bluuebunny/crossref_metadata_2025_split huggingface dataset:
Splicing Variants of Osteocalcin-mRNA in Haematological Malignancies.
Authors: Wihlidal Peter , Karlic Heidrun , Varga Franz , Nosslinger Thomas , Pittermann Elisabeth , Pfeilstocker Michael 
Published: April 2020

Abstract
Osteocalcin (OCN) is defined as a marker for osteoblasts but incompletely spliced variants as characterised by intron retention may also occur in non-osseous tissues including tumour cells. Bone marrow of haematological diseases was shown to harbour a stem cell candidate common to MSC (mesenchymal stem cells) and HSC (haematopoietic stem cells), which expressed OCN. In this study, we analysed cells from bone marrow samples obtained from 4 patients with AMLs, one patient with CML at blast crisis, one CML patient in chronic phase and 7 patients with myeloproliferative disease (MPD), as well as stem cells from peripheral blood and bone marrow obtained from healthy donors. In attempt to find out whether the presence of such stem cells is associated with disease status, sequential bone marrow samples from a patient with acute myeloid leukaemia (AML/M2Eo) were analysed at 6 time points including diagnosis, remission (= months 4, 5 and 17 after diagnosis) and relapse (months 29 and 41 after diagnosis). RT-PCR analyses were performed to characterise spliced and unspliced mRNA variants of OCN and to quantify mRNA levels of c-KIT and the Runt-transcription factors AML1 and AML3, known to regulate gene expression of c-KIT and OCN. Results of immunostaining for c-KIT and OCN showed that positive signals for OCN were detected only in those bone marrow smears, which expressed spliced OCN-mRNA, i.e. the bone marrow samples from the AML/M2Eo-patient in the relapse phase, the 2 samples of AML/M2, the sample of CML at blast crisis. The same samples and all others, which were positive for the unspliced OCN-mRNA, were also positive for c-KIT (both at the protein and at the mRNA level) as well as for AML1- and AML3-mRNA, however, at lower quantities. Our data indicate an association of OCNs expression with haematological malignancies at disease stages with activated bone marrow stem cells.
Cultured CD41a+ Megakaryocytes Derived from Cord Blood CD34+ Cells Are Activated Via STAT5 but Not STAT3.
Authors: Smith Stephen 
Published: April 2020

Abstract
Lengthy periods of thrombocytopenia and prolonged platelet recovery (typically, a side-affect of high-dose chemotherapy and/or radiation) have been associated with cord blood transplants. It maybe possible to alleviate the period of thrombocytopenia using cultured megakaryocytes derived from CD34+ stem cells however, platelet development invitro is delayed. Using immuno-magnetic beads, CD34+ stem cells were isolated from Cord Blood (CB) units and then cultured in serum-free culture medium (X-VIVO-10®, BioWhittaker Co. Walkersville, MD) supplemented with Thrombopoietin Peptide Agonist (TpoA), Stem Cell Factor (SCF), Flt-3L, and Vascular Endothelial Growth Factor (VEGF). A series of culture experiments were setup to investigate the activation of the JAK-STAT pathway following cytokine stimulation. Using flow cytometry, CB CD34+ cell cultures were evaluated weekly for the expression of CD41a, STAT3, STAT5, and Proliferating Cell Nuclear Antigen (PCNA) at week 1–6 of culture. Typically, CD41a expression peaked at week 3 of culture. At week 3, the total cells were 14.1 ±4.9 x106 cells per culture with 48.8 ±12.5% CD41a+ cells or 6.4 ±1.2 x106 CD41a+ cells per culture. Similarily, PCNA activation peaked at week 3–4 (64.1 and 62.4%, respectively). The level of PCNA activation increased incrementally from weeks 1, 2 and 3 of culture however declined at 5 and 6 weeks of culture. However, there was an incremental increase in the activation of STAT5 during the 6 weeks of culture. The STAT5 expression did not peak until week 5–6 (80.9 and 73.8%, respectively). Conversely, STAT3 expression declined between week 1 and 6 from 3.2. to 0.4%, respectively. With the exception of weeks 1 and 2, there was only minor activation or no activation of STAT3. These results suggest that STAT5 but, not STAT3, is activated during CB megakarycyte differentiation. These results may provide incite as to CB megakaryocyte development and might explain the delayed production of platelets following transplant. The PCNA results suggest that the cultured cells reach their greatest proliferative potential at week 3 and 4 of culture. This might provide useful information as to the optimal time period for the transplant of the cultured cells and suggest that cells transplanted from 5 and 6-week cultures have less proliferative capacity post-transplant. Additional studies are needed to determine the differentiation of megakaryocytes and platelet development from CB CD34+ cells invitro.
