bluuebunny/crossref_metadata_2025_split huggingface dataset:
Presence of Factor V Leiden or Prothrombin Mutation Influence Inhibitor Development in Children with Severe Hemophilia A: Data of a Multicenter Cohort Study
Authors: Kurnik Karin , Bidlingmaier Christoph , During Christine , Halimeh Susan , Schobess Rosemarie , Nowak-Gottl Ulrike 
Published: April 2020

Abstract
It has been recently suggested that the clinical phenotype of severe hemophilia A (HA) is influenced by co-inheritance with the factor V (FV) G1691A mutation. In addition, we have recently demonstrated that the first symptomatic bleeding onset in children with severe HA carrying prothrombotic risk factors, namely the FV Leiden or the factor II (FII) G20210A variant, is significantly later in life than in non-carriers. The course of bleeding episodes, however, is also controversially discussed with respect to individual therapeutic regimens performed, and recently a protective effect of early use of prophylaxis on inhibitor development in hemophilic children compared with on-demand therapy has been reported. Thus, the present non-concurrent cohort study was performed to investigate the impact of FV and FII (prothrombin) mutations on clinical meaningful inhibitor development (outcome variable) in white children with severe HA. 122 patients with HA consecutively ascertained from four German catchment areas were followed over a median (min-max) period of 15 years (1–24) from HA onset. 19 of 122 children (15.6%) additionally carried either the FV or the FII variant. During the follow-up period 28 of 122 children (22.9%) developed a clinical meaningful inhibitor: 9 out of 19 in carriers of thrombophilia (47.4%) compared with 19 of 103 (18.44%) in the non-thrombophilia affected cohort [p=0.01]. Multivariate analysis (Cox regression), adjusted for age at first bleeding onset, substitution regimen (start of prophylaxis latest at second joint bleed versus more than two joint bleeds), FVIII dosage (≤ 30 IU/kgbw versus &gt; 30 IU/kgbw), and first-line use of plasma-derived versus recombinant FVIII concentrations revealed that the presence of thrombophilia independently increases the risk of clinical meaningful inhibitor development to a hazard of 2.4 [95%CI: 1.1–5.5]. The cumulative high-responder-free survival with respect to FVIII exposure days was significantly reduced in carriers of the factor V or II mutation (logrank p=0.009). None of the confounders adjusted for, showed a statistically significant influence on clinical important inhibitor development. Data presented here support the hypothesis that clinical meaningful inhibitor development is of multifactorial origin and that FV and FII mutations should be included in the aetiology research in future studies.
Notch-Dependent Control of Blood Lineage Development is Modified by Fucosylation
Authors: Zhou Lan , Yan Quanjian , Yao David , Li Lebing W , Gerson Stanton L. , Lowe John B 
Published: April 2020

Abstract
Notch receptors are conserved cell surface molecules essential for hematopoietic cell fate determination. Activated Notch enhances self-renewal of hematopoietic stem cells and promotes T lymphopoiesis. O-linked fucose moieties attached to the EGF domains of Notch receptors and its modification by Fringe can strongly modulate Notch signaling. Our recently published results indicate that Notch-dependent signaling controls myelopoiesis both in vitro and in vivo, and identify a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in hematopoietic progenitor cells. In the current study, we tested the hypothesis that fucosylation controlled Notch signaling regulates hematopoietic lineage homeostasis. Genetically-modified mouse embryonic stem (ES) cells deficient in Notch1 receptor (NOTCH1−/−) or pofut1 (POFUT1−/−) that controls O-fucose modification of Notch receptor EGF repeats are studied in an in vitro co-culture assay with Notch ligand-expressing OP9 cells. Activation of Notch in wild type ES cells promotes T lymphopoiesis, while exposure of NOTCH1−/− or POFUT1−/− ES cells to Notch ligand failed to generate T lymphocytes but sustained granulocytic production. When probed with recombinant Notch ligand Dll1 or Dll4, hematopoietic cells derived from wild type ES line displayed robust Notch ligand binding, but cells from NOTCH1−/− or POFUT1−/− ES lines showed completely absent or reduced Notch ligand interaction, respectively. In comparison, ES cells deficient in pofut2 (POFUT2−/−) that controls O-fucose modification on thrombospondin repeats (TSR) displayed a wild type lineage development phenotype and normal Notch ligand binding ability. When examined for their in vivo hematopoietic reconstitution, blood cells derived from NOTCH1−/− or POFUT1−/− ES lines, but not POFUT2−/− ES line, showed enhanced granulocytic but suppressed T and B lymphoid lineage development. These results are consistent with our bone marrow transplantation findings that hematopoietic reconstitution by fucosylation-deficient marrow progenitor cells exhibited increased granulocytopoiesis while wild type or fucosylation-intact marrow cells have normal lineage distribution. Our observations indicate that Notch signaling maintains blood lineage homeostasis by promoting lymphoid lineage development and suppressing overt myeloid development. O-fucose modification of EGF repeats on Notch receptor is essential for this Notch-dependent control of blood lineage homeostasis as deficiency of fucose on Notch receptor results in enhanced myeloid development.
IL7 Counteraction with Notch Signaling Followed by EBF1 Expression Marks the B-Cell Commitment in CLP Stage
Authors: Zandi Sasan , Tsapogas Panagiotis , Månsson Robert , Sigvardsson Mikael 
Published: April 2020

