bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gamma Prime Fibrinogen Does Not Cause Arterial Thrombosis
Authors: Walton Bethany L , Getz Todd M , Bergmeier Wolfgang , Uitte de Willige Shirley , Wolberg Alisa S. 
Published: October 2019

Abstract
Elevated levels of the acute phase plasma protein fibrinogen are associated with increased risk of thrombosis. Using a mouse model of arterial thrombosis, we have previously shown that elevated total fibrinogen levels increase the rate of fibrin formation, increase thrombus fibrin content, and directly promote arterial thrombosis (Machlus et al. 2011 Blood 117:4953-63). Fibrinogen is composed of two copies each of three polypeptide chains: Aα, Bβ, and γ. The fibrinogen γ chain undergoes alternative splicing, resulting in a dominant form (γA/γA) that comprises ∼90% of circulating fibrinogen and a minor species (γA/γ’) that comprises 8-15% of circulating fibrinogen. Epidemiologic studies have detected elevated levels of γA/γ’ fibrinogen in patients with a history of arterial thrombosis, indicating that this isoform associates with thrombotic risk. However, in vitro data show that γA/γ’ fibrinogen has anticoagulant properties due to its ability to bind and sequester thrombin, and suggest that its expression is upregulated in response to a proinflammatory/procoagulant process. To determine whether γA/γ’ fibrinogen is pro- or antithrombotic in vivo, we purified γA/γA and γA/γ’ fibrinogen from human plasma by anion exchange chromatography, raised circulating levels in mice by intravenous infusion, and determined the effects of increased levels of these proteins on arterial thrombus formation in vivo. We also determined the effects of γA/γA and γA/γ’ fibrinogen on whole plasma clot formation, platelet aggregation, and endogenous procoagulant activity. Compared to buffer controls (7.3 [4.6-40.0] minutes, median [range]), γA/γA fibrinogen shortened the time to carotid artery occlusion (5.5 [4.0-8.0] minutes, P<0.05), whereas γA/γ’ fibrinogen did not (7.6 [4.9-40.0] minutes, P=0.9). Both γA/γA and γA/γ’ fibrinogen could be cleaved by murine and human thrombin, were incorporated into murine and human clots, and supported murine and human thrombin-induced platelet aggregation. When γA/γA or γA/γ’ fibrinogen was spiked into normal human or murine plasma, both γA/γA and γA/γ’ fibrinogen accelerated the fibrin formation rate (P<0.02). However, γA/γA fibrinogen increased the rate to a greater extent than γA/γ’ fibrinogen (P<0.02). Interestingly, compared to buffer controls (18.9 [9.8-126.2] ng/mL), mice infused with γA/γ’ fibrinogen had lower levels of circulating plasma thrombin-antithrombin complexes (6.2 [0.9-89.4] ng/mL, P<0.01) following arterial injury, whereas mice infused with γA/γA fibrinogen did not (13.2 [10.7-80.1] ng/mL, P=0.7). These data suggest γA/γ’ fibrinogen exhibits “antithrombin I” activity in vivo by binding thrombin and decreasing circulating prothrombotic activity. Together, these findings indicate that elevated levels of the γA/γA fibrinogen isoform promote arterial thrombosis in vivo, whereas the γA/γ’ isoform does not.

Disclosures:
No relevant conflicts of interest to declare.
Anti-Factor VIII A2 Epitopes In a Murine Bleeding Model
Authors: Eubanks Joshua , Baldwin Wallace Hunter , Markovitz Rebecca , Parker Ernest T , Meeks Shannon L. 
Published: October 2019

