bluuebunny/crossref_metadata_2025_split huggingface dataset:
Herpes Simplex Virus Infection of Endothelial Cells Is Enhanced by Thrombin.
Authors: Sutherland Michael R. , Friedman Harvey M. , Pryzdial Edward L. 
Published: October 2019

Abstract
Herpes simplex virus type 1 (HSV1) and type 2 (HSV2) are highly prevalent and form life-long infections. While acute effects are not often critical, chronic infection has been linked to vascular disease. To better understand the underlying basis for the clinical correlations, our lab has demonstrated that HSV can initiate and sustain clot formation directly on the virus surface, bypassing the normal cellular requirements. This is facilitated by anionic phospholipid and tissue factor on HSV1 and HSV2, and the participation of at least HSV1- encoded glycoprotein C (gC) in factor VIIa-dependent factor X (FX) activation, emulating tissue factor. The evolution of the HSV surface as a procoagulant agent suggests that generation of thrombin is advantageous to the viral lifecycle. The current study therefore examines the hypothesis that thrombin enhances infection. Several purified HSV1 strains were evaluated, including the wild-type lab strain, MacIntyre, and the clinical isolate, NS. These viruses were compared to gC-deficient HSV1, ns-1, and the gC-restored strain, rns. As an example of HSV2, strain G, was also examined. ns-1 has been shown previously to generate less FXa per particle than the gC-containing viruses. To restrict in situ thrombin production, human umbilical vein endothelial cells were inoculated in serum-free culture media. Consistent with our hypothesis, standard plaque-forming assays revealed that co-incubation of the virus and cells with purified thrombin enhanced infection by up to 400%. The observed half-maximal concentration was approximately 20 nM thrombin for all viruses studied. As a demonstration of specificity, hirudin attenuated the effects of purified thrombin. The involvement of the protease activated receptor 1 (PAR1) in this mechanism was shown using the functional signaling analogue, thrombin receptor activating peptide (TRAP). In the absence of serum, TRAP resulted in increased infection comparable to that of purified thrombin. Approximately half the effect of TRAP was observed at 10uM, while a PAR4-activating peptide had no effect. Enabling the production of thrombin during the infection process, plaque-forming assays were also conducted with serum in the cell culture media. The presence of serum enhanced infectivity by as much as 20-fold. At a constant hirudin concentration of 5 U/ml, infection was attenuated by approximately 50% for the gC-deficient strain, ns-1, whereas the other viruses were only marginally or insignificantly affected, supporting a role for thrombin generation in situ and the participation of gC. Collectively, these data show that thrombin production by HSV enhances infection through a mechanism involving PAR1-mediated cell modulation.
Differential Binding of Plasminogen, Plasmin, and Angiostatin4.5 to Cell Surface β-Actin: Implications for Cancer-Mediated Angiogenesis.
Authors: Soff Gerald A. , Wang Hao , Doll Jennifer A. , Jiang Keyi , Cundiff Deborah L. 
Published: October 2019

Abstract
Angiostatin4.5 (AS4.5), the naturally occurring human angiostatin isoform, is the product of plasmin autoproteolysis and consists of kringles 1 to 4 and ~85% of kringle 5. An important question related to tumor angiogenesis is the mechanism by which tumor cells convert plasminogen to angiostatin locally and yet the angiostatin acts systemically. We recently reported that cancer cell surface globular β-actin mediates plasmin autoproteolysis to AS4.5. We now characterize the binding of plasminogen, plasmin and AS4.5 to globular β-actin. Plasminogen and plasmin bind comparably to immobilized globular β-actin, with a KD of approximately 140 nM. The binding is inhibited by ε-aminocaproic acid (EACA), a lysine analog, indicating the requirement for a lysine-kringle domain interaction. Using a series of peptides derived from β-actin in competitive binding studies, we show that the actin domain necessary for binding of plasminogen is within “GDEAQSKRGILTLKY”, (amino acids 55 to 69), which includes lys61 and lys68. Substitution of lys61 or Lys68 with arginine results in the loss of the peptide’s ability to block plasminogen binding, indicating that lys61 and Lys68 are necessary for plasminogen binding. Other actin peptides, including peptides with other lysines, did not inhibit the plasminogen-actin interaction. While plasminogen and plasmin bind to actin efficiently, AS4.5 did not bind, even at high concentrations (500 nM). Plasminogen, plasmin, and AS4.5 all contain kringles 1–4, however, kringle 5 is truncated in AS4.5, suggesting that the kringle 5 is the domain by which plasminogen and plasmin bind to actin. We thus tested pure recombinant kringle 5 for its ability to specifically bind actin. Isolated kringle 5 was shown to bind to actin supporting the hypothesis that intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface β-actin, and the truncated kringle 5 in AS4.5 results in its release from β-actin. We propose the following paradigm: Plasminogen binds cell surface globular β-actin, where uPA mediates activation to plasmin which remains bound to actin. Actin then mediates plasmin autoproteolysis, with an intra-kringle 5 cleavage, resulting in AS4.5 formation. With the truncation of kringle 5, the AS4.5 dissociates from the surface actin and enters the systemic circulation. These data may help explain why AS4.5 is formed locally, on cancer cell surfaces, and yet acts systemically.
Difference in Outcome of Adolescents with Acute Lymphoblastic Leukemia (ALL) Enrolled in Pediatric (AIEOP) and Adult (GIMEMA) Protocols.
Authors: Testi Anna Maria , Valsecchi Maria Grazia , Conter Valentino , Vignetti Marco , Paoloni Francesca , Giona Fiorina , Moleti Maria Luisa , Nigro Luca Lo , Locatelli Franco , La Dogana Saverio , Specchia Giorgina , Fabiano Francesca , Recchia Anna , Mandelli Franco , Foa’ Robert , Masera Giuseppe 
Published: October 2019

