bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of Fetal Hemoglobin among Adult Human Erythroblasts Is Associated with an Altered Expression Pattern for the Transcription Factor c-Myb.
Authors: Rudrabhatla Parvathi , Miller Jeffery L. 
Published: April 2020

Abstract
The c-Myb transcription factor plays a central role in regulating growth and differentiation during definitive erythropoiesis. In addition, c-Myb expression has been previously linked to GATA-1 expression and hereditary persistence of fetal hemoglobin. Here we examined a potential role for c-Myb in signaled expression of fetal hemoglobin (HbF) among primary adult human erythroblasts. Initial experiments were performed to determine the expression pattern of c-Myb under conditions of low fetal hemoglobin production (CD34 cells cultured over 14 days in media supplemented with erythropoietin (EPO) alone). Quantitative PCR performed on cells sampled on days 2, 5, 8, 11, and 14 demonstrated highly regulated c-Myb expression. A dramatic increase (&gt;30 fold) in c-Myb mRNA was measured between days 5 and 8 as the committed progenitor cells differentiated to the proerythroblast stage. That increase was followed by a 3-fold reduction in c-Myb during terminal differentiation. Western blotting of nuclear extracts obtained on days 7 and 14 in EPO cultures corroborated with the quantitative PCR analyses. Electrophoretic mobility shift assays performed using day 7 and day 14 nuclear extracts showed a shifted major band with a c-Myb consensus probe. That major band was competed by unlabeled c-Myb probe on both days. Moreover, day 14 extracts repeatedly revealed two additional bands that were not competed suggesting the possibility of other complexes binding to the Myb motif or changes in c-Myb conformation during terminal differentiation. Chromatin immunoprecipitation assays also revealed preliminary evidence for Myb binding directly to beta globin locus. When compared to low HbF culture conditions (EPO; HbF &lt;1%), cells grown in high HbF conditions (erythropoietin + stem cell factor + TGF-beta (EST); HbF&gt;35%) demonstrated a distinct pattern of c-Myb expression. Relative to the low HbF conditions, high HbF expressing cells had reduced c-Myb levels on day 7 followed by increased levels on day 14. These results suggest that c-Myb possesses highly regulated patterns of expression and DNA binding during erythroid maturation. In addition, the association of changes in c-Myb expression patterns with HbF production suggests that this growth-related transcription factor may help regulate fetal hemoglobin synthesis in patients with beta-hemoglobinopathies.
Antagonistic Roles of ERK1/2 and p38 MAP Kinases in Hemoglobin Synthesis.
Authors: Blank Volker , Lehalle Damien , Mardini Louay , Boudia Mansouria Merad , Derjuga Anna , Moore Amy 
Published: April 2020

Abstract
Murine erythroleukemia (MEL) cells provide a valuable model to study the molecular events leading to erythroid differentiation. Maturing erythroid cells synthesize large quantities of hemoglobin, a process requiring the coordinated synthesis of heme and globin. Here, we investigated the role of the ERK and p38 mitogen-activated protein kinase (MAPK) signaling pathways in the differentiation of MEL cells. We determined the effect of the MEK1/2 inhibitor U0126 that blocks the ERK1/2 pathway, and the p38 inhibitor SB202190 on the differentiation potential of MEL cells induced by hexamethylene bisacetamide (HMBA). We found that treatment of HMBA induced MEL cells with the ERK1/2 pathway inhibitor U0126 results in higher hemoglobin levels. Using a fluorometric assay, we determined that intracellular heme levels also increased. Immunoblot studies showed an increase in globin protein levels. In contrast, treatment of MEL cells with the p38 inhibitor SB202190 has the opposite effect, leading to decreased amounts of heme and hemoglobin. In addition, inhibition of the p38 pathways results in lower transferrin receptor levels. Our results suggest that the ERK1/2 and p38 pathways play antagonistic roles in HMBA induced erythroid differentiation in MEL cells. This data also provides a novel link between MAPK signaling and the regulation of heme biosynthesis and iron uptake in erythroid cells.
Surveyor™ Nuclease: A New Rapid and Effective Strategy for Detection of Single Nucleotide Changes in Alpha Globin Genes.
Authors: Chandy Mammen , Sindhuvi Eunice E. , Shaji R.V. , Mathews Vikram , George Biju , Vishwabandya Auro , Srivastava Alok 
Published: April 2020