Abstract
Up to 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). In addition, autoimmune antibodies to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. Patients with congenital hemophilia who develop inhibitors usually have a polyclonal antibody response directed against the A2 and C2 domains of fVIII. Patients with acquired hemophilia typically have a more limited B-cell epitope response with antibodies directed against the A2 or C2 domain but not both. We have shown that within the C2 domain of fVIII antibody epitope is more important than inhibitory titer in predicting pathogenicity in a murine in vivo bleeding model. In this project we investigated the pathogenicity of a diverse panel of anti-A2 monoclonal antibodies (Mabs) in the murine in vivo bleeding model. We have previously characterized anti-A2 antibodies into groups A, AB, B, BCD, C, D, DE, and E based on the pattern of overlap on the B-cell epitopes in a competition ELISA. Table 1 shows the characteristics of the anti-A2 Mabs. Mabs were injected retro-orbitally into Exon 16 hemophilic (E16) mice at a dose of 0.5 umg per g body weight (∼ 65nM plasma concentration). Fifteen minutes later, mice were injected with B-domain deleted human fVIII at a dose of 180U/kg (∼ 2.5nM plasma concentration). In addition, a subset of Mabs has also been tested at a high dose of 360U/kg (∼5 nM). Two hours after fVIII injection, the mice were anesthetized and a 4mm tail snip was performed. Blood was collected in a tube of normal saline over 40 minutes and measured. 4A4, 2-76, and 1D4 are all high inhibitory titer, type I Mabs that produced significant bleeding with 180 U/kg fVIII when compared to control. In addition, 2-76 and 4A4 were tested at the higher dose of fVIII and significant bleeding was again seen. In comparison, the high titer type II Mab 2-54 had a similar inhibitory titer but no significant bleeding at either dose of fVIII. B94 is a type II inhibitor with a similar inhibitory profile to 2-54, but maximum inhibition is 45% as compared to 82% for 2-54. B94 also was not pathogenic at either fVIII dose tested. Both 4C7—a non-inhibitory Mab—and B25—a very low titer Mab that would be predicted to have residual fVIII activity at the Mab concentration tested—did not produce significant bleeding. The inhibitory titer alone did not predict bleeding phenotype within a diverse panel of anti-A2 Mabs. This discrepancy combined with similar findings in the C2 domain stress the importance of inhibitor properties not detected in the standard Bethesda assay in predicting response to fVIII therapy.

Disclosures:
No relevant conflicts of interest to declare.
Venous Thromboembolism (VTE) Risk Assessment & Prophylaxis In Irish Hospitalized Obstetric Patients: A Nationwide Cross-Sectional Study
Authors: Noone Caroline M , O'Shea Susan , Crowley Maeve P , Higgins John 
Published: October 2019

Abstract

Background
Pulmonary embolism continues to be the second leading cause of mortality in pregnancy and the puerperium. VTE complicates 1 - 2/1000 pregnancies, and the risk increases with age, mode of delivery, and presence of co-morbidities. Literature has shown that the use of low molecular weight heparin (LMWH) is safe in this patient cohort.

Aims
Assessment of the prevalence of VTE risk in hospitalised women in both the antenatal and postnatal groups to determine the proportion of at-risk patients who receive LMWH prophylaxis appropriately.

Methods
The study period was September 2011 to November 2012. All inpatients in the participating hospitals on the day of investigation were assessed for risk of VTE on the basis of hospital chart review. Risk profile was assessed in accordance with the 2009 Royal College of Obstetricians and Gynaecologist Guidelines. Patients undergoing procedures or on the labour ward at the time of review were excluded. Ethical approval was obtained from the ethics committees governing all centres.

Results
610 pregnancies were reviewed across 19 centres. The average age of was 31+/- 5.65yrs (Range 16-47), with 21.87% aged over 35. 22% had a parity of 3 or more. The average weight was 71.51kg (Range 42-134kg, SD 14.482kg). Data on BMI was available for 77% - 34% were overweight and 21% were obese. 1% had a BMI>40. 31% were antenatal and 69% were postnatal.
63% of antenatal patients were low risk (<2 risk factors), 35% were intermediate risk (2 or more risk factors, prophylaxis should be considered) and 2% were high risk. All the high risk patients were on prophylaxis at an appropriate dose. 4% of the low risk patients were on prophylaxis unnecessarily. Only 7% of the intermediate risk patients were on correctly dosed prophylaxis.
Among postnatal patients, 41% were low risk (<2 risk factors), 58% were intermediate risk (2 or more risk factors, require prophylaxis) and <1% were high risk. 80% were appropriately risk stratified and put on LMWH if necessary. 59% of patients should have been on LMWH but only 42% were (92% Tinzaparin and 8% Enoxoparin). This included 8 patients who were on LMWH unnecessarily. 38% were on too low a dose.

