bluuebunny/crossref_metadata_2025_split huggingface dataset:
The EGFR-Inhibitor Erlotinib Induces Apoptosis in Neoplastic Cells of Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), an Effect Determined by the Expression Level of Nucleophosmin (NPM).
Authors: Boehrer Simone , Ades Lionel , Fabre Claire , Fenaux Pierre , Kroemer Guido 
Published: April 2020

Abstract
Background: the epidermal-growth-factor-receptor (EGFR)-inhibitor erlotinib was rationally designed to antagonize the deregulated EGFR-activity in solid tumors. Abundant studies in these entities not only demonstrated clinical efficacy, but also a favorable toxicity profile. In particular the absence of hematopoietic toxicity prompted us to investigate the therapeutic potential of erlotinib in MDS and AML cells.
Methods: We incubated ex vivo cells from patients with MDS (n=4, 2 lower risk and 2 higher risk) and AML (n=6, de novo: 3; post MDS: 3), as well as a broad spectrum of myeloid cell lines (P39, KG-1, HL-60, MV4-11, MOLM-13) with increasing dosages of erlotinib (1μM to 10μM). As controls (n=4) we used non-malignant CD34 + bone marrow cells. Before incubation, all ex vivo cells underwent CD34 + selection. Serial FACS-analyses of parameters determining apoptosis (DIOC/PI and AnnexinV/PI) were carried out over a maximum of 6 days.
Results: We found that erlotinib was able to induce a considerable degree of apoptosis in MDS and AML cells. Although there was a high interindividual difference in sensitivity towards erlotinib, “responders” treated with 10μM erlotinib showed an increase of apoptotic cells between 20–30% after 72h, which reached a maximum of 60% on day 6. This apoptosis-inducing effect was achieved in a dose-dependent manner and not restricted to a specific entity. Noteworthy, erlotinib exhibited no toxicity towards non-neoplastic progenitor cells. Evaluating the molecular mechanisms determining sensitivity we showed that the apoptosis-inducing effect of erlotinib critically depended on the expression level of NPM. Thus erlotinib-resistant myeloid cell lines (i.e. P39) exhibited a higher epression of NPM than sensitive cell lines (i.e. KG-1). In addition, down-regulation of NPM by small-interfering RNA not only increased the apoptosis-inducing effect of erlotinib in sensitive cells, but moreover established sensitivity in otherwise erlotinib-resistant cells. Accordingly, siRNA-induced down-regulation of NPM in P39 cells elevated the percentage of apoptotic cells upon treatment with 10μM erlotinib by about 30% as compared to mock-transfected controls. Conclusion, we showed an off-target effect of erlotinib, as evidenced by its ability to induce apoptosis in EGFR-negative cells. Of particular interest is the observation that erlotinib induced apoptosis exclusively in neoplastic myeloid cells while sparing non-malignant progenitors. To the best of our knowledge, this is the first report providing evidence for the therapeutic potential of erlotinib in MDS and AML.
CD28 Costimulation Induces Transcription of SKP2 and CKS1, the Substrate Recognition Components of the Skp1-Cullin-F-box Ubiquitin Ligase, SCFSkp2.
Authors: Appleman Leonard J. , Chernova Irene , Li Lequn , Boussiotis Vassiliki 
Published: April 2020

Abstract
Cellular immune responses require expansion of antigen-specific T cell clones from the pool of resting T lymphocytes that perform immune surveillance. Regulation of this proliferative potential is critical for defense against pathogens and avoidance of autoimmunity. Costimulation through the T cell receptor and accessory molecule, CD28, triggers replication of T lymphocytes in response to antigen that is mediated via both IL-2-dependent and IL-2-independent mechanisms. This event is associated with decreased abundance of the cyclin-dependent kinase inhibitor p27kip1 and increased cyclin D/cdk4 and cyclin E/cdk2 enzymatic activity. We have previously reported that in contrast to p27kip1 protein, the abundance of P27KIP mRNA transcript is unchanged in response to TCR/CD28 T cell activation. Ubiquitin-targeted degradation by the proteasome regulates the abundance of p27kip1 protein and ubiquitination of p27kip1 occurs in primary human T cells activated through the TCR and CD28. Ubiqutination of p27kip1 is mediated by a