bluuebunny/crossref_metadata_2025_split huggingface dataset:
Measurement of Blood-Borne Tissue Factor Procoagulant Activity in Healthy Individuals and Cancer Patients Using a Novel Assay.
Authors: Tilley Rachel E. , Holscher Todd , Belani Rajesh , Nieva Jorge , Mackman Nigel 
Published: October 2019

Abstract
Tissue factor (TF) is the cellular initiator of the extrinsic pathway of coagulation. Recent studies have reported circulating blood-borne TF (BBTF) antigen by methods including ELISA and flow cytometry. Whether this BBTF is active has been rarely addressed due to the lack of a simple functional assay. We have developed a new assay that measures BBTF procoagulant activity. Whole blood was collected into tubes containing citrate and corn trypsin inhibitor (FXIIa inhibitor). First, we compared the procoagulant activity obtained in detergent lyzed platelets and microparticles with peripheral blood mononuclear cells (PBMCs) following a 5 hour ex vivo stimulation of whole blood with LPS. Platelets and microparticles had 3% (23 ± 10 mU/mL) and 0.1% (1.0 ± 0.4 mU/mL), respectively, of the procoagulant activity of PBMCs (1048 ± 200 mU/mL). The procoagulant activity of PBMCs and platelets was inhibited by >90% in the presence of an anti-TF polyclonal antibody. Next, we determined the time course of TF-dependent procoagulant activity of microparticles and platelets after ex vivo LPS stimulation of whole blood. In both cases, a dramatic increase in microparticle and platelet procoagulant activity was observed after 12 hours of LPS stimulation, with a maximum activity observed at 48 hours (48 mU/mL for microparticles and 1028 mU/mL for platelets at 48 hours). The majority (>90%) of this procoagulant activity was TF-dependent. Therefore, we subsequently analyzed combined platelet and microparticle (P+MP) fractions for BBTF activity. We determined functional BBTF activity in P+MP fractions from the blood of healthy individuals with and without LPS ex vivo stimulation. In healthy individuals, very low levels of BBTF procoagulant activity was detected in the absence of LPS stimulation (0.22 ± 0.09 mU/mL) and this activity was decreased on average by 28% with an anti-TF antibody (0.16 ± 0.1 mU/mL; p = 0.01). In contrast, P+MP from LPS stimulated blood demonstrated on average 30 fold higher procoagulant activity, of which >90% was TF-dependent. Cancer is associated with an increased susceptibility to develop pathological thrombosis. Using our new assay, we demonstrate a significant elevation of the procoagulant activity of P+MP fractions from advanced (stage IV) solid tumor patients of varying histology’s (n=14; 0.88 ± 0.55 mU/mL) compared with normal subjects (p <0.001). Furthermore, we showed that on average 50% of the procoagulant activity was TF-dependent (procoagulant activity was reduced to 0.57 ± 0.35 mU/mL in the presence of an anti-TF antibody; p < 0.01), suggesting that circulating BBTF in cancer patients has the potential to contribute to thrombosis in vivo. In summary, we have developed a novel assay that measures BBTF activity. This assay may be useful in the detection of a pre-thrombotic state in cancer patients.
Long-Term Follow-Up of Patients Treated by Gene Therapy for X-Linked Chronic Granulomatous Disease.
Authors: Ott Marion G. , Grez Manuel , Stein Stefan , Siler Ulrich , Koehl Ulrike , Kuehlcke Klaus , Schmidt Manfred , Schilz Andrea , Kunkel Hana , Naundorf Sonja , Brinkmann Andrea , Hassan Moustapha , von Kalle Christof , Trasher Adrian , Seger Reinhard , Hoelzer Dieter 
Published: October 2019

Abstract
Chronic granulomatous disease (CGD) is a primary immunodeficiency in which phagocytic cells of affected patients have impaired antimicrobial activity due to a defect in the production of reactive oxygen species (ROS). CGD is caused by mutations in any one of four genes encoding for the subunits of the NADPH oxidase complex. Although curable by HSC transplantation, this strategy is usually limited only to patients with HLA-matched sibling or unrelated donors, as mismatched transplantation is associated with high morbidity and mortality due to graft failure and slow immune reconstitution. A therapeutic alternative for CGD patients is the genetic modification of autologous HSC. In January 2004 we initiated a Phase I/II clinical trial for X-CGD patients including conditioning with busulfan (8 mg/kg/total dose) prior to infusion of genetically modified HSC. G-CSF mobilized CD34+ cells from 2 adult patients (25 and 26 years) were transduced ex-vivo with a monocistronic gp91phox retroviral vector.
