bluuebunny/crossref_metadata_2025_split huggingface dataset:
Diverse Myeloid Lineage Potential Arises in the Yolk Sac of the Mammalian Embryo.
Authors: Palis James , Koniski Anne , Bushnell Timothy P. , McGrath Kathleen E. 
Published: October 2019

Abstract
The emergence of the hematopoietic system in the mammalian embryo is characterized by two temporally overlapping waves of distinct “primitive” and “definitive” erythroid progenitors that peak in the murine yolk sac at E8.5 and E10.5, respectively. We have recently determined that each wave contains megakaryocyte potential (Tober, et al., submitted), but the initial emergence of distinct myeloid lineages is less well understood. Temporal and spatial analysis of hematopoietic progenitors in the murine embryo suggest that the macrophage lineage is associated with both waves, while neutrophil and mast cell lineages are restricted to the second, definitive wave (Palis, et al., Development126:5073, 1999). To better define the emergence of myelopoiesis in the murine embryo, we cultured dissociated E9.5 yolk sac cells in a broad array of cytokines (SCF, IL3, IL5, IL6, IL7, IL9, IL11 and GM-CSF) for seven days and identified morphologically distinguishable macrophages, neutrophils, eosinophils, basophils, and mast cells. The presence of eosinophils was further confirmed by expression of eosinophil peroxidase transcripts. As expected, this multilineage myeloid potential was restricted to c-kit++ cells. However, unlike the bone marrow, these c-kit++ cells in the E9.5 yolk sac were predominately a single population that also express CD41 and Fc gamma receptor (FcγR, CD16/32). FcγR+ cells first emerge in the yolk sac between E8.5 and E9.5 and are not found in the embryo proper until E10.5, when they are restricted to the liver. These findings suggest that the expression of FcγR is restricted in the yolk sac to definitive hematopoiesis and tracks its transition to the fetal liver. While mature definitive erythroid cells first emerge from the fetal liver at E12.5, to our knowledge the onset of granulopoiesis has not been investigated in the early murine embryo. We found approximately 1000 GR1+/Mac1+ cells in the fetal liver at E12.5 and this population expands 100-fold during the next two days of development. These GR1+/Mac1+ cells consisted predominantly of progressively maturing neutrophils. We conclude that the potential to generate multiple myeloid lineages first arises in the yolk sac along with definitive erythroid progenitors and that a robust granulocyte population subsequently emerges in the fetal liver at midgestation.
The Effect of Von Willebrand Factor Glycans on the Interaction with ADAMTS13.
Authors: McKinnon Thomas A.J. , Chion Alain C.K. , Laffan Mike A. 
Published: October 2019

Abstract
Von Willebrand factor (VWF) is a large plasma glycoprotein that mediates platelet tethering at sites of vascular injury. This function is dependent upon its multimeric size, which is modulated in plasma through proteolysis by ADAMTS13. Terminal ABO blood group sugars presented on the N-linked glycans of VWF alter the susceptibility of VWF to cleavage by ADAMTS13. Two predicted N-linked glycan sites (N1515 & N1574) are in close proximity to the ADAMTS13 cleavage site (Y1605-M1606). However, is it not known whether these predicted sites are occupied, or indeed, present the ABO sugars. In this study, purified plasma VWF was digested with trypsin, and the resulting fragments separated by ion-exchange chromatography. A 55kDa fragment observed under reducing SDS-PAGE was identified as VWF residues 1493–1849 by mass spectrocosopy and N-terminal sequencing. This fragment encompassed the predicted N-linked glycan sites at N1515 & N1574. Incomplete PNGase F digestion produced three protein bands, indicating that both glycosylation sites were occupied. Furthermore, analysis with blood group specific lectins demonstrated presentation of the ABO sugars on these glycan chains. This data suggests that the N1515 and N1574 glycosylation sites and subsequent ABO(H) sugar modification may influence VWF proteolysis. To further elucidate the role of other VWF glycan structures on susceptibility to ADAMTS13 cleavage, purified VWF was treated with neuraminidase to remove terminal sialic acid residues, producing asialo VWF (As-VWF), and PNGase F to remove whole N-linked glycan chains. (Nless-VWF). Lectin analysis demonstrated removal of >95% of terminal sialic residues and >75% whole N-linked glycan chains. Both AsVWF and Nless-VWF bound to type III collagen with similar affinity to wild type VWF (KD 1.9nM, 1.6nM, 1.4nM respectively) and demonstrated similar multimeric composition. Interestingly, As-VWF had decreased susceptibility to ADAMTS13 cleavage, but bound ADAMTS13 with comparable affinity to wild type VWF (KD 11nM & 12nM respectively). Nless-VWF was cleaved faster by ADAMTS13 and bound ADAMTS13 with ~ 4-fold increased affinity (KD 3.4nM).
