bluuebunny/crossref_metadata_2025 huggingface dataset:
EVI1 Blocks Apoptosis in DA-1 Myeloid Leukemia Cells Via Enhanced Transcription of the Prosurvival Gene Bcl2a1 (A1)
Authors: Perkins Archibald S. , del Campo Jacob J. , Xiao Ying-Yi , Zhang Yi , Lin Sharon J. , Dudley Jonathan , Tuck David , Yatsula Bogdan
Published: October 2019

Retroviral insertion at the Evi1 locus causes high level of expression of the gene in myeloid neoplasms in mice and in nonmalignant expansions of myeloid cells in humans and monkeys. In these settings, it is suggested that EVI1 confers a survival advantage on myeloid cells. Here, we investigate the survival phenotype in DA-1 cells, a leukemic cell line with provirally activated Evi1. We report that short hairpin-mediated suppression of Evi1 in DA-1 cells induces apoptosis via the intrinsic/mitochondrial pathway: DNA fragmentation and histone release are both induced, as is reduction in mitochondrial membrane potential. In addition, procasapses 3 and 9, but not caspase 8 or Bid, are cleaved following Evi1 knockdown; phosphoAKT remains unchanged. Furthermore, mRNA expression profiling following Evi1 suppression show transcriptional changes in several apoptotic regulators, including a 3.5-fold decrease in Bcl2a1 (bfl/A1), a prosurvival member of the Bcl-2 family. To assess whether EVI1 regulates Bcl2a1 expression in a cell type other than DA-1 cells, we transduced primary murine Lin-/Sca-1+/c-Kit+ cells with EVI1 via retroviral vector, and then assessed the expression of Bcl2a1. This revealed that EVI1 induced a more than five-fold increase in Bcl2a1 mRNA expression, as measured by quantitative PCR. Furthermore, transduction of primary murine bone marrow cells with retroviruses bearing either Bcl2a1 or Evi1 resulted in a significant decrease in spontaneous apoptosis, as assessed by the activity of caspases 3 and 7. To further test if Bcl2a1 is necessary for leukemic transformation by Evi1, we assessed the ability of Evi1 to confer serial replating ability on primary bone marrow cells from Bcl2a1−/− mice. Bone marrow was harvested from Bcl2a1−/− and C57BL6 mice and transduced with retrovirus containing either no gene or Evi1. While in C57BL6 mice, Evi1 induced a significant increase in colonies, bone marrow from Bcl2a1−/− mice were resistant to transformation by Evi1. To show that this was due to the lack of Bcl2a1, the gene was added back via retrovirus. While Bcl2a1 by itself did not induce significant number of colonies over vector, when introduced into Bcl2a1−/− cells together with Evi1, there was a significant increase in colony formation. These data indicate that transformation of bone marrow cells by Evi1 depends on the presence of the Bcl2a1 gene. We further show EVI1 can transcriptionally upregulate a BCL2A1::luc reporter that harbors 1.37 kb of the human BCL2A1 upstream sequence. Our analysis of the Bcl2a1 promoter indicates that the effect of EVI1 is likely indirect. Based on our findings, we propose that EVI1 acts to block apoptosis in DA-1 cells by transcriptionally activating Bcl2a1.
PML Controls PTEN Localization and Function in APL through a Novel Deubiquitination Pathway
Authors: Carracedo Arkaitz , Song MIn-Sup , Salmena Leonardo , Egia Ainara , Lo-Coco Francesco , Teruya-Feldstein Julie , Pandolfi Pier Paolo
Published: October 2019