Abstract
Development of B-cell lineage from hematopoietic Stem cells in bone marrow is a stepwise process associated with a gradual loss of myeloid and T cell potential. This process involves a complex interaction of transcription factors like EBF1 and E2A, and extrinsic signals including IL7. It has been suggested that IL7 plays an inductive role in B-cell commitment through EBF activation in early B-Cell development. Mice deficient in Il-7 signaling show a dramatic reduction in the number of B-cell progenitors and reduced expression of EBF1 in the common lymphoid progenitor (CLP) compartment and ectopic expression of EBF can partially rescue the B-cell phenotype. However, the rather limited ability of EBF1 to rescue the phenotype as well as the powerful function of Il-7 in the expansion of committed cells creates a complex situation with an inherent difficulty to separate instructive and permissive actions of Il-7. Using transgenic mice carrying a reporter gene under the control of the EBF1 dependent Igll1 promoter, we were able to identify a B220−CD19− committed B-cell progenitor likely to represent the earliest committed population in the mouse bone marrow. This has opened the possibility to investigate lineage commitment in cells not expressing classical surface markers creating increased possibilities to study lineage choices. In order to investigate the inductive role of Il-7 we crossed the Igll1 reporter mice to Il-7 deficient mice. Analysis of reporter gene expression, gene expression by multiplex single cell PCR as well as functional analysis by in vitro differentiation assays, all supported that the committed lineage negative population was dramatically decreased in the absence of Il-7. These data all support the idea that Il-7 is critical not only for expansion of B-lineage progenitors but also for commitment per se. Investigation of the expression of EBF-1 by Real time and single cell PCR suggested that the expression level of EBF was on an average 50% lower in Il-7 deficient progenitors as compared to wild type cells. This expression level was recapitulated in mice heterozygote for a mutation in the EBF1 gene but since the formation of the early committed cells was not as dramatically effected in these mice, we found a need to look for a further function of Il-7 in its instructive role in B-cell development. This prompted us to investigate a potential function of Il-7 in the modulation of Notch signals known to counteract B-cell commitment and EBF function. This revealed that the addition of Il-7 largely inhibits the Notch response in pro-B cells in vitro. Therefore we suggest that Il-7 is directly involved in B-cell commitment and that this function is achieved by modulation of EBF1 both at the transcriptional and functional level.
In Vivo Cellular Imaging Pinpoints the Role of Reactive Oxygen Species in the Early Steps of Adult Hematopoietic Reconstitution
Authors: Lewandowski Daniel , Barroca Vilma , Ducongé Frédéric , Tavitian Bertrand , Roméo Paul-Henri 
Published: April 2020

Abstract
Progress in regenerative medicine will be greatly helped by the characterization of the in vivo processes that lead to tissue reconstitution by adult somatic stem cells. Many major advances have been achieved in the purification and the ex vivo amplification of the adult somatic stem cells, but very few data are available on the pre-requisites that will enhance their in vivo biological activities. The best studied model of tissue reconstitution is the long-term reconstitution of hematopoiesis after transplantation of adult hematopoietic stem cells (HSCs) following lethal irradiation and imaging strategies are amongst the most promising techniques to reveal the in vivo cellular dynamics of this process. Here, we present a new imaging technique to track labeled cells in living animals and we show that this technique can visualize the hematopoietic reconstitution within the femoral cavity after lethal irradiation. We use a fibered in vivo confocal fluorescence imaging system that can navigate inside the femoral cavity from the knee to the femoral head. Imaging is done in living mice, does not alter their viability, does not interfere with hematopoietic reconstitution and can easily be used to follow the temporal dynamics of tissue reconstitution in the mouse at a cellular level. Using this imaging system, we track the temporal and topological cellular dynamics of in vivo engraftment and proliferation of Lin-Sca+c−Kit+CD34− cells (LSKCD34−), enriched in HSCs, transplanted into lethally irradiated mice. Homing and engraftment of LSKCD34− hematopoietic cells first occur in the two femoral epiphyses where these cells are found associated with vascularized structures as early as one hour after transplantation. Reactive oxygen species (ROS) regulate
the initial homing of transplanted HSCs through the modulation of VCAM-1 expression by the endothelial cells and the proliferation of transplanted HSCs.
Preclinical Studies Using Polyethylenimine (PEI) Nanoparticles Complexed with Eukaryotic Translation Initiation Factor 5A (eIF5A) siRNA and eIF5AK50R Plasmid DNA Demonstrates Significant Anti-Myeloma Activity in Vitro and in Vivo
Authors: Lust John A. , Thompson John E. , Taylor Catherine A. , Ye Bin , Liu Zhongda , Sun Zhong , Dondero Richard S. , Moon-Tasson Laurie L. , Kline Michael P. , Donovan Kathleen A. 
Published: April 2020