Conclusion
VTE prophylaxis remains a central issue in obstetric care given its prominent role in maternal morbidity and mortality and the increasing prevalence of risk factors such as obesity and increasing maternal age. It is clear that while there is good awareness of the risk in the postnatal period, there is less emphasis on risk assessment in antenatal patients where prophylaxis is rarely used. Those on prophylaxis are also likely to be on an incorrect dose. There is a clear role for a national guideline to standardize care for all pregnant women. On the basis of these findings, we have authored a guideline and this is now in use as a reference in all Irish maternity units.

Disclosures:
No relevant conflicts of interest to declare.
The RNA Environment Of Hematopoietic Stem Cells
Authors: Wendt George R , Oshima Motohiko , Iwama Atsushi 
Published: October 2019

Abstract
Recent reports in RNA biology have suggested that long non-coding RNAs, edited RNAs, and alternatively spliced RNAs (henceforth referred to as non-canonical RNAs) play a relatively unclear but critical role in numerous cells, tissues, and diseases. Of interest to hematology, several recent studies have also shown that these non-canonical RNAs all play various roles in both normal and malignant hematopoiesis, but no study yet has comprehensively examined these non-canonical RNAs in hematopoietic stem cells (HSCs).
To this end, we conducted microarray and RNAseq analyses of mouse hematopoietic cells at various stages of differentiation. Microarray analysis detected several thousand possible lincRNAs expressed in hematopoietic stem and progenitor cells (HSPCs), several hundred of which were significantly enriched in CD34-LSKs compared to more differentiated populations. RNAseq analysis provided a more detailed look at these non-coding RNAs, as well as a slightly more conservative estimate of the abundance and structure of the long non-coding RNAs expressed and significantly enriched in HSPCs. In addition to examining lincRNAs, RNAseq data allowed us to profile alternative splicing events and RNA editing events in HSPCs. Interestingly, several hundred potentially significant HSC-specific alternative splicing events and RNA editing events were also discovered.
In order to test whether these non-canonical RNAs play a functional role in HSPCs we performed shRNA mediated knockdown of several interesting HSC-specific lincRNAs and alternatively-spliced transcripts. We found that knockdown of several of these candidates was incompatible with hematopoiesis, both in vitro and in vivo. To better understand how these particular non-canonical RNAs function in HSCs, we are currently carrying out RNA FISH studies.
In this study, we explored the RNA environment of HSCs and compared it to their downstream progenitors. We discovered several hundred novel non-coding transcripts, hundreds of alternative splicing events and hundreds of RNA editing events unique to HSCs. Functional investigation of some of these events revealed that these non-canonical RNAs do indeed play a critical role in HSC maintenance. The data presented here should eventually be an asset to other HSC researchers interested in the RNA biology of HSCs.

Disclosures:
No relevant conflicts of interest to declare.
G-CSF Treatment Induces Toll-Like Receptor Signaling and Regulates Hematopoietic Stem Cell Function
Authors: Schuettpelz Laura G. , Borgerding Joshua N. , Gopalan Priya , Christopher Matt , Romine Molly , Woloszynek Jill , Greenbaum Adam , Link Daniel C. 
Published: October 2019

Abstract
Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte-colony stimulating factor (G-CSF) is often induced with infection and plays a key role in the stress granulopoiesis response. However, its effects on HSCs are unclear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect. RNA profiling and flow cytometry studies of HSCs from G-CSF treated mice show that multiple toll- like receptors (TLRs) are upregulated in HSCs upon G-CSF treatment, and gene set enrichment analysis shows enhancement of TLR signaling in G-CSF-treated HSCs. G-CSF-induced expansion of phenotypic HSCs is reduced in mice lacking the TLR signaling adaptors MyD88 or Trif, and the induction of quiescence is abrogated in mice lacking these adaptors. Furthermore, loss of TLR4 mitigates the G-CSF-mediated HSC repopulating defect. Interestingly, baseline HSC function is also dependent on TLR signaling. We show that HSC long-term repopulating activity is enhanced in Tlr4-/- and MyD88-/- mice, but not Trif-/- mice. One potential source of TLR ligands affecting HSC function in the bone marrow is the gut microbiota. Indeed, we show that in mice treated with antibiotics to suppress intestinal flora, G-CSF induced HSC quiescence and hematopoietic progenitor mobilization are attenuated. Moreover, in germ free mice, HSC long-term repopulating activity is enhanced. Collectively these data suggest that low level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling. Our finding of enhanced TLR signaling upon G-CSF treatment, and the mitigation of G-CSF’s effects in mice deficient for TLR signaling or commensal organisms, suggest that TLR antagonists and/or agonists may ultimately be used clinically to enhance engraftment following bone marrow transplantation or applied toward the treatment of patients with bone marrow failure.

