bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inducible Caspase 9 as a Safety Switch in Genetically Modified Cytotoxic T Cells.
Authors: Straathof Karin C. , Pule Martin A. , Yotnda Patricia , Dotti Gianpietro , Vanin Elio F. , Brenner Malcolm K. , Heslop Helen E. , Spencer David M. , Rooney Cliona M. 
Published: October 2019

Abstract
T cell therapy has proven effective to prevent and treat viral infections and immunogenic tumors. Application of this strategy to less immunogenic tumors that employ tumor evasion mechanisms may require genetic modification of the infused cells. However, oncogenic events due to vector/transgene integration, and toxicities due to the infused cells themselves have tempered enthusiasm. A safe and efficient means of destroying aberrant cells in vivo would ameliorate these concerns. The xenogeneic Herpes Simplex Virus I-derived thymidine kinase gene has been used as a suicide strategy for this purpose, but its immunogenicity results in immune elimination of the infused T cells and precludes administration of anti-viral drug ganciclovir for other indications. We adapted an alternative strategy using inducible human death molecules. Human caspase 9 was fused to a modified FK506 binding protein that allows for conditional dimerization using a chemical inducer of dimerization (CID). Administration of this small molecule pharmaceutical that has proven safe in healthy volunteers (Iuliucci et al, J Clin Pharmacol 2001, 41:870–879), activates the caspase molecule resulting in apoptosis of the cell. This system consists of human gene products with low potential immunogenicity. We chose caspase 9 as the death molecule since it is highly sensitive to dimerization and acts late in the apoptosis pathway, thereby reducing the risk of impairment of its function by cellular inhibitors of apoptosis. However, only low-level expression of the full-length inducible Caspase 9 (iCasp9) construct was compatible with survival of CTL. Deletion of the caspase recruitment domain (CARD) and removal of one of the two FKBPs allowed expression of iCasp9 in >70% of primary human T cells after a single transduction with a retrovirus vector. In vitro a single 10 nM dose of CID induced apoptosis in > 99% of T cells sorted for high transgene expression. A similarly high percentage of iCasp9-expressing T cells underwent apoptosis after CID administration in vivo in a mouse SCID human xenograft model. This functional activity is a log greater than previously available self-destruction systems. Importantly, even without sorting, activation of iCasp9 when co-expressed with transgenic IL-12 (a cytokine with potent anti-tumor activity) resulted in elimination of all IL-12 secreting cells. Unlike inducible Fas, iCasp9 maintained its function in MT-2 cells that over-express the anti-apoptotic molecule bclxl, demonstrating the importance of using a death molecule that acts downstream of the death inhibitors. Since iCasp9 does not impair T cell phenotype or function, these characteristics favor incorporation of inducible caspase 9 as safety feature in human T cell therapies.
Transduction and Expansion of HIV+ CD4 T Cells with an HIV-1 Based Lentiviral Vector and Immobilized CD3/CD28 Antibodies Maintains the Diversity of the TCR Vβ Repertoire.
Authors: Lemiale Franck , Pereira Mario , Humeau Laurent , Dropulic Boro 
Published: October 2019

Abstract
Recently, we initiated the first ex vivo HIV-based gene therapy trial in humans with HIV+ CD4+ T cells. In this protocol, a modified lentiviral vector carrying an anti-HIV payload is used to modify CD4+ T cells isolated from HIV-infected patients by apheresis and CD8 negative selection. The T cells are activated in the presence of vector and expanded using immobilized CD3/CD28 antibodies, and then infused back into the patient. T cell receptor (TCR) repertoire analysis has value for safety monitoring of adoptive T cell transfers in the detection of aberrant clonal expansions or deletions. In this study, the TCR Vβ repertoire was assessed using a flow cytometry based assay at various time points in the selection/transduction/expansion process of CD4+ T cells. PBMC isolated from whole blood of HIV+ patients were CD4-selected using a CD8 negative selection, followed by enrichment by CD3 antibody. CD4+ purified cells were transduced with the lentiviral vector, VRX496, in the presence of retronectin, and then co-cultured with CD3/CD28 coated M450 Dynabeads for ten days. The TCR Vβ repertoire was assessed in throughout the process using a FACS-based assay that employs a panel of 20 monoclonal antibodies recognizing most of the 24 Vβ families in PBMC and CD4+ T cells. Repertoires from subjects with normal polyclonal TCR profiles were conserved, as shown by the absence of any significant change in any Vβ family. Moreover, the transduction/expansion of CD4+ T cells from a patient with a previously skewed TCR profile allowed the improvement of the TCR Vβ repertoire. Finally, no significant difference was observed in the repertoire of cells transduced with VRX496 versus mock-transduced cells. These data demonstrate stability of the repertoire diversity and thus provide important support information in favor of the safety of a gene therapy approach involving lentiviral vector mediated modification and expansion of CD4+ T-cells.
Fondaparinux in the Treatment of Heparin-Induced Thrombocytopenia.
Authors: Bradner James , Hallisey Robert K. , Kuter David J. 
Published: October 2019