Therapeutically significant gene marking levels were detected in neutrophils of both patients with up to 60% functionally corrected phagocytes 14 months after gene therapy. This high correction resulted from an unexpected but temporarily restricted expansion of gene transduced myeloid cells in vivo. In contrast gene marking levels in B-cells has remained constant at a level of 20%, while gene marking in T-cells is below 5%. Gene marking in bone marrow was detected at levels between 30% and 40% one year after transplantation of gene modified cells. Killing assays in vitro have demonstrated antibacterial and antifungal activity in gene transduced phagocytes and both patients recovered of Staph. aureus and Aspergillus fumigatus infections after gene therapy. Our results suggest that gene therapy in combination with bone marrow conditioning is a therapeutic option for inherited diseases affecting the myeloid compartment and can be successfully used to treat CGD.
Exosite-Specific Inhibition of Thrombin Using Photo-Crosslinked Fluorescent Reporter Peptides.
Authors: Yegneswaran Subramanian , Tiefenbrunn Theresa , Fernandez Jose’ A. , Dawson Philip E. 
Published: October 2019

Abstract
Multi-functional enzymes such as thrombin acquire specificity from exosite-dependent interactions. Peptide inhibition studies of protein-protein interactions are useful to identify exosite functions. We developed a novel general approach for exosite analysis using photocrosslinking of exosite inhibitory peptides that also carry a fluorescent tag and reporter. Here we demonstrate the method’s feasibility in studying the thrombin-hirugen (enzyme-peptide) complex. Thrombin contains two positively-charged exosites, named exosites I and II. Hirugen, the C-terminal residues 54 to 65 of hirudin, binds exosite I of thrombin. A series of desulfo-hirugen analogs were synthesized with either an additional fluorescein-(benzoyl) phenylalanine-(abbreviated Fl-bF-), or Fl-bF-mercaptopropionic acid or Fl-bF-lactic acid moiety conjugated to the N-terminal end of the peptide; i.e. a fluorescent label followed by a photocrosslinker and a cleavable linker in succession were conjugated to the N-terminus of hirugen analogs. Each hirugen analog was bound and photocrosslinked to thrombin, and the resulting covalent complex was purified from the reactants to homogeneity. These thrombin-hirugen adducts hydrolyzed small oligopeptide substrates (CBS 34-47 or S2366) with an efficiency similar to that of native thrombin, showing that the active site was unaffected by labeling. However, these adducts were significantly inhibited in assays of fibrinogen clotting and of thrombomodulin-mediated protein C activation, implying that exosite I was blocked, causing reduced accessibility for the substrate, fibrinogen, or for the cofactor, thrombomodulin. Exosite II mediates thrombin binding to heparin. The thrombin-hirugen adduct bound to and eluted from heparin-Sepharose like native thrombin, suggesting that exosite II was unaffected by the photocrosslinked hirugen analog. Treatment of the thrombin-(Fl-bF-mercaptopropionic acid) hirugen adduct that contains a thioester bond with the mild nucleophile hydroxylamine released the amino acids C-terminal to the thioester bond from the adduct, effectively releasing hirugen residues 54–65 from the complex while retaining the fluorescein reporter near exosite I (Fl-thrombin). This treatment did not affect the amidolytic activity of Fl-thrombin. Additionally, Fl-thrombin could be inhibited by unlabeled hirugen suggesting that exosite I was uncovered by release of the peptide by hydroxylamine treatment. In summary, our results show that exosites of clotting factors, e.g., thrombin, can be specifically targeted and labeled with fluorescent reporters. This novel technology may have broad applicability for studies of protein-protein interactions that regulate coagulation.
The CXCR4-Antagonist AMD3100 Augments the Number of Mobilized Peripheral Blood Progenitor Cells (PBPC) When Added to a G-CSF Standard Mobilization Regime and AMD3100-Mobilized PBPC Result in Rapid Hematopoietic Reconstitution after Autologous Transplantation.
Authors: Fruehauf Stefan , Seeger Timon , Topaly Julian , Herrmann Doris , Dillmann Falk , Humpert Per , Calandra Gary , Laufs Stephanie , Goldschmidt Hartmut , Ho Anthony D. 
