bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of NPM1-Mutated/FLT3 ITD-Negative Acute Myeloid Leukemia with Normal Karyotype by Gene Expression Profiling.
Authors: Bullinger Lars , Dohner Konstanze , Kranz Raphael , Rucker Frank G. , Frohling Stefan , Schlenk Richard F. , Pollack Jonathan R. , Dohner Hartmut
Published: October 2019

Acute myeloid leukemia (AML) with normal karyotype comprises a large number of molecularly distinct variants. For example the presence of internal tandem duplications (ITDs) of the FLT3 (fms-related tyrosine kinase 3) gene is associated with poor outcome, whereas mutations of the NPM1 (nucleophosmin) gene are prognostically favorable. However, this effect is mainly attributed to the NPM1-mutated/FLT3 ITD-negative AML cases. While NPM1-mutated cases are characterized by a distinct gene expression pattern, it remains unclear whether NPM1-mutated/FLT3 ITD-negative cases also display a characteristic signature, which might provide additional insights into the molecular basis for the good clinical outcome. Thus, we sought to identify a molecular profile for AML cases with NPM1-mutated/FLT3 ITD-negative normal karyotype disease. Towards this goal, we profiled gene expression of 138 samples of adult AML patients with normal karyotype using DNA microarray technology. All samples analyzed were derived from AML patients entered within the randomized multicenter treatment trial HD-98A of the German-Austrian AML Study Group (AMLSG). Based on supervised data analyses we were able to identify a 116-genes comprising expression pattern correlated with NPM1-mutated and FLT3 ITD-negative AML cases. In accordance with previous findings in NPM1-mutated cases (Alcalay et al. 2005, Verhaak et al. 2005), the NPM1-mutated/FLT3 ITD-negative pattern was also in part characterized by a prominent HOX gene cluster, which clearly separated the NPM1-wildtype from the NPM1-mutated cases. Similarly, the expression levels of BAALC and MN1 were correlated with the NPM1 mutational status, with NPM1-unmutated cases displaying higher BAALC and MN1 expression in our data set. However, as expected the newly defined signature also defined a NPM1-mutated group that did not contain many FLT3 ITD-positive samples. This group was characterized by several interesting genes including for example TLE1, which encodes a Groucho/TLE family protein. Groucho/TLE family proteins are transcriptional co-repressors, which mediate repression essential in embryonic development and are involved in regulation of Wnt signaling in adult tissue. Moreover, we identified several other genes of potential pathogenic relevance which also have been previously shown to be predictive in normal karyotype AML. Our findings support a distinct molecular mechanism associated with the favorable outcome of NPM1-mutated/FLT3 ITD-negative AML cases. Furthermore, the reported signature might contribute to improved risk stratification and clinical management of AML patients with normal karyotype disease.
High Oxygen Environment during Pregnancy Rescues Sickle Cell Animia Mouse from Prenatal Death.
Authors: Ye Lin , Chang Judy C. , Chan Kalee , Lu Ronghua , Kan Yuet Wai
Published: October 2019

Several mouse models for sickle cell disease have been developed for the studies of the pathophysiology of sickle cell disease and the investigations of drug and gene therapies. In previous years, we have also succeeded in producing a sickle cell anemia mouse model. In this model the endogenous mouse α and β globin genes were knocked out and replaced by the human α and βs globin transgenes. The βs globin gene is contained in a 240kb YAC that preserves the entire native genomic context of the β-globin locus. These mice have anemia, reticulocytosis and irreversible sickle cells in the peripheral blood, as well as other pathological features of sickle cell disease. However, their γ globin switches to βs globin expression from around 12 days of gestation. The low level of fetal hemoglobin expression in utero led to intrauterine sickling and fetal death so that very few live-born sickle cell anemia mice could be obtained. To rescue these mice from intrauterine death we investigated the effect of placing the pregnant mothers into a high O2 environment. From the tenth day of gestation onwards, we placed the mothers into a chamber containing 50% O2 and kept the newborn pups in it for another 10 days after birth. The frequency of sickle cell anemia mice we obtained was increased from 0.2% to 32%. Moreover, 55% to 88% of the newborn sickle cell anemia mice survived in the oxygen treated group, whereas none of the sickle cell anemia mice survived in regular air breeding conditions. The survived sickle cell anemia mice develop congestion, atrophy, and infarcts in multiple organs by histological analysis. These pathological finding are very similar to those find in patients with sickle cell disease. We conclude that a high oxygen environment can be used to obtain more sickle cell anemia mice in those models that have a high perinatal mortality. The higher yield of these mice will facilitate physiological and therapeutic studies of sickle cell anemia.
Contribution of Coagulation Pathways and Fibrinolysis in Generation of Kinins.
Authors: Eve Moreau Marie , Désormeaux Anik , Fortin Jean-Philippe , Rivard Georges E. , Lepage Yves , Brown Nancy J. , Gauthier Francis , Marceau François , Adam Albert
Published: October 2019

BACKGROUND. Classically, there are two main pathways by which kinins are generated: the plasma and tissue kallikrein-kinin systems. Each of this complex multi-protein system includes tissue or plasma kallikrein, which generate kinins from high or low molecular weight kininogen. In addition to plasma and tissue kallikreins, other serine proteases may have a kinin-forming capacity.

OBJECTIVES. The main purpose of this study was to characterize the kininogenase activity of plasma kallikrein, thrombin (factor IIa), factor Xa, and plasmin in a purified system. We also aimed at characterizing the kinetic profile of generation of kinins in a whole blood system where coagulation and fibrinolysis were monitored by thromboelastography.