Expression of Runx1 Isoforms in Sub-Populations of Human Blood and Bone Marrow Cells.
Authors: Yankelevich Maxim , Margolin Judith F. , Goodell Margaret A. 
Published: April 2020

Abstract
Runx1 is a pivotal regulator of hematopoiesis. Deficiency of Runx1 leads to complete block of definitive hematopoiesis in mouse embryos. Runx1 protein forms a heterodimer with CBFβ and binds to the specific DNA sequence TGT/cGGT to regulate the expression of a number of hematopoietic genes. Runx 1 has been recognized to have three different isoforms (a, b, and c) due to differential splicing and promoter utilization. The distal (Runx1c) promoter region was shown to have binding sites mediating repression of Runx1 transcription. Data on the pattern of Runx1 isoforms expression in different hematopoietic lineages are scant. The functional impact of different Runx1 isoforms on commitment and differentiation of blood cells is also essentially unknown. To further explore this, we studied Runx1 isoform expression in different subpopulations of human hematopoietic cells. Total RNA was extracted from peripheral blood and unstimulated bone marrow. Also, Lin-CD34+CD38+, and Lin-CD34+CD38− cell populations were sorted from G-CSF-stimulated bone marrow and total RNA extracted. Expression of Runx1 isoforms was assessed by semiquantitative RT-PCR analysis, using GAPDH as a control. All 3 isoforms were present in all studied cell populations. Runx1a was uniformly a minor form with lowest expression. Runx1c isoform was a dominant one in peripheral blood as opposed to Runx1b in bone marrow. Most interestingly, the ratio of Runx1b to other isoforms was significantly increased in Lin-CD34+CD38- subpopulation compared to Lin-CD34+CD38+ cells. The prevalence of Runx1c isoform in peripheral blood as compaired to bone marrow is most likely related to a different cellular composition of these tissues and suggests that Runx 1c isoform may be predominantly expressed in some of the mature cell lineages. Since Lin-CD34+CD38− cells represent most primitive progenitor cell population with multi-lineage repopulating ability, the observed overexpression of Runx1b may be related to transcriptional accessibility of these cells. Upregulation of Runx 1b with G-CSF in this subpopulation can not be ruled out as well. These preliminary data suggest that the expression of Runx1 alternative splicing isoforms is both lineage and differentiation stage-specific in human hematopoietic cells. We will also test whether Runx1 isoform expression has a consistent pattern in different types of leukemia and if it is resembles such of mature blood counterparts or primitive progenitors. These studies, as well as the studies of differential expression of Runx1 isoforms in different mature blood lineages and progenitor cell sub-populations are underway and results will be presented. These data may further shed a light on functional differences in Runx1 isoforms and mechanisms of hematopoiesis.
PMA Responsive Sequence (s) in Gαq Promoter during Megakaryocytic Differentiation.
