bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evidence for an Enhanced Fibrinolytic Capacity in Liver Cirrhosis Measured with a New Global Fibrinolysis Test in Whole Blood.
Authors: Leebeek Frank W.G. , Kock Edith L. , Guimaraes Ana H. , Murad Sarwa D. , Janssen Harry L. , Rijken Dingemans C. 
Published: October 2019

Abstract
It is known that liver cirrhosis is associated with hyperfibrinolysis, which might contribute to bleeding problems of patients with this disease. However, hyperfibrinolysis has been assessed by using tests (such as euglobulin, plasma or diluted whole blood clot lysis assays), which do not reflect all changes that might occur in the fibrinolytic parameters in blood. A recent study even questioned the presence of a hyperfibrinolytic state in nonbleeding cirrhotic patients, since no evidence of increased fibrinolysis was observed with a new plasma-based clot lysis assay (Gastroenterology2001;121:131). Therefore we decided to re-investigate the fibrinolytic state of cirrhotic patients with a recently developed test for global fibrinolytic capacity (GFC) using undiluted whole blood. Non-anticoagulated blood was collected from 30 healthy controls and 75 patients with cirrhosis of varying severity (34 Child-Pugh A, 28 Child-Pugh B and 13 Child-Pugh C). The GFC was determined immediately after collection by clotting the blood, incubating the clots during 3 hours at 37 degrees and then measuring the generated fibrin degradation products. Various haemostatic parameters were determined in plasma samples by using functional activity assays. The median GFC (25th–75th percentile) increased from 1.7 (0.8–3.5) mg/l in the controls to 4.0 (1.6–13.0) mg/l in Child-Pugh A (n.s.), to 11.1 (2.3–31.9) mg/l in Child-Pugh B (P<0.001) and to 22.5 (3.1–89.6) mg/l in Child-Pugh C (P<0.01). Plasma samples of the cirrhotic patients showed significantly decreased levels of antithrombin, plasminogen, alpha-2-antiplasmin and thrombin-activatable fibrinolysis inhibitor (TAFI), significantly increased levels of tissue-type plasminogen activator (tPA) and no significant change in plasminogen activator inhibitor-1 (PAI-1). Spearman correlation tests in the whole group (controls plus patients) showed significant correlations between GFC and antithrombin (r=−0.44), plasminogen (r=−0.38), alpha-2-antiplasmin (r=−0.45), TAFI (r=−0.40), tPA (r=0.77) and PAI-1 (r=−0.51) (all p-values <0.0001). In conclusion, the new GFC test, which includes all components present in blood, indicates that the fibrinolytic capacity is strongly increased in patients with severe cirrhosis. The anti-fibrinolytic effects of decreased plasminogen levels in cirrhosis are apparently overruled by the pro-fibrinolytic effects of decreased alpha-2-antiplasmin and TAFI levels and particularly of increased tPA activity levels.
Mutational Analysis of VKOR.
Authors: Jin Da-Yun , Tie Jian-Ke , Stafford Darrel W. 
Published: October 2019

Abstract
More than 21 million prescriptions for warfarin are written yearly in the US for Vitamin K epoxide reductase (VKOR), the target of warfarin The vitamin K epoxide reductase, VKOR, apparently uses cysteines, 132 and 135 as active sites. In addition to cysteines 132 and 135, cysteines 43 and 51 are conserved throughout evolution. Rost et al. have mutated each of the cysteines in VKOR and found that, in whole cell lysates, mutations in of C43 result in less than 20% of wild-type activity while mutation of C53 eliminates activity. We have repeated these experiments and mutated all of the cysteine residues in VKOR. Our results in microsomes are similar to the results of Rost et. al. (2005) Thromb. Haemost. 94, 780–786. However, when the mutated enzymes are purified we find that the activity of C43 has 30% residual activity while C51 has 60% residual activity. Mutation of both residues in the same molecule results in an enzyme that is similar to the C51 mutation. In addition, we find that a portion of purified VKOR has a disulfide bond between residues 43 and 51. This suggested that we might be able to remove the loop between C43 and C51 and retain activity. Indeed, the mutated enzyme with this loop removed also has substantial activity. Warfarin inhibition studies suggest that these mutations do not materially affect warfarin sensitivity. Our results stress the importance of utilizing purified enzyme for interpreting the results of mutations in VKOR.
High Molecular Weight Kininogen Fragments Stimulate the Secretion of Interleukin-1β through the NFκ B, JNK and p38 MAP Kinase Signaling Pathways in Monocytes.
Authors: Khan Mohammad M. , Bradford Harland N. , Isordia-Salas Irma , Liu Yuchuan , Wu Yi , Espinola Ricardo G. , Ghebrehiwet Berhane , Colman Robert W. 
Published: October 2019