METHODS. Bradykinin (BK) released from both low and high molecular weight kininogens and quantified with a specific competitive enzyme immunoassay was used to calculate the kinetic parameters for the different kininogenase activities. Physicochemical and pharmacological characterization of immunoreactive kinins confirm their nature. The kinetic profile of kinin generation was studied during the process of coagulation which was initiated with kaolin or with tissue factor and monitored by thromboelastography.

RESULTS. BK was released not only by plasma kallikrein; both plasmin and factor Xa hydrolyzed high molecular kininogen to produce BK. In the case of thrombin, the release of both BK and kallidin was detected from low and high molecular weight kininogen. When applied to whole blood, this analytical approach indicated that the kinin-forming capacity of the coagulation intrinsic pathway was higher than that of the extrinsic pathway. The triggering of fibrinolysis by tissue plasminogen activator dramatically potentiated the release of des-Arg9-BK.

CONCLUSIONS. Although plasma kallikrein is considered as being the main kinin-forming enzyme, other serine proteases of the coagulation and the fibrinolysis pathways are able to generate kinins and could play a role in different pathologies where kinins are potentially involved.
Amelioration of Inflammation, Angiogenesis and CTGF Expression in an Arthritis Model by a TSP1-Derived Peptide Treatment.
Authors: Rico Mario C. , Castaneda Julian L. , Manns Joanne M. , Uknis Audrey B. , Safadi Fayez F. , Popoff Steven N. , De La Cadena Raul A.
Published: October 2019

Objective. To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis.

Methods. Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-APS). Animals were divided into four groups:negative control and groups receivingno treatment,treatment with a TSP1-derived peptide andtreatment with a scrambled peptide.

Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR.

Results. Histological data indicated that TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunohistochemical and RT-PCR analyses showed an increase of the CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to TSP1-derived peptide treated animals. By immunohistochemistry, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals.

Conclusion. Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues.

Vasculature measurements at the perisynovial tissue of the TSP1-derived peptide treated (DT) and scramble peptide treated (DS) animals after day 16 PG-APS-induced erosive arthritis. Vascular Measurements Disease TSP1-derived peptide treated (DT) Disease scrambled peptide treated (DS) P Value Vascular area mean±SEM (μm2) 77.41 ± 7.96 μm2 336 ± 49.21 μm2 p=0.0008 Vessel wall thickness (μm) 3.53 ± 0.21μm 8.92 ± 0.48;μm p<0.0001 Vascular index (VI) ± SEM (VI=total number of vessels/mm2) 5.6x10−3 ± 0.37x10−3 vessels/mm2 17.9x10−3 ± 1.33x10−3 vessels/mm2 p=0.006
Human Embryonic Stem Cell-Derived “NET-Like” Structure Serves as a Hematopoietic Progenitor Niche and Favors Generation of Mature Megakaryocytes and Functional Platelets.
Authors: Takayama Naoya , Nishikii Hidekazu , Hiroyama Takashi , Nakamura Yukio , Tsukui Hiroko , Eto Koji , Nakauchi Hiromitsu
Published: October 2019

Human embryonic stem cells (hESCs) are proposed as an alternative source in regenerative medicine while being a promising tool to trace earliest ontogeny in vivo. Anucleate platelets can be irradiated to eliminate residual ESCs that potentially cause immunological rejection and teratoma formation in transplantation. Herein, we describe for the first time the generation of functional platelets from hESCs (Kyoto hES1, -2, and -3) by a novel method that is distinct from previously established OP9 co-culture. Exogenous combination of TPO, VEGF and IGF-II allowed hECSs to facilitate the differentiating kinetics of hematopoietic marker emergence when cultured on feeder cells, 10T1/2, whereas independent single administration had little effect on hematopoietic differentiation (P<0.01). Consequently, unique “NET-like” structures expressing VE-cadherin low+, PECAM-1 high+, CD34 high+, CD45 + and VEGF-R2 + were observed around 14–15 days of culture. These structures contained hematopoietic progenitors capable of forming hematopoietic colonies in semisolid culture, or of differentiating into mature megakaryocytes in the presence of TPO, IL-6, and IL-11 (around 45 % of total cells displayed GPIbα +GPIX + by day 23). While totally 24–25 days culture was required for production of hESC-derived integrin αIIb +GPIX + platelets in this way, the platelets exhibited specific fibrinogen-bound to integrin αIIbβ3 in the presence of G-protein coupled receptor agonists in a dose-dependent manner. The efficiency of platelet production appeared to be well correlated with the frequency of “NET-like” structure. Thus, this structure may be a useful hallmark in our newly obtained culture method that may serve as an invaluable tool for not only generation of hESC-derived functional platelets but also studying niche machinery in hematopoiesis.
Anti-Factor IX Antibodies Displaying FVIIIa-Like Activity.
Authors: Kerschbaumer Randolf J. , Dockal Michael , Jan Rosing , Scheiflinger Fritz
Published: October 2019