PML was discovered as a gene frequently involved in the t(15:17) translocation of acute promyelocytic leukemia (APL). Since then, consequences of PML-loss of function in APL and other solid tumors have been extensively studied. Numerous pathways influenced by this tumor suppressor, including the PI3K pathway, have been identified but their importance for the pathogenesis APL is still unclear. We have now discovered that PTEN, the main tumor suppressor opposing PI3K signaling, is aberrantly delocalized to the cytoplasm in APL, but not in AML. Moreover, we show that drugs such as arsenic trioxide and retinoic acid, currently used for the treatment of APL and known to target the oncogenic PML-RARalpha fusion protein, rescue the nuclear localization of PTEN. This discovery has critical biological implications since nuclear localization of PTEN is central to its tumor suppressive activity. Utilizing an APL cell line, genetically engineered cells and transgenic mice, we have delineated the mechanism by which PML sustains nuclear PTEN. PML opposes the activity of a novel PTEN-deubiquitinating enzyme, HAUSP, towards PTEN through a framework involving the adaptor protein DAXX (death domain-associated protein). We demonstrate that PML-RARalpha translocation results in overactive HAUSP and as a consequence, nuclear-excluded PTEN. In support of this notion, we show that HAUSP is overexpressed in human cancer, (e.g. in prostate cancer) which is in turn associated with a nuclear exclusion phenotype of PTEN. Overall our results describe a novel role for PML in the maintenance of PTEN function that is perturbed by oncogenic cues in human leukemia and solid tumors. Significantly, our findings open new avenues for the design of targeted therapies focused on potentiating the activity of the PML-PTEN tumor suppressive network.
Differing Clot Distribution in Black Compared to White Patients Presenting with Acute Lower Extremity Deep Venous Thrombosis
Authors: Buckner Tyler W , Puvvada Soham , Amin Chirag , Key Nigel S.
Published: October 2019

Venous thromboembolism (VTE) is a major cause of morbidity and mortality in the United States. There is growing evidence that VTE incidence and mortality rates differ among ethnic/racial groups. Specifically, it has been demonstrated previously that blacks with VTE have a higher proportional rate of pulmonary embolism (PE), as well as a higher case fatality rate from PE compared to whites (Arch Int Med2003;163(15):1843–8). We therefore compared the location of DVT (proximal vs. distal) in blacks and whites at the time of diagnosis. The null hypothesis was that there is no difference in the location of DVT between white and black patients at the time of diagnosis. We reviewed all lower extremity Doppler ultrasound studies that were positive for acute DVT at our Institution over a 3-year period to create a retrospective case series of 941 patients. Subjects were further classified by self-reported race (black or white), disposition at diagnosis (inpatient or outpatient), and clot location (proximal or distal). Distal DVT was defined as thrombus in one or more deep veins distal to -- but not including -- the popliteal vein. Results were analyzed using Stata version 10. We found that the rate of any proximal (proximal or both proximal and distal) DVT compared with distal only was significantly higher in black patients (OR = 1.4; 95%CI: 1.03, 1.8; P = 0.032), both for 637 inpatients (OR = 1.3; (0.94, 1.9); P = 0.11) and for 304 outpatients (OR = 2.0; (1.01, 3.8); P = 0.048). These differences persisted after controlling for sex and age. We conclude that blacks with acute lower extremity DVT are more likely to present with proximal DVT than whites. These results confirm earlier suggestions that the proportion of proximal lower extremity DVT is higher in blacks, which might account in part for the higher rate of PE (Arch Int Med2004;164(12):1348–9). We are further examining the possible causes of this disparity. Possibilities include genetic factors (such as factor V Leiden which is associated with more distal DVT presentation and is rare in blacks), co-morbid conditions (such as diabetes which is more prevalent in blacks), and access to healthcare and diagnostic services, which may lead to more delayed evaluation. Efforts will also be made to determine rates of idiopathic and provoked DVT in these sub-groups.
Risk of Venous Thromboembolism with Thalidomide in Cancer Patients: A Systematic Review and Meta-Analysis of Randomized Controlled Trials
Authors: Gray Keriann N , Chu David , Wu Shenhong , Lin Richard Z.
Published: October 2019

Background: Thalidomide, an oral angiogenesis inhibitor with immunomodulatory activity, is effective in the treatment of multiple myeloma, and also undergoing evaluation for other malignancies. The use of thalidomide is associated with significant side effects, including venous thromboembolism (VTE). Currently the overall risk of VTE with thalidomide is not well-defined.

