound plasma, do not activate fVII. Stimulated mononuclear cells convert nearly all available fVII (10 nmoles/L) to fVIIa with 3.5 pmoles/L pTF. Unstimulated mononuclear cells convert small amounts of fVII with as little as 1 pmole/L rsTF but no VIIa is produced by platelets, neutrophils or erythrocytes under these conditions. At all concentrations (0.001 to 1m moles/L) mononuclear cells more efficiently convert fVII to fVIIa than platelets. This study shows that stimulated mononuclear cells provide the most efficient phospholipid platform for activation of truncated or long length molecules of soluble TF at low concentrations of TF antigen. The results suggest a new mechanism by which soluble TF, which is increased in clinical inflammatory diseases, may lead to a prothrombotic state.
Prodefensin in Plasma of Patients with Chronic Myeloproliferative Disorders. A Parameter of Myelopoietic Activity.
Authors: Kamp Søren , Jensen Morten Krogh , Borregaard Niels 
Published: October 2019

Abstract
Prodefensins (proHNPs) are the 74 aa antimicrobially inactive precursor peptides of defensins (HNPs). ProHNPs are synthesized in neutrophil precursors from the late promyelocytic stage and until the band cell stage. In promyelocytes, proHNP is proteolytically cleaved to yield 29–30 aa HNPs that are efficiently stored in primary (azurophil) granules. Beyond the promyelocytic stage, proHNPs are no longer proteolytically processed and significant amounts of proHNP escape the cell through constitutive exocytosis. The fate and biological implications of proHNP constitutively secreted from myeloid cells is unclear. We have raised antibodies against a synthetic propiece of proHNP (45 N-terminal amino acids) and developed an accurate, sensitive and specific ELISA for proHNP using these antibodies, with a detection limit of 4.0 ng/ml. We found levels of pro HNP in bone marrow plasma of 9 μg/ml and levels in blood plasma of 1.7 μg/ml. This indicates that the bone marrow is an important organ for generation of this pro-antibiotic molecule giving rise to plasma concentrations in the same order of magnitude as lysozyme and hCAP-18. Studies of plasma from patients with ph- chronic myeloproliferative disorders showed a mean concentration of 3.5 μg/ml indicating that proHNP is a parameter of myelopoietic activity. ProHNP in plasma was apparently not complexed to higher molecular weight structures, yet it was not present in urine. This indicates that proHNP from bone marrow is processed in circulation.
Hemoglobin and All-Cause Mortality: Results from the Baltimore Longitudinal Study of Aging (BLSA).
Authors: Longo Dan L. , Ble Alessandro , Metter Jeffrey E. , Guralnik Jack M. , Woodman Richard C. , Ferrucci Luigi 
Published: October 2019

Abstract
Whether mild-to-moderate hemoglobin (Hb) reduction is associated with increased mortality independent of underlying diseases is still debated. Two epidemiological studies addressing this issue in high risk populations (disabled older women, and persons 85 years or older) reported conflicting results. The BLSA offers the opportunity to investigate this association in an adult generally healthy population. The BLSA is a large cohort study on aging started in 1958. The participants are volunteers living in the Baltimore-Washington area (USA), well educated and self-describing as generally healthy persons. Participants are examined every 1-2 years. In the present analysis, all participants evaluated at least one occasion for Hb were included. Subjects were not excluded for any pre-existing conditions or specific outcomes. Hb levels were measured in 2979 participants recruited from 1958 to 2002 (1793 men and 1186 women, 78.7% Caucasians, age range: 18–96 years). Mean age at initial evaluation was 52.3 (±17.4) years for women and 52.1 (±17.9) for men, and the average time from baseline to death/censoring was 17.6 years for men and 12.4 years for women. Crude mortality rates were 21/1000 person years for men and 9.7/1000 person years for women. After adjusting for age, a U-shaped relationship (obtained fitting a Cox model with a Poisson regression approach according Therneau & Grambsch, 2000) was observed between Hb concentration and mortality with nadir at Hb=12.8g/dL in women and 15.0g in men. In the