One of the key events during hemostasis is the conversion of the zymogen factor X (FX) into its enzymatically active form FXa by the protease factor IXa (FIXa). FIXa assembles on a phospholipid surface together with its cofactor factor VIIIa (FVIIIa) which transforms FIXa from a very poor enzyme into a highly active protease. In order to mimic the stimulating effect of coagulation FVIIIa on FIXa, we identified a series of antibodies specific for FIX that exhibit FVIII-like activity. Upon binding to human FIXa these antibodies increased the turnover number (kcat) of FIXa-catalysed FX activation approximately 10-fold. Procoagulant activity of these anti-FIXa antibodies could be detected in model systems containing purified proteins as well as in FVIII-depleted plasma and FVIII-inhibitor plasma. In plasma-assays contact activators and tissue factor were applied as a trigger, and the antibodies were effective in both cases. Our findings demonstrate that FVIII can be at least partially replaced by an antibody which might open up a new strategy for improving the treatment of hemophilia.
Antigen Recognition Induces Phosphatidylserine Exposure on the Cell Surface of Human CD8+ T Cells.
Authors: Fischer Karin , Voelkl Simon , Berger Jana , Andreesen Reinhard , Pomorski Thomas , Mackensen Andreas
Published: October 2019

In eukaryotic cells the phospholipid phosphatidylserine (PS) is restricted to the inner plasma membrane leaflet. This lipid asymmetry which is maintained by the concerted action of phospholipid transport proteins is mainly lost during apoptosis. Here, we demonstrate that primary human CD8+ cytotoxic T lymphocytes (CTL) expose PS upon T-cell receptor (TCR)-mediated antigen recognition: antigen-specific CTL, recognizing the HLA-A2 binding Melan-A peptide, demonstrated a marked exposure of PS as determined by annV-FITC staining, a highly specific PS-binding protein, after 4 h of stimulation with Melan-A-loaded antigen presenting cells (APC). PS exposure was Ag-specific (>80% annV-positive T cells) but control peptide (gp100)-pulsed and unpulsed APC also induced a slight background PS exposure on the CTL. To follow more precisely the fate of annV-positive CTL after antigen-specific stimulation, we labeled Melan-A-specific CTL with the membrane dye PKH-26 and isolated all annV+ PKH-26+ CTL 4 h after stimulation with peptide-pulsed APC cells by cell sorting. Results demonstrate that the annV-positive T-cell population is heterogenous: while the annV-high T-cell population retained the annV-high phenotype and consisted of apoptotic T cells, the annV-intermediate(int) T-cell population revealed a constant decrease of annV binding and became annV-negative at 54 to 72 h after stimulation. Using three independent assays for apoptosis we found that annV-int T cells are propidium iodide negative, do not exhibit DNA strand brakes and contain no active caspase 3. In contrast, annV-int CTL revealed a strongly activated phenotype indicated by an upregulation of CD69 expression and downregulation of the TCRαβ chain expression. Fluorescence microscopic analysis demonstrated that PS is distributed inhomogenously over the plasma membrane and concentrated in membrane lipid raft domains at the immunological synapse. By studying the activity of PS transport proteins using a fluorescence-labeled PS analogue, we determined a constitutive outward transfer of PS molecules in Melan-A-specific CTL. The constitutive PS outward transport was not further accelerated after antigen re-stimulation. In sharp contrast, the inward transporting flippase was strongly inhibited in stimulated CTL resulting in an accumulation of PS molecules on the cell surface. Shielding of exposed PS by annexinV protein during antigen recognition diminished cytokine secretion, activation and cell-cell clustering of antigen-specific CTL. In summary, our data demonstrate for the first time that externalized PS on antigen-stimulated CTL is linked to T-cell activation and probably involved in cell-cell contact formation at the immunological synapse.
Placenta Derived Stem Cells (PDAC) Suppress the Allo-MLR and the EBV Regression Assay.
Authors: Paludan Casper , Harbacheuski Ryhor , Ann Murray Rose , Mendillo Megan , Soler Jorge , Wang Jia-Lun , Ye Qian , Edinger James , Hofgartner Wolfgang , Kaplan Gilla , Hariri Robert
Published: October 2019

The placenta is a readily available and ethically non-controversial source of large amounts of therapeutic stem cells. Placenta Derived Adherent Cells (PDACs) are isolated from the placenta by one of several methods including physical disruption of tissue from several different anatomical sites within the placenta that include the amniotic membrane, chorion, placental cotyledons, or any combination thereof. Flow cytometry analysis showed that PDACs isolated from certain sites exhibit phenotypes, including for example CD200+ CD105+ CD73+ CD34− CD45− at percentages ≥70%. Furthermore, PDACs demonstrate in vitro pluripotency. Here, we report on the immune suppressive capabilities of PDACs.

PDACs were introduced in a six-day mixed leukocyte reaction, using purified CD8+ T cells as effectors and allogeneic dendritic cells (DC)s. The ratios were T/DC/PDAC=10/1/2. Alternatively, an EBV regression assay, measuring T cell proliferation against EBV expressing antigen presenting cells (APCs) generated from healthy donors was performed. Here, the T/APC/PDAC ratios were 10/1/2. In this system, PDACs inhibited the proliferation, as measured by CFSE dilution, of CD8+ T cells >50% (range: 0–95%, SEM 20%). The suppression was the same whether only CD4+ or CD8+ cells were used, or whether equal amounts of CD4+ and CD8+ T cells were added. When Natural Killer (NK) cells were used, with 50 U/ml IL-2, as effectors instead of T cells, the suppressive effect was 45% (range: 40–65%, SEM 5%). Different PDAC preparations suppressed the reactions equally well. Separation of certain PDACs and MLR by a transwell membrane reduced the suppression by 21%, suggesting that part of the suppression was contact dependent. Suppression by other types of PDACs, on the other hand, was 75% contact dependent. When CD4+CD25+ T regulatory cells were removed by magnetic bead separation, the suppression in the regression assay was unchanged.