This data demonstrates that the glycan moiety of VWF plays an important role in mediating the interaction with ADAMTS13.
Protein S Down-Regulates Factor VIIIa by Competing with Factor IXa.
Authors: Takeyama Masahiro , Nogami Keiji , Tatsumi Kohei , Fujita Yuri , Tanaka Ichiro , Yoshioka Akira , Shima Midori 
Published: October 2019

Abstract
Factor VIII functions as a cofactor in the factor Xase complex responsible for phospholipid surface-dependent conversion of factor X to factor Xa by factor IXa. Factor VIIIa, activated form by thrombin and factor Xa, is down regulated by activated protein C (APC), and the reaction is enhanced by the presence of protein S, a cofactor for APC. It was previously reported that protein S inactivated directly factor Xa or factor Va, however, the direct regulation of factor VIII by protein S remains to be investigated. In the present study, surface plasmon resonance (SPR)-based assay showed that factor VIII bound directly to immobilized protein S (Kd; 70 nM). The isolated A2 and A3 domains also bound to protein S with similar modest affinity (Kd; 15 and 17 nM, respectively), whilst the isolated A1 and C2 domains failed to bind, suggesting the presence of protein S-binding sites within the A2 and A3 domain. Since it is known that factor IXa also interacts with the A2 and A3 domains in factor VIII, we examined the inhibitory effect of factor IXa on the factor VIII and protein S interaction in a SPR-based assay. Active-site modified (EGR−) factor IXa competitively inhibited the binding of protein S to both the A2 and A3-C1-C2 domains dose-dependently. Furthermore, Western blotting analysis using an anti-A1 monoclonal antibody revealed that Arg336 cleavage in factor VIII by factor IXa in the presence of protein S was slower with an ~1.8-fold lower cleavage rate than that in its absence, supporting that protein S competed the factor IXa interaction with factor VIII. Of interest, the reaction with protein S to factor VIII inhibited the generation of factor Xa dose-dependently in a factor Xa generation assay (IC50; 150 nM). The Km value for factor X obtained with factor Xase complex in the presence of physiological concentration of protein S was 19 nM, which was ~2-fold lower than that in its absence (45 nM). Whilst, the Km value for factor IXa in the presence of protein S was greater than 100 nM, which was ~5000-fold higher than that in its absence (21 pM). We demonstrate that protein S not only contributes to down-regulate factor VIIIa activity as a cofactor for APC, but also impairs the factor Xase complex by competing the binding of factor IXa to factor VIII.
Non-Hodgkin Lymphoma B-Cells Induce Intratumoral CD4+CD25− T Cells To Express Foxp3 and Gain Regulatory Function.
Authors: Yang Zhi-Zhang , Novak Anne J. , Witzig Thomas E. , Ansell Stephen M. 
Published: October 2019

Abstract
We have previously shown that CD4+CD25+Foxp3+ regulatory T cells from NHL tumors suppress the function of infiltrating CD4+ T cells and cytolytic CD8+ T cells. Expression of Foxp3 has been demonstrated to be crucial to the development and function of CD4+CD25+ regulatory T cells. However, the mechanistic details that drive development of Foxp3 expression in T cells, in both the normal and malignant scenario, remains to be fully elucidated. Previous studies suggest that Foxp3 expression in CD4+CD25− T cells can be upregulated by tolerizing stimuli such as activation through TCR, corticosteroids, estrogen, and TGF-beta. Because lymphoma B cells have been shown to induce T-cell tolerance, we postulated that lymphoma B cells may play a role in the generation of regulatory T cells by inducing Foxp3 expression in CD4+CD25− T cells.