Skp1-Cullin-F-box (SCF) family ubiquitin ligase, SCFskp2. The SCFskp2 holoenzyme is comprised of the core components Roc1, Cul1, Skp1, and the substrate recognititon components, an F-box (cyclin F homology) protein, Skp2, that in cooperation with a smaller subunit, Cks1, mediates recognition of p27kip1 substrate. Targeted deletion of the murine Skp2 or Cks1 genes results in accumulation of p27kip1 in multiple cell types. T cell proliferation in response to TCR/CD3-plus-CD28 costimulation is reduced in Skp2−/− mice in comparison to wild-type controls, confirming the role of Skp2 in CD28 costimulation mediated degradation of p27kip1 and T cell proliferation. Here, we examined the mechanisms that regulate Skp2 and Cks1 abundance in primary T lymphocytes in response to TCR/CD3-plus-CD28 costimulation. Using primary human T lymphocytes we observed that the SCFskp2 core components Roc1, Cul1 and Skp1 are constitutively expressed and their levels remain unchanged upon TCR/CD3-plus-CD28 costimulation. In contrast, TCR/CD3-plus-CD28 costimulation lead to striking induction of SKP2 and CKS1 mRNA, and this event is dependent on PI3K/PKB and MEK/ERK signaling pathways. We determined that the SKP2 promoter lies directly upstream of the translation start site and contains binding sites for SP1, Elk-1 and E2F transcription factors. Mutagenesis of SP1 and Elk-1 sites abrogated TCR/CD3-plus-CD28-mediated SKP2 promoter-driven reporter activity, whereas mutagenesis of E2F site enhanced reporter activity, suggesting that SKP2 promoter may act as a node of integration for mitogenic and anti-mitogenic signals. Thus, in primary T lymphocytes CD28 costimulation can directly regulate cell cycle progression by inducing transcription of the substrate recognition components of SCFskp2 ubiquitin ligase that targets p27kip1 for degradation.
Outcomes during and after Pregnancy in Patients with Primary Hypercoagulable States.
Authors: Khalid Aysha , Cohen Alice J. 
Published: April 2020

Abstract
Pregnant women with primary hypercoagulable states have an increased risk of recurrent fetal loss, fetal growth retardation, preclampsia, and placental abruption, as well as thromboembolism in both the antepartum and postpartum periods. Conditions that have been associated with adverse pregnancy outcomes include inherited gene mutation disorders such as Factor V Leiden G1691A (FVL); prothrombin gene mutation G20210A (PGM); hyperhomocysteinemia with C677T mutation (MTHFR); deficiencies of protein S (PS), protein C (PC), antithrombin III (ATIII); and anticardiolipin antibodies/lupus anticoagulants (LA). Screening by obstetricians for these disorders has led to an increase in diagnosis yet there are no established guidelines and therapeutic interventions are variable. A retrospective chart review was conducted on 59 women and 197 pregnancies (1–12 per pt) in whom primary hypercoagulable state was diagnosed: FVL (n=7), PGM (n=11), MTHFR (n=3), ATIII (n=2), PC (n=1), PS (n=8), LA (n=10), and those with more than one thrombophilic risk (TR) (n=12) or lupus (n=2). Of the 197 pregnancies, only 106 (54%) were carried to term in 50 pts (85%). Of the remaining 91 pregnancies, there were 16 terminations, 5 ectopic pregnancies and 4 preterm live births. 66 fetal losses occurred: 45 in the first trimester (26 with single TR/19 with &gt;1 TR), 19 in the 2nd–3rd trimester (14 with single TR/5 with &gt; 1 TR) and 2 unknown. See Table for risk of fetal loss by class of hypercoagulable state. Venous thromboembolism occurred in 8/106 (8%) of term pregnancies, including 5 postpartum. Of women with previous fetal losses, management with anticoagulation (A/C) subsequently was utilized in 20 pregnancies: 9 low molecular weight heparin (LMWH), 6 LMWH+aspirin (ASA), 3 heparin (H), 1 ASA, and 1 H+ASA. 18/20(90%) of these pregnancies resulted in live term births; 1 loss due to intracranial hemorrhage at 27 weeks in a patient on L+ASA. Compared to 146 pregnancies untreated with A/C, successful completion of pregnancy was significantly greater on A/C, 90% vs. 58% (p=0.006).
Conclusions: Fetal losses secondary to TR occurred in both the first and late trimesters in high proportion of pregnancies in women with hypercoagulable states. Outcomes may be improved with the use of A/C therapy.