These results suggest that PDACs may have potential therapeutic use as universal immune suppressive donor cells. The suppression is independent of T regulatory cells.
Expression of Alternatively-Spliced Human Tissue Factor, but Not Full-Length Tissue Factor, Enhances Tumor Growth and Tumor-Associated Vasculature.
Authors: Soff Gerald A. , Hobbs Jennifer , Hyman Emily , Cundiff Deborah L.
Published: October 2019

It is well-established that cancer is associated with activation of the blood coagulation system, with associated thrombosis as a major cause of morbidity and mortality. Increased expression of Tissue Factor (TF) by cancer cells correlates with a more aggressive grade and clinical course. It is widely presumed that activation of coagulation facilitates cancer growth, and in mouse models, anticoagulation can reduce development of lung metastases. Yet primary tumors are not reduced in a fibrinogen knock-out mouse host, and most importantly, anticoagulation has not been shown to reduce tumor growth in cancer patients. We therefore studied the effect of expression of full-length Tissue Factor (FLTF) and alternatively-spliced human Tissue Factor (asHTF) in a mouse model of human pancreatic cancer. Due to the loss of exon 5, asHTF has a truncated extracellular domain with incomplete procoagulant activity. And due to a frame shift, exon 6 does not code for the transmembrane domain and cytoplasmic tail of FLTF, but codes for a novel peptide sequence. asHTF is soluble and of unknown function. We show that 5 of 6 human pancreatic cancer cell lines tested expressed both FLTF as well as asHTF. The MiaPaca-2 line did not express detectable mRNA or protein of either TF isoform. We generated mammalian expression vectors for both FLTF and asHTF, and established Miapaca-2 clones, stably expressing FLTF, asHTF, or control clones with an empty vector. As anticipated, conditioned media from all FLTF clones shortened the whole blood clotting times by approximately 75%. Conditioned media from control cells and asHTF expressing cells had no effect on clotting times. To evaluate the effect of the TF isoforms on primary tumor growth, 5 X 106 cells from three independent clones of stably transfected clones of FLTF, asHTF, or control clones were injected into the flanks of nude mice (4 mice per clone). At 31 days, the mice were sacrificed and tumor mass measured. Tumors grew in 10 of 12 control mice, but were small (mean tumors 90 mg, SEM 21 mg). Interestingly, FLTF was associated with reduced primary tumor growth; only 4 of 12 developed measurable tumors (mean tumors 10 mg, SEM 4 mg, p = 0.002). In contrast, asHTF expression was associated with enhanced tumor growth; 12 of 12 animals developed tumors (mean tumors 390 mg, SEM 102 mg, p=0.018). In animals with asHTF expressing tumors, circulating asHTF protein was observed in the plasma. The asHTF tumors had increased vascular density compared with controls, suggesting a role of asHTF promoting angiogenesis. In contrast to the prevailing paradigm, our data suggest that FLTF, with procoagulant activity, not only fails to promote primary tumor growth, but may actually inhibit tumor growth. In contrast, asHTF, may be the more important TF isoform in the enhancement of tumor growth.
Identifying Novel Modifier Genes Regulating von Willebrand Factor Levels in Mice.
Authors: Shavit Jordan A. , Lemmerhirt Heidi L. , Ginsburg David
Published: October 2019

Plasma von Willebrand factor (VWF) levels are highly variable in the normal human population, and twin studies suggest that two-thirds of this variability is heritable. Only one-third of this genetic component is explained by ABO blood group, with the factors responsible for the majority of the effect still unknown. These putative VWF modifier genes contribute to the wide variability in bleeding severity among patients with von Willebrand disease (VWD), as well as the frequent difficulty in establishing this diagnosis. We previously mapped four loci responsible for variable plasma VWF levels among several inbred mouse strains, which we termed Mvwf1-4 (modifier of Vwf). We have identified the mutant alleles for Mvwf1, a variant in a glycosyltransferase with homology to a human blood group antigen, and Mvwf2, a mutation in the Vwf gene itself. For the current study we chose two inbred strains with 3.5-fold divergent VWF plasma levels, C57BL/6J and WSB/EiJ, neither of which carries the Mvwf1 or Mvwf2 alleles. F1 hybrid mice were backcrossed onto C57BL/6J to generate 200 N2 progeny, followed by determination of plasma VWF levels by ELISA. A dense genome scan of 149 markers, an average of one marker every 10 centimorgans, was performed on genomic DNA from all 200 N2 mice by the Mammalian Genotyping Service at the Marshfield Clinic Research Foundation. Analysis of the data with the R/qtl statistical package identified two major candidate loci with significant evidence for linkage to VWF levels. The first locus (Mvwf5) mapped to a region containing the Vwf gene itself on chromosome 6, with a logarithm of the odds (LOD) score of 12.1. Preliminary studies of Vwf mRNA from F1 mice by primer extension SNP (single nucleotide polymorphism) analysis suggest that Mvwf5 exerts its effect at the level of Vwf transcription or mRNA stability. A second potential modifier (Mvwf6) localized to chromosome 10 with a LOD score of 4.6, and displayed additive effects with Mvwf5. Two additional suggestive loci mapped to murine chromosomes 4 and 5, with LOD scores of 2.4 and 3.4, respectively. The former locus maps to the same region of chromosome 4 as Mvwf3, a candidate modifier previously identified in an F2 intercross between the strains A/J and CASA/RkJ. In summary, we have identified a 2nd natural Vwf gene variant among inbred mice (Mvwf5), a novel VWF regulatory locus on murine chromosome 10 (Mvwf6), and 2 other possible VWF modifiers on chromosomes 4 and 5. Surprisingly, two of six Mvwf loci characterized to date correspond to hypomorphic mutations at the Vwf gene itself and appear to interact with modifier loci on other chromosomes. Similar interactions are likely to explain the extensive variability in plasma VWF levels observed in the general human population as well as among patients with VWD.
Formation of Tissue Factor-Factor VIIa Complex and Generation of Factor Xa Induce Activation of the mTOR Pathway Which Regulates Migration of Human Breast Cancer Cells.
Authors: Jiang Xiaofeng , Zhu Shimei , Panetti Tracee S. , Bromberg Michael E.
Published: October 2019