Abstract
The development of new non-heparin anticoagulant therapies has increased interest in the treatment of type II heparin-induced thrombocytopenia (HIT). Standard therapy involves administration of an intravenous direct thrombin inhibitor (DTI), either argatroban or lepirudin. Due to a lack of cross-reactivity to HIT antibodies in vitro, the synthetic pentasaccharide fondaparinux sodium is an appealing alternative, but its use in this disorder has not been comprehensively examined. Here, we report our experience with fondaparinux in HIT and an analysis of safety and efficacy in this setting. We reviewed all in-patient cases of HIT at the Massachusetts General Hospital that were confirmed by the heparin-platelet factor 4 antibody test and were treated with fondaparinux. Twenty patients satisfied diagnostic criteria for HIT. Each patient studied lacked another competing, compelling etiology for thrombocytopenia and was treated with fondaparinux for more than five days within one month of diagnosis. The average duration of therapy was 17 days and 16 patients (80%) were treated within two weeks of diagnosis. Patients were grouped according to whether they received fondaparinux after initial therapy with a DTI (n=10) or as initial treatment following the diagnosis of HIT (n=10). The initial dose of fondaparinux administered was 2.5 mg in 18 patients. Neither absolute nor relative thrombocytopenia was identified in either group following treatment. Patients with HIT treated with fondaparinux while thrombocytopenic experienced platelet recovery on average in 3.7 days, as represented in Figure 1. In addition, there were no observed thrombotic complications among patients treated with fondaparinux. In this analysis of a high-risk group of inpatients with type-II HIT, fondaparinux was tolerated without the continuation or recurrence of thrombocytopenia. This suggests that cross-reactivity between the heparin-PF4 antibody and fondaparinux is not likely to be clinically significant. Additionally, the absence of thrombotic sequelae among fondaparinux-treated patients argues that fondaparinux may be a well-tolerated, effective treatment for this morbid, prothrombotic disorder. A prospective trial of fondaparinux in the treatment of type-II HIT is planned.
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Does All-Transretinoic Acid (ATRA) Have a Role in Non-APL Acute Myeloid Leukaemia?: Results from 1666 Patients in Three MRC Trials.
Authors: Burnett Alan K. , Milligan Donald , Hills Robert K. , Goldstone Anthony H. , Prentice Archie G. , Wheatley Keith , Webb David C. , Gibson Brenda 
Published: October 2019

Abstract
ATRA has a key role in the treatment of APL where the presence of the PML-RARα protein is predictive of response. Pre-clinical data and some non-randomised clinical studies suggested that ATRA may enhance the effect of chemotherapy particularly based on its potential to reduce the Bcl2 expression which may thereby improve apoptotic responses to chemotherapy. We have evaluated this possibility in 3 randomised trials in remission induction in patients under 60 years (AML12) n=1097; in relapse (AML-HR) n=362; and as a non-intensive approach in older patients (AML14-non-intensive) n=207. In AML12 patients were randomised to receive standard induction chemotherapy options (ADE vs DAT or MAE vs DAT with Ara-C at either Ara-C 200mg/m2/day or 400mgs/m2/day). There was no significant difference in CR rate (83% vs 85%) or survival at 5 years (40% vs 36%) on ATRA or no ATRA overall or in any demographic, risk or treatment subgroup.
Patients with high risk disease (adverse cytogenetics or less than PR after course 1 of induction) or relapse were randomised to receive Fludarabine/Ara-C or ADE with or without G-CSF, with or without ATRA for 60 days. The addition of ATRA made no overall impact on remission rate (61% vs 61%) or survival at 3 years (23% vs 27%). There was no subgroup where an advantage was seen.
In AML14 patients not considered fit for intensive treatment were randomised to receive Hydroxyurea or Low Dose Ara-C with or without ATRA for 90 days. There was no evidence that patients who received ATRA derived any benefit either overall or within the treatment arms.
In this extensive assessment, no advantage of adding ATRA to chemotherapy - as a continuous daily dose during induction - could be detected in any trial or in any subgroup within each trial.
Reaction of Fibrin-Bound Thrombin with a Covalent Antithrombin-Heparin Complex Results in an Anticoagulant Surface.
Authors: Smith Lesley J. , Berry Leslie R. , Chan Anthony K. 
Published: October 2019