Authors: Jalagadugula Gauthami S. , Dhanasekharan Danny , Rao A.Koneti 
Published: April 2020

Abstract
Human erthroleukemia cells (HEL) differentiate towards megakaryocytic (MK) phenotype when stimulated with phorbol 12-myristate-13-acetate (PMA). We observed that the expression of Gq, a protein that plays a major role in platelet signal transduction, is increased in PMA-treated HEL cells. Western blotting revealed that Gq is upregulated in PMA-treated cells relative to untreated cells. Gq gene induction by PMA treatment was investigated with respect to transcriptional control. Serial 5′-truncations of the upstream region (upto 2727 bp from the ATG) of Gq gene were fused to a luciferase (Luc) reporter gene vector, PGL-3 Basic, and were transiently transfected into HEL cells in the absence and presence of PMA (10 nM). After 24 h, reporter gene activities were measured using Dual Luciferase Reporter Assay System (Promega). A reporter plasmid −1042 bp-Luc with a genomic region −1042/−1 showed a 12 fold activity in PMA treated cells and 4 fold activity in untreated cells. Its truncated plasmid with the genomic region −1036/−1 showed a decrease in luciferase activity by 50% in treated cells; and the activity became identical to that in untreated cells. Further truncation between −1036 and −1011 caused a complete loss of activity in both the cells. Thus, a PMA responsive element was localized to a region between −1042 and −1037 bp. Transcription factor data base search (TFSEARCH) predicted two consensus sites for early growth response factor EGR-1 at -1042/−1031 and −1026/−1015. Gel shift studies were performed with two oligos, −1042/−1012 and −1036/−1012, and nuclear extracts from PMA- treated and untreated cells. The studies with −1042/−1012 probe and extracts from treated cells showed that there was nuclear protein binding, which was abolished by competition with the consensus EGR-1 sequence. In extracts from untreated cells, the protein binding was observed but was not competed with consensus EGR-1 sequence. This suggests EGR-1 binding to the region −1042/−1012 in PMA-treated cells and role for this transcription factor in inducing Gq promoter activity. Moreover, studies on the region −1036/−1012 showed nuclear protein binding that was identical between extracts of untreated and treated cells, and it was not competed with consensus EGR-1 sequence. These findings suggest that, EGR-1 binding is localized to −1042/−1037, but not to −1036/−1012.
Conclusion: A PMA responsive sequence (−1042/−1037) was identified in the Gq promoter. Our studies suggest that EGR-1 binding to this sequence confers the PMA responsive activity. These studies provide further evidence that EGR-1 plays an important role in the upregulation of Gq expression during PMA induced megakaryocytic differentiation.
Morbid Risks of Unnecessary Red Blood Cell Transfusion in Stable Coronary Artery Bypass Graft Patients.
Authors: Moehnle Patrick , Snyder-Ramos Stephanie A. , Weng Yi-Shin , Kulier Alexander , Boettiger Bernd W. , Wang Shirley , Levin Jack , Mangano Dennis T. 
Published: April 2020

Abstract
The persistent variability in red blood cell transfusion practice in coronary artery bypass graft (CABG) patients, despite established guidelines, suggests inappropriate use. Our objective was to determine the impact of postoperative red blood cell (RBC) transfusion in entirely stable CABG patients. We investigated a cohort of 940 stable CABG patients from the 5,065 patients enrolled in the Multicenter Study of Perioperative Ischemia Epidemiology II (EPI II) Study with (1) low to moderate risk profile; (2) postoperative hemoglobin levels ≥ 10 g/dL; (3) minimal postoperative blood loss, and (4) no evidence of any morbid event on the day of surgery. RBC transfusion was assessed during the first 24 postoperative hours and multiorgan outcomes as well as markers of resource utilization from postoperative day one to hospital discharge. Transfused patients (N =190, 20.2 %) were more likely to suffer myocardial infarction (OR, 1.89; 95 % CI, 1.08 to 3.29; P = 0.02); renal dysfunction (OR, 3.35; 95 % CI, 1.01 to 11.1; P = 0.04); renal failure requiring dialysis (OR, 4.01; 95 % CI, 0.99 to 16.2; P = 0.05); and/or harvest site wound infection (OR, 5.45; 95 % CI, 1.87 to 15.9; P = 0.001). RBC transfusion was shown to be an independent predictor of composite morbidity outcome, cardiac morbidity, and harvest site wound infection in multivariate analysis. In transfused patients, the mean duration of hospitalization was increased by 2.5 days (14.4 days vs. 11.9 days; P &lt; 0.001). In CABG patients with low to moderate mortality risk profiles, adequate hemoglobin values and low bleeding rates, postoperative RBC transfusion is associated with an increased risk for cardiac, renal and infectious morbidity, as well as increased health care expenses, without any detectable benefit.