FoxP3 expression was initially thought to be restricted to CD4+CD25+ regulatory T cell population. However, recent literature suggests that Foxp3 may also be expressed in CD4+CD25− T cells. Using biopsy specimens from patients with B-cell NHL, we found that a subset, 15%, of infiltrating CD4+CD25− T cells express Foxp3 and are capable of suppressing the proliferation and granule production of infiltrating cytotoxic CD8+ T cells. These initial studies suggest that CD4+CD25−Foxp3+ T cells have regulatory function. To explore the underlying mechanism by which Foxp3 expression is regulated, we determined the effect of costimulatory signals on Foxp3 expression in CD4+CD25−Foxp3− T cells. Activation with OKT3/anti-CD28 Ab as well as DC-mediated activation induced Foxp3 expression in a subset of CD4+CD25− T cells. We also found that the presence of lymphoma B cells during activation augmented the induction of Foxp3 expression in CD4+CD25− T cells and that NHL B cell-mediated Foxp3 expression was cell contact-dependent. To better understand the contribution of NHL B cells in Foxp3 expression, we explored the possibility that CD27-CD70 interaction may be involved in Foxp3 expression. Lymphoma B cells express CD70, but not B7-1 and B7-2, which have been shown to be important in protecting tumor cells from lysis and contributing to cancer pathogenesis. Ligation of CD27 by receptor cross-linking enhanced Foxp3 expression in infiltrating CD4+CD25− T cells in B-cell NHL.
Taken together these studies reveal a novel role for NHL B cells in development of regulatory T cells. Our data show that lymphoma B cells induce expression of Foxp3 in infiltrating CD4+CD25− T cells and may result in development of T cells with regulatory function within the tumor microenvironment. Our results also suggest a potential role for CD27-CD70 interactions in this process. The ability of malignant B cells to drive development of regulatory T cells may be one mechanism by which lymphoma B cells protect themselves from anti-tumor immunity. (Supported in part by the Iowa/Mayo Lymphoma SPORE CA97274).
Analysis of Peripheral Blood CD4+/CD25high Treg Cells and FOXP3 mRNA Expression in Patients Treated with Ipilimumab (Monoclonal Human Anti-CTLA4, MDX-010) for Relapse of Malignancy Following Allogeneic Hematopoietic Transplantation.
Authors: Zhou Jiehua , Zhong Rui-kun , Kalcheva Iveta , Medina Bridget , Ball Edward D. , Bashey Asad 
Published: October 2019

Abstract
CTLA-4 is expressed upon activation of T-cells,and serves as an important negative regulator of their effector function. It is also expressed constitutively on CD4+/CD25+ regulatory T cells (Treg), where its function is not clear. Following allogeneic hematopoietic stem cell transplantation (allo-HCT), CTLA-4 function may be involved in suppression of alloreactive T cells that mediate the graft-versus malignancy effect and GVHD. We have studied the administration of a single dose of Ipilimumab (MDX-010), a fully human monoclonal anti-CTLA-4 antibody, in a dose escalation trial in patients with relapse/progression of malignancy following allo-HCT. Here we report effects of ipilimumab on peripheral CD4+/CD25+ cell levels and FOXP3 mRNA expression in these patients. Seventeen patients with a variety of malignancies were enrolled in this study. Ipilimumab was given intravenously at a dose level of 0.1, 0.33, 0.66, 1, and 3mg/kg. The blood samples were obtained prior and after infusion at day 7, 14 and 30. The immunophynotyping of peripheral blood mononuclear cells (PBMC) was analyzed by flow cytometry. CD4+/CD25+ cells from nine patients at day 0, 7, and five normal donors were separated using a Dynal CD4+/CD25+ Treg kit. FOXP3 mRNA expression on CD4+/CD25+ cells were analyzed by a quantitative RT-PCR. Expression level of FOXP3 was normalized to 18S rRNA. Within CD4+ cell population, the percentage of CD4+/CD25high cells was significantly higher in patients at day 0 (11.6±6.7%, n=17), compared with normal donors (3.8±1.6%, n=12; P<0.001). There was no significant change in CD4+/CD25high Treg cells in 17 patients after infusion. However, there was a transient 55±24% decrease of CD4+/CD25high Treg cells in 6 patients at day 7. FOXP3 mRNA expression in CD4+/CD25+ cells was significantly higher in patients at day 0 (2451±1731, n=9) compared with normal donors (918±348, n=5; P<0.001). After ipilimumab infusion, 3/9 patients showed a greater than 50% decrease, 4/9 patients showed no significant change, and 2/9 patients showed a 3–10 fold increase of FOXP3 mRNA expression at day 7. There was no correlation between the dose of ipilimumab and the percentage of CD4+/CD25high cells, or the FOXP3 mRNA expression. We did not observe a correlation of CD4+/CD25high Treg cells and FOXP3 mRNA expression in patients who had a clinical response or immune breakthrough adverse events in response to ipilimumab. These data suggest that in-vivo CTLA-4 blockade does not consistently impact the number of CD4+ CD25high Treg cells and that the clinical effects observed are probably related to the effects of ipilimumab upon activated effector T-cells.