Disclosures:
No relevant conflicts of interest to declare.
MiR-29a Maintains Hematopoietic Stem Cell Self-Renewal and Is Required For Myeloid Leukemogenesis
Authors: Hu Wenhuo , Dooley James , Chung Stephen S. , Yalcin Safak , Shin Yu Sup , De Strooper Bart , Liston Adrian , Park Christopher Y. 
Published: October 2019

Abstract
microRNAs (miRNAs) are important regulators of both embryonic and adult tissue stem cell self-renewal. We previously showed that ectopic expression of miR-29a, a miRNA highly expressed in HSCs as well as in human acute myeloid leukemia (AML) stem cells, in immature mouse hematopoietic cells is sufficient to induce a myeloproliferative disorder that progresses to AML. During the early phase of this disease, miR-29a induces aberrant self-renewal of committed myeloid progenitors, strongly suggesting a role for miR-29a in regulating HSC self-renewal. In order to determine the role of miR-29a in HSC function, we have evaluated our recently described miR-29a/b1 null mouse. Homozygous deletion of miR-29a/b1 resulted in reduced bone marrow cellularity and reduced colony forming capacity of hematopoietic stem and progenitor cells (HSPCs). The phenotype was mediated specifically by miR-29a since miR-29b expression was not significantly altered in HSCs and reconstitution of miR-29a/b1 null HSPCs with miR-29a, but not miR-29b, rescued in vitro colony formation defects. Self-renewal defects were observed in miR-29a deficient HSCs in both competitive and non-competitive transplantation assays, and these deficits were associated with increased HSC cell cycling and apoptosis. Gene expression studies of miR-29a deficient HSCs demonstrated widespread gene dysregulation including a number of up-regulated miR-29a target genes including DNA methylation enzymes (Dnmt3a, -3b) and cell cycle regulators (e.g. Cdk6, Tcl1, Hbp1, Pten). Knockdown of one of these targets, Dnmt3a, in miR-29a deficient HSCs resulted in partial restoration of colony formation, providing functional validation that Dnmt3a mediates part of miR-29a null HSPCs functional defects. miR-29a loss also abrogated leukemogenesis in the MLL-AF9 retroviral AML model. Together, our results demonstrate that miR-29a positively regulates HSC self-renewal and is required for myeloid leukemogenesis.

Disclosures:
No relevant conflicts of interest to declare.
Comparative Analysis Of Cytogenetic Evolution Patterns During Relapse In The Hematopoietic Stem Cell Transplantation and Chemotherapy Settings Of Patients With Acute Leukemia
Authors: Cho Young-Uk , Chi Hyun-Sook , Park Sang Hyuk , Kim Young Jin , Jang Seongsoo , Seo Eul-Ju , Park Chan-Jeoung 
Published: October 2019

Abstract

Background
Despite significant therapeutic progress, many patients with acute leukemia die from deteriorating disease after relapse. Relapse of acute leukemia has repeatedly been associated with cytogenetic clonal evolution. However, only a few studies focused on the direct comparison of cytogenetic evolution patterns during relapse of leukemia after hematopoietic stem cell transplantation (HSCT) and conventional chemotherapy. Thus, we performed comparisons of the cytogenetic patterns of patients with acute leukemia both at diagnosis and relapse after HSCT or standard chemotherapy (SC). Cytogenetic patterns with FLT3 mutational instability were also compared.

Methods
This retrospective analysis was based on a total of 516 patients. Among them, 349 were diagnosed with acute myeloid leukemia (AML) who developed relapse after HSCT (n = 125) or SC (n = 224); 167 were diagnosed with acute lymphoblastic leukemia (ALL) and developed relapse after HSCT (n = 63) or SC (n = 104). Cytogenetic analysis and FLT3 mutation detection were performed according to standard methods.