Table - Risk of Fetal Loss by Class of Hypercoagulable State LA PGM MTHFR PS FVL ATIII FVL/other Trimester1 9/38 (24%) 7/44 (16%) 3/9 (33%) 3/27 (11%) 1/27 (4%) 2/5 (40%) 17/42 (40%) Trimester 2–3 5/38 (13%) 1/36 (3%) 0% 5/16 (31%) 2/27 (7%) 1/5 (20%) 4/44 (9%)
Thrombomodulin-Dependent Protein C Activator Treatment Improves the Short-Term Outcome of Experimental Ischemic Stroke in Mice.
Authors: Gruber Andras , Hurn Patricia D. , Hurst Sawan , Bush Leslie , Marzec Ulla M. , Tucker Erik , Hanson Stephen R. , Di Cera Enrico 
Published: April 2020

Abstract
High dose human or mouse activated protein C (APC) treatment is neuroprotective in the transient intraluminal middle cerebral artery occlusion (MCAO) and reperfusion model of ischemic stroke, in mice. However, the therapeutic potential of systemic APC treatment in stroke might be limited because it can disable hemostasis. Low dose thrombomodulin-dependent protein C activator (PCA) enzyme treatment can generate antithrombotic APC on the intraluminal surface without profound systemic anticoagulation and antihemostatic effects in primates. We thus explored whether PCA treatment could produce short term outcome benefits in comparison to fibrinolysis with tPA in the mouse stroke model. Stroke was induced by MCAO between 0 and 60 min using an intraluminal filament that causes progressive and occlusive secondary thrombosis in the MCA region. Cortical perfusion as a measure of thrombotic occlusion was monitored from start until 75 min, i.e., following removal of the intraluminal filament. Cerebral infarct size (% brain volume, TTC stain), edema (% brain volume; calculated), and neurological damage scores (scale 0 – 5) were assessed in survivors at sacrifice next day. All animals (N=12 to 16 in each treatment group) were evaluated for macroscopic hemorrhage (intracerebral or other sites) on autopsy after death. Treatments with vehicle (saline bolus), recombinant PCA thrombin analog W215A/E217A (0.025 mg/kg IV bolus) or plasminogen activator (2.5 mg/kg Bolus, [tPA-B], or 10 mg/kg Infusion for 45 min, [tPA-I]) were started 15 min after initiation of MCAO. One day mortality rates were similar: 29% in the PCA, 38% in the tPA-B, and 23% in the tPA-I treatment groups vs. 23% in vehicle controls (P&gt;0.05 each). Cerebral infarct size was reduced by 62%, 43%, and 73% in the PCA bolus, tPA-B, and tPA-I treated groups, respectively (p&lt;0.05 for each). Macroscopic hemorrhage (intracranial and/or other sites) was found in 15% (2/13), 14% (2/14), 44% (7/16), and 31% (4/13) in the vehicle, PCA, tPA-B, and tPA-I-treated groups, respectively. One day neurological scores averaged 1.5 in PCA-treated (p&lt;0.05), 3.2 in tPA-B (NS), and 2.2 in tPA-I treated (p&lt;0.05) versus 3.9 in saline vehicle-treated animals. All enzyme treatments significantly reduced secondary (thrombotic) vascular occlusions, as evidenced by 62% (PCA), 66% (tPA-B), and 72% (tPA-I) improvement of early cortical reperfusion vs. vehicle-treated controls (p&lt;0.05 each). The means and SEM are illustrated in the figure below. The apparent efficacy and safety of intermediate dose PCA in this model suggests that PCA treatment might have therapeutic utility in acute ischemic stroke, e.g. when reperfusion of the MCA is achieved by tPA treatment or other modalities.
Figure Figure
Erythrocyte Coupled tPA Improves Outcomes of Percussion Brain Trauma in Rats.
Authors: Ganguly Kumkum , Stein Sherman C. , Groff Robert F. , Zhang Jun , Smith Douglas H. , Cines Douglas B. , Muzykantov Vladimir R. 