Published: October 2019

Abstract
Sufficient mobilization of peripheral blood progenitor cells (PBPC) is pivotal for successful autologous transplantation. G-CSF has gained a confirmed and dominant role in standard mobilization regimens. Recent reports provided evidence for the importance of the SDF1/CXCR4 axis in hematopoietic stem cell trafficking. AMD3100 is a CXCR4 antagonist that induces rapid mobilization of CD34+ cells in healthy volunteers. We initiated a phase II study assessing the safety and potential of AMD3100 in patients with multiple myeloma (MM) and non-Hodgkin’s lymphoma (NHL). At the time of the report 6 patients with MM and 4 patients with NHL were enrolled (5 female, 5 male; age median 44, range 44–71 yrs; prior chemotherapy regimens median 3, range 1–8). All patients with MM were in stage IIA or IIIA. Patients with NHL were in stage IIB, IIIA, IIIE or IV. Mobilization treatment consisted of 5 days G-CSF (10 μg/kg, s.c. AM) and a single dose of AMD3100 (240 μg/kg, s.c.) in the evening of day 4, 10–11 hours prior to leukapheresis. As expected, following 4 days of G-CSF treatment the CD34+ cell count in the peripheral blood increased 22-fold (range 7,8–33) and there was a correlation between baseline and day 4 PB CD34+ counts (r=0,88). Addition of AMD3100 led almost to a tripling of circulating CD34+ cells within 10 h after administration (2,8-fold increase, range 1,85–4,74). On the other hand, there was no mobilization of B-cells (CD19) -thus giving no indication for the co-mobilization of tumor cells- and no mobilization of NK/T-cell subsets (CD2, CD3, CD4, CD8). Patients with low starting PB CD34+ counts profited most. There was no association between the SDF1 1-3A polymorphism and the mobilization efficiency following AMD3100+G-CSF vs. G-CSF mobilization: 21% of patients showed the heterozygous G/A phenotype and the remainder the G/G phenotype. Interestingly, SDF1-serum levels in patients increased significantly after addition of AMD3100. Per leukapheresis procedure 4,3 (range 2,6–12,1) * 10e6 CD34+ cells/kg body weight (bw) were collected. Adverse effects were mild, one patient reported of nausea and emesis, WHO grade I. To date, four patients have been transplanted after high-dose chemotherapy (Melphalan 200mg/m2 or BEAM) with 4,7 (range 2,4–6,05) * 10e6 CD34+ cells/kg bw. Hematopoietic reconstitution (leukocytes >1/nl and thrombocytes >20/nl) was observed within a median of 14 (range 12–17) and 13 (range 10–15) days, respectively and is sustained in all patients. Thus CD34+ cells mobilized with AMD3100 appear to be fully functional. In conclusion AMD3100 is a seminal new drug development in the field of stem cell transplantation with the highest potential in poorly mobilizing patients.
Mobilization of Human Hemopoietic Progenitor Stem Cell Via Modulating CXCR4/SDF-1 Using Galactofucan Sulfate.
Authors: Irhimeh Mohammad R. , Lowenthal Ray M. , Fitton J. Helen 
Published: October 2019

Abstract
Mobilization of peripheral blood progenitor cells for hematopoietic rescue following autologous transplantation is usually achieved with the chemokine G-CSF. Engraftment potential is increased when higher levels of the receptor CXCR4 are noted on the hematopoietic progenitor stem cells (HPCs). It was previously reported that intravenous treatment with fucoidan or dextran sulfate, which are sulfated high molecular weight compounds, increased numbers of circulating mature white blood cells and HPCs in mice and nonhuman primates. This treatment also led to an increase in the pro-inflammatory cytokines IFN-γ and IL-12 levels in mice. In vitro treatment of bone marrow mononuclear cells with IFN-γ can up-regulate the expression of CXCR4 on granulocyte precursors and monocytes. We obtained ethics approval and informed consent to study the mobilization effect of orally ingested GFSTM (Galactofucan Sulfate), a seaweed-derived fucoidan, in healthy human volunteers in a single blinded placebo controlled phase I/II clinical study. Flow cytometry was used to monitor CXCR4 receptor on CD34+ stem cells. When moderate quantities (3 g/day) of seaweed containing 10% GFSTM were ingested, a slight increase in the total number of HPCs (CD34+) in the peripheral blood (PB) was observed, from 1.38 to 1.69 cells/μL (p=0.22, n=6). In addition, there was a small increase in the percentage of HPCs that expressed CXCR4 surface receptor, from 0.59 to 1.47 cells/μL, which is equivalent to 43% to 63% (p=0.19, n=6). Moreover, when 3 g/day of 75% GFSTM was ingested, a greater increase in the total number of HPCs (CD34+) in PB was observed, from 1.65 to 1.84 cells/μL (p=0.04, n=23). Furthermore, the percentage of HPCs that expressed CXCR4 increased from 0.746–1.652 cells/μL, which is equivalent to 45% to 90% (p=0.0002, n=23). Cytokine analysis, which was performed using ELISA to test for SDF-1 and IFN-γ, showed a significant increase in the plasma level of these cytokines. SDF-1 level was elevated from 1979 to 2068 pg/mL (p=0.051, n=10) and the level of IFN-γ from 9.04 to 9.90 pg/mL (p=0.007, n=10). These results suggest that GFSTM may modulate CXCR4 or disturb the SDF-1 gradient between bone marrow and PB. IFN-γ might play a role in the up-regulation of the expression of CXCR4 on CD34+ cells. To the authors’ knowledge, this is the first report of mobilization of HPCs by disruption of CXCR4/SDF-1 interaction using oral fucoidan.
