bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reversal of Anticoagulation as Assessed by Thrombin Generation Measurement.
Authors: Gatt Alex , van Veen Joost , Cooper Peter , Kitchen Steve , Makris Michael 
Published: April 2020

Abstract
Antidotes to the ever-growing number of anticoagulants are always desirable in order to placate bleeding in emergencies. In general, the older anticoagulants like coumarins and unfractionated heparin (UFH) have proven reversal agents, whereas the newer ones do not. We studied the in vitro effect of 5 different heparinoids (UFH, Tinzaparin, Enoxaparin, Fondaparinux and Danaparoid) on the calibrated automated thrombogram. The assay uses a fluorogenic substrate that is cleaved by the thrombin formed after the addition of plasma to 5pM tissue factor, 4μM phospholipids and calcium chloride. We investigated all the parameters generated by dedicated software (Thrombinoscope™) ie the lag time (LT), the time to peak (ttpeak), the endogenous thrombin potential (ETP) and the peak thrombin. All the five anticoagulants tested inhibited thrombin generation (TG) in a concentration dependent manner.
We subsequently analysed the in vitro effect of different concentrations of six potential reversal agents on correcting TG parameters of maximally inhibited plasma for each anticoagulant. These were protamine sulphate at 2.5, 5 & 8μg/ml, activated FVIIa (Novoseven®) at 5, 10 & 50μg/ml, FEIBA® at 0.5,1 & 2U/ml, Beriplex® at 0.3, 0.6 & 1.2U/ml, Prothromplex® TIM4 at 0.4, 0.8 & 1.8U/ml and fresh frozen plasma (FFP) at 250, 500 & 750μl/ml. The three concentrations reflect the recommended therapeutic doses for each agent together with lower and higher doses than normally used.
As predicted, UFH (final concentration 0.527U/ml) was completely reversed with a standard protamine concentration of 5μg/ml. However, the highest dose of protamine gave slightly lower TG, indicating that higher concentrations of protamine sulphate can have a paradoxical ‘anticoagulant’ effect. High doses of FEIBA (2U/ml) and FVIIa (50μg/ml) restored ~50% of thrombin generation parameters. Tinzaparin (at 1antiXaUnit/ml) was also completely neutralised by protamine. However, a higher concentration of 8μg/ml protamine was required. This effect was not seen with Enoxaparin with this higher concentration of protamine reversing only ~40% of the ETP, 21% of the peak thrombin, 71% of ttpeak and 72% of the LT. There was no positive effect of protamine on Fondaparinux (3μg/ml) and Danaparoid (1antiXa U/ml)-treated plasma. Whereas Danaparoid seemed relatively resistant to all six reversal agents, Fondaparinux effect was completely neutralised by FVIIa at concentrations between 10–50μg/ml.
This study highlights the differences in neutralisation of different low molecular weight heparins and UFH. In particular, Tinzaparin was much more readily reversed with protamine sulphate than Enoxaparin. It also indicates that high doses of FVIIa could completely reverse Fondaparinux anticoagulation.
Evaluation of a VENOus-Return Assist Device (Venowave™) To Treat Severe Post-Thrombotic Syndrome (VENOPTS): A Randomized Controlled Trial.
Authors: O’Donnell Martin , McRae Simon , Kahn Susan , Julian Jim , Kearon Clive , MacKinnon Betsy , Magier Debbie , Strulovitch Carla , Lyons Theresa , Hirsh Jack , Ginsberg Jeffrey 
Published: April 2020

Abstract
Severe post-thrombotic syndrome (PTS) is responsible for considerable disability, reduced quality of life and increased health care costs. Current therapies are limited and often ineffective.
Methods: We performed a two-center, randomized, cross-over controlled trial to evaluate Venowave™, a novel lower-limb venous return assist device, for the treatment of severe PTS. Eligible subjects were allocated to receive, in randomized order, Venowave™ for 8 weeks and control-device for 8 weeks. Participants were crossed-over after a 4-week (wash-out) period. The primary outcome measure was a clinical success, defined as; 1) reported benefit from the device with; 2) moderate (or greater) improvement in symptoms of PTS; and 3) willingness to continue using the device long-term. Secondary outcome measures included venous disease-specific quality of life (QOL) (measured by VEINES-QOL questionnaire; higher scores indicate better QOL) and PTS severity (measured by the Villalta PTS scale; higher scores indicate more severe PTS). The study was registered with ClinicalTrials.gov (NCT00182208).