Abstract
Thrombin bound to fibrin is protected from inhibition by mixtures of antithrombin (AT) and heparin (H). Resistance of thrombin to reaction with AT + H when fibrin is present results from the formation of a ternary fibrin:thrombin:H complex. However, recent investigations using an antithrombin-heparin conjugate (ATH) showed that the covalent ATH complex rapidly inhibits fibrin-bound thrombin, with the thrombin-ATH product remaining attached to the fibrin. One intriguing possibility from these findings was that the heparin chain of ATH (or thrombin-ATH) on the fibrin surface might still be available to interact with fluid-phase molecules. We studied the anticoagulant activity of fibrin-bound ATH by determining the ability to catalyze the inhibition of added thrombin + AT. Excess fibrin monomer (polymerization inhibited by GPRP) was mixed with thrombin to form soluble complexes. Fibrin:thrombin was then incubated in the absence or presence of AT, H, AT + H or ATH, at various concentrations relative to the thrombin. Following treatment with anticoagulant, a vast excess of exogenous thrombin + AT was added and the ability of fibrin:thrombin-bound heparin to catalyze inhibition of the exogenous thrombin was assessed by measuring the loss of thrombin activity (chromogenic substrate assay). In the absence of heparin, reaction of exogenous thrombin + AT was essentially unaffected by the presence of soluble fibrin:thrombin. When fibrin:thrombin complex was mixed with AT + H (concentrations equimolar to thrombin), an insignificant ability to catalyze inhibition of excess thrombin by AT was observed (remaining thrombin activity = 49 +/− 4 vs 43 +/− 3 mOD/min for reaction with AT + H vs without AT + H, respectively). However, when similar concentrations of ATH were combined with fibrin:thrombin, a marked catalytic activity was seen (remaining thrombin = 34 +/− 3 mOD/min). Furthermore, the significant catalytic effect of ATH could be demonstrated over a wide range of concentrations relative to fibrin:thrombin. At these same concentrations, AT + H had little effect on exogenous thrombin + AT reactions. We conclude that although AT + H is unable to effectively inhibit fibrin-bound thrombin, covalently linked ATH not only overcomes this defect but can convert the fibrin:thrombin into a new site for anticoagulant activity to catalyze thrombin inhibition by exogenous AT. If clot-based antithrombin activity is demonstrated in vivo, we speculate that ATH may be useful for transforming thrombi from procoagulant to anticoagulant entities.
Antithrombotic and Hemorrhagic Effects of DU-176b, a Novel, Potent and Orally Active Direct Factor Xa Inhibitor: A Wider Safety Margin Compared to Heparins and Warfarin.
Authors: Furugohri Taketoshi , Honda Yuko , Matsumoto Chikako , Isobe Koji , Sugiyama Nobutoshi , Morishima Yoshiyuki , Shibano Toshiro 
Published: October 2019

Abstract
DU-176b is a novel potent, orally active and selective direct inhibitor of factor Xa (FXa). Direct FXa inhibitors have been reported to exert little effect on bleeding time at antithrombotic doses in animal studies. The aim of the present study was to compare the antithrombotic and hemorrhagic effects of DU-176b with unfractionated heparin (UFH), low molecular weight heparin (LMWH; dalteparin) and warfarin in rat models of thrombosis and hemorrhage. Rats were treated with DU-176b, UFH and LMWH by continuous intravenous infusion for 2 – 2.5 h, and with warfarin orally once daily for 4 days before thrombosis or hemorrhage. Thrombosis was induced by the insertion of a platinum wire into the inferior vena cava and left for 60 min. Tail template bleeding time was measured after an incision on the tail. DU-176b dose-dependently inhibited thrombus formation in the venous thrombosis model. The dose required for 50% inhibition (ED50) was 0.076 mg/kg/h. In contrast, the dose of DU-176b to double template bleeding time (BT2) was 0.75 mg/kg/h, indicating 10-fold dissociation of the doses of antithrombotic and hemorrhagic effects. UFH, LMWH and warfarin also prevented thrombus formation (ED50 = 56 U/kg/h, 66 U/kg/h and 0.16 mg/kg/day, respectively), but prolonged bleeding time at slightly higher doses (BT2 = 73 U/kg/h, 135 U/kg/h and 0.21 mg/kg/day, respectively) than the effective doses. The dissociation of the doses for these compounds was only 1.3, 2.0 and 1.3-fold, respectively. Moreover, the slope of dose-antithrombotic response curve of DU-176b was significantly slighter than those of UFH, LMWH and warfarin, indicating that the therapeutic dose range of DU-176b would be wider than those of the other anticoagulants. These results suggest that direct and selective inhibition of FXa by DU-176b is preferable for the treatment of thrombotic diseases in the aspect of lack of compromising primary hemostasis.
Relationships between Risk Factors for Coronary Artery Disease and Aspirin Resistance.
Authors: Schwartz Kenneth A. , Schwartz Dianne E. , Barber Kimberly R. , Reeves Mathew R. , Hall Janette M. , De Franco Anthony C. 
Published: October 2019