Molecular Profiling of CD34+ Cells from Diagnosis in CML Patients Identifies Low Expression of CD7 with High Expression of Proteinase 3 or Elastase as Predictors of Longer Survival.
Authors: Yong Agnes S.M. , Szydlo Richard M. , Goldman John M. , Apperley Jane F. , Melo Junia V. 
Published: October 2019

Abstract
Although most patients with chronic myeloid leukemia (CML) have the same initial molecular abnormality, the BCR-ABL fusion gene, the duration of chronic phase (CP) varies widely. To identify the possible molecular basis of this heterogeneity, we studied CD34+ cells collected at diagnosis from 68 CML-CP patients. In all cases the Philadelphia chromosome was the only cytogenetic abnormality identified. Cryopreserved cells had all been collected by leukapheresis within 3 months of diagnosis, when the patients still had high leukocyte counts prior to definitive treatment. Although the majority of patients were diagnosed in the pre-imatinib era and treated with hydroxyurea and/or interferon-α, 32 (47%) received imatinib during the course of their disease. The median age at diagnosis was 45.2 years (range 17.6–68.3), and the male: female ratio was 1.8: 1 (44 males, 24 females). Total RNA was extracted from CD34+ purified cells. Gene expression profiling using Affymetrix HG-U133A microarrays was performed on two subsets of patients, one comprising patients with an ‘aggressive disease’ who developed blastic transformation (BT) within 3 years of diagnosis (n=10) and, at the other extreme, patients with an ‘indolent disease’ whose BT occurred 7 or more years from diagnosis (n=9). There was no difference in the proportion of the CD34+ subsets of CD38+ Lin+, CD38+ Lin− or CD38− Lin− cells between patients in the indolent as compared to the aggressive CML groups. Microarray data were analysed using a combination of MAS 5.0, dChip, RMA and GeneSpring 5.0 software, and revealed 20 genes differentially expressed in patients with aggressive and indolent disease, which were validated by quantitative real-time polymerase chain reaction (qPCR). The correlation between microarray and qPCR expression measures was highly significant for 18/20 genes (p<0.005, Spearman’s Rho). In the complete cohort of 68 patients, a multivariate Cox regression model identified the combination of low CD7 expression with high expression of proteinase 3 (PR-3) or elastase as associated with longer survival (p=0.0004 and p=0.0006, respectively). The levels of cytoplasmic PR-3 and surface CD7 protein expression by flowcytometry were also of prognostic value (p=0.029 and p=0.031, respectively). Patients who had more than 11% CD34+ cells expressing surface CD7 protein at diagnosis had a poor survival. Conversely, patients with more than 2% CD34+ cells expressing cytoplasmic PR-3 had a superior survival. This differential pattern of gene expression probably reflects the intrinsic heterogeneity of the disease; if so, assessing expression levels of selected genes at diagnosis may be valuable in predicting duration of survival in patients treated upfront with imatinib and the newer tyrosine kinase inhibitors.
Fetal Hematopoietic Development in the Mouse Aorta-Gonad-Mesonephros Region Is Inhibited by Homeodomain-Interacting Protein Kinase 2.