same model, significant excess of mortality was associated with Hb ≤ 11.2g/dL (95%CI 9.8g/dL–12.3 g/dL) and ≥ 14.0 g/dL (95%CI 13.5g/dL–14.9g/dL) in women, and ≤ 13.4 g/dL (95% CI 12.5g/dL–14.4g/dL) and ≥ 16.2g/dL (95% CI 15.4g/dL–17.2g/dL) in men. After adjusting for demographics, anthropometrics, smoking status, radiation exposure, and co-morbid diseases (heart disease, stroke, pulmonary disease, cancer, diabetes, hypertension), men with Hb ≤ 13.4 g/dL (mean: 12.7 g/dL, SD: 0.6) were at 30% higher risk (RR: 1.31, 95%CI: 1.08–1.58) compared to the reference group (Hb: 13.4–16.2). Accordingly, women with Hb ≤ 11.2 g/dL (mean: 10.2 g/dL, SD: 1.0) showed 100% greater risk compared to the reference group (Hb: 11.2–14.0), however this association was not statistically significant (RR: 2.01, 95% CI: 0.88–4.63). When men with Hb levels <10g/dL and women with <9 g/dL were excluded the results did not substantially change. Our findings showed that in the general population moderately-low Hb levels per se were associated with increased mortality risk. The impact of anemia correction on mortality in the elderly remains to be determined.
Homozygous JAK2 V617P Gain-of-Function Mutation Is Responsible for Constitutive JAK2/STAT5 Activation and Proliferation of HEL.92.1.7. Cell Line.
Authors: Estes Myka L. , Ozawa Yuki , Williams Ann H. , List Alan F. , Sokol Lubomir 
Published: October 2019

Abstract
HEL (Human erythroleukemia) is a growth factor independent erythroleukemic cell line established from the bone marrow of a patient with relapsed Hodgkin disease after autologous bone marrow transplantation (Martin P & Papayannopoulou T: Science1982;216:1233–1235). HEL cells display a block in differentiation at the level of common erythroid-megakaryocytic progenitor and have been commonly used as a model to study erythroid and megakaryocytic differentiation. Liu RY et al. (Blood1999;93:2369–2379) reported that constitutive activation of JAK2 tyrosine kinase in Dami/HEL cell line correlated with factor independent growth. Recently, several groups of investigators described a point mutation V617P in the autoinhibitory regulatory (JH2) domain of JAK2 tyrosine kinase in patients with myeloproliferative disorders (MPD). This acquired mutation results in constitutive activation of the JAK2/STAT5 pathway in hematopoietic cells. To date, the V617P mutation represents the most common causative genetic defect detected in patients with Philadelphia chromosome negative MPD. Interestingly, rare cases of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) were found to have the same mutation. We tested several hematopoietic cell lines including HL-60, HEL, KG-1, K-562, TF-1, U937 and UT-7 for the presence of V617P mutation using RFLP and automatic DNA sequencing. We found the homozygous mutation V617P only in HEL cells. This cell line has a very complex karyotype suggesting that a defect of multiple oncogenes or antioncogenes could be responsible for sustained cell proliferation. We employed the JAK2 inhibitor, AG490 at concentrations ranging from 0 to 100 mM and measured proliferation of HEL and control K562 cell lines using MTT assay. We observed dose-dependent inhibition of HEL cell proliferation with low micromolar concentrations of AG490 in contrast to K562 control cell line, harboring bcr-abl fusion tyrosine kinase. IC50 was 13 mM for HEL cells and 65 mM for control K562 cells, respectively. Low concentrations of AG490 significantly decreased phosphorylation of JAK2 and STAT5 in HEL cells but not in control K562 cell line, where bcr-abl activates separately JAK2 and STAT5 pathway. Exposure of HEL cells to AG490 [0–50mM] induced apoptosis as measured by annexin V labeling in a concentration dependent fashion. No significant increase in apoptosis was detected in K562 cells using similar concentrations of the JAK2 inhibitor. Overall, these results suggest that the mechanism of proliferation of the HEL cell line is driven by constitutive activation of JAK2 tyrosine kinase secondary to V617P gain-of-function mutation. Our data provides further evidence that the JAK2/STAT5 intracellular signaling pathway is preserved in this cell line. Thus, HEL cells can serve as a model to test novel JAK2 specific inhibitors in preclinical studies.