Abstract
Aspirin prevents or delays atherogenesis and subsequent arterial occlusions. Patients who are relatively resistant to aspirin have a higher rate of vascular occlusive events. We evaluated the hypothesis that in patients with a previous myocardial infarction (MI) relative resistance to aspirin was related to risk factors for coronary heart disease (CHD). We studied 192 patients with a history of at least 1 MI including 136 males and 64 females. Mean age was 62. Compliance was assured by studying patients 2 hours after they were observed to ingest 325mg aspirin and confirmed with a qualitative assay (arachidonic acid 1.0mM stimulated light aggregometry) that detected aspirin inhibition of platelet aggregation in all of the study patients. To assess amount of aspirin inhibition of platelet aggregation (aspirin resistance) platelet prostaglandin agonist (PPA) 30mM stimulated light aggregation was performed (Schwartz J. Lab Clin Med2002;139:227). A CHD risk assessment score that includes diabetes, HDL, total cholesterol, high blood pressure (HBP) smoking and age was used as a measure of 10 year risk for CHD. Statistical comparisons were evaluated using a univariate general linear model. A significant relationship was observed between platelet resistance to aspirin inhibition and increased CHD risk (p=0.03). Evaluation of individual CHD risk factors showed that diabetic patients were more resistant to aspirin as demonstrated by an increased slope of aggregation (p=0.02) a decreased time to 50% aggregation (p=0.04) and a linear relationship between an increase in fasting blood sugar and an increased aggregation slope (p=0.05). A higher (BMI) (p=0.05) or a lower HDL (p=0.02) were also related to aspirin resistance. Although decreased aspirin inhibition of platelets was observed in patients who smoked or had HBP, these differences were not significant. We conclude that in patients with CHD a relative resistance to aspirin inhibition of platelet aggregation correlates with a commonly used risk assessment equation and with the presence of diabetes, increasing BMI or decreasing HDL. This suggests the hypothesis that CHD patients who have a higher risk for CHD, or those who have diabetes or obesity or a decreased HDL, may benefit from additional platelet inhibiting medications.
Specific Inhibition of BCR-ABL Gene Expression in Acute Lymphoblastic Leukemia by Catalytic DNAzymes.
Authors: Taube Tillmann , Shalapour Shabnam , Seifert Georg J. , Pfau Madlen , Henze Guenter , Seeger Karlheinz 
Published: October 2019