Role of Fas / FasL in Regulation of Basophillic Erythroblast Homeostasis.
Authors: Capocasale Renold J. , Makropoulos Dorie A. , Arlen Jeffrey , Achuthanandam Ram , Quinn John , Volk Amy L. , Bugelski Peter J. 
Published: April 2020

Abstract
Many studies have shown Fas- Fas Ligand (FasL) mediated apoptosis to be important in maturation and differentiation of erythroid precursors in vitro. To determine if there is a similar process regulating erythropoietic homeostasis in vivo, we studied erythropoiesis in Fas(lpr) and FasL (gld) deficient mice. We postulated that deficiency of Fas or FasL should result in changes in red blood cell (RBC) parameters and/or decreased levels of apoptosis of erythroblasts. To test this hypothesis under steady state conditions, blood and bone marrow were collected from 10-week old C57Bl/6 control mice, B6.MRL-Tnfrsf6 lpr /J CD95 deficient mice, and B6Smn.C3-Tnfsf6 gld /J CD95L deficient mice. Hematology was studied using a Bayer Advia 120 and femoral bone marrow was analyzed by 6-color flow cytometry using a Becton Dickinson FACSAria. Hematologic analysis revealed no differences in reticulocyte counts, RBC counts or hemoglobin (Hgb) in either lpr or gld mice compared to C57Bl/6 controls. Similarly, analysis of bone marrow revealed no differences in % of Ter-119bright CD71bright basophilic erythroblast (BEB), % apoptotic BEB (annexin V+, 7-AADdim) or % FasL+ BEB in either gld or lpr mice compared to control. As expected, lpr mice expressed 10 fold fewer Fas+ BEB while similar levels were observed in gld mice compared to controls. To test our hypothesis under stimulated conditions, control, lpr and gld mice received a single s.c. dose of 10,000 units of recombinat human erythropoietin (rhEPO). Bone marrow samples were collected 48 hours after dosing and blood samples 4, 8 and 16 days after dosing. Hematologic analysis revealed no differences in the erythropoietic response among the three strains of mice tested. Moreover, treatment with rhEPO had no effect on % Fas+ BEB in any strain, but induced a 2–5 fold increase in the % FasL+ BEB and a 2–3 fold increase in apoptotic BEB in all three strains. Based on our observations, we conclude Fas/FasL is unlikely to play a pivotal role in regulating erythroid homeostasis.
Off-Label Use of Recombinant FVIIa: Clinical Characteristics That May Influence Outcomes.
Authors: Saito Yoriko , Healy Brian , Kaufman Richard M. , Dzik Walter H. 
Published: April 2020

Abstract
Recombinant activated factor VII (rFVIIa) is increasingly being used in off-label settings. To understand patient characteristics that may affect outcomes in patients who receive off-label rFVIIa, a retrospective review of off-label rFVIIa use from 1/1999 through 3/2005 was conducted at two tertiary-care academic hospitals. Through a hospital IRB-approved protocol, patient charts were examined for all 117 consecutive patients who received off-label rFVIIa. Overall 30-day mortality was 75/117 (64%; 95% CI [0.55 - 0.72]). Overall incidence of new renal dysfunction (serum creatinine&gt;1.3mg/dL for females, &gt;1.5mg/dL for males) or worsening renal dysfunction (&gt;50% increase in creatinine in patients with pre-existing renal dysfunction) was 31/108 (28.7%; 95% CI [0.21 - 0.38]). Thromboembolic complications were documented in 9/117 (7.6%; 95%CI [0.04 - 0.14]). Multiple clinical characteristics were recorded and the Fisher’s exact test was used to determine which characteristics were associated with either an increased (or decreased) frequency of renal dysfunction within 72 hours of rFVIIa administration; or with an increased (or decreased) frequency of 30-day mortality. The characteristics that showed statistically significant association in univariate analysis are listed in the table. The incidence of renal failure was significantly increased over baseline (29%) for patients with either one of 2 characteristics (rows g,h). The incidence of death was significantly increased over baseline (64%) for patients with any of 6 clinical characteristics (a – f) and decreased compared to baseline patients with 3 characteristics (i–k). Characteristics which showed no statistically significant association for either outcomes were: intracranial hemorrhage, subdural hematoma, gastrointestinal bleeding, post-procedural bleeding, trauma, surgical intensive unit, hx of solid tumor, hx of coronary artery disease, hx of atrial fibrillation, hx of diabetes mellitus, gender and age. This analysis helps identify covariant clinical features to be considered when selecting a matched control group to examine patient outcomes resulting from off-label rFVIIa use.