Tissue factor (TF) is a 47 kDa transmembrane glycoprotein that initiates blood coagulation when complexed with factor VIIa (FVIIa). TF is constitutively expressed in a variety of tumor cells and has been shown to have roles in cellular signaling and tumor progression. We showed previously that formation of TF-FVIIa-Factor Xa (FXa) complex induces cellular signaling in the Adr-MCF-7 cell line, a multidrug-resistant subline of the human breast cancer cell line, MCF-7, that leads to enhanced cell migration and inhibition of apoptosis. The Adr-MCF-7 cell line has high endogenous expression of TF and expression of protease-activated receptor 1 (PAR1) and protease-activated receptor 2 (PAR2). Treatment of the Adr-MCF-7 cells with the combination of FVIIa (10 nM) and FX (150 nM) induces phosphorylation of p44/42 mitogen-activated protein kinase and protein kinase B. In the present study, we investigated the role of TF-FVIIa-mediated signaling in activation of the mammalian target of rapamycin (mTOR) pathway. Treatment of the Adr-MCF-7 cells with the combination of FVIIa (10 nM) and FX (150 nM) induced phosphorylation of mTOR and p70 S6 kinase (p70S6K) by 2 and 2.5 fold, respectively, compared with untreated cells. Phosphorylation of these proteins could be inhibited by pretreatment of the cells with either anti-TF antibody (TF85G9) or TAP, a specific FXa inhibitor. No increase in the basal level of phosphorylation of these proteins occurred with treatment of the cells with FVIIa (10 nM) alone. Moreover, phosphorylation of mTOR and p70S6K was probably mediated by PAR1 and/or PAR2 activation. Using a modified Boyden chamber chemotaxis assay, activation of the mTOR pathway with the combination of FVIIa (10 nM) and FX (150 nM) promoted migration of the Adr-MCF-7 cells. Inhibition of this pathway with the specific mTOR inhibitor, rapamycin, markedly decreased cell migration. Results from these studies suggest that TF-FVIIa-mediated signaling modulates mTOR pathway activation, which in turn regulates breast cancer cell migration.
Long-Term Effects of Deferasirox (Exjade®, ICL670) on Serum Ferritin: Outcome of Dose Adjustments in Achieving Maintenance or Reduction in Body Iron Stores.
Authors: Porter J.B. , Cohen A. , Agaoglu L. , Ganser A. , Cappellini M.D. , Maseruka H. , Glimm E. , Alberti D.
Published: October 2019

During 1-year core phases of two deferasirox trials, doses were initially assigned by baseline liver iron concentration (LIC). In these regularly transfused patients (mean iron intake 0.37 mg/kg/day), a clear dose response was observed: deferasirox 20/30 mg/kg/day effectively maintained/reduced body iron, 10 mg/kg/day maintained body iron in patients with low transfusion rates, whereas 5 mg/kg/day removed less iron than added by ongoing transfusions. This post-hoc analysis evaluates the impact on serum ferritin (SF) of dose increases during the extension phases, in patients who initially received 5/10 mg/kg/day.

In patients who initially received deferasirox 5/10 mg/kg/day with increasing iron burden, doses were generally increased to 20–30 mg/kg/day during the first 6 months of the extension phase. Dose modifications for patients on 20/30 mg/kg/day were based on safety and efficacy parameters. SF was measured monthly.

480 patients (β-thalassemia, n=381; myelodysplastic syndromes, n=47; other anemias, n=52) initially received deferasirox 5/10 (n=119), 20 (n=136) or 30 (n=225) mg/kg/day in the core phase. Most continued on treatment in the extension phase, receiving deferasirox for a median of 2.6 years.

Median baseline SF values in the three dose cohorts were 1932, 2527 and 4158 ng/mL, respectively. During the core and extension phases, SF levels decreased in patients who received deferasirox 30 mg/kg/day and were maintained in patients who received 20 mg/kg/day. In contrast, SF steadily increased in patients who initially received 5/10 mg/kg/day. Subsequent dose escalation during the extension phase generally resulted in decreased SF levels (see shaded area of Table), returning close to baseline by data cut-off. A similar pattern was observed irrespective of age or underlying anemia.

Table. Median change from baseline in S:F (ng/ml) during deferasirox treatment of up to 2.6 years (n=480)

A further 258 patients (deferoxamine arm in core phase) crossed over to deferasirox during the extension phase, receiving deferasirox for a median of 1.6 years. Comparable responses were observed in these patients versus those in the deferasirox cohort at corresponding doses.