Abstract
Progress in the treatment of acute lymphoblastic leukaemia (ALL) has led to better survival rates; however, children have had a greater benefit from improved treatment modalities than adolescent who show an overall lower event-free survival (EFS) compared to younger patients. Some differences in the clinical and biologic characteristics of adolescents compared to childhood ALL may partly account for the different outcome, but adolescents treated on pediatric ALL trials seem to have a significantly better EFS than those treated on adult trials. We retrospectively compared the results obtained in a series of 245 patients ranging in age from 14 to 18 years diagnosed and enrolled in specific Italian children and adult ALL trials, between 4/1996 and 10/2003. One hundred and fifty patients, from 30 pediatric centers, underwent the childhood AIEOP ALL 95 and 2000 protocols; the other 95, from 28 adult centers, were enrolled in the GIMEMA ALL 0496 and 2000 protocols. The AIEOP 95 and 2000 trials are BFM-like protocols with a 7 drug induction followed by risk-modulated post-remission therapy that includes high-dose MTX and reinduction for low and intermediate groups, and intensive blocks (high-dose MTX and cytarabine) for high-risk patients. Standard maintenance therapy is administered up to a total of 2 years. Cranial radiotherapy is limited to high-risk patients. Stem cell transplantation is planned for very high-risk patients. The GIMEMA regimens are instead based on an induction with high-dose anthracyclines (cumulative dose 550 mg/m2), high-dose cytarabine as consolidation and do not include high-dose MTX and the reinduction phase. Standard maintenance with vincristine + daunorubicin/cyclophosphamide pulses is given for 2 years. Cranial radiotherapy is administered to all patients. The main patients characteristics at diagnosis, in the two groups under examination, were comparable except for age: median age was 15 and 16 years, respectively in the AIEOP and GIMEMA trials.Poor risk cytogenetic translocations and T-immunophenotype were equally dinstributed. Adolescents in the AIEOP protocols had a higher CR rate (94% vs 89%) and a lower relapse rate (17% vs 45%) compared to the adolescents enrolled in the GIMEMA trials. The 2-year overall survival rate was 80% in the AIEOP protocols and 71% in the GIMEMA trials. Detailed results according to the different clinical and biologic features of the adolescents analyzed will be presented. The results of our comparative study indicate that adolescents enrolled in pediatric trials have a more favourable clinical outcome.
AMN107: In Vitro Profile of a New Inhibitor of the Tyrosine Kinase Activity of Bcr-Abl.
Authors: Mestan Juergen , Ellen Weisberg , Sandra Cowan-Jacob , Doriano Fabbro , Pascal Furet , Gabriele Fendrich , Gerard Goutte , Dominique Kempf , Markus Gaugler , James Griffin D. , Paul Manley W. 
Published: October 2019