Abstract
Background: Hypusine is a unique amino acid present in all eukaryotes but found in only one highly conserved protein, eukaryotic translation initiation factor 5A. It is formed during a two-step post-translational modification of eIF5A. eIF5A has been identified as a critical factor for cell proliferation in yeast, as a modulator of p53, and recent studies indicate that the unhypusinated form of eIF5A is a pro-apoptotic protein. Because myeloma remains incurable, we hypothesized that induction of apoptosis by modulation of eIF5A may be a useful therapeutic approach. In this study we utilized PEI nanoparticles to deliver plasmid DNA and siRNA to myeloma tumor cells in vitro and in vivo.
Methods: An siRNA was used to suppress levels of endogenous hypusinated eIF5A in tumors while an RNAi-resistant plasmid expressing a mutant of eIF5A (eIF5AK50R), that cannot be hypusinated, was used to increase levels of unhypusinated eIF5A which is proapoptotic. In vitro, KAS-6/1 myeloma cells were incubated with control siRNA/control plasmid, eIF5A siRNA alone, or eIF5A siRNA/eIF5AK50R for 72 hours in the presence or absence of IL-6. Apoptosis was quantitated using flow cytometry to detect annexin V/propidium iodide stained cells; results were corrected for percent apoptosis of the mock control to enable comparison between experiments. In vivo, SCID mice (3/group) were injected with 10 million KAS-6/1 cells SQ in the right flank and approximately 6 weeks later injected intratumorally twice weekly as follows: Group 1 control mice received 20 μg empty vector and 10 μg control siRNA. Group 2 received 20 μg eIF5AK50R plasmid and 10 μg control siRNA. Group 3 received 20 μg eIF5AK50R plasmid and 10 μg eIF5A siRNA. Tumor volumes were quantitated in mm3 using a2b/2 where a is the smaller dimension.
Results: The in vitro results showed the following % apoptosis: Absence of IL-6 - Control: 6.5%, eIF5A siRNA/eIF5AK50R 30.3%. Presence of IL-6–Control:8.2%; eIF5A siRNA alone 36.5%; eIF5A siRNA/eIF5AK50R 45.2%. Western analysis demonstrated that the siRNA successfully inhibited the production of endogenous eIF5A. In vivo, after 24 days of monitoring tumor volume: group 1 mice showed that the KAS-6/1 cells grew from a mean tumor volume of 7±1 up to 102±16 mm3; tumors in group 2 mice remained relatively stable in size from 21±9 to 26±10 mm3. Group 3 mice showed a mean 92% tumor regression from 99±21 down to 7±1 mm3.
Conclusions: Delivery of eIF5A siRNA and eIF5AK50R plasmid by PEI nanoparticles results in significant anti-tumoral reponses in vitro and in vivo. These studies may provide a novel therapy for myeloma patients with end stage disease that have failed standard treatments.
Role of Hypoxia Induced Factor (HIF-1 alpha) in Pulmonary Hypertension in Sickle Cell Disease
Authors: Kumar Mukta , Zhu Xuhui , Buch Shilpa 
Published: April 2020