Conclusions: SF steadily decreased following dose increases in regularly transfused patients who initially received deferasirox 5/10 mg/kg/day. This analysis confirms that 30 mg/kg/day effectively reduces body iron over the long term, whereas 20 mg/kg/day is effective in maintaining body iron levels in regularly transfused patients. Deferasirox dose can therefore be effectively tailored depending on the goal of therapy (maintenance or reduction in body iron levels).
Influence of Iron Chelation Therapy on R1 and R2 Calibration Curves in Gerbil Liver and Heart.
Authors: Wood John C. , Aguilar Michelle I. , Otto-Duessel Maya , Nick Hanspeter , Nelson Marvin D. , Moats Rex
Published: October 2019

Introduction: MRI is gaining increasing importance for the noninvasive quantification of organ iron burden. To date, MRI validation studies have not systematically examined the effects of different iron chelators. Since transverse relaxation rates depend on iron distribution as well as iron concentration, physiologic and pharmacologic processes that alter iron distribution could change MRI calibration curves. This paper compares the effect of three iron chelators, deferoxamine, deferiprone, and deferasirox on R1 and R2 calibration curves according to two loading and chelation strategies.

Methods: 33 Mongolian gerbils underwent iron dextran 500 mg/kg/wk for 4 weeks followed by 4 weeks of chelation therapy using deferoxamine, deferiprone and defersirox. An additional 32 animals received less aggressive iron loading (200 mg/kg/week) for 10 weeks, followed by 12 weeks of chelation therapy. R1 and R2 measurements were obtained immediately post euthanasia using an NMR relaxometer. Calibration curves from 28 unchelated animals loaded with 200 mg/kg/week from 2 to 48 weeks were used as the reference standard for both chelated groups, using Bland-Altman analysis.

Results: In the liver, R2-iron calibration became more variable over time regardless of whether chelation was performed or not (mean COV 28% versus 12%); no significant changes were observed in the heart R2-iron relationship. Variability in R1 measurements did not change for either heart or liver. Two systematic chelator-specific changes in liver iron calibration curves were noted:deferiprone treated animals exhibited signficantly higher R1 values (Figure 1) anddeferasirox treated animals demonstrated lower R2 values for given iron concentration (Figure 2).

Both changes were associated with obvious changes in water content or iron distribution.

Discussion: The acuity of the iron loading process affects the variability but not the bias of MRI-iron calibration curves. In contrast, iron chelation can produce systematic shifts in MRI calibration curves compared with the unchelated state, reflecting gross changes in tissue hydration and iron distribution. Since the rate of iron-loading and extraction performed in animals is more extreme than occurs in humans, limiting tissue requilibration, it is possible that the present studies overestimate the potential for chelator-specific calibration bias. Nonetheless, caution should be used in extrapolating calibration curves derived from patients using deferoxamine therapy to others being treated with deferiprone and deferasirox. Careful, longitudinal assessment of MRI calibration curves of patients receiving oral chelation therapies is warranted.

Figure Figure

Figure Figure
von Willebrand Factor and Factor VIII Are Independently Required To Form Stable Occlusive Thrombi in Injured Veins.
Authors: Chauhan Anil K. , Kisucka Janka , Lamb Colin B. , Bergmeier Wofgang , Wagner Denisa D.
Published: October 2019

von Willebrand factor (VWF) is a large adhesive glycoprotein synthesized in megakaryocytes and endothelial cells and stored in platelet a-granules and Weibel-Palade bodies respectively. It protects Factor VIII (FVIII) from proteolysis and mediates the initial contact of platelets with the injured vessel wall thus playing an important role in hemostasis and thrombosis. VWF is crucial for the formation of occlusive thrombi at arterial shear rates. However, with only a few conflicting studies published, the role of VWF in venous thrombosis is still unclear. Therefore in order to understand the in vivo role of VWF and FVIII in experimental thrombosis under venous flow conditions, we decided to evaluate thrombosis in VWF−/−, FVIII−/− and transgenic mice lacking the GPIbα extracellular domain which was replaced by human Interleukin-4 receptor (IL4Rα/GPIbα-tg). In ferric chloride-injured veins, platelet adhesion to subendothelium is decreased and thrombus growth is impaired in the VWF−/− mice when compared to wild-type (WT). In the WT mice, thrombi grew to occlusive size with a mean time of 18 min and all injured venules occluded, whereas in VWF−/− mice none of the vessels occluded by 40 min after injury, when observation was terminated. Venules of mice deficient in FVIII treated similarly also did not occlude because of embolization. The infusion of recombinant human-FVIII (r-hu-FVIII) in FVIII−/− mice normalized the occlusion time to WT values. We also observed thrombus instability in the VWF−/− mice, which was due to lower FVIII levels in these mice since r-huFVIII restored thrombus stability i.e. prevented breaking of the thrombi with large platelet aggregates moving downstream. Despite normalization of blood clotting time and thrombus stability after r-FVIII infusion, the VWF−/− thrombi grew at a slower rate than WT and the venules did not occlude. In transgenic mice lacking the GPIbα extracellular domain, all injured venules occluded. Thus, VWF uses other adhesion receptors besides GPIbα in thrombus growth under venous shear conditions. Our studies document crucial independent roles for VWF and FVIII in experimental thrombosis under venous flow conditions in vivo.
A Sensitive Risk Score for Directing Treatment in Younger Patients with AML.
Authors: Burnett Alan K. , Hills Robert K. , Wheatley Keith , Goldstone Anthony H. , Prentice Archie G. , Milligan Donald
Published: October 2019