Abstract
Alpha thalassemia is the most common globin gene disorder in the world and the disease is caused by mutations in either the alpha2 or alpha1 genes. Deletions of one (-α) or both (--) of these genes are the most common cause of alpha-thalassemia, while point mutations and small insertions/deletions are present but at a much lower frequency (5%). The number of point mutations described in these genes has been steadily increasing, with greater than 40 identified to date. Identifying these point mutations acquires importance as patients with non-deletional hemoglobin (Hb) H disease are more severely affected. Comprehensive evaluation of α globin genes is also essential while determining the molecular basis of the thalassemia intermedia phenotype. Mutations in the alpha globin gene can be detected either by direct DNA sequencing or by screening methods. Although small (about 1kb, 3 exons and 2 introns), the alpha-globin genes are duplicate and highly G-C rich, which makes them difficult to denature and therefore reducing sequencing efficiency and causing frequent artifacts. We have developed a simple and robust method based on mismatch-specific DNA endonuclease, SurveyorTM Nuclease. Genomic DNA control samples (n=21) harboring 12 different nucleotide changes characterized by direct DNA sequencing in either the alpha2 or alpha1 samples were used to validate this method. These samples were heterozygous or homozygous for each point mutation, or compound heterozygous for a deletion and the point mutation. Controls were amplified by gene specific primers designed by Primer3 software followed by digestion with SurveyorTM Nuclease. This endonuclease is a member of the CEL nuclease family of mismatch-specific nucleases derived from celery. It recognizes all insertions, deletions and base substitutions, and cleaves the mismatch of any heteroduplex DNA. After digestion, cleavage products are analyzed by agarose gel electrophoresis. The size of the digestion products indicates the location of the mutation, which is then confirmed and characterized by sequencing. All the 12 different nucleotide changes [α1 CD 14 TàC, α2 CD 104 TàG, α2 IVS II-55 TàG, α1 IVS II 141 TàC, α1 IVS I 117 GàT, α2 IVS I 1 GàA, Poly A (-AA), α1 CD 119 CàT, α2 IVS I 116, α2 CD 22 CàT, α2 CD 29 TàC, α2 +15 CàG] in the alpha globin genes were detected by this assay indicating a high sensitivity and specificity of this method. Mutation screening by SurveyorTM Nuclease is rapid (~ 5 hours), cost effective ($8/sample) and sensitive (100%). This assay in combination with the multiplex-PCR for screening the common alpha globin gene deletions will be the most comprehensive and economical approach for genetic diagnosis of alpha thalassaemia. Mismatch specific nucleases may have increasing application as a mutation detection strategy for human genetic diseases.
CREB Transgenic Mice Develop Myeloproliferative Disease/Myelodysplastic Syndrome after a Prolonged Latency.
Authors: Shankar Deepa B. , Kinjo Kentaro , Chang Jenny , Sakamoto Kathleen M. 
Published: April 2020

Abstract
The cyclic AMP Response Element Binding Protein, CREB, is a basic leucine zipper transcription factor that induces genes that regulate cell proliferation and survival. CREB is a downstream target of GM-CSF signaling pathways in myeloid cells, resulting in transactivation of critical target genes. We previously demonstrated that both CREB protein and mRNA levels are increased in the bone marrow cells of patients with AML at diagnosis. In contrast, CREB is expressed at low levels in normal bone marrow cells. To examine the expression of CREB in specific bone marrow and leukemia stem cell populations, we performed quantitative real-time PCR. AML blast cells were sorted and were shown to have increased CREB mRNA expression in CD34+CD33+ and CD34-CD33+ population compared to normal bone marrow progenitor cells. To understand the role of CREB in normal hematopoiesis and leukemogenesis, we created transgenic mice in which the myeloid specific hMRP8 promoter controls CREB expression. Within months, these mice developed increased monocytes and neutrophils in the peripheral blood. The increased cells in the peripheral blood and bone marrow were Gr1+Mac1+. We did not observe increases in other cell lineages using FACS analysis and the markers, B220, CD3, and Ter119. Bone marrow cells from CREB transgenic mice displayed increased colony size, greater numbers of colonies, and immortalization in colony replating assays with methylcellulose containing IL-3, IL-6, and Stem Cell Factor, compared to age-matched littermate controls. Bone marrow from CREB transgenic mice grew in the absence of cytokines, demonstrating factor-independent growth. Increased numbers of CFU-M was observed with CREB transgenic mouse bone marrow in colony assays with methylcellulose containing M-CSF. Although the mice did not develop acute leukemia, 7 out of 8 CREB transgenic mice compared to 0 out of 13 control mice developed enlarged spleens and myeloproliferative disease (MPD) after 12 months of age. Histology of the spleens showed destruction of the normal architecture with aberrant myeloid cells, suggestive of a myeloproliferative disease/myelodysplastic syndrome. Both the indolent course and phenotype of our mice were similar to Chronic Myelomonocytic Leukemia (CMML) observed in humans. Interestingly, myeloid cells from patients with CML in chronic phase also express higher levels of CREB. Studies are underway to characterize CREB expression in peripheral blood or bone marrow cells from patients with MPD/MDS, including CML, CMML, and transient myeloproliferative disease of Down Syndrome. We are also analyzing the spleens from CREB transgenic mice that develop MPD/MDS. Our results demonstrate that overexpression of CREB is sufficient to induce myeloid cell transformation to a preleukemic state in vivo. Therefore, the CREB transgenic mouse provides a useful model to test novel therapies to treat MPD/MDS.
Allogeneic Stem Cell Transplantation (SCT) with Reduced-Intensity Conditioning (RIC) in Children with Malignancies: A Retrospective Study from the Societe Francaise de Greffe de Moelle et de Therapie Cellulaire (SFGM-TC).
Authors: Sirvent Anne , Baruchel Andre , Paillard Catherine , Esperou Helene , Bordigoni Pierre , Bay Jacques-Olivier , Michel Gerard , Tabrizi Reza , Vannier Jean-Pierre , Galambrun Claire , Buzyn Agnes , Dalle Jean-Hugues 
Published: April 2020