Abstract
Background. Pulmonary hypertension is a leading cause of mortality in sickle cell disease (SCD), however, the mechanisms leading to the development of pulmonary hypertension remain poorly understood. The pathogenesis of SCD revolves around chronic hypoxia and hypoxic events that are mediated through the upregulation of hypoxia-inducible factor-1alpha (HIF-1alpha), a transcriptional factor, that is involved in regulation of several redox sensitive genes including vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and ApoE. The objective of this study was to examine the role of HIF-1 and its target genes in SCD-associated pulmonary lung disease.
Methods. In this study, 3 groups of age-matched homo and hemizygous sickle and control C57/Bl6 mice (Jackson Labs) were sacrificed and organs harvested for RNA isolation in Trizol. Using quantitative RT-PCR we assessed the RNA expression of HIF-1 and its target genes, VEGF and PDGF-B from the lungs of homozygous sickle mice and compared these with the C57/BL6 and the hemizygous sickle controls. HPRT was used as the housekeeping gene control.
Results. Homozygous sickle mice demonstrated an upregulation of HIF-1 alpha RNA (almost 2.3 fold) compared with the hemizygous sickle and the C57/Bl6 controls. VEGF RNA expression was elevated ~8.3 to 10.9 fold in sickle mice compared to the two control groups. Interestingly there was a down regulation of PDGF-B expression (0.3 fold) in the sickle mice group compared to the control groups. In contrast, expression of all the HIF-related genes tested was down-regulated in the spleens and brains of sickle mice compared with the respective control tissues.
Discussion. These findings support the possibility that complex molecular pathways, including dysregulation of HIF pathway, may contribute to the development of pulmonary hypertension in sickle cell disease and underscore role of oxidative stress pathways in the onset of disease pathogenesis.
Integration of DNA Copy Number and Gene Expression Alterations Reveal Novel Insights into the Molecular Pathogenesis and Prognosis of Multiple Myeloma
Authors: Zhou Yiming , Barlogie Bart , Herman Damir , Stephens Owen , Tian Erming , Williams David , Cartron Marie-astrid , van Rhee Frits , Pineda-Roman Mauricio , Alsayed Yazan , Anaissie Elias , Sawyer Jeffrey , Shaughnessy John D. 
Published: April 2020

Abstract
Multiple myeloma (MM) is characterized by a profound genomic instability and a highly variable clinical course. To comprehensively analyze the effects of copy number alterations on gene activity and prognosis, we applied novel bioinformatics and computational approaches to integrate high-resolution aCGH data (Agilent 244K array) and gene expression profiling data (Affymetrix U133Plus2.0 array) from CD138-selected plasma cells from 92 newly diagnosed MM treated with high-dose therapy and stem cell transplantation. We identified what we have termed the “atom region” (AR). ARs are found throughout the genome and define recurrent breakpoint boundaries of sub-chromosomal loss or gain. Genes coding for miRNAs were significantly enriched in regions of the genome defined by ARs. Although many AR-related breakpoints lie in intergenic regions, and thus capture whole genes, a substantial number were identified within genes, making these genes strong candidate disease genes. We found that gain of 1q and loss of 1p represent the most significant copy number alterations linked to early disease-related death. Importantly, 1q gains and 1p loss were significantly linked to a recently described 70-gene expression model of high-risk disease predominated by increased expression of genes mapping to 1q and reduced expression of genes mapping to 1p (1). Gain of 1q and loss of 1p were also highly correlated with a gene-expression-based proliferation index widely used across many tumor types. We provide evidence that gain of 8q24 was also highly correlated with the 70-gene high-risk score, but not the proliferation index. 8q24 gains and survival were linked to elevated expression of EIF2C2/AGO2, a master regulator of microRNA expression and maturation and an overexpressed gene previously identified in our 70-gene high-risk model (1). Interestingly, although the expression of MYC, which also maps to 8q24, was related to copy number changes, its expression was not related to survival. Given the importance of AGO2 in B-cell development (2), and miRNAs in cancer, detailed functional studies of the potential role of AGO2 in MM are underway.
The CD34+CD38LowCD19+ Candidate Leukemic Stem Cell Phenotype Revisited: Useful for Flow MRD Monitoring?
Authors: Wilson Kerrie , Case Marian , Minto Lynne , Bailey Simon , Vormoor Josef , Irving Julie 
Published: April 2020