Abstract
Plasma high molecular weight kininogen (HK) is cleaved in inflammatory diseases by kallikrein to cleaved HK (HKa). We have reported that HKa releases cytokines (TNFα , IL-1β , IL-6) and chemokines (IL-8 and MCP-1) from isolated human peripheral blood monocytes. At a concentration of 600nM, Glutathione -S- transferase (GST) fusion proteins of kininogen domain 3 (D3), E7P - an active fragment of domain 3 (aaG255-Q292), HK domain 5 (D5), and D5 recombinant peptide HG (aa K420-D474) each stimulated secretion of IL-1β from monocytes. Receptors on monocytes including Mac-1, LFA-1, uPAR and gC1qR are required for IL-1β secretion from monocytes. We now report the signaling pathways and evidence for synthesis of IL-1 β in monocytes stimulated by HKa. Inhibitors of signaling pathways initiated by NFkB, JNK and p38, but not ERK, decreased IL-1b release from monocytes. A specific inhibitor of NFkB, MG-132 (1, 10 and 100 μM) significantly reduced IL-1β release from monocytes by 69.7, 67.3, 88.5% respectively, when stimulated by GST-E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 83.4% by MG-132 (100 μM). A specific inhibitor of JNK, SP 600125 (1, 10 and 100 μM) significantly reduced IL-1β release from monocytes by 55.7, 76.3, 78.9% respectively, when stimulated by GST-E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 90.23% by SP 600125 (100 μM). A specific inhibitor of p38, SB202190 (1,10 and 100 μM) significantly reduced IL-1β release from monocytes by 27.8, 14.2, 91.0% respectively, when stimulated by E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 76.8 % by SB202190 (100 μM). In contrast, the ERK activation inhibitor U0126 (1, 10 and 100 μM) failed to inhibit GST-E7P-induced release of IL-1β from monocytes but the release of IL-1β with LPS (10 μg/ml), was blocked by 77.4% at 100 μM. After monocytes (4 X 106/ml) were treated with HKa (600 nM) or LPS (10 ng/ml), total RNA was extracted and RT-PCR reactions for expression of IL-1β and gC1qR mRNA using specific primers were carried out. PCR products was separated in 4% ethidium bromide-stained agarose gels and photographed. No IL-1b mRNA was detected prior to exposure to HKa or LPS but both were detected at 1 hour. In contrast, gC1qR mRNA was present without stimulation by HKa. HKa domains 3 and 5 may contribute to the pathogenesis of inflammatory diseases by stimulating the synthesis and release of IL-1β from human monocytes using intracellular signaling pathways initiated by uPAR, β 2 integrins and gC1qR receptors.
The Ligand of Glucocorticoid-Induced TNF Receptor Is Expressed on Human Macrophages and Stimulates Proinflammatory and Anti-Tumor Effector Functions.
Authors: Kloss Mercedes , Krusch Matthias , Baltz Katrin M. , Peterfi Andrea , Kumbier Ingrid , Kanz Lothar , Salih Helmut R. 
Published: October 2019

Abstract
Macrophages are involved in inflammatory and anti-tumor responses by antigen presentation, release of immunoregulatory cytokines and cellular cytotoxicity. Among the molecules influencing macrophage functions are many members of the TNF superfamily. The TNF receptor family member Glucocorticoid-induced TNF Receptor (GITR) costimulates effector T cells, modulates apoptosis and nuclear factor kappa B and abrogates suppression of murine but not human regulatory T cells. Expression of its cognate ligand GITRL has, in humans, been detected in dendritic cells and various healthy nonlymphoid tissues. Up to now, nothing is known regarding the function of human GITRL, while in mice, reverse signaling through GITRL has been shown to influence the activation and survival of macrophages. Here we report for the first time that GITRL is expressed in human macrophages as determined by FACS and RT-PCR analysis. Reverse signaling through GITRL using a recombinant GITR-Ig fusion protein differentially affects expression of costimulatory molecules like CD80, CD86 and B7-H1, death ligands like TNF, CD178 and TRAIL as well as MHC class I and II on the macrophage surface. Furthermore, stimulation of GITRL enhances phagocytosis and respiratory burst of macrophages as determined by analysis of ingestion of PE-labeled polystyrene microspheres and oxidation of dichlor-fluorescein diacetate, respectively by FACS. Interaction of GITRL with its receptor also leads to significantly increased production of proinflammatory cytokines like TNF, IL-6 and IL-8 as determined by ELISA. In addition, stimulation of GITRL significantly increases the killing of various human tumor cell lines by macrophages as determined by cytotoxicity assays. Taken together, our data demonstrate that GITRL plays an important role in the stimulation of macrophage-mediated inflammatory responses and immune responses against tumors.
Delineation of the Common Developmental Pathway for Granulocyte/Monocyte and Lymphoid Lineages by Using an Expression Reporter for PU.1.
Authors: Arinobu Yojiro , Mizuno Shin-ichi , Shigematsu Hirokazu , Ozawa Hidetoshi , Chong Yong , Iwasaki Hiromi , Kastner Philippe , Chan Susan , Akashi Koichi 
Published: October 2019