Abstract
RIC regimens followed by allogeneic SCT have demonstrated to induce complete donor chimerism and long-term survival with decreased transplant-related mortality in adults with poor-risk hematological malignancies. Pediatric experience of RIC regimens is mostly reported in children with non malignant diseases in whom late long-term complications should be reduced. There are only limited data in children with malignancies, ineligible for conventional SCT. We conducted a retrospective survey of 28 children who underwent a RIC regimen for a malignant hematological disease (18 AL, 2 RAEBt, 1 CML, 3 NHL, 2 HD) or a solid tumor (1 Ewing sarcoma, 1 rhabdomyosarcoma) from 1998 to 2004 in 11 participating hospitals. All children had contraindications against myeloablative regimen. Median age was 14 years. Fourteen patients (pts) had an advanced disease (&gt; CR2, refractory or progressive).Ten pts had already undergone at least one transplantation. Most RIC were fludarabine-based (n= 26). Nineteen children received additional anti-thymocyte globulin and 4 were transplanted with a T-depleted graft. Source of hematopoietic stem cell was marrow (n= 17), peripheral blood (n= 9) or cord blood (n= 2). Donor was matched related (n= 12) or unrelated (n= 8), mismatched related (n= 2) or unrelated (n= 6). Median follow-up after SCT is 142 days (range: 22–2345). Engraftment was observed in all 25 valuable pts. Day-90 chimerism analysis was performed in 18 pts and showed complete donor cells in 12 and autologous reconstitution in 3 pts. Grade II–IV aGVHD and cGVHD developed in 8/25 and 3/16 valuable pts, respectively. The day-100 transplant-related mortality rate was 18%. Disease progression or relapse occurred in 13 pts. Nineteen pts died: 11 from initial disease, 2 from infection, 2 from GVHD, 4 from other toxic complication. Four pts underwent a second transplantation for relapse or autologous reconstitution (3 myeloablative, 1RIC SCT). Five pts are alive in CR 145, 336, 782, 993, 2345 days after a first transplantation, 2 pts 272 and 1062 days after a second transplantation. Only 1 child among the 14 with an advanced disease is alive versus 6 among those with a lower-risk disease. In conclusion, allogeneic SCT with RIC is feasible in heavily pre-treated children. In order to reduce the high incidence of relapse, further prospective studies are needed to determine best pts selection criteria (initial diagnosis, treatment prior to transplant, chemosensitivity of the disease) and the best transplantation strategy (timing, type of the RIC, post-transplant additional treatment).
Lymphoid Chimerism (LC) Kinetic after Reduced Intensity Conditioning (RIC) Allogeneic Stem Cell Transplantation (allo-SCT).
Authors: Mohty Mohamad , Avinens Odile , Faucher Catherine , Furst Sabine , Blaise Didier , Eliaou Jean-Francois 
Published: April 2020