Treatment strategies for younger patients (<60 years) with AML who enter complete remission (CR) are frequently decided on a risk assessment based on cytogenetic characterisation. Most collaborative groups have devised prognostic cytogenetic groupings of favourable, intermediate or poor risk which have relapse risks of 25–35%, 50–60%, and 80–85% respectively. The precise lesions in each group might vary by study group, but the overall discrimination is consistent. Favourable and possibly intermediate risk cases may not receive transplantation, whereas poor risk cases will. Outcomes for poor risk cases have failed to improve over the years, and if not transplanted these patients are candidates for experimental therapy. There is concern that cytogenetic segregation may not on its own be sufficiently sensitive on an individual patient level, and the inclusion of other factors may better customise risk. We performed a Cox regression analysis on 1937 non-APL cases with complete data who entered CR in the MRC AML10 and 12 trials. Increasing age, cytogenetic risk group higher WBC, male sex, secondary disease, and incomplete response to course 1 were all identified as significantly (p= 0.05) associated with poorer survival. These respectively contributed 0.013 per year, 0.651 per cytogenetic group, 0.002 per WBC unit (/nl), 0.170 for males, 0.221 for secondary disease, and 0.195 for PR/ 0.390 for resistant disease following course 1 to an individual’s total risk score. The groups were separated into scores of <2.00 (good risk), 2.00–2.667 (standard) and >2.667 (poor risk) which gave excellent discrimination for survival from CR at 5 years of 63%, 47% and 24% (p<0.00001). The score was prospectively validated in 897 patients who entered the MRC AML15 trial and was confirmed to be predictive with survivals from CR of 69%, 61% and 42% (p<0.00001). The value of the new score was to move patients out of the old cytogenetic category. The major effect was to move 274 patients from intermediate risk to poor risk and 42 poor risk patients to intermediate risk (table). This score has implications for treatment approach since, relatively, 60% more patients were identified as high risk and are therefore candidates for experimental therapy, or may, from Mantel-Byar analysis, benefit from transplantation.

MRC Risk Group Total Good Standard Poor Crosstabulation of old and new risk group classification New Risk Classification Good 309 28 0 337 (14%) Standard 51 1288 42 1381 (59%) Poor 2 274 353 629 (27%) Total 362 (15%) 1590 (68%) 395 (17%) 2347
Critical Role of CIB1 in Endothelial Cell Function and In Vivo Ischemia-Induced Angiogenesis.
Authors: Zayed Mohamed A. , McFadden Andrew , Yuan Weiping , Hartnett Mary E. , Chalothorn Dan , Faber James , Parise Leslie V.
Published: October 2019

CIB1, a 22kDa EF-hand containing calcium binding protein, was originally identified in a yeast two-hybrid screen as a binding partner for the cytoplasmic tail of the platelet integrin αIIb. CIB1 also associates with a number of kinases and modulates their activity, suggesting that CIB1 is an important regulatory molecule. Recently, we found that CIB1 is expressed in multiple endothelial cell (EC) types. We therefore tested the role of CIB1 in EC function in vitro, and in angiogenesis both ex vivo and in vivo. To test the role of CIB1 in EC function in vitro, we reduced endogenous CIB1 levels in ECs by RNA interference with an shRNA-delivered by lentivirus. CIB1 depletion significantly decreased EC haptotaxis on fibronectin and EC vascular tube formation on growth factor-reduced Matrigel. Treatment with FGF-2, an angiogenic factor, did not counter the observed inhibition of haptotaxis and tube formation by shRNA against CIB1. However, CIB1 overexpression enhanced FGF-2-induced EC haptotaxis relative to control cells. Similarly, ECs derived from CIB1 null mice exhibited a significant decrease in haptotaxis, tube formation, and proliferation compared to ECs isolated from wild-type littermate controls. In ex vivo aortic ring and tibialis anterior muscle culture assays, CIB1 null cultures supplemented with serum or FGF-2 demonstrated reduced blood vessel sprouting compared to wild-type littermate control cultures. Finally, in vivo assays for hyperoxic retinal angiogenesis and hind-limb induced-ischemia revealed a decrease in post-ischemia retinal neovascularization and Doppler hind-limb blood perfusion recovery, although developmental retinal angiogenesis in CIB1 null mice appeared normal. In conclusion, these findings support a critical role for CIB1 in EC function that appears to be important for ischemia-induced angiogenesis.
Identification of Genomic Classifiers That Distinguish Induction Failure in T-Lineage Acute Lymphoblastic Leukemia in COG Study 9404.
Authors: Winter Stuart S. , Khawaja Hadya , Jiang Zeyu , Griffin Timothy , Asselin Barbara , Larson Richard S.
Published: October 2019