Abstract
Understanding how multipotent cells commit to each of their terminal fate potentials is an important aspect of stem cell biology. Hematopoietic stem cells (HSCs) of Lin −Sca-1+c-Kit+ (LSK) phenotype have been purified, which were further divided into CD34-long-term and CD34+ short-term (ST)-HSCs. The existence of phenotypically isolatable common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) downstream of ST-HSCs suggests that the first commitment step after the HSC stage is the bifurcation of lymphoid vs. myeloid pathway. Recent studies, however, suggest that the loss of MegE potential could be an early event in HSC stage. For example, LSK cells activating RAG-1 or Flt-3 expression retained granulocyte/monocyte (GM) but not megakaryocyte/erythrocyte (MegE) potential together with lymphoid potential, suggesting the existence of common progenitor for GM and lymphoid lineages. Here we report that a fraction of ST-LSK cells expressing high levels of PU.1, a transcription factor necessary for GM and lymphoid development, represents GM/lymphoid bipotent progenitors. In mice harboring knock-in GFP reporter for PU.1, LSK cells were divided into GFP high and GFP low subpopulations. Although PU.1low LSK cells were multipotent, PU.1high LSK cells differentiated only into GM and lymphoid cells in vitro and in vivo. We also found that single PU.1high LSK cells differentiated into GM, T and B cells in vivo. These data formally prove the existence of the third major early progenitor population activating PU.1 at a high level, the granulocyte/monocyte/lymphoid progenitor (GMLP). The existence of prospectively isolatable GMLPs strongly suggests that HSCs sequentially lose MegE then GM potential during their lymphoid commitment.
FGF-2 Supplementation Enhances Hematopoietic Differentiation of Rhesus ESC’s.
Authors: Shaaban Aimen , Gaur Lasya , Thomson James A. , Rajesh Deepika 
Published: October 2019

Abstract
Progress toward clinical application of embryonic stem cells (ESC) derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model such as the rhesus macaque. However, in contrast to human ESC’s (hESC’s), efforts to induce conclusive hematopoietic differentiation from rhesus ESC’s (rESC’s) have been unsuccessful. Despite their close phylogenetic relationship, subtle differences exist between the hematopoietic differentiation of rESC’s and hESC’s. We recently reported that although rESC’s have the potential for hematopoietic differentiation; they exhibit an arrest at the hematoendothelial precursor stage of hematopoietic development in culture conditions developed for hESC’s. One possible difference may be in the requirement for fibroblast growth factor (FGF) signaling. Despite documentation of its contribution to the maintenance ESC’s in an undifferentiated state, the role for FGF-2 in the hematopoietic differentiation of hESC’s and rESC’s has not been similarly examined. Given its critical role for the formation and subsequent hematopoietic differentiation of murine ESC-derived hemangioblasts, we wondered if enhanced hematopoietic differentiation from rESC’s could be achieved by culture supplementation with FGF-2. To answer this question, undifferentiated rESC’s were subjected to embryoid body (EB) differentiation with daily FGF-2 supplementation of the cytokine-rich media. Cultures were analyzed by flow cytometry after 16 days of EB culture. We found that the FGF-2 supplemented cultures appeared more robust with an overall higher numbers of cells. More importantly, a dramatic expansion of hematoendothelial precursors (Flk1hi+ VE-cadherin- CD45−), committed hematopoietic progenitors (CD34+CD45+Lin−), and hematopoietic cells (CD45+) was seen in FGF-2 supplemented cultures when compared to controls. These effects were consistent in two separate lines of rESC’s (R420 and R456). Next we wondered if the observed effect of FGF-2 on hematopoietic development was concentration-dependent. Therefore, we compared serial increases in FGF-2 concentration (0, 10, 50 and 100 ng/ml) of the EB differentiation media and found the effect to be concentration-dependent. From these results, we conclude that FGF-2 appears to play a critical role in the hematopoietic differentiation of rESC’s. Both the development of hematoendothelial precursors and the differentiation of committed hematopoietic cell types are augmented. To study this further, the significance of FGF signaling at various stages of rESC-derived hematopoietic differentiation must be evaluated. A better understanding of the requirements for FGF-2 in EB development will likely lead to improved protocols for the production of human and rhesus ESC-derived hematopoietic progenitors.
Abcg2 Expression Marks Neural Stem Cells in the Murine Cerebellum.
Authors: Zhou Sheng , Fatima Soghra , Sorrentino Brian P. 
Published: October 2019