Molecular Dissection of Human Tissue Factor Pre-mRNA Processing: Identification and Characterization of Functional Exonic Splicing Enhancers in Variable Exon 5.
Authors: Tardos Jonathan G. , Bogdanov Vladimir Y. 
Published: October 2019

Abstract
Tissue Factor (TF), the regulatory co-factor of FVII/VIIa, is the physiologic trigger of blood coagulation. Two naturally occurring isoforms of TF differ in their structure: the full length TF (flTF) is a membrane-spanning molecule, whereas the recently discovered alternatively spliced TF (asTF), generated by exclusion of exon 5, lacks the transmembrane domain, and is soluble. Biosynthesis of the soluble TF variant appears to be evolutionarily advantageous, as it exists in mice. Both isoforms are present in plasma and produced predominantly by circulating monocytes. The biologic role of asTF is unknown; it has, however, been proposed that precise regulation of the TF isoform ratio in blood is critical to normal hemostasis. This study aims to identify molecular mechanisms governing the fate of variable exon 5 during the processing of the human TF pre-mRNA, and to apply these findings to generate mini-genes expressing altered human flTF/asTF isoform ratios in animal models to elucidate the role of TF isoforms in normal hemostasis and/or development of coagulopathies. In silico analysis of the human TF exon 5 revealed 14 putative exonic splicing enhancer (ESE) motifs for SF2/ASF and SC35, essential SR proteins involved in pre-mRNA splicing. A segment of the TF gene comprising the end of exon 4 through the beginning of exon 6 (including exon 5 and the entire introns 4 and 5) was inserted into the GFP open reading frame of the pGL expression vector. The resultant construct (pGL-hTF) was transfected into a human monocytic cell line (THP-1) and shown to faithfully recapitulate the pattern of TF mRNA isoform production. Site-directed mutagenesis of each putative ESE motif was then performed, and the mRNA isoform levels were assayed by RT-PCR. Removal of four ASF/SF2 sites at positions 100–117 favored exclusion of exon 5. In contrast, removal of either of the two SC35 motifs at positions 33 and 81 favored inclusion of the exon. A pSPL-3b-hTF expression vector, featuring the above mutations in the context of a different promoter and a smaller segment of the TF gene, was generated to confirm these findings: similar shifts in isoform ratio production were observed. Results were further validated by introducing both synergistic and antagonistic double mutations of the functional ESEs in pGL-hTF. Production of the two naturally occurring human TF isoforms appears to be regulated at the post-transcriptional level by SR proteins ASF/SF2 and SC35. Further elucidation of the molecular mechanisms involved in TF isoform production is very likely to provide effective means for creating state-of-the-art animal models to characterize the biologic importance of flTF/asTF ratio in circulating blood. In addition, this study is the first to show that SF2/ASF and SC35 can exert opposite effects on pre-mRNA splicing.
In Vitro and In Vivo Antioxidant Effect of Fermented Papaya Preparation (FPP) on Blood Cells of Beta-Thalassaemia Patients.