Effect of Bax G125A Single Nucleotide Polymorphism (SNP) on Bax Gene Expression in Diffuse Large Cell Lymphoma.
Authors: Saxena Anurag , Bosch Mark , Qureshi Tania , Nokhrina Kateryna , Moshynska Oksana 
Published: October 2019

Abstract
Altered expression of Bax protein has been shown to be predictive of overall and disease free-survival of patients with diffuse large B-cell lymphoma (DLCL)(Am J Hematol, 2003; 73:101–7). Further, absence of Bax and low levels of Bcl-2 have been shown to be associated with a more aggressive phenotype (P<0.05) (Blood, 1997; 90:3173–8) in patients with DLCL. We have previously reported a single nucleotide polymorphim (SNP), G125A, in the 5′-untranslated region of the BAX promoter and its association with reduced protein expression and treatment resistance in B-cell chronic lymphocytic leukemia (Cancer Lett2002; 187:199–205). The aim of the present study was to evaluate BAX SNP incidence and its association with the protein expression in lymphomas. DNA was extracted from fifty lymphoma samples (31 DLCL, seven follicular lymphomas (FL), three marginal zone lymphomas (MZL), four T-cell lymphomas, three Hodgkin’s lymphomas, and two cases of Mantle cell lymphoma (MCL), and from 55 control samples. BAX SNP was detected by restriction enzyme analysis (J Clin Pathol, 2003; 56: 205–9). Fourteen out of 50 (28%) samples had G125A SNP, in all cases only one allele was replaced. It was present in eight (26%) DLCL, two (29%) FL, in two MZL, in one T-cell lymphoma, in one HL, and none of MCL had this SNP. Eight out of 55 (14.5%) controls had the SNP. The presence of BAX promoter SNP was associated with reduced Bax protein expression as detected by Western blot analysis (P=0.024). Two plasmids with G and A nucleotides at position 125 of the BAX promoter linked to luciferase as a reporter were constructed and used for transfection, as well as the pGL3-Basic and pGL3-Control plasmids; pRL-SV40 plasmid was used for co-transfection. Dual-Glo luciferase assay was performed and Firefly luciferase activity was normalized to Renilla luciferase. In transfection experiments, the G125A SNP reduced expression (P=0.0202) of BAX promoter. BAX SNP, G125A, present in approximately a quarter of DLCL has a direct negative effect on BAX gene expression.
Point Mutations Found in the Juxtamembrane Domain of FLT3 Define a New Class of Activating Mutations in AML.
Authors: Reindl Carola , Bagrintseva Ksenia , Schnittger Susanne , Ellwart Joachim W. , Wenig Katja , Hopfner Karl-Peter , Hiddemann Wolfgang , Spiekermann Karsten 
Published: October 2019

Abstract
In acute myeloid leukemia (AML) FMS-like tyrosine kinase-3 (FLT3) has been shown to be mutated in about one third of patients. Until now, two distinct activating mutations are known: FLT3-length mutations (FLT3-LM) in the juxtamembrane (JM) domain in 20–25% and FLT3-point mutations in the tyrosine-kinase domain (FLT3-TKD) in 7–10% of patients. Here, we have characterized a new class of activating point mutations (PM) that cluster in a 16 amino acid stretch of the juxtamembrane domain of FLT3 (FLT3-JM-PM).