Abstract
The assessment of minimal residual disease during therapy is a powerful prognostic marker in childhood acute lymphoblastic leukemia but current techniques, whether molecular analysis of antigen receptor gene rearrangements or flow cytometric analysis of aberrant immunophenotypes, track the blast population(s) that predominate(s) at diagnosis. Recent evidence from diagnostic TEL/AML1 positive cases (Hong et al, 2008) suggests that the candidate leukemic stem cell population may be characterised by the immunophenotype CD34+CD38LowCD19+ and thus the identification and quantitation of this cell population at diagnosis and during therapy may have greater clinical significance.
Cytogenetic subgroup Number with candidate population Number without candidate population TEL/AML1 8 1 High Hyperdiploid 6 5 Other 15 16
Therefore, we have sought this proposed leukemic stem cell population in 51 consecutive diagnostic precursor-B acute lymphoblastic leukaemia samples using multi-parametric flow cytometry. Patient blasts were stained with the antibodies CD34PerCP, CD38FITC and CD19APC and 50,000 events acquired on a FACSCalibur. The postulated cancer stem cell population, CD34+CD38LowCD19+, was found in 57% of patients (29 of 51). Correlation with major cytogenetic subgroups showed that 89% (8 of 9) of TEL/AML1 positive cases had evidence of the population, compared to 55% (6 of 11) of high hyperdiploid and 48% (15 of 31) of the remainder (see Table). There was no evidence of the population in ‘normal marrow’ samples which were from children in the latter stages of therapy for ALL i.e. end of treatment bone marrow samples (n=4). We also examined bone marrow samples taken 28 days after the start of treatment in a cohort of patients scoring positive for the candidate population at diagnosis (n=11) and found evidence for persistence of the cells in 4 of them.
CD38 under-expression, in relation to normal B cell precursors, is a common feature of ALL blasts and classification of CD38 expression levels revealed that 92% (24/26) of these under-expressers unsurprisingly housed the population because their blasts were also CD34+ and CD19+. Most importantly, since 43% of the diagnostic samples analysed show no evidence of this population (or it exists below the limits of detection of the assay i.e.&lt;0.1% ), it seems unlikely that this represents a definitive cancer stem cell population in childhood ALL per se and would, therefore, not be appropriate as a generic marker of minimal residual leukemic stem cells.
Integrin Alpha M Chain Expression at Diagnosis Is Inversely Correlated with Cytoreduction Rate and Is Consistently Up-Regulated during Therapy in Acute Lymphoblastic Leukemia (ALL)
Authors: Rhein Peter , Ratei Richard , Mitlohner Rita , Basso Giuseppe , Gaipa Giuseppe , Dworzak Michael N , Stanulla Martin , Schrappe Martin , Ludwig Wolf-Dieter , Karawajew Leonid 
Published: April 2020

Abstract
In childhood ALL, detection and quantification of minimal residual disease (MRD) is of crucial prognostic significance. Gene expression pattern of MRD cells undergoes treatment-related changes which provide insights into the mechanisms of therapy response and resistance. Our recent genome-wide analysis of blasts isolated from peripheral blood (PB) of pediatric patients with B-cell precursor (BCP-) ALL at early stages of induction therapy (day 8) revealed an up-regulation of the gene encoding for integrin alpha M chain (ITGAM, CD11b), and pointed to the involvement of adhesion receptors in the therapy response. In the present study we investigate expression of CD11b at protein level and its clinical relevance in BCP-ALL. At diagnosis, ALL cells heterogeneously express CD11b both in PB (mean 1839+/−515 MESF, 160 pts) and in bone marrow (BM) (655+/−114 MESF, 79 pts). At day 8, increased expression of CD11b has been observed in the majority of PB samples (77 of 100 pts, up-regulation range 1.5- to 74-fold, mean 8.2-fold). In BM, the CD11b expression has been consistently up-regulated at the clinically significant MRD timepoints of induction therapy (days 15, 33 and 78; ALL-BFM 2000 protocol). Moreover, since normal CD19+/CD10+ precursor cells are CD11b negative, the detection of CD11b improved discrimination of normal and leukemic blasts in the MRD positive cases with regenerating BM at day 78. Of particular interest, evaluation of the CD11b expression at diagnosis in context of the early therapy response disclosed its potential clinical relevance. In the bivariate data analysis, expression of CD11b significantly correlated with the blast reduction rate (BRR) and blast count (BC) in PB at day 8 (p=0.007 and 0.001, 103 pts). Similarly, the CD11b expression in BM significantly correlated with the BRR and BC in BM at day 15 (p=0.024 and 0.012, 58 pts). In the multivariate regression analysis, excluding BCR-ABL and MLL-AF4 positive ALL cases and using the additional clinical parameters at diagnosis (age, white blood cell count in PB, DNA index), CD11b expression at diagnosis was the most significant variable contributing to BRR at day 8 (p&lt;0.001, standardized beta coefficient 0.61; 98 pts) and to BRR at day 15 (p&lt;0.001, standardized beta coefficient 0.68; 47 pts). Taken together, our data indicate that the expression of CD11b contributes to the early therapy response and has a promising potential as an MRDspecific marker in BCP-ALL.