Authors: Fibach Eitan , Amer Johnny , Goldfarb Ada , Rachmilewitz Eliezer 
Published: October 2019

Abstract
In sickle cell anemia (SCD) and thalassemia, although the basic lesions are mutations in the globin genes, the pathophysiology involves oxidative stress-mediated cell damage in the bone marrow (ineffective erythropoiesis due to apoptosis of early erythroid precursors) and in the peripheral blood (chronic hemolysis of mature RBC). In addition, some patients develop thromboembolic complications and recurrent bacterial infections, the etiology of which is related at least in part, to documented oxidative stress in platelets and neutrophils (PMN), respectively. To study the presence and the role of oxidative stress in thalassemia and SCD, we adapted flow cytometry techniques for measuring the generation of Reactive Oxygen Species (ROS), the content of reduced glutathione (GSH), membrane lipid peroxidation and externalization of phosphatidylserine (PS) moieties in RBC, platelets and PMN. Cells derived from the peripheral blood of patients with beta-thalassemia major, intermedia or SCD showed increased oxidative status (increased ROS, lipid peroxidation and PS externalization, and decreased GSH) compared with their normal counterparts. Incubating fresh blood samples from patients with thalassemia major and thalassemia intermedia with 10 mg/ml FPP for 16 hours at 37oC reduced the oxidative status of RBC as well as platelets and PMN. Experiments carried out in normal and thalassemic mice (Th3/+, a mouse model of human beta-thalassemia intermedia demonstrated that mice treated for one week with 10 mg/ml FPP (dissolved in the drinking water) had reduced oxidative stress compared to control mice. The in-vivo effect of FPP was tested on 9 patients with beta-thalassemia (6 - major and 3 - intermedia) treated with 3 gr FPP per os three times a day for 12–15 weeks. Following the treatment, the ROS in RBC, platelets and PMN decreased and the GSH increased in all patients (see table). Six of these patients responded by a modest increase in RBC, reticulocytes and hemoglobin levels. These results suggest that FPP may have an important clinical efficacy as an antioxidant in thalassemia and sickle cell anemia.
The in vivo effect of FPP treatment of beta-thalassemia patients Baseline After treatment n Mean ± SE Mean ± SE P-value* * Paired samples t-test RBC 9 324.07 ± 29.19 209.55 ± 23.65 0.001 ROS Platelets 9 223.73 ± 26.49 109.11 ± 8.71 0.001 PMN 9 222.72 ± 46.42 117.61 ± 8.98 0.045 RBC 9 55.37 ± 5.37 94.88 ± 3.71 0.001 GSH Platelets 9 59.41 ± 4.98 97.55 ± 5.26 <0.0001 PMN 9 58.29 ± 5.35 90.06 ± 5.87 0.005
A Threshold Level of Von Willebrand Factor Is Required for Disease Pathogenesis in a Mouse Model of TTP.
Authors: Motto David G. , Zhu Guojing , Wagner Denisa D. , Ginsburg David 
Published: October 2019

Abstract
Thrombotic Thrombocytopenic Purpura (TTP) is a life threatening systemic illness associated with inherited or acquired deficiency of the ADAMTS13 metalloprotease. ADAMTS13 deficiency results in the appearance of ultra-large multimers of von Willebrand Factor (UL-VWF) in the circulation, which are thought to play a central role in the formation of the platelet-rich thrombi which are the pathological hallmark of this disease. Despite the critical role postulated for VWF in TTP pathogenesis, direct experimental evidence in support of this hypothesis is lacking. Recently we demonstrated that a TTP-like syndrome can be induced in ADAMTS13-deficient mice of a mixed CASA/Rk genetic background (Adamts13CASA−/−) by the bacterial agent shigatoxin. As pure CASA/Rk mice exhibit VWF levels 5 to 10-fold higher than most of the common strains of laboratory mice, we hypothesized that increased plasma VWF levels in the Adamts13CASA−/− mice (which ranged from 200–600% of C57BL/6) were responsible for their increased TTP susceptibility. Surprisingly however, plasma VWF levels did not correlate with the degree of thrombocytopenia or hemolytic anemia induced in Adamts13CASA−/− mice by shigatoxin challenge, with mice inheriting the lowest VWF levels having TTP pathology equal to those inheriting the highest levels. To further examine the requirement for VWF in TTP pathogenesis, we generated ADAMTS13-deficient mice on a susceptible CASA/Rk genetic background that were also either haploinsufficient (Vwf+/−) or completely deficient (Vwf−/−) in VWF. Absolute deficiency of VWF resulted in complete protection from shigatoxin-induced thrombocytopenia. However, thrombocytopenia was consistently observed in Vwf+/− mice similar to that previously reported in Adamts13CASA−/− mice with wild-type VWF. Importantly, again no correlation was observed between VWF level and degree of thrombocytopenia among the ADAMTS13-deficient mice that were Vwf haploinsufficient (Vwf+/−), confirming our previous observation. In summary, we now have demonstrated an absolute requirement for VWF in the pathogenesis of TTP in this mouse model system. Together with the observations that increasing VWF levels do not correlate with increasing TTP pathology, these results suggest that a threshold level of VWF is required for induction of TTP, but that further increases of VWF level do not result in worsening disease. Though these findings remain to be confirmed in humans, our data suggest that the wide variation in plasma VWF levels in the human population will not be a significant determinant of TTP susceptibility or disease severity.