Authors: Ohtsu Naoki , Nobuhisa Ikuo , Mochita Miyuki , Taga Tetsuya 
Published: October 2019

Abstract
Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of mouse embryo. Our previous studies showed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine 727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in the hematopoietic development. HIPK2 transcripts were found in the fetal hematopoietic tissues such as the AGM region at embryonic day (E) 10.5 and E11.5 and the E14.5 fetal liver as detected by RT-PCR and whole mount in situ hybridization. The functional analysis of HIPK2 was done by introducing wild-type and its mutant constructs into the primary culture of the mouse AGM region. Retrovirus mediated forced expression of HIPK2 with green fluorescent protein (GFP) in the AGM culture remarkably inhibited the emergence of GFP+ nonadherent cells. In coculture of virus-infected GFP+ nonadherent cells with OP9 stromal cells, no cobblestone-like area-forming colonies were detected in the HIPK2-infected cells, while control GFP virus had no effect on the colony formation. Similarly, when GFP+ nonadherent cells were sorted and cultured in semisolid medium, colonies were not formed from the HIPK2-GFP virus-infected AGM culture. These data suggest that HIPK2 suppresses differentiation of adherent endothelial-like cells to colony-forming hematopoietic cells or, alternatively HIPK2 inhibits proliferation of newly developed hematopoietic progenitors. Development of cells positive for CD45 (an immature hematopoietic cell maker) cells was suppressed by forced expression of HIPK2 in the AGM culture. To determine the domain important for the inhibitory activity of HIPK2, we constructed a series of point and deletion mutants. Overexpression of HIPK2 mutants in the AGM culture showed that a certain structure of the kinase domain as well as its C-terminally located structure but not the kinase activity is essential for the HIPK2-mediated inhibition of hematopoiesis. When nonadherent cells in the AGM culture were fractionated for their expression levels of CD45 and c-Kit (a receptor for stem cell factor), CD45lowc-Kit+ cells have a potential to form hematopoietic cell colonies. In the HIPK2-overexpressed AGM culture, emergence of CD45lowc-Kit+ cells was significantly inhibited. These data indicated that HIPK2 plays a negative regulatory role in the development of AGM hematopoiesis in mouse embryo.
Inhibition of Hypusination of Eukaryotic Initiation Factor 5a (eIF-5A) as a Novel a Synergistic Treatment Strategy in Imatinib-Treated BCR-ABL Positive Leukemias Identified by a Global Proteomics Approach.
Authors: Balabanov Stefan , Ziegler Patrick , Hartmann Ulricke , Kammer Winfried , Copland Mhairi , Priemer Martin , Wilhelm Thomas , Bokemeyer Carsten , Kanz Lothar , Holyoake Tessa , Nordheim Alfred , Bruemmendorf Tim 
Published: October 2019

Abstract
Imatinib (IM) is highly effective in the treatment of CML and remains the gold standard non-transplant therapeutic option. However, resistance to IM develops frequently, particularly in late stage disease. The aim of this study was to discover novel cellular Bcr-Abl targets leading to the identification of potential novel treatment options that would synergise with IM. Protein expression analysis of K562 leukemic cells after IM treatment was performed using 2-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF) and nano electrospray ionization tandem mass spectrometry (ESI-MS/MS). Differential protein expression of selected proteins was confirmed by Western blot analysis. Among others, we detected the IM-dependent down-regulation of the eukaryotic translation initiation factor 5A (eIF-5A), which is essential for cell proliferation. EIF-5a represents the only known eukaryotic protein activated by a unique, 2-step post-translational modification called hypusination, a process that can be specifically inhibited by hypusination inhibitors (HI), e.g. ciclopirox and GC7. We demonstrated that selective HI elicit a strong synergistic effect with IM on both cellular cytotoxicity (using MTT assay) and apoptosis (using PI staining) of Bcr-Abl positive leukemic cell lines and even more strikingly on primary CD34+ cells from CML patients at diagnosis. The effect of HI on CD34+ cells from healthy donors and CML patients was compared by short-term expansion in serum free medium using high resolution cell cycle analysis and carboxy-fluorescein succinimidyl ester (CFSE) labelling to track cell division. We observed a substantial anti-proliferative effect of the drug combination on Bcr-Abl positive as compared to Bcr-Abl negative cells. However, we were unable to demonstrate a significant cytotoxic effect of the combination on the quiescent Ph+ stem cell population. Despite this, HI exerted strong antiprolifertive effects not only on Ba/Fp210 but also on resistant Ba/F3p210 mutants T315I, E255V and M351T.
In conclusion, IM induced significant changes of global cellular protein expression in K562 cells, including down-regulation of eIF-5A. Inhibition of eIF-5A hypusination represents a promising target for combined therapy in Bcr-Abl positive leukemias. Ongoing studies are investigating whether novel, more specific, HI in combination with IM (and/or 2nd generation Bcr-Abl inihibitors e.g AMN107, BMS-354825) will be able to exert these effects on the quiescent Bcr-Abl+ stem cell compartment, and if these novel combinations are effective in IM-resistant cells.