Published: April 2020

Abstract
Coupling tissue plasminogen activator (tPA) to carrier red blood cells (RBC): i) restricts tPA’s permeation into tissues and pre-existing hemostatic clots, minimizing hemorrhage; ii) protects it from plasma inhibitors; and iii) prolongs its circulation, permitting incorporation into nascent clots and their lysis from within. These features support the thromboprophylactic utility of RBC coupled tPA (RBC/tPA). In this study we explored the utility of RBC/tPA in traumatic brain injury (TBI), a disorder in which both cerebral thrombosis leading to cerebral ischemia and intracerebral hemorrhage (ICH) have been implicated. Eleven male, Sprague-Dawley rats (340–400g) were subjected to a moderate (avg. peak pressure 2.6atm) lateral fluid percussion injury to the left hemisphere. Rats were given a single intravenous dose of RBC/tPA (0.05mg/Kg, n=5) or vehicle (n=4), 15 min post-injury. Animals were sacrificed 48h later for histopathology and staining for fibrin. The lesions in control animals occupied 8.3 ± 2.8% (mean±SD) of the hemispheric volume. Animals treated with RBC/tPA had a significant decrease in mean lesion volume (1.4±0.7%; p&lt;0.001). Thrombotic burden was reduced from a mean of 10 clots in vehicle-treated to 1 per RBC/tPA-treated rats p&lt;0.001). These data indicate that RBC/tPA attenuates post-traumatic thrombosis without aggravating hemorrhage induced by TBI. These observations support the safety of RBC/tPA in thromboprophylaxis even in the context of brain trauma, due to intravascular containment of RBC/tPA, among other factors. Durable lysis of post-traumatic thrombi by long-circulating RBC/tPA may alleviate secondary brain ischemia and resultant ICH. Correlation between reduction of thrombotic burden and lesion size implicate thrombosis in the pathophysiology of parenchymal brain damage after head trauma. Supported by PENN Research Foundation, HL66442, HL60169 and in part by NS38104.
The Effect of BIBT 986, a Novel Small Molecule Dual Inhibitor of Thrombin and Factor Xa, on Coagulation in a Model of Endotoxin Induced, Tissue Factor Triggered Coagulation Activation in Humans.
Authors: Jilma Bernd , Leitner Judith M. , Cardona Francesco , Mayr Florian B. , Firbas Christa , Rathgen Karin , Staehle Hildegard , Schuehly Uwe , Graefe-Mody Eva U. 
Published: April 2020

Abstract
Background: BIBT 986 is a novel potent anticoagulant that dually inhibits Factors Xa and IIa. We hypothesized that BIBT 986 would dose-dependently decrease endotoxin-induced, tissue factor triggered coagulation activation. Hence it was the aim of the study to compare with placebo the anticoagulant activity of three dosages of BIBT 986 on parameters of coagulation, platelet activation and inflammation and to examine the safety of BIBT 986 in this setting.
Methods: This study was a prospective, randomized, double-blind, placebo-controlled, parallel-group dose escalation trial in 48 healthy male volunteers. Participants were randomised to receive bolus primed continuous infusions of one of the three doses of BIBT 986 or placebo. All of them received a bolus infusion of 2ng/kg body weight lipopolysaccharide (LPS).
Results: BIBT dose-dependently increased anti-Xa activity, activated partial thromboplastin time (APTT), ecarin clotting time (ECT), thrombin time (TT) and the international normalisation ratio (INR). Importantly, BIBT 986 dose-dependently blocked the LPS-induced coagulation as assessed by the in vivo markers of thrombin generation and action: BIBT 986 doses that prolonged APTT by 25% were already effective. The BIBT dose that prolonged APTT by 100%, completely suppressed the increase in prothrombin fragment (F1+2), thrombin-antithrombin complexes (TAT) and D-dimer. BIBT 986 had no influence on activation markers of inflammation, fibrinolysis, endothelial or platelet activation.
Conclusion: Infusion of BIBT 986 was safe and well tolerated. BIBT 986 specifically and dose-dependently blocked LPS-induced, tissue factor trigger coagulation. When compared to different anticoagulants tested previously in this standardized model, BIBT 986 was more effective in suppressing thrombin generation (F1+2 levels) than standard doses of danaparoid, dalteparin or lepirudin. BIBT 986 represents the first drug of a new class of dual FXa and FIIa inhibitors, and displays high potency.
Murine NK Cells Require Activation-Dependent Expression of Granzyme B and Perforin To Become Potent Cytotoxic Effectors.