Abstract
In contrast to standard myeloablative allo-SCT, the kinetic of lineage-specific chimerism of donor origin is an important issue after RIC-allo-SCT, because of its major impact on transplant-related events and outcome. Predictive factors for full CD3+ T cell LC are not yet well defined in this setting. Here, we investigated the impact of different factors on the establishment of full LC after RIC-allo-SCT. 102 patients from a single center receiving allo-SCT from an HLA-identical sibling were analyzed. Patients and graft characteristics are: age 49 y. (range, 18–67), diagnoses: 41 myeloid malignancies (40%), 43 lymphoid malignancies (42%) and 18 metastatic solid tumors (18%). 83 patients (81%) were considered as high risk. 65 patients (64%) received a fludarabine, busulfan and ATG-based RIC, while 36 pts (36%) received a low dose irradiation-based RIC. All patients received a PBSC graft, with 55 (54%) receiving CSA alone for GVHD prophylaxis and 47 (46%) receiving CSA and MMF. Chimerism of immunomagnetically CD3+ T cells was assessed by VNTR at day 30, 60, 90 and &gt;90 after allo-SCT. Full LC was defined as &gt;95% of CD3+ sorted T cells of donor origin. At day 30, only 30% (95%CI, 21–39%) of patients achieved a full donor LC, and this increased to 50% (95%CI, 40–60%) by day 60. At day 90, a majority of 77% of the patients (95%CI, 69–85%) had a full donor LC. In this series, none of the patients’, graft, RIC regimen or disease characteristics could be predictive of establishment of a full LC at day 30 after allo-SCT. When looking for predictive factors for full donor LC at day 90, univariate analysis including patients and disease characteristics, RIC type and GVHD prophylaxis regimen, PBSC graft composition (CD34+, CD4+, CD8+, CD19+, NK cells), and early transplant-related events such as acute GVHD and time to engraftment, showed that a female donor, diagnosis (lymphoid malignancy or solid tumor vs. myeloid malignancy), CD34+ stem cell dose, and CD4+ T cell dose infused with the graft, were significantly associated with the establishment of full LC by day 90. In the multivariate analysis, a diagnosis other than a myeloid malignancy, was the strongest parameter significantly predictive of establishment of full LC at day 90 after RIC-allo-SCT (P=0.007; OR=3.82; 95%CI, 1.4–10.1). Overall, these results suggest that graft composition may represent an attractive tool towards harnessing chimerism of donor origin after RIC allo-SCT. Most importantly, disease-related features (type, previous courses of chemotherapy, previous autologous transplantation,≡) are the strongest predictors of the kinetic of donor LC that may impact transplant-related events after RIC allo-SCT.
αvβ3 Integrin Activation and Matrix Binding Mediate Cellular Responses to Mechanical Stretch.
Authors: Katsumi Akira , Matsushita Tadashi , Naoe Tomoki , Schwartz Martin A. 
Published: April 2020

Abstract
Mechanical tension is a critical determinant of vascular cell growth, differentiation, apoptosis, migration and development. Integrins have been implicated in sensing force but little is known about how forces are transduced to biochemical signals. We now show that mechanical strain stimulates conformational activation of integrin αvβ3 in NIH3T3 cells by using conformation specific antibody WOW-1. LM609, an antibody against αvβ3 integrin that is insensitive to integrin conformation, showed no change in binding after stretch, excluding changes in surface expression. Stretch also induced an increase of LIBS-6 binding, evidence that conformational activation leads to new binding of integrins to ECM. PI3K and Akt are both rapidly activated by strain, and PI3K inhibition decreases integrin activation. These data strongly suggest that PI3K mediates αvβ3 integrin activation. The blocking anti-fibronectin antibody 16G3 strongly inhibited the stretch-induced activation of JNK under conditions where existing adhesions did not appear to be disrupted, indicating that new integrin-ECM binding is required for JNK activation. These data define a pathway by which early activation of PI3K, through induction of integrin activation and ECM binding, stimulates a cytoplasmic signaling pathway implicated in cellular responses.
In Vitro Protection of Human Endothelial Cells from Adenovirus Vector Toxicity by Activated Protein C.
Authors: Lozier Jay Nelson , D’Agnillo Felice 
Published: April 2020