Abstract
Imatinib mesylate (Gleevec®, Novartis Pharma AG), an inhibitor of the c-Abl/Bcr-Abl, c-Kit, and PDGFR kinases, is an effective therapy for CML, GIST and HES. However, advanced stage CML and Ph+ ALL patients frequently relapse due to the development of resistance. In up to 50% of cases, imatinib resistance is the result of the clonal expansion of cells expressing Bcr-Abl kinase domain mutants and more than 30 such mutations have been detected. Different mutations display varying degrees of resistance, and although some are sensitive to increased concentrations of imatinib, new Abl kinase inhibitors with higher potency against wild-type and imatinib-resistant mutants of Bcr-Abl could have substantial clinical utility. Rational drug design based on the structural information obtained from the crystal structure of the Abl-imatinib complex and medicinal chemistry optimization of drug-like properties, resulted in the discovery of AMN107. The potencies of AMN107 and imatinib were compared with cellular assays using CML lines (K562 and KU812F) and Ba/F3 cells expressing either ‘wt’- or imatinib-resistant mutants of Bcr-Abl. AMN107 effectively reduced the cellular Bcr-Abl autophosphorylation in K562, KU812F and Ba/F3 Bcr-Abl cells with IC50s of 42, 60 and 23 nM, respectively (Gleevec: 470, 399 and 231 nM) The proliferation of these cells was inhibited with IC50s of 11, 8 and 23 nM (Gleevec: 272, 80 and 634 nM). Autophosphorylation of the Bcr-Abl mutants M351T, F317L and E255V was reduced by AMN107 with IC50s of 33, 43 and 245 nM (Gleevec: 595, 818 and 6499 nM), resulting in inhibition of cell proliferation with IC50s of 30, 77 and 684 nM (Gleevec: 1285, 1583 and 6294 nM). No effect could be observed below 8 μM against the T315I and G250E bcr-abl mutants. Similar to imatinib, AMN107 inhibited the cellular tyrosine kinase activity of PDGFR-β (A31 cells) and c-Kit (GIST882 cells), with IC50s of 85 and 192 nM (Gleevec: 74 and 96 nM). Control (Ba/F3) cell proliferation was unaffected by 10 μM AMN107, and added IL-3 abrogated the antiproliferative effects on Bcr-Abl expressing Ba/F3 cells. Furthermore, AMN107 (< 10 μM), did not effect unrelated kinases, e.g. c-erbB2 (BT474), Ins-R (A14) and IGF-1R (NWT-21), in cellular assays, consistent with its specificity and lack of general cytotoxicity. Based on these in vitro assays and preclinical in vivo and pharmacokinetic data to be presented separately, AMN107 represents a promising new tyrosine kinase inhibitor for testing in human clinical trials.
Identification of Candidate Oncogenes in Acute Megakaryoblastic Leukemias with Gain of Chromosome 19.
Authors: Alvarez Sara , Largo Cristina , Blesa David , Gomez-Pineda Alfonso , Martin M. Carmen , MacGrogan Donal , Garcia Jose F. , Roncador Giovanna , Benitez Javier , Nimer Stephen D. , Cigudosa Juan C. 
Published: October 2019

Abstract
Acute Megakaryocytic leukemia is a rare subtype of AML that is often difficult to diagnose. Although GATA1 mutations are always found in AML-M7 associated with Down’s syndrome, such mutations are a rare event in adult AML-M7 leukemia. In 2001, we reported that Comparative Genomic Hybridization (CGH) identifies gain of chromosome 19 as part of the spectrum of nonrandom abnormalities characterizing megakaryoblastic proliferations, being its frequency underestimated when only conventional cytogenetics is used (1,2).
To identify candidate genes amplified and overexpressed on chromosome 19, we selected 10 cell lines derived from AML-M7 patients, that were previously characterized and classified by CGH according to their chr. 19 gain status. For each cell line, two hybridizations were performed: one with total RNA and one with DNA. Both hybridizations were done against the same c-DNA CNIO Oncochip, which allowed us to compare the results. As controls, 2 hybridizations with RNA obtained from CD34 cells selected from different leukapheresis were used to normalize the expression pattern from the cell lines.
First, we analyzed the expression values (normalized by the CD34+ cells) of the 201 c-DNA clones localized on chr. 19. The total number of overexpressed clones was higher in cell lines with gain or high level amplification of chr. 19 ( 38 to 79 of c-DNAs up) than in cell lines with normal chr. 19 or a discrete gain of 19q13.2 (29 to 31 of c-DNAs up). Second, examining the copy number changes and expression data allowed us to exclude some functionally interesting genes as MLL4 and to identify 36 clones that were recurrent and simultaneously gained and overexpressed. Eight out of these 36 clones showed at least three times greater expression in at least two cell lines. Six of these genes (KCNK6, APOC1, CD33, CEACAM5, CCNE1, and PPF1A3) are located on the q arm and two of them (NR2F6 and ASF1B) on the p arm. Interestingly, 80% of these 8 genes have at least one Gene Ontology annotated term relative to cell growth and/or maintenance. The dendogram of the unsupervised analysis of these 8 genes gives three arms that discriminate between 1) the cell lines with gain of chr. 19, 2) the cell lines with high level amplification of 19q, and 3) cell lines with normal chromosome19 or a discrete gain of 19q13.2. Moreover, the supervised analyses of the expression pattern of these 8 genes could identify significant differences among the four different states of chr.19 in the cell lines: high level amplification of 19q, gain of 19q13.2, gain of the whole 19, or normal 19, based on the expression of three genes: KCNK6, NR2F6, and APOC1. Further analyses, to identify the function of these genes in acute megakaryocytic leukemia cells are in progress.
Drug-Response Signature Predicts Outcome in Adult Acute Myeloid Leukemia and Associates Poor Response with Molecular Characteristics of Hematopoietic Stem Cells.
Authors: Heuser Michael , Wingen Luzie U. , Steinemann Doris , Cario Gunnar , von Neuhoff Nils , Tauscher Marcel , Bullinger Lars , Dohner Hartmut , Schlegelberger Brigitte , Ganser Arnold 
Published: October 2019