Abstract
The BCR-ABL fusion protein p190 resulting from the translocation t(9;22) exhibits dysregulated tyrosine kinase activity and was shown to cause acute lymphoblastic leukemia (ALL). Detection of the BCR-ABL fusion gene in childhood ALL is associated with an adverse prognosis and defines a group of high risk patients. Because the BCR-ABL gene fusion is specific for leukemic cells it represents an ideal target for leukemia specific treatment approaches.
Catalytic DNAzymes are able to cleave mRNA in a sequence specific manner, causing inhibition of protein translation from the DNAzyme targeted mRNA both in vitro and in vivo. In order to cut off the BCR-ABL driven malignant proliferation, we designed DNAzymes to impede the expression of p190 BCR-ABL by cleaving the BCR-ABL mRNA adjacent to the fusion site. One construct was found that cleaved the target mRNA efficiently and specifically leaving BCR and ABL, relevant for normal cell survival and proliferation, unaffected. Activity and specificity of the BCR-ABL DNAzyme was investigated in cleavage assays with in vitro transcribed BCR-ABL, BCR and ABL mRNA. DNAzymes were delivered to cultured BCR-ABL+ ALL cells by lipid transfection. The efficiency of cellular delivery reached 90% as studied by flow cytometry, fluorescence microscopy and confocal microscopy after transfection of FITC labeled DNAzymes. To control for unspecific effects of DNAzyme delivery as well as for antisense effects, a catalytically inactive DNAzyme still exhibiting BCR-ABL antisense activity was designed. Fourty-eight hours after a single treatment of BCR-ABL+ ALL-cells with DNAyzmes the BCR-ABL mRNA concentration, as measured by quantitative real-time RT-PCR, was significantly reduced by 56% and 66% compared to controls treated with the inactivated DNAzyme and to untreated cells, respectively. Western blot analysis showed a decrease in p190 protein levels after DNAzyme treatment in comparison to the control treated with inactive DNAzyme as well as to the untreated cells. Most noteworthy, four days after a single DNAzyme treatment the net growth of BCR-ABL+ ALL cells treated with the active DNAzyme was inhibited by 68% compared to the untreated control.
From these data we conclude, firstly, DNAzymes targeting mRNA coding for the minor BCR-ABL variant are able to significantly reduce the amount of fusion mRNA in the cells, leading to a reduction in protein expression, followed by the inhibition of BCR-ABL driven proliferation of ALL cells. Secondly, this exemplified setting gives a hint that DNAzymes might be of therapeutic use in hematopoietic malignancies associated with specific mutations, expressing oncogenic fusion genes or overexpressing oncogenic genes.
DDAVP at a Maximal Dose of 15 ug Administered Subcutaneously Is a Safe and Effective Alternative to 20 ug IV for Patients > 50 kg with von Willebrand Disease or Mild Hemophilia A.
Authors: Mangel Joy , Laudenbach Lori , Schembri Sheila , Jardine Lawrence , Lohmann Reinhard 
Published: October 2019

Abstract
Background: DDAVP (desmopressin) is commonly used for the prophylaxis and treatment of bleeding in patients with mild forms of von Willebrand disease (VWD) and Hemophilia A (HemA). The standard dose is 0.3 ug/kg IV, to a maximum of 20 ug. In 1995, our Bleeding Disorders Program began to use a maximum dose of 15 ug both for DDAVP challenges and for therapeutic purposes. This dosing change was triggered by the new availability of 15 ug (1 ml) vials of DDAVP. We also switched to subcutaneous administration rather than intravenous, given the smaller volume (1 ml), and the body of evidence supporting this route of administration (De Sio et al, Thrombosis and Hemostasis1985; 54:387–9). With this strategy, patients weighing <50 kg continue to receive a weight-based dose, but those weighing ≥ 50 kg receive 15ug.
Method: In order to evaluate this new dosing regimen, a retrospective review was performed of 62 patients with VWD (n=36) or mild hemophilia (n=26) weighing ≥ 50kg who underwent DDAVP challenges using a 15 ug s/c dose of DDAVP. Data was also collected on the 9 patients (5 VWD, 4 HemA) tested prior to 1995 who received 20 ug IV of DDAVP.
Results: Levels of von Willebrand factor antigen (vWFag), ristocetin cofactor activity (RCof) and factor VIII (FVIII) were measured immediately prior to and 1 hour after administration of DDAVP. In the VWD patients (14M, 22F), median baseline levels of vWFag, RCof and FVIII were 31, 27 and 45 U/ml respectively, and rose to a median of 93, 90 and 177 U/ml following a 15 ug dose of DDAVP. This resulted in a median increase of 64 for vWFag (range 0–180), 55 for RCof (24–191) and 121 for FVIII (21–237). Results were comparable in the 5 pts who received 20 ug doses of DDAVP, achieving median increases of 77 (range 17–125), 53 (40–91) and 98 (79–184) respectively. In the HemA patients (19M, 7F), median baseline level of FVIII was 23 U/ml, and rose to a median of 57 U/ml following 15 ug DDAVP. Median increase in FVIII levels was 33 U/ml (range 2–108). These results compare favorably to the 4 patients who received 20 ug DDAVP and achieved a median FVIII increase of 19 U/ml (range 9–60). Chart review identified 26 patients (14 VWD, 12 HemA) who successfully received 15 ug doses of DDAVP in 44 clinical settings, either for the treatment of an acute bleeding episode or as prophylaxis prior to a planned surgical or dental procedure.
Conclusions: DDAVP given subcutaneously to a maximum dose of 15 ug appears to be a safe and effective alternative to 20 ug IV for pts > 50 kg with VWD or mild hemophilia A.