Patient Clinical Characteristics n Renal Dysfunction; Odds Ratio [95% CI] Death; Odds Ratio [95 % CI] *p&lt;0.005; **p&lt;0.05; †p&gt;0.05; ¶One-sided Odds Ratio is reported because no patient survived. a Sepsis 23 0.49 [0.11–1.68]† &gt;4.73 *,¶ b DIC 15 0.16 [0.004–1.2]† &gt;2.67 *,¶ c BMT/Stem Cell Transplant 15 0.64 [0.11–2.7]† &gt;2.67 *,¶ d Heme malignancy 21 0.41 [0.07–1.6]† 14.9 [2.2–632.6]* e Medical ICU 36 1.2 [0.45–3.2]† 10.2 [2.8–55.2]* f Pre-existing renal dysfunction 53 0.42 [0.16–1.1]† 4.7 [1.8–12.7]* g Liver disease/Ped liver transplant 23 3.19 [1.02–9.78]** 0.54 [0.19–1.5]† h Cardiac/thoracic surgery 18 3.1 [0.95–9.9]** 0.86 [0.28–2.9]† i Anticoagulant-related bleeding 19 0.64 [0.11–2.7]† 0.19 [0.06–0.62]* j Surgical prophylaxis 6 0.48 [0.01–4.6]† 0 [0–0.32]*
The Target Receptor Siglec-3 (CD33) Is Expressed on AML Stem Cells in a Majority of All Patients with AML.
Authors: Hauswirth Alexander W. , FLorian Stefan , Krauth Maria-Theresa , Schernthaner Gerit-Holger , Selzer Edgar , Wacheck Volker , Printz Dieter , Fritsch Gerhard , Sotlar Karl , Sperr Wolfgang R. , Valent Peter 
Published: April 2020

Abstract
The cell surface antigen Siglec-3 = CD33 is becoming increasingly important as target of therapy in acute myeloid leukemia (AML). In particular, a conjugate consisting of the humanized CD33 antibody P67.6 (gemtuzumab) and the cytostatic drug calicheamicin has been developed for clinical use and was found to work as an effective antileukemic agent (Mylotarg®) in patients with CD33+ AML. In normal myelopoiesis, expression of CD33 is restricted to advanced stages of differentiation, whereas primitive stem cells do not express CD33 (Siglec-3). In line with this notion, CD33-targeting therapy is a non-myeloablative approach. In the present study, we asked whether leukemic stem cells in patients with AML express CD33. For this purpose, a multicolor-staining technique was applied in eleven patients with AML. Leukemic stem cells were defined as CD34+/CD38−/CD123+ cells. In all patients in whom the majority of myeloblasts expressed CD33 (=CD33+ AML, n=8), the AML progenitor cells reacted with the CD33 antibody P67.6. Repopulation experiments utilizing NOD/SCID mice confirmed that the AML stem cells in these patients reside within the CD33+ subpopulation of leukemic cells. Moreover, AML stem cells (CD34+/CD38−/CD123+ cells) highly purified (&gt;98% purity) from patients with (CD33+) AML by cell sorting, were found to express CD33 mRNA in RT-PCR analyses. To demonstrate that AML stem cells can also reside within the CD33-negative fraction of the AML clone, we purified CD33-negative cells in a patient with AML in whom a majority of leukemic stem cells were found to lack CD33. In this particular patient, the CD33-negative cells were found to repopulate NOD/SCID mice with leukemias in the same way as the entire leukemic clone did. The CD33 antigen was neither detectable on CD34+/CD38− cells in the normal bone marrow nor on leukemic stem cells in patients with CD33-negative AML. In summary, our data show that leukemic stem cells in patients with CD33+ AML frequently express the target receptor CD33. This observation is in favor of novel treatment concepts employing CD33-targeting antibodies (Mylotarg®) in acute myeloid leukemia.