Abstract
Resistance to induction chemotherapy is of independent prognostic value in acute myeloid leukemia (AML). DNA microarrays were used to determine the gene-expression profile of AML blasts in 38 patients with good or poor response to induction chemotherapy. We selected an 11-sample training set, applying prediction analysis of microarrays (PAM) to devise a drug-response predictor which was tested on the remaining 27 samples and an independent set of samples recently published (Bullinger et al. 2004). Our drug-response predictor with 46 clones divided the 27 samples into two prognostic subgroups, the poor response group having a significantly shorter overall survival (P= .021). A subset of these 46 clones was sufficient to divide the published 62-sample test-set with intermediate risk cytogenetics into prognostically relevant subgroups (P= .028), adding prognostic information to that of known risk factors in multivariate analysis (hazard ratio, 2.8; 95 percent confidence interval, 1.4 to 5.8; P= .005). Thirteen of 39 drug resistance-enriched genes are known to be upregulated in hematopoietic stem/progenitor cells, and the expression pattern in normal CD34+ cells is highly correlated to the drug-resistance signature. This suggests that drug resistant AMLs show molecular features of hematopoietic stem/progenitor cells and can be identified by a characteristic gene-expression profile.
Pharmacology and Safety of SB-497115-GR, an Orally Active Small Molecular Weight TPO Receptor Agonist, in Chimpanzees, Rats and Dogs.
Authors: Sellers Teresa , Hart Timothy , Semanik Michael , Murthy Krishna 
Published: October 2019

Abstract
SB 497115-GR is a small molecular weight Tpo receptor (TpoR) agonist that has properties similar to thrombopoietin (TPO), primarily inducing proliferation and differentiation of megakaryocytes from bone marrow progenitor cells. SB-497115-GR is being developed for the treatment of thrombocytopenias, such as immune thrombocytopenic purpura. In vitro and in vivo studies have demonstrated that SB-497115-GR has very distinct species specificity. SB-497115 or other molecules in this class induced dose dependent STAT activation in platelets from humans and chimpanzees but not in platelets from laboratory animal species commonly used in drug safety studies. In order to demonstrate in vivo activity of SB-497115-GR, a single dose and 5 daily dose pharmacology and safety study in chimpanzees was conducted. To support initiation of clinical trials, a comprehensive package of toxicology studies was conducted including studies up to 14 days duration in rats and dogs. All procedures involving the care and use of animals in these studies were reviewed and approved by the appropriate Institutional Animal Care and Use Committees. Female chimpanzees (1–3/group) were administered vehicle or SB-497115-GR at doses of 0.1 to 10 mg/kg/day by oral gavage. For toxicology studies, SB-497115-GR was administered orally to rats (10/sex/group) by gavage at doses of 3 to 40 mg/kg/day and to dogs (3/sex/group) by capsule at doses of 3 to 30 mg/kg/day for 14 days. SB-497115-GR was well tolerated in chimpanzees, rats and dogs at all doses tested. In chimpanzees, no treatment related increases in platelet counts were observed after administration of single doses of up to 10 mg/kg or 5 daily doses of up to 3 mg/kg/day. However, following 5 daily doses of 10 mg/kg/day SB-497115-GR, there was a 1.3- to 2.4-fold increase in circulating platelet counts in 3 chimpanzees. A similar change in reticulated platelet counts was observed preceding this increase. In contrast, there was no effect of treatment for up to 14 days on platelet counts in rats or dogs. In conclusion, SB-497115-GR, an orally bioavailable small molecular weight agonist of the TpoR, has been shown to increase platelet counts in chimpanzees. These in vivo data confirm the in vitro data demonstrating the unique species-specific effects of this novel Tpo receptor agonist on platelets and were predictive of a pharmacodynamic effect currently being observed in human clinical trials.
Comparison of Three Tests for Confirmation of the Diagnosis of Heparin Induced Thrombocytopenia.
Authors: Messmore Harry L. , Choudhury Abdul M. , Fabbrini Nancy J. , Bird Mary L. , Moritz Thomas , Thottapurathu Lizzy , Wehrmacher William H. , Prechel Margaret , Walenga Jeanine M. , Crosby Thelma J. , Love Betty C. , Zarr Courtney A. , Griffin Beth E. , Balbale Sanober S. 
Published: October 2019