Results: Thirty-two patients were enrolled. Of these, 26 (80%) were also using graduated compression stockings. Twenty-six participants completed both trial periods. The primary outcome measure occurred in 10 participants receiving Venowave and 4 participants receiving the control-device (P=0.1). Two participants reported a clinical success with both devices. Mean Villalta scale score at the end of treatment period was significantly lower for Venowave™ (12.2; SD 6.3) compared to control (15.0; SD 6.1). (P=0.004) Mean VEINES-QOL score at the end of treatment period was significantly greater for Venowave™ (52.5; SD 5.8) compared to control (50.2; SD 6.2). (P=0.004)
Conclusions: Venowave ™ is a very promising new therapy for patients with severe PTS, which may be used alone or in combination with graduated compression stockings. In our study population of patients with severe PTS, refractory to standard therapies, almost one-third of patients reported clinical improvement with Venowave™.
Prostaglandin E2 (PGE2) Regulates Osteoblastic Jagged1 and Expands Primitive Hematopoietic Cells In Vivo.
Authors: Calvi Laura M. , Frisch Benjamin J. , Gigliotti Benjamin J. , Christianson Christina A. , Weber Jonathan M. , Jordan Craig T. , O’Keefe Regis J. 
Published: April 2020

Abstract
Parathyroid Hormone (PTH) targets osteoblastic cells (OBs) in the bone marrow microenvironment and expands hematopoietic stem cells (HSC) through Notch activation. Since PTH stimulates the Notch ligand Jagged1 (J1) in OBs, we have focused on the signaling pathways involved in this PTH effect in order to identify novel activators of the HSC niche. Osteoblastic Protein Kinase A (PKA) activation is required for the PTH-dependent J1 increase in OBs. Therefore, we hypothesized that alternative PKA activators could also regulate osteoblastic J1, alter the HSC niche, and provide additional pharmacologic tools to expand HSC in vivo. Consistent with this hypothesis, direct PKA agonists 8-bromo-cAMP and dibutyryl-cAMP stimulated J1 in osteoblastic UMR106 cells. In addition, PGE2, a member of the prostaglandin family known to stimulate PKA in OBs, was studied in vivo and in vitro. By real-time RT-PCR analysis, J1 mRNA was increased up to 5 fold at 2 hours in UMR106 cells when treated with PGE2 (10−7 M) compared to vehicle. J1 protein was also increased after treatment with PGE2. The PGE2-dependent J1 increase was blocked in the presence of the specific PKA inhibitors H89 and myristoylated PKA Inhibitory Peptide (14–22)(PKI) (200ug/ml), demonstrating that PKA is necessary for osteoblastic J1 stimulation by PGE2. Since systemic PGE2 is known to have bone anabolic effects in both humans and animal models, adult wild-type FVB/N male mice were treated with PGE2 (6mg/kg/day i.p.) for 12 days. This regimen has previously been shown to have bone anabolic effects in rats. At day 12, histologic analysis demonstrated an anabolic effect mainly on cortical bone, as was evident in the femurs and tibiae of PGE2-treated mice compared to control. This histologic finding was confirmed by histomorphometry (trabecular bone area means 41% vs 12%,p=0.0916, n=3 in both groups; cortical thickness means 138 vs 85 μm, p=0.0071, n=3 in both groups). Frequency of hematopoietic stem cells (c-Kit+, Sca1+, lin−) was increased in bone marrow from PGE2-treated vs control mice by over 20% (p=0.0018, n=8 in both groups). In summary, PGE2 stimulates J1 in osteoblastic cells through PKA activation and increases mainly cortical bone in vivo. Ongoing studies will confirm whether in vivo PGE2 treatment expands HSC, and whether osteoblastic J1 regulates this process. This study identifies PGE2 as a novel regulator of osteoblastic J1, and as a potential new microenvironmental modulator of HSC, which could be used for in vivo therapeutic HSC niche manipulation.