Authors: Fehniger Todd A. , Cai Sheng F. , Cao Xuefang , Bredemeyer Andrew J. , Presti Rachel M. , French Anthony R. , Ley Timothy James 
Published: April 2020

Abstract
NK cells predominantly utilize the granule exocytosis pathway to kill virus-infected and malignant target cells. Current paradigms suggest that resting NK cells have pre-formed granules containing granzymes A, B, and perforin and are ready to kill targets immediately upon proper recognition by NK receptors. Here, we report that resting murine NK cells in the spleen exhibit poor cytotoxicity (5.4±1.6% target cell death, 20:1 E:T ratio and 4 hour incubation), compared with cytokine-activated (IL-15, 48 hours) splenic NK cells (59.7±10.6% target cell death), against the RMAS tumor cell line in vitro as measured by a flow-based killing assay. In addition, using intracellular flow cytometric analysis with monoclonal antibodies specific for granzymes A, B, and perforin, we find that resting murine NK cells express abundant granzyme A (86.2±1.9% positive), but little or no granzyme B (4.4±5.4% positive) or perforin (2.6±1.8% positive). Activation of murine NK cells with IL-15 induces robust expression of both perforin (59.1±2.0% positive) and granzyme B (91.5±7.9% positive), which correlates with increased cytotoxicity. Further, granzyme B cluster −/− (26±6.7% target cell death) and perforin −/− (5.7±1.3% target cell death) NK cells have poor cytotoxicity in vitro despite IL-15 activation. Poly I:C simulates RNA virus infection and activates NK cell cytotoxicity in vivo through TLR3 and cytokine cascades. NK cell granzyme B and perforin expression is induced in vivo 24 hours after poly I:C injection, correlating with increased in vitro NK killing of tumor targets. In wild type mice infected with murine cytomegalovirus (MCMV), NK cell expression of both perforin (83.5±4.9% positive) and granzyme B (89.3±2.1% positive) is upregulated in the spleen, peaking 2–4 days post-infection and returning to baseline by 8 days post-infection. In addition, MCMV titers are significantly elevated at day 3 post-infection in both granzyme B cluster −/− (P&lt;0.01) and perforin −/− (P&lt;0.01) mice, compared to wild type mice. Moreover, survival following MCMV infection was significantly lower in granzyme B cluster −/− and perforin −/− mice, compared with wild type mice (P&lt;0.001, see survival curve). Thus, our findings show that murine NK cells require the activation of granzyme B and perforin to become potent cytotoxic effectors. We also demonstrate for the first time that granzyme B is critical for early host defense against MCMV. These findings explain the long-standing observation that murine NK cells require prior activation for potent natural killing of tumor targets in vitro. Further, this requirement for activation-dependent granzyme B and perforin expression in NK cells may influence outcomes in murine models of innate immune anti-tumor and anti-viral responses.
Figure Figure
Down-Regulation of Glucocorticoid-Induced TNF Receptor (GITR) Ligand on Human Tumors by Proteolytic Shedding Increases Anti-Tumor Reactivity of NK Cells.
Authors: Baltz Katrin M. , Krusch Matthias , Baessler Tina , Kloss Mercedes , Kumbier Ingrid , Peterfi Andrea , Kanz Lothar , Salih Helmut R. 
Published: April 2020

Abstract
Members of the TNF superfamily mediate multiple cellular functions, including cellular proliferation, differentiation and cell death. Many members of this protein family are shed from the cell surface as soluble forms, which affects cell-cell interactions by reduction of ligand densities and distally modulates effector cells bearing the respective receptor. The TNF family member Glucocorticoid-induced TNF Receptor (GITR) costimulates effector T cells, modulates apoptosis and NFkappaB and abrogates suppression of murine but not human regulatory T cells. Its cognate ligand GITRL has been found in various healthy tissues. Recently we reported that NK cells express GITR, while tumor cells express GITR ligand (GITRL), and GITR/GITRL interaction downregulates NK cell-mediated anti-tumor effector mechanisms like cytotoxicity and IFN-gamma production. Here we report that human tumor cells spontaneously release a soluble form of GITRL (sGITRL) detectable in culture supernatants by ELISA. Furthermore, we found elevated levels of sGITRL in sera of patients with various malignancies compared to healthy controls. We demonstrate that the release of GITRL from tumor cells can be blocked by inhibition of metalloproteinases, concomitantly causing accumulation of GITRL on the tumor cell surface as determined by FACS analysis. Upregulated GITRL surface expression further increased inhibition of NK cell anti-tumor effector mechanisms, while, in contrast, presence of sGITRL in cocultures of GITRL-expressing tumor cells and GITR-positive NK cells enhanced NK cell cytotoxicity and IFN-gamma production. Thus, in line with the results obtained with other TNF family members, conversion of membrane bound GITRL to its soluble form is associated with downregulation of its function, potentially due to blocking its cognate receptor. Thus, release of sGITRL substantially influences the interaction of tumor cells with NK cells. In addition, determination of sGITRL levels may be implemented as a diagnostic marker in patients with malignancies. Further prospective studies are currently being conducted addressing the value of GITRL as a tumor marker in different tumor entities.