Objective: We conducted a systematic review and meta-analysis of published randomized controlled trials (RCT) to determine the risk of VTE with thalidomide in cancer patients, and assess whether it is affected by anti-thrombosis prophylaxis.

Methods: We searched databases including PUBMED, the Web of Science (January, 1966–July, 2008), and abstracts presented at recent American Society of Clinical Oncology and American Society of Hematology conferences to identify relevant studies. Eligible studies included prospective RCT in which a standard anti-cancer therapy or placebo was employed with or without thalidomide with available data on VTE for analysis. The summary incidence, relative risk (RR), and 95% confidence interval (CI) were calculated using a random- or fixed-effects model based on the heterogeneity of the included studies.

Results: From 17 RCTs, a total of 3977 patients with multiple myeloma and a variety of solid tumors (prostate, breast, renal cell, melanoma, ovarian) were included for analysis. The overall incidence of VTE was 11.7% (95% CI: 8.1–16.5%). Patients treated with thalidomide had a significantly increased risk of VTE with a RR of 2.4 (95% CI: 1.9–3.0, p<0.001) when compared to controls. The risk was significantly decreased with prophylaxis from a RR of 3.5 (95%CI: 2.5–4.9, p<0.001) in the absence of prophylaxis to 1.9 (95%CI: 1.4–2.5, p<0.001) in the presence of prophylaxis. The risk of VTE may vary with tumor types; in patients with multiple myeloma, the incidence and RR were 15.7% (95%CI: 10.9–22.1) and 3.1 (95%CI: 2.1–4.5, p<0.001) respectively; while for patients with solid tumors, the incidence and RR were 5.3% (95% CI 2.1–12.8) and 3.5 (95% CI: 1.1–10.6, p=0.028) respectively.

Conclusion: Thalidomide therapy is associated with a significantly increased risk of VTE in cancer patients. The risk varies with anti-thrombosis prophylaxis and tumor type. It is strongly recommended to have surveillance for VTE in all patients receiving thalidomide, and prophylaxis to reduce the risk in patients with multiple myeloma.
Total Tissue Factor Pathway Inhibitor and Incident Venous Thrombosis: The Longitudinal Investigation of Thromboembolism Etiology
Authors: Zakai Neil A , Lutsey Pamela , Folsom Aaron R , Heckbert Susan , Cushman Mary
Published: October 2019

Introduction: Tissue Factor Pathway Inhibitor (TFPI) inhibits tissue factor, a potent coagulation initiator. Limited evidence suggests that low TFPI levels are associated with increased risk of venous thromboembolism (VT).

Methods: VT was ascertained in the Longitudinal Investigation of Thromboembolism Etiology, which combines data from the Atherosclerosis Risk in Communities Study and the Cardiovascular Health Study. Total TFPI was measured in a nested case-control sample. With 12 years of follow-up, 534 cases of new VTE were age-, sex-, and race-frequency matched with 1091 controls. The odds ratio for VT was determined for quartiles of TFPI, and for the bottom 5% versus the top 95% in logistic regression models adjusting for demographics (age, race, gender, study), coagulation factors (Factors VII, VIII, IX, XI, D-dimer), and other VT risk factors. To evaluate for greater than additive interactions we calculated the percent relative excess risk of interaction between TFPI and other VT risk factors.

Results: Mean TFPI in ng/mL (standard deviation) in cases and controls was 36.4 (12.8) and 35.0 (11.1), respectively. Increasing quartiles of TFPI were associated with male gender and increasing age, body mass index, factors VII, VIII, IX, XI, and D-dimer (all p<0.05). TFPI quartiles were not related to ethnicity, factor V Leiden, or the prothrombin gene polymorphism. Compared with those in the upper 95%, those in the bottom 5% of TFPI (32 cases, 56 controls) had an age-, sex-, race-, and cohort-adjusted odds ratio of 1.35 (0.86, 2.12) for VT. Adjusting for factors VII, VIII, IX, XI, and D-dimer increased the odds ratio to 1.93 (1.05, 3.53). Further addition of BMI to the coagulation factor adjusted model slightly attenuated the odds ratio to 1.70 (0.98, 2.93). There were no associations of TFPI in quartiles or as a continuous variable with VT. TFPI in the bottom fifth percentile did not demonstrate greater than additive interaction with factor V Leiden or with higher BMI.