Increased Levels of Inflammatory Mediators in Lung Cancer and Their Modulation by Oral Anticoagulant Treatment.
Authors: DeMir Muzaffer , Hoppensteadt Debra A. , Cunanan Josephine , Iqbal Omer , Fareed Jawed 
Published: April 2020

Abstract
Venous thromboembolic events (VTE) are associated with lung cancer and significantly contributes to the increased mortality in these patients. While the increased prevalence of VTE is fully recognized in lung cancer, its pathogenesis is not fully understood. Even the patients undergoing therapeutic interventions are at high risk to develop VTE. These patients provide a unique clinical setting to investigate the pathogenesis of lung cancer associated thrombosis. In a prospective, randomized, controlled study patients with inoperable lung cancer (n=100) were randomized to receive chemotherapy, radiation and warfarin (INR 1.5–2.5) or chemotherapy, radiation without warfarin (n=50). Blood samples were drawn prior to and after the second treatment cycle with warfarin. All samples were analyzed for tumor necrosis factor alpha (TNF a), CD 40 ligand (CD 40L), C-reactive protein (CRP), interleukin 1 beta (IL-1b), asymmetric dimethylarginine (ADMA) and nitric oxide (NO) as summarized in Table 1. All of the surrogate markers of inflammation showed a decreased trend (13–50%) in the warfarin treated group, whereas the non-warfarin treated group exhibited an increase (18–46%) in all markers except CRP and ADMA. The levels of various inflammatory markers are upregulated in lung cancer suggesting a pathogenic role of this process in lung cancer. Warfarin down regulated the inflammatory process in contrast to the non-warfarin treated group. The clinical relevance of these observations require additional analysis.
Table 1 Test Warfarin Treated Warfarin Treated Warfarin Treated Non-Warfarin Treated Non-Warfarin Treated Non-Warfarin Treated Pre Post % Change Pre Post % Change TNF α (pg/ml) 63±22 44.4±18.2 ↓30 50.7±18 62.9±19.2 ↑24 CD 40L (ng/ml) 671±212 470±123 ↓29 563±176 712±234 ↑26 CRP (μg/ml) 19.1±8.5 9.6±2.8 ↓50 18.2±9.2 9.9±3.1 ↓46 NO (μM) 33.4±9.8 29±8.5 ↓13 29.9±8.8 35.2±8.2 ↑18 IL-1β (pg/ml) 14.6±5.7 11.1±5.3 ↓24 15.3±6.2 15.2±5.8 NC ADMA (μ/ml) 2.0±0.8 1.53±0.8 ↓24 2.0±0.8 1.9±0.7 ↓18
The Tumor Suppressor Gene SLC5A8 Is Selectively Silenced in MLL Partial Tandem Duplication-Positive Acute Myeloid Leukemia with Normal Cytogenetics.
Authors: Whitman Susan P. , Wen Jing , Muto Gina , Hackanson Bjoern , Yu Li , Liu Chunhui , Sanfilippo Joseph L. , Marcucci Guido , Rush Laura J. , Plass Christoph , Caligiuri Michael A. 