Results
In patients with AML as shown in Table 1, differences in the karyotypes between diagnosis and relapse were more frequent in the HSCT cohort than in the SC cohort (53.2% vs. 40.1%, respectively; P = 0.035). Development of more than three new cytogenetic changes was also more frequent in the HSCT cohort than in the SC cohort (31.4% vs. 15.3%, respectively; P = 0.046). Overall, FLT3 mutation instability did not correlate to the clonal cytogenetic changes. However, restricted to patients showing FLT3 mutation instability, tyrosine kinase domain (TKD) mutation was more frequently involved in patients with cytogenetic changes compared to those without (46.2% vs. 5.0%, respectively; P = 0.008) (Table 2). Conversely, in patients with ALL, frequency and pattern of the cytogenetic alterations between diagnosis and relapse did not differ between HSCT and SC cohorts. For both types of acute leukemia, the median number of cytogenetic alterations increased from 1.0 at diagnosis to 2.0 at relapse (P < 0.001) in the HSCT cohort. The increasing number of cytogenetic alterations was also seen in the SC cohort (P = 0.011).

Conclusions
Higher frequencies of clonal cytogenetic changes and more complex cytogenetic patterns were observed in the HSCT cohort compared to the SC cohort. This finding may be associated with an adverse outcome for relapse patients after HSCT. FLT3 TKD mutation could have a certain role in promoting cytogenetic progression in a subset of AML patients. In addition, our data extends the findings of recent previous studies, which described patterns of clonal evolution in acute leukemia patients.

Disclosures:
No relevant conflicts of interest to declare.
Exosomes as Markers Of Therapeutic Response In Patients With Acute Myeloid Leukemia
Authors: Boyiadzis Michael , Hong Chang-Sook , Whiteside Theresa L 
Published: October 2019

Abstract

Objective
Exosomes are virus-size (50-100 nm) double membrane-bound microvescicles formed within the endocytic compartments and released into the extracellular space via the fusion of multivescicles bodies with the cell membrane. While all cells release exosomes under physiologic conditions, tumor cells are avid exosome producers. In this study, we isolated exosomes from plasma of acute myeloid leukemia (AML) patients at diagnosis, during and after chemotherapy and studied their protein and TGF-β1 content. The hypothesis was tested that these exosomal components may serve as biomarkers of response to chemotherapy in AML.

Methods
Exosome fractions were isolated from plasma by exclusion chromatography on Sephadex G50 columns followed by ultracentrifugation. Exosome protein content was determined and expressed in μg protein/mL plasma. Exosome-associated TGF-β1 was measured by ELISA following exosome sonication/acidification and expressed in pg/exosome protein/mL plasma.

Results
Exosomes isolated from AML patients’ plasma (n=16) at diagnosis and prior to any treatment had a higher mean protein content (55.2 ± 13.8 μg protein/mL) than those isolated from sera of normal controls (n=7), (13.1 ± 2.4 μg protein/mL, p<0.009). Following a course of induction chemotherapy, a reduction of the exosome protein level (23.8 ± 4.4 μg protein/mL, p<0.05) was observed in AML patients’ plasma (n=9). The exosome protein levels in the patients who at 14 days after the initiation of induction chemotherapy had a significant reduction of their blasts (to <5%) were lower compared to those in the patients with residual leukemia (18.5 ± 5.8 vs 30.8 ± 7.5 μg protein/mL, p=0.2). The exosome protein levels during consolidation therapy with high dose cytarabine in patients who had achieved complete remission (n=10) was higher (52.4 ± 8.9 μg protein/mL) compared to levels measured immediately after induction chemotherapy (p<0.02) and approaching those seen at diagnosis. Protein levels of exosomes isolated from plasma of patients (n=5) in long-term remission (>2 years after completing consolidation therapy) normalized to the levels which were not different from those of normal controls. Levels of active TGF-β1 carried by exosomes obtained at AML diagnosis were high and then decreased (p<0.005) following induction chemotherapy. Similar to protein levels, TGF-β1 levels in plasma-derived exosomes were elevated (p<0.004) during consolidation chemotherapy. In AML patients in long-term remission, TGF-β1 levels carried by exosomes normalized, approaching levels seen in exosomes of normal controls.

Conclusions
Our results indicate that exosomal protein and TGF-β1 levels in AML plasma could serve as biomarkers of response to chemotherapy and might reflect the presence/absence of residual disease after therapy.

Disclosures:
No relevant conflicts of interest to declare.