bluuebunny/crossref_metadata_2025_split huggingface dataset:
Anti-Factor IX Antibodies Displaying FVIIIa-Like Activity.
Authors: Kerschbaumer Randolf J. , Dockal Michael , Jan Rosing , Scheiflinger Fritz 
Published: October 2019

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.
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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

Abstract
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.