Published: April 2020

Abstract
The Mixed Lineage Leukemia Partial Tandem Duplication (MLL PTD) defect, present in 4–10% of adults with acute myeloid leukemia (AML) and normal cytogenetics (NC), predicts for shorter complete remission duration. Functionally, the MLL PTD is associated with aberrant histone methylation. Since posttranslational histone modifications are also frequently associated with changes in the methylation status of gene promoters, we evaluated whether AML with the MLL PTD is associated with a pattern of DNA methylation distinct from AML with NC and wild type MLL (MLL WT). Significant differences in DNA methylation patterns of over 2,000 CpG island fragments, most of them located in the 5′-region of genes, were observed between NC AML with the MLL PTD (n=9) and NC AML with MLL WT (n=23) using Restriction Landmark Genomic Scanning (RLGS) (P&lt;0.001). This analysis revealed 201 RLGS spots were lost, corresponding to DNA methylation events, at a higher frequency in MLL PTD+ AML compared with MLL WT AML. Among the non-polymorphic, methylated spots, spot 3D41 corresponding to the SLC5A8 gene was found methylated in 78% of MLL PTD+ AMLs compared with only 17% of MLL WT AMLs (P&lt;0.003). The SMCT1 protein, encoded by SLC5A8, is a known tumor suppressor reported to be silenced by DNA methylation in colorectal, glioma, thyroid and gastric cancers. This plasma membrane protein mediates intracellular transport of substrates such as butyrate, a histone deacetylase inhibitor (HDACi), and when SLC5A8 is underexpressed, this predicts worse prognosis in colon cancer patients. In MLL PTD+ AML samples exhibiting 3D41 loss by RLGS, bisulfite-PCR sequencing confirmed CpG hypermethylation within the SLC5A8 proximal promoter (range: 24–78% promoter methylation) compared with those AML samples (0%–12% promoter methylation) exhibiting no 3D41 loss. Furthermore, SLC5A8 transcript levels were significantly reduced in MLL PTD+ AML patient samples compared to MLL WT+ AML patient samples (P&lt;0.002). The demethylating agent, decitabine (Dacogen®, MGI Pharma), alone or in sequential combination with one of the following HDACis- depsipeptide, valproic acid or MS275, induced expression of SLC5A8 and cell death in the MLL PTD+ EOL−1 cell line. These results suggest that aberrant promoter DNA methylation and the reduced expression of the tumor suppressor gene SLC5A8 may be associated with the short remission duration observed in MLL PTD+ AML patients. These data also support further investigation of DNA demethylating agents, such as recently FDA-approved Dacogen® and the appropriate timing of administration of HDACi, such as butyrate analogs, as a therapeutic approach for this poor prognostic AML subgroup.
Preclinical Pharmacokinetic and Metabolism of Apixaban, a Potent and Selective Factor Xa Inhibitor.
Authors: He Kan , He Bing , Grace James E. , Xin Baomin , Zhang Donglu , Pinto Donald J. , Luettgen Joseph M. , Knabb Robert M. , Lam Patrick Y.S. , Wexler Ruth R. , Humphreys William G. , Shyu Wen-Chyi , Grossman Scott J. 
Published: April 2020

Abstract
Apixaban is a potent, orally available, highly selective, and reversible factor Xa inhibitor, and currently under development for prevention and treatment of thrombosis. The preclinical pharmacokinetic and metabolism attributes of apixaban feature a small volume of distribution, a low systemic clearance, good oral bioavailability, multiple elimination pathways and minimal potential for drug-drug interactions. Apixaban is well absorbed in chimpanzees, dogs and rats with a mean oral bioavailability of 51, 88 and 34%, respectively. The mean volume of distribution of apixaban is 0.17, 0.29 and 0.31 L/kg in chimpanzees, dogs and rats, respectively, suggesting apixaban is primarily distributed (30–50%) to blood where the therapeutic action resides. The small volume is not due to extensive plasma protein binding, but possibly attributed to limited extravascular tissue distribution, given that the unbound fraction is approximately 13, 5, 8 and 4% in human, chimpanzee, dog and rat serum, respectively. The systemic clearance is &lt;3% of hepatic blood flow in chimpanzees (0.018 L/h/kg) and dogs (0.052 L/h/kg), and &lt;10% in rats (0.26 L/h/kg). Consistent with this low clearance, the in vitro metabolic clearance of apixaban is low, as indicated by the lack of significant metabolism in human liver microsomes and hepatocytes, and in chimpanzee and dog liver microsomes. The primary metabolite identified in vitro is the O-demethylated product which is formed mainly by CYP3A4 in human liver microsomes. The elimination of apixaban involves multiple pathways including renal and intestinal excretion of the parent and metabolism. The biliary clearance is low in dogs, accounting for approximately 2% of the systemic clearance. Apixaban did not inhibit CYP1A2, 2C8, 2C9, 2C19, 2D6 and 3A4 activities in cDNA-expressed enzyme systems and human liver microsomes, nor induced 1A2, 2B6 and CYP3A4 in human hepatocytes. No glutathione adduct with apixaban was formed in dog and rat, and in human hepatocytes and liver microsomal incubation in the presence of glutathione, suggesting low potential for the formation of reactive metabolites. In conclusion, apixaban shows excellent pharmacokinetic and metabolic properties for clinical development.