Conclusions: Associations of high TFPI levels with VT were masked by concurrent higher procoagulant levels. After adjusting for procoagulant factors and D-dimer, TFPI ≤ 5% was associated with a 1.9-fold increased odds of VT. These data suggest high total TFPI may be a response to procoagulant states.
Iron Sufficient to Cause Hepatic Fibrosis and Ascites Does Not Cause Cardiac Arrhythmias in the Gerbil
Authors: Kaiser Lana , Davis John , Patterson Jon , Johnson Abby , Bohart George , Schwartz Kenneth A.
Published: October 2019

Chronic iron overload associated with hereditary hemochromatosis or repeated red cell transfusions is known to cause cardiac failure. Cardiac arrhythmias have been incidentally noted in patients with iron overload, but often times dismissed as being caused by other co-morbid conditions. Studies with iron-loaded gerbils suggest a role for iron in the development of cardiac arrhythmias, however these studies utilized short duration recordings of anesthetized gerbils. Furthermore, we were unable to reproduce these loading protocols without significant morbidity and mortality. Our goal was to characterize iron-induced arrhythmias in the chronically instrumented, untethered, telemetered gerbil. Monitored gerbils were divided into 2 groups: iron-loaded (n=23) and control (n=8). Iron loaded gerbils received iron dextran intraperitoneally at a dose of 1.7 (n=4), 3.0 (n=5) or 6.2 (n=14) g/kg; control gerbils received dextran. Gerbils were weighed and given a physical exam weekly. Electrocardiograms were recorded for 10 seconds every 30 minutes for approximately 6 months (DSI Ponehma) and reviewed daily. Quantitative analysis was completed on 6 iron loaded (6.2g/kg) and 3 control gerbils. Heart rate and intervals were calculated and arrhythmias were characterized and counted. Cardiac and hepatic histology and tissue iron concentration were assessed.

All gerbils showed evidence of frequent sinus arrhythmia (more than one episode per hour). However, except for two control gerbils that showed frequent unifocal PVCs, no significant arrhythmias were noted in daily review. There was no difference in heart rate, P duration, PR interval, QRS duration or QT interval between groups. Neither total number of arrhythmias nor arrhythmias per minute were different between groups. One iron-loaded gerbil had a single episode (11 beats) of supraventricular tachycardia. Two iron-loaded gerbils had PVCs, one had only a single beat and the other had 9 unifocal PVCs over the duration of the study. Iron-loaded gerbils rarely showed other arrhythmias One control gerbil had 260 unifocal PVCs over the duration of the study. Other arrhythmias were noted rarely.

Body weight and heart weight was not different between groups, while liver weight increased with increasing iron dose. Cardiac and hepatic iron were significantly increased in iron loaded gerbils when compared to control. Liver weight increased as iron dose increased. Seven of 14 gerbils loaded to 6.2 g/kg developed ascites as assessed both by physical examination and necropsy.

We conclude that an iron load sufficient to cause clinical liver disease does not, in the absence of co-morbid conditions, cause cardiac arrhythmias in the gerbil model of iron overload. This suggests that iron alone is insufficient to cause cardiac arrhythmias.
The GM-CSF Receptor Utilises Members of the Canonical Wnt-Signaling Pathway during Macrophage Differentiation
Authors: Brown Anna , Salerno Diana , Wilkinson Christopher , Perugini Michelle , Kok Chung , Goodall Greg , Solomon Patricia , Gonda Thomas , D’Andrea Richard
Published: October 2019

Over the past several years we have used a factor-dependent murine bi-potential cell line, FDB1, in