Abstract
Neural stem cells have been identified in both the cerebellum and forebrain of fetal and adult mice. These cells can form neurospheres in culture and differentiate into both glia and neurons either in vitro or in vivo. In embryonic day 14 forebrain, neural stem cells are found to exist exclusively in a subpopulation with the Side Population (SP) phenotype, and express Abcg2, a member of the ABC transporter family that is responsible for the SP phenotype in hematopoietic stem cells (HSCs). The expression of Abcg2 in stem cells in the cerebellum has not been characterized. We have generated an Abcg2/GFP knock-in mouse model in which expression of GFP is under control of the endogenous Abcg2 locus and used this model to demonstrate that Abcg2 expression can be used for HSC enrichment. Here we report the use of this mouse model to explore the relationship between Abcg2 expression and neural stem cell function in neonatal cerebellum. Single cells were prepared from cerebellum of 4–9 day old mice by digesting with papain. We then stained the cells with anti-CD45 and anti-Ter119 antibody to exclude the resident hematopoietic cells in subsequent flow cytometry analysis and cell sorting. We found that a small but consistent subpopulation of cells, comprising 0.7±0.12% of total CD45−Ter119- single cell preparations, expressed the Abcg2/GFP allele. To determine whether these GFP+ cells were enriched for neural stem cells, we sorted the CD45−Ter119- cells into GFP+ and GFP− subpopulations and analyzed for their neurosphere forming activity in the presence of epidermal growth factor and basic fibroblast growth factor. We found that the GFP+ subpopulation formed 21 fold more neurospheres compared with the GFP− subpopulation. These neurosphere forming cells can self-renew as evidenced by their capacity to form secondary neurospheres when replated. These results demonstrate that similar to what is seen with HSCs and with embryonic forebrain cells, Abcg2 is expressed in the neural stem cells in neonatal cerebellum, and Abcg2/GFP expression in this mouse model could also be used as a marker to prospectively purify neural stem cells from cerebellum. Ongoing studies are focused on defining the in vivo multilineage differentiation potential of the Abcg2/GFP+ cells and determining whether Abcg2 expression could be used as a marker for purification of medulloblastoma stem cells.
Contribution of HGF to Liver Regeneration by Strong Mobilization of Bone Marrow Stem Cells.
Authors: Tajima Fumihito , Tsuchitya Hiroyuki , Nishikawa Kenichi , Okazaki Toshirou , Shiota Goushi 
Published: October 2019

Abstract
Cell plasticity of bone marrow stem cells to hepatocytes is known, however, the details are still unclear. hepatocyte growth factor (HGF) promotes an increase in liver stem cells in severely injured liver, but intervention to bone marrow stem cell is unclear. We examined a role of HGF in bone marrow stem cell-mediated liver regeneration in order to obtain effective liver regeneration.
First, we found that the phenotype of stem cells, which can differentiate into hepatocytes, is Lin−c-kit+Sca-1+CD34− in bone marrow or Lin−c-kit+Sca−1+CD34+ in peripheral blood mobilized by G-CSF. We transplanted single Lin−c-kit+Sca-1+CD34− bone marrow cell harvested from male EGFP mouse to female wild type mouse, and then, using this GFP-chimera-mouse, we found that bone marrow origin GFP+ and Y chromosome+ hepatic cells were present in liver after acute liver damage. Next, single Lin−c-kit+Sca-1+CD34+ peripheral blood cell, which was mobilized in peripheral blood by administration of G-CSF, was transplanted into the portal vein of the wild type mouse which was given hepatic damage. We found that the GFP-positive cells also expressed albumin.
Second, we investigated whether the HGF can mobilize stem cells from bone marrow to peripheral blood. In peripheral blood of HGF transgenic mice, 1.1% developed CD34+ cells and 20±3 colony forming cells of 1X106 peripheral blood mononuclear cells were shown. Colony forming cells were found in the mouse into which an HGF-expressing adenovirus was administered. After injection of rHGF to mice, a significant time-dependent increase of percentage of CD34+ cells in the PB was noted at the first 3 hours and CD34+ cells were increased in dose-dependent manner of rHGF and reached plateau level at 100 m/kg. The mice having transplantation with PB cells from 100 mg HGF-treated mice for 4days showed engraftment 2 months after transplantation. Upon activation of rHGF in mouse MS-5 stromal cells, phosphorylation c-Met and SCF were up-regulated, while VCAM -1, MMP-9, SDF -1 or CXCR4 were not changed. SCF level in conditioned media was also increased after the HGF stimulation.
Finally, we examined whether the bone marrow stem cells in PB mobilized by HGF transdifferentiate into hepatocytes. Using GFP-chimera-mice given acute liver injury after administration of retorolusine and CCl4, the levels of GFP+ cells in liver of GFP-chimera-mice 2 months after treatment by PBS and HGF were 2.2±1.4% and 12.7±3.6%, respectively (p<0.01). In conclusion, HGF can mobilize stem cells with long-term engraftment capabilities from bone marrow to peripheral blood, resulting in contribution to liver regeneration.
Hematopoietic Stem Cells Neither Reconstitute Satellite Cells nor Myofibers after Direct Injection into Dystrophic Muscle.
Authors: Kuhr Christian S. , Lupu Marilena , Storb Rainer 
Published: October 2019