Role of 4-1BB as a Regulator for NKG2D Costimulation in Human CD8+ T Cells.
Authors: Kim Young-June , Han Myung-Kwan , Broxmeyer Hal E. 
Published: April 2020

Abstract
NKG2D is a potent costimulatory receptor which is involved in activating various CD8+ T cell responses. Cells undergoing various stresses including tumor formation and virus infection express MHC class I related chain (MIC), a ligand for NKG2D. Thus, MIC acts as a signature molecule of the target cells for CD8+ T cells to recognize in killing. In contrast, MIC also plays a role in impairing effector CD8+ T cell responses because it rapidly internalizes NKG2D from the cell surface. It is unclear how these opposing effects of NKG2D ligation on CD8+ T cell fate are regulated. In this study, we demonstrated that 4-1BB, another costimulatory receptor, might be an important regulator for induction and maintenance of NKG2D expression after its internalization, thereby enhancing effector CD8+ T cell responses. Whereas NKG2D is constitutively expressed on naïve human CD8+ T cells, 4-1BB is induced only by activation. Costimulation of NKG2D with anti-NKG2D and suboptimal anti-CD3 eliminated NKG2D but efficiently induced 4-1BB on naïve CD8+ T cells isolated from human umbilical cord blood, even in the presence of TGF-ß1 which inhibited 4-1BB expression in the absence of NKG2D costimulation. In vitro chromium release assays indicated that the CD8+ T cells costimulated with NKG2D remained low in their cytotoxic activity against C1R B lymphoma cell line expressing MIC which was used as target cells. We found these results despite the fact that the CD8+ T cells were fully activated and expressed 4-1BB and granzyme B at high levels. This is correlated with a lack of NKG2D on the cell surface. Subsequent 4-1BB costimulation restored NKG2D to the cell surface, even in the presence of TGF-ß1. The resulting CD8+ T cells expressing both NKG2D and 4-1BB showed extremely potent cytotoxic activity. Importantly, the NKG2D co-expressed with 4-1BB was unique for being highly refractory to the downregulation by TGF-ß1 and internalization from the cell surface. IL-4 markedly promoted downregulation of NKG2D and 4-1BB expression induced by TGF-ß1, generating a CD8+ T cell type lacking both NKG2D and 4-1BB on the cell surface. These cells were inert and unable to gain cytotoxic as well as proliferative activity, suggesting that CD8+ T cells lacking both NKG2D and 4-1BB may represent terminally incapacitated cells. In summary, NKG2D may be involved in inducing 4-1BB expression and in turn 4-1BB may play a critical role for stabilizing surface NKG2D expression to overcome tumor immune evasion especially in tumor microenvironments enriched with soluble MIC and TGF-ß1. Due to such coupled supports, co-expression of NKG2D and 4-1BB may be a key biomarker for defining effective tumor infiltrating CD8+ T cells that can be used in a clinical setting of immune therapeutic strategies.
Interleukin-10 Severely Impairs TLR Mediated Activation of Antigen Presenting Cells.
Authors: Knödler Anja , Bringmann Anita , Weck Markus M. , Brauer Katharina M. , Kanz Lothar , Grünebach Frank , Brossart Peter 
Published: April 2020

Abstract
Danger signals provided by pathogens are sensed by specialized receptors, Toll-like receptors (TLR), on the cell surface of antigen presenting cells (APC) leading to activation of dendritic cells (DC) accompanied by their phenotypic and functional changes. Activation of DC via the TLR is believed to be a critical event regulating the strength and outcome of an immunological response. IL-10 is an immunosuppressive cytokine shown to have inhibitory effects on function and differentiation of APC by interfering with signaling pathways involved in DC activation like the PI3-kinase or NFκB pathway.