The clinical features of age, white count, and presence of extramedullary disease cannot predict risk for induction failure (IF) in patients who present with T-cell acute lymphoblastic leukemia (T-ALL). On the basis of recent observations that gene expression profiles can distinguish clinicopathologic cohorts of patients with acute leukemia, we hypothesized that microarray analyses performed on diagnostic T-ALL bone marrow samples might identify a genomic classifier for IF patients. Using a case-control study design for children and young adults treated for T-ALL on Children’s Oncology Group Study 9404, we analyzed 50 cryopreserved T-ALL samples using Affymetrix U133A Plus 2 genechips, which have 54,000 genes, ESTs and genomic classifiers. Following RMA normalization, we used Prognostic Multi-array Analysis (PAM) to identify a 116-member genomic classifier that could accurately identify all 6 IF cases from the 44 patients who achieved remission. Within the IF cohort, 37 genes were up-regulated and 79 were down-regulated in comparison to other outcome groups. To further investigate the genetic mechanisms governing IF, we developed four cell lines with acquired drug resistance: Jurkat and Sup T1; each having resistance to daunorubicin (DNR) and asparaginase (ASP). Using a comparative analysis for fold-change in gene expression among 6 IF patients and the T-ALL DNR and ASP-resistant cell lines, we identified seven genes that were up-regulated, and another set of seven genes that were commonly down-regulated. To validate the potential use of our 116-member gene set in predicting IF in T-ALL, we tested our genomic classifier in 42 cases which were treated on COG study 8704 and hybridized to the Affymetrix U133Av.2 chip. Because only 85 probes were shared between U133A Plus 2 and U133Av. 2 chips, we employed shrunken class centroids to constrain our classifier to 25 rank-ordered probes. This smaller classifier correctly identified the single IF case in 8704, as well as another patient who was an early treatment failure, indicating that similar genomic classifiers may identify IF patients in different clinical trials. These results indicate that genetic profiling may be useful in prospectively identifying IF patients in T-ALL. In addition, we identified genes that were commonly upregulated in IF patients and T-ALL cell lines with intrinsic drug resistance.
Bcl2 Accelerates Onset but Not Progression of MYC-Induced T-Cell Leukemia in Transgenic Zebrafish.
Authors: Feng Hui , Langenau David M. , Kilgore Jennifer A. , Quinkertz Andre , Jette Cicely A. , Neuberg Donna S. , Campos-Ortega Jose A. , Kanki John P. , Look Thomas
Published: October 2019

MYC is a potent proto-oncogene aberrantly expressed in over 70% of human cancers. Our laboratory has previously generated transgenic zebrafish models that overexpress the mouse c-Myc gene fused to green fluorescent protein and develop T-cell Acute Lymphoblastic Leukemia (T-ALL) that recapitulates the human disease both molecularly and pathologically. These previous models have been limited by the inability to breed non-conditional transgenic animals due to disease onset prior to sexual maturity and by the low disease penetrance when conditional transgenic embryos are injected with Cre RNA. In order to improve these zebrafish T-ALL models to make modifier screens feasible, we have generated a new stable Cre transgenic line in which Cre expression is regulated by a heat-shock promoter, and have established a conditional compound transgenic zebrafish model by breeding this pzhsp70-Cre line with conditional rag2-lox-dsRED2-lox-EGFP-mMyc transgenic fish. Upon heat-shock treatment, 81% of compound transgenic fish developed tumor by 197 days of life (mean latency: 120 ± 43 days). Using this model, we showed that overexpression of zebrafish Bcl-2 strikingly accelerates the disease onset, suggesting that suppression of apoptosis is critical for zebrafish Myc-induced tumorigenesis and serving as a proof of principle for subsequent modifier screens. Paradoxically, overexpression of Bcl-2 delays the progression of T-ALL, implying functional roles for Bcl-2 in addition to the inhibition of apoptosis.
Clinical Significance of Free Circulating CD4 and CD8 in Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome.
Authors: Abdool Adam , Giles Francis , Hodge Menna , O’Brien Susan , Keating Michael , Jilani Iman , Kantarjian Hagop , Estey Elihu , Albitar Maher
Published: October 2019

CD4 is a 55-kDa glycoprotein expressed mainly on the surface of helper/inducer T-cells and monocyte-derived cells. CD4 responds to antigen associated with MHC class II. CD8 is a disulfide-linked dimer of either an alpha- and a beta-chain or two alpha-chains (34 kDa each). CD8 is expressed on T-cytotoxic/suppressor cells and binds to antigen associated with MHC class I. Since leukemias are known to elicit an immune response we speculated that, owing to turnover of the responding T-cells, free circulating CD4 (cCD4) and CD8 (cCD8) could be detected in the plasma of patients with leukemias. We have developed a bead-based sandwich immunoassay to measure cCD4 and cCD8 in the plasma. Using this assay, we measured cCD4 and cCD8 in the plasma of 98 patients with AML and 50 patients with MDS and assessed associations with clinical behavior. Whereas cCD4 levels did not differ significantly between AML (median, 918 U/μl; range, 0–30038 U/μl) and MDS (median, 1029 U/μl; range, 0–48338 U/μl in MDS), cCD8 levels were significantly higher in AML (median, 120390 U/μl; range, 0–694820 U/μl) than in MDS (median, 105340 U/μl; range, 0–810110 U/μl) (P=0.03). The ratio of cCD4 to cCD8 was significantly higher in AML and MDS than in 51 normal controls (P <0.01). There was no correlation between cCD4 or cCD8 and clinical response or survival in AML patients. In MDS, Cox regression analysis showed that higher levels of cCD4 correlated with longer survival when considered as a continuous variable (P = 0.03) and when the median was considered as a cut-point (P = 0.04). MDS patients with high levels of cCD4 (>929 U/μl) also had longer remission duration (P = 0.01); there was no correlation between cCD8 levels and clinical behavior. These findings confirm that the immune system plays a significant role in the clinical behavior of MDS and shows that CD4 cells are more important immunomodulator in MDS.

Figure Figure