Abstract
In a recent two year period only 14% of platelet aggregation tests performed on 230 patients suspected of having heparin induced thrombocytopenia (HIT) were positive. The test (control platelet-rich plasma 0.14 mL, fresh heated patient serum 0.22 mL plus heparin 0.04 mL (1 Unit/mL final concentration) was performed using suitable controls on a commercial 4 channel aggregometer. 20% or greater increase in light transmission was a positive result. We then started a prospective study in which three different tests were done on the serum of each of 96 consecutive Hines Veterans Affairs Hospital patients who were clinically suspected of having HIT. The three tests were: platelet aggregation (PA) (same method as in the earlier study), C14 serotonin release (standard washed platelet method-SRA) and ELISA (GTI-PF4, Waukeshaw, Wisconsin), all performed on the same serum sample. The SRA and ELISA tests were performed on serum stored at −70°C. Blinded to the test results, a clinical score was determined from the record as HIGH (30% or greater fall in platelet count with recovery within 15 days off heparin), MEDIUM (same criteria but other cause present, or failure to recover platelet counts on continued heparin therapy) or LOW probability (failure to fall at least 30% or to recover from 30% or greater fall within 15 days observation). Results of the clinical scoring and laboratory tests were as follows: Clinical Probabilities: High (n=23), Medium (N=33), Low (n=40). Of the 56 who were High or Medium probability, 41% were High and 59% were Medium probability. Analysis of the results of the laboratory tests for sensitivity and specificity for each category revealed the following:
Clinical Probability PA SRA ELISA Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity High and Medium (n=56) 19.6% 97.5% 10.7% 97.5% 10.7% 90% High (n=23) 30.4% 93.1% 17.4% 95.9% 8.7% 89%
Conclusions: Sensitivity of the PA test was greater than either of the other two tests, with specificities for the PA and SRA being nearly equal. The ELISA test was relatively insensitive and less specific than then other two.
Novel Lentiviral Vectors Displaying ‘Early-Acting-Cytokines’ Preferentially Promote the Survival and Transduction of NOD/SCID Repopulating Human Hematopoietic Stem Cells.
Authors: Verhoeyen E.L.S. , Wiznerowicz Maciej , Olivier Delphine , Izac Brigitte , Trono Didier , Dubart-Kupperschmitt Anne , Cosset Francois L. 
Published: October 2019

Abstract
A major limitation of current generation lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent target cells which hampers their application for hematopoietic stem cell gene therapy. Human CD34+ cells that reside into G0 phase of the cell cycle and thus are quiescent, are indeed higly enriched in hematopoietic stem cells. Here, we designed novel lentiviral vectors that overcome this type of restriction by displaying early-acting-cytokines on their surface. Presentation of a single cytokine, thrombopoietin (TPO), or co-presentation of TPO and stem cell factor (SCF) on the lentiviral vector surface improved gene transfer into quiescent CD34+ cord blood cells by 45-fold and 77-fold, respectively, as compared to conventional lentiviral vectors. Moreover, these new LVs preferentially transduced and promoted the survival of immature resting cells rather than cycling CD34+ cells. Most importantly, the new early-cytokine-displaying lentiviral vectors allowed highly efficient gene transfer in CD34+ immature cells with long-term in vivo NOD/SCID mice repopulating capacity, a hallmark of bona fide HSCs. In conclusion, the novel ‘early-acting cytokines’ displaying LVs described here provide simplified, reproducible gene transfer protocols that ensure efficient gene transfer in hematopoietic stem cells. As such, these novel reagents bring us one step closer to selective in vivo gene therapy.