Stable expression of four distinct FLT3-JM-PM in IL-3 dependent murine Ba/F3 cells led to factor-independent growth, hyperresponsiveness to FLT3-ligand and resistance to apoptotic cell death compared to FLT3-WT-expressing cells. As a molecular mechanism, we could show activation of STAT5 and upregulation of Bcl-x(L) by all FLT3-JM-PM. A selective FLT3-inhibitor, PKC412, was able to abrogate the factor-independent growth of FLT3-JM-PM. Mapping of the FLT3-JM-PM on the crystal structure of FLT3 showed that these mutations probably reduce the stability of the JM domain in the autoinhibitory conformation, and provide a structural basis for their transforming capacity.
Our results show that point mutations in the autoinhibitory JM domain represent a new class of gain-of-function mutations able to activate the transforming potential of FLT3.
Aberrant Pre-mRNA Processing Due to Novel Splicing Mutations in the CBL Proto-Oncogene in Acute Myeloid Leukemia.
Authors: Arnoczky Kristy J. , Marburger Trent B. , Vukosavljevic Tamara , Caligiuri Michael A. , Whitman Susan P. 
Published: October 2019

Abstract
Ubiquitin (Ub)-dependent proteolysis is a highly controlled pathway that regulates almost all cellular processes by modulating the levels of key proteins. Several E3 Ub ligases are functionally abnormal in cancer. The E3 ubiquitin ligase, Casitas B-lineage lymphoma (CBL) protein, negatively regulates many receptor tyrosine kinases (RTKs) that are frequently over-expressed and/or constitutively activated (e.g., FLT3) in acute myeloid leukemia (AML). The CBL linker region, encoded in part by exon 8, lies between the tyrosine kinase binding and the RING finger domains and, mutations introduced into this linker region were previously shown to confer transforming ability. Herein, we describe novel splicing mutations in the c-CBL gene that lead to aberrant pre-mRNA processing in AML. DNA PCR revealed a splice site mutation whereby 14 nucleotides were deleted at the exon 8-intron 8 boundary in one CBL allele of the MOLM-13 cell line. Out of 18 patient samples, one primary AML blast sample had a 4 base pair deletion (16 bases upstream of the intron 7-exon 8 junction) which was replaced by a 12 base pair GC-rich insertion. We hypothesized that this mutation was an intronic splicing silencer element. RT-PCR/sequencing confirmed aberrant pre-mRNA processing in both the AML patient sample and MOLM-13 cells as they expressed a wild-type CBL transcript and an in-frame variant lacking exon 8. The splice variants are reminiscent of the oncogenic Cbl intragenic deletion mutation present in the murine pre-B cell line, p70Z. Such Cbl mutants are suggested to result in a structural alteration, allowing displacement of wild-type Cbl from the RTK complex thereby abrogating Cbl’s negative regulatory function. Thus it is possible that this mutant may enhance proliferation and survival of AML cells through attenuation of ubiquitin/proteosome-dependent degradation of receptor tyrosine kinases. Further investigation into the role of CBL and other players in the Ub-mediated proteolytic pathway is likely to provide additional insight into a subset of AML patients that in turn could lead to discovery of new therapeutic targets and strategies.
Increased Superoxide Levels, HO-1 and Fyn Protein Expression in Bcr/abl Overexpressing Cells: Implications for Adaptation and Survival in an Oxidatively Challenging Environment.