Abstract
High dose adenovirus vector administration in vivo has been associated with toxicity toward many cell types, including endothelial cells. Some of the prominent pathological features of an adenovirus vector death in a gene therapy trial included capillary leak syndrome and disseminated intravascular coagulation (DIC). We investigated the hypothesis that activated protein C (APC) might have a protective effect on primary human microvascular endothelial cells exposed to a first-generation adenovirus vector. We exposed primary human endothelial cells to a first-generation (E1, E3 deleted) adenovirus vector, AVC3FIX5 at concentrations ranging from 103 to 105 vector particles per cell and showed dose-dependent cell death as early as 6 hours (40% cell death at the highest dose). Phase contrast and immunofluorescence microscopy revealed that some cells died rapidly by primary necrosis while others died by apoptosis over a longer time course. By 40 hours, only 40% of the cells were viable. We then tested the effect of pretreatment of endothelial cells with APC concentrations ranging from 1 nM to 100 nM. Dose-dependent protection was seen in which cell death was reduced to 9 and 2 % at APC concentrations of 50 and 100 nM, respectively. We also tested the effect of timing of the APC treatment and showed that 1 hour pre-treatment or concurrent APC treatment were protective, but APC administered one hour after the adenovirus exposure was substantially less protective. This suggested that APC exerts its protective actions on endothelial cells either by interfering with early steps in the interaction of the vector with the cells, (e.g., vector entry) or by modulating death signaling pathways. It has been proposed that APC protects against cell damage in sepsis by interaction with the endothelial cell protein C receptor (EPCR) and protease activated receptor 1 (PAR1) on the endothelial cell surface to induce MCP-1 and other immunomodulatory genes by proteolytic signaling (Riewald et al., Science296:1880–1882, 2002). Other investigators have shown protective effects of APC for endothelial cells subjected to hypoxia through normalization of levels of p53, Bax, and Bcl-2 gene expression (Cheng et al., Nat Med9:338–342, 2003). The APC concentrations in our experiments that were maximally protective (50–100 nM) were of the same order of magnitude as was shown to be protective in vitro by these investigators. If APC can be shown to have a protective effect against adenovirus-induced endothelial cell toxicity and DIC in vivo, this may be a useful therapeutic strategy to explore as treatement of gene therapy vector toxicity.
Cell Viability vs. APC Concentration Cell Viability vs. APC Concentration
γ-Carboxylation Is Not Required for gas6-Axl Binding but Is Necessary for gas6-Dependent Endothelial Cell Survival.
Authors: Blostein Mark D. , Rajotte Isabelle , Hasanbasic Ines 
Published: April 2020

Abstract
Gas6 is a novel member of the vitamin K-dependent family of γ-carboxylated proteins and is a ligand for the receptor tyrosine kinase Axl. Gas6-Axl interactions have been shown to mediate cell survival in vascular endothelium. Although the receptor-binding portion of gas6 lies in the C-terminus, the significance of the N-terminal γ-carboxylated residues (Gla domain) is not clear. To address this question, we recombinantly produced both carboxylated and decarboxylated gas6 and confirmed that the latter is not γ-carboxylated by both Western blotting and fluorescence spectroscopy. Using DAPI staining and flow cytometry, we show that carboxylated gas6 mediates protection of endothelial cells from serum starvation-induced apoptosis whereas decarboxylated gas6 does not. Furthermore, carboxylated gas6, but not decarboxylated gas6, activates Axl and phosphorylates Akt during gas6-Axl-mediated protection of endothelial cells. Interestingly, although decarboxylated gas6 cannot activate Axl and rescue endothelium from serum starvation-induced apoptosis, it can inhibit the survival effect of carboxylated gas6. To further explore the properties of Gla domain of gas6, the binding each form of gas6 to endothelial cells was determined. Both carboxylated and decarboxylated gas6 bind to endothelial cells with an equal affinity of 50 nM. The binding of both forms of gas6 to endothelium is inhibited by an antibody to the extracellular domain of Axl thereby demonstrating that the Gla domain is not required for the direct binding of the C-terminus of gas6 to Axl. These findings support the conclusion that inhibition of gas6-mediated survival by decarboxylated gas6 is not mediated through inhibition of binding of gas6 to its receptor Axl. Rather, the Gla domain of gas6 has a key role by impacting Axl activation via heretofore unknown mechanism(s). Taken together, the results of this study suggest a novel role for γ-carboxylation in gas6 function.
RBC Histogram Predictive of HE.
Authors: Chakmakjian Carl , Rappaport Ed 
Published: April 2020

Abstract
Hereditary elliptocytosis (HE) encompasses a family of inherited erythrocyte disorders characterized by red blood cells with an oval, elongated shape. Diagnosis is most commonly made upon peripheral smear examination. Although the disorder is usually clinically silent, these patients may have some degree of chronic hemolysis that may necessitate splenectomy on occasion. We report a consistent finding in HE common to most electronic cell counters. The red blood cell histogram reliably has a two to three millimeter elevation above baseline at the extreme left of the graph. This “foot” on the histogram is predictive of possible red blood cell elliptocytes. Please see example.
Figure Figure