Complex Translocation (8;16;21); a New Variant of t(8;21), with t(13;22) in Acute Myeloid Leukemia.
Authors: Gupta Shiphali , Brody Judith , John Veena , Koduru Prasad 
Published: April 2020

Abstract
The translocation (8;21)(q22;q22) is a common recurrent chromosome aberration present in 10% to 15% of all acute myeloid leukemias. In approximately 3% of all cases with t(8;21), a variant of t(8;21) involving chromosomes 8,21 and other chromosomes is present. We report here a case of AML-M2 with a new complex translocation (8;16;21)(q22;p16.3;q22) associated with additional abnormality t(13;22)(q22;q13) not described before. Fluorescent in-situ hybridization analysis with probes for ETO and AML1 genes, and probes for the subtelomeric regions of chromosome 16 demonstrated an ETO-AML1 fusion signal on der(8). The 5′ region of ETO gene with chromosome arm distal to translocation breakpoint had moves to der(16) while region of AML1 probe proximal to translocation brealpoint stayed on der(21). The signal for the subtelomeric probe for 16p was present on der(21). Use of painting probe for chromosome 13 confirmed t(13;22). In a hematopoietic cell the expression of chimeric AML1- ETO protein resulting from t(8;21) plays a key role in leukemic transformation by targeting AML1-CBFB transcription factor complex, an essential regulator of genes required for normal hematopoietic cell development, by dominant negative effect on normal AML1 protein. However, the expression of AML1- ETO does not block normal differentiation of stem cells. Additional mutational events must occur in hematopoietic progenitor cell to block normal path of differentiation. Although clinical features of variant translocation is same as cases with conventional t(8;21), prognostic implications of variant (8;21) are yet to be established. Chromosome region 16p13 has been involved in clinically important translocations with poor prognosis such as t(8;16)(p11;p13) and t(11;16)(q23;p13) and may have similar implications. Translocation (8;21) is frequently accompanied by additional chromosome abnormalities i.e. loss of sex chromosome, trisomy 8 and structural abnormalities of 9q. Additional reports on secondary genetic alterations accompanying t(8;21) are needed to understand their cumulative effect. Chromosome region 16p13, 13q22 and 22q13 may harbor putative genes influencing normal path of differentiation.
Study on Gene Exppression Profiling Associated with Prognosis in AML (M2a).
Authors: Meng Fan Yi , Tang Jiaming , Ma Wenli 
Published: April 2020

Abstract
Objective: to explore genes related to the prognosis of AML (M2a).
Methods: 6 newly diagnosed AML were involved in this experiment and were divided into 2 groups. Group A: 3 AML (M2a) patients with continuous complete remission (CCR1) less than 6 months; Group B: 3 AML (M2a) patients with CCR1 more than 12 months. Bone marrow mononuclear cells were separated and mRNA was purified and labeled with Cy3 and Cy5 respectively, which were used to hybridize against the Agilent human 1B 60mer oligonucleotide microarrays.
Results: in the 20173 genes tested, 22 genes were found to be expressed differently between these two groups, among them 10 genes were up-regulated in group A, and 12 genes were up-regulated in group B.
Conclusion: with microarray assay, 22 genes including APP were found to be differently expressed in AML (M2a) treated with standard chemotherapy. These genes may be early indicators for the diagnosis as well as prognosis of the refractory AML.