Authors: Chandra Joya , Ban Kechen 
Published: October 2019

Abstract
The bcr/abl oncogene is present in the majority of CML and in a subset of ALL patients. In addition to anti-apoptotic properties, proliferative advantages and growth factor independence, the presence of the Bcr/abl kinase raises intracellular levels of reactive oxygen species (ROS). Increased ROS in CML have been linked to DNA damage and DNA repair deficiencies with implications for promoting genomic instability and secondary mutations associated with disease progression. Here we show that superoxide is the specific ROS entity that is elevated when Bcr/abl is overexpressed in an IL-3 dependent pre-B cell line, BaF3. Strikingly, treatment with classical oxidants, such as hydrogen peroxide or tert-butyl peroxide, demonstrated an inherent resistance to oxidative stress induced apoptosis in Bcr/abl-containing cells, suggesting that antioxidant defenses activated by Bcr/abl as a response to the increased endogenous ROS production may promote survival in Bcr/abl positive populations.
In an effort to characterize the redox environment in Bcr/abl containing cells, we examined several antioxidant enzymes that are known downstream substrates of c-abl or bcr/abl, including peroxiredoxin, catalase and heme oxygenase-1 (HO-1). Of these, HO-1 protein expression was three fold higher in Bcr/abl transductants, consistent with a reported role for HO-1 as a Bcr/abl dependent survival factor. Interestingly, the Src family kinase, Fyn, which can be activated by oxidative stress, was increased four fold in Bcr/abl overexpressing cells. This is consistent with reported microarray data in which fyn mRNA levels are higher in Philadelphia positive leukemias than non-Philapdelphia positive leukemias. Other components of ROS responsive signaling pathways were also interrogated including the Mapk pathway and Akt pathway, with no difference noted between parental and Bcr/abl overexpressing cells.
Our data suggest a relationship between Bcr/abl-induced superoxide elevation, resistance to oxidative stress, elevation of HO-1 (which has antioxidant properties) and elevated Fyn (a putative downstream target for ROS production). These redox alterations and their consequences may equip Bcr/abl positive cells with a survival and/or proliferative advantage in an oxidatively challenging environment, thus promoting disease progression and blast crisis.
Prognostic Significance of Multidrug Resistance Gene 1 (MDR1), Multidrug Resistance-Related Protein (MRP) and Lung Resistance Protein (LRP) mRNA Expression in Acute Leukemia.
Authors: Huh Hee Jin , Park Chan-Jeoung , Jang Seongsoo , Seo Eul-Ju , Chi Hyun-Sook , Lee Je-Hwan , Lee Kyoo-Hyung , Seo Jong Jin , Moon Hyung Nam , Ghim Thad 
Published: October 2019

Abstract
The prognostic significance of multidrug resistance (MDR) gene expression is controversial. We investigated whether multidrug resistance gene 1 (MDR1), multidrug resistance-related protein (MRP) and lung resistance protein (LRP) mRNA expression are associated with outcomes in acute leukemia patients. At diagnosis we examined MDR1, MRP and LRP mRNA expression in bone marrow samples from 71 acute leukemia patients (AML 39, ALL 32) using nested reverse transcription polymerase chain reaction (RT-PCR). The expression of each of these genes was then expressed as a ratio in relation to ß-actin gene expression, and the three genes were categorized as being either not expressed (0), weakly expressed (1+), moderately expressed (2+) or strongly expressed (3+). MDR1, MRP and LRP mRNA expression was detected in 23.9%, 83.1% and 45.1%, respectively, of acute leukemia patients. LRP mRNA expression was significantly associated with resistance to induction chemotherapy in acute leukemia patients, and in the AML proportion of these patients, compared to LRP-negative patients (p=0.02 and p=0.03, respectively). MRP and high MDR1 mRNA expression was associated with poorer 2-year survival (P=0.049 and p=0.04, respectively) compared to MRP-negative and non-high MDR1 mRNA-expressing patients, respectively. Patients expressing both MRP and LRP mRNA had poorer outcomes in response to induction chemotherapy and had worse 2-year survival compared to patients not expressing both genes. The present data suggest that MDR gene expression affects CR and survival rates in acute leukemia patients. Thus, determination of MDR gene expression at diagnosis appears likely to provide useful prognostic information for acute leukemia patients.