In our study we analyzed the function of human dendritic cells generated in the presence of IL-10 upon activation by Toll-like receptor ligands (TLR2, TLR3, TLR4, TLR7/8). Exposure of DCs to IL-10 resulted in a skewed phenotypic maturation in response to stimuli provided by TLR ligands, a reduced production of cytokines like IL-12, IL-6 or TNF-α and an impaired capacity to stimulate T cell activation. Furthermore, CCR7 upregulation in DC exposed to TLR stimulation as well as migration toward CCL19/MIP-3β were strongly reduced. As a suitable mechanism for these effects, IL-10 was found to downregulate MyD88, an essential adaptor molecule for TLR signaling, and to decrease TLR-induced nuclear expression of the NFκB transcription factor members c-Rel and Rel-B as well as IRF-3 and IRF-8. This was not due to the inhibition of the MAP kinase pathway as phosphorylation of p38 and ERK was not affected but was rather mediated by inhibitory effects on the PI3-kinase pathway. IL-10 treatment of APC resulted in a reduced TLR induced mTOR phosphorylation. In summary, our results demonstrate that IL-10 can inhibit TLR-mediated activation of APC via a MyD88-dependent and independent pathway.
Transcriptional Activation of Mdm2 in Germinal-Center B Cells Is Induced by IRF8.
Authors: Zhou Jeff X. , Lee Chang-Hoon , Qi Chen-Feng , Maghashfar Zohreh , Zhao Ming , Morse Herbert C. 
Published: April 2020

Abstract
The transcription factor, interferon regulatory factor 8 (IRF8), is expressed at low levels in both naïve and terminally differentiated B cells but at high levels in germinal center (GC) B cells where it contributes to transcriptional activation of two genes critically involved in the GC reaction: BCL6, a BTB/POZ-zinc finger transcriptional repressor; and AICDA (also known as AID) a single stranded DNA deaminase (Lee et al. J. Exp. Med.201(1):63–72, 2006). AID is responsible for the generation of double stranded DNA breaks (DSDB) that occur physiologically in B cells during the processes of somatic hypermutation (SHM) of immunoglobulin (Ig) gene variable region sequences and Ig class switch recombination (CSR). The response of most cell types to DSDB is marked by activation of p53 (also termed TP53) that induces cell cycle arrest or apoptosis. p53 also functions at a critical checkpoint to prevent aberrant repair of DSDB in Ig genes that can result in chromosomal translocations that activate proto-oncogenes. GC B cells, however, must be able to tolerate these breaks without experiencing p53-dependent apoptosis. Recent studies showed that functional inactivation of p53 in GC cells is governed in part by transcriptional repression of the p53 gene by BCL6 (Phan and Dalla-Favera Nature432(7017):635–639, 2004). Here we report that IRF8 also suppresses p53 function via transcriptional activation of MDM2. MDM2 is a p53-binding protein and E3 ubiquitin ligase that blocks the transcriptional activity of p53 and stimulates p53 degradation. The levels of both Mdm2 mRNA and its encoded protein were decreased in GC B cells of Irf8 knockout mice as well as in a B lymphoma cell line with siRNA-induced knockdowns of IRF8 expression. Conversely, MDM2 protein levels were increased in cells induced to overexpress IRF8. Transfection of a B lymphoma cell line with an IRF8 expression vector resulted in marked activation of a Mdm2 promoter reporter construct. Oligonucleotide pull-down assays using sequences from the Mdm2 promoter similar to the interferon stimulated response element, a known IRF8 target sequence, were found to bring down IRF8. IL-21, produced by GC T helper cells, promotes the differentiation of B cells activated through the BCR, while inducing apoptosis of unengaged cells. Studies of a B lymphoma cell line treated with IL21 showed that the rate of apoptosis was significantly increased when IRF8 expression was suppressed by siRNAs. The same pattern was true for cells treated with etoposide, a drug that induces DSDB. These results indicate that IRF8 shepherds B cells through the GC reaction by stimulating expression of BCL6 and MDM2 thereby suppressing p53-mediated cell cycle arrest or death in response to DNA breaks.