Abstract
We sought to determine whether wild-type hematopoietic stem cells directly injected into muscle could restore dystrophin expression in a relevant pre-clinical canine model of Duchenne muscular dystrophy (DMD). Recipients that possessed the dystrophin mutation and thus exhibited the DMD phenotype were chosen. They were rendered tolerant to their dog leukocyte antigen (DLA)-matched unaffected littermates through myeloablative conditioning followed by hematopoietic stem cell transplantation (HCT). Donor bone marrow cells were collected (2.19e8 cells/kg) and injected directly under the aponeurosis of the superspinatus muscle of the DMD affected animals. Excisional muscle biopsies 5, 9 and 15 weeks following injection were examined with immunofluorescence and reverse-transcription-PCR for dystrophin protein and mRNA expression (figure), respectively, and failed to demonstrate increases over background. Consistent with these results, clonal satellite cell cultures from these biopsies at the initiation of myotube formation showed no detectable donor contributions. These findings lessen the enthusiasm for strategies relying on hematopoietic stem cell transdifferentiation to repair or regenerate skeletal muscle.
Figure Figure
Identification of Hepatic Progenitors Having No Hematopoietic Potential in Murine Bone Marrow.
Authors: Yamada Yasuhiro , Miyake Hirosada , Nishimoto Eishi , Mitsuya Hiroaki , Yonemura Yuji 
Published: October 2019

Abstract
We recently reported that when Sca-1+ c-Kit− bone marrow cells (BMCs) were co-cultured with fetal liver cells (FLCs) on laminin-coated dishes, alpha-fetoprotein (AFP)-expressing BMCs became completely adherent by day 3 and expressed albumin as assessed with immunochemistry and RNA-PCR (Yamada et al., Exp Hematol. 34: 97, 2006). In the current study, we attempted to further delineate the characteristics of BMCs that differentiate into hepatic-like cells. It was found that AFP-expressing cells were in CD5+ or B220+ lymphoid lineage, mostly Sca-1+CD5+ lineage, expressing AFP. When cKit+Sca-1+ lineage− BMCs (KSLs), which did not express AFP, were cocultured with CD5+ BMCs, both from green fluorescence protein (GFP)-expressing transgenic mice, in the presence of FLCs from ROSA26 mice (X-gal+ FLCs), fractionated cells gave rise to adherent hepatic-like cells, which expressed albumin and cytokeratin 8 (assessed with immunochemistry) and AFP, albumin, transthyretin and dipeptidylpeptidase IV (examined with RNA-PCR). The hepatic-like cells from KSLs and CD5+ BMCs emerged at the frequency of 1 in 50 and 1x103 as assessed with titration assay. However, CD5+ Mac-1− Gr-1− Ter119− BMCs did not differentiate into hepatic-like cells. CD5+ CD45− cells differentiated into hepatic-like cells without fusion at the frequency of 1 in 300 but CD5+ CD45+ cells did not. CD5+ CD45− cells hardly produced hematopoietic colonies as compared with CD5+ CD45+ cells did.
In conclusion, we have shown that KSLs and CD5+ CD45− cells exposed to FLCs are capable of generating hepatic-like cells, which expressed albumin as assessed with immunochemistry and RNA-PCR. We should further explore whether CD5+ CD45− cells emerge from hematopoietic stem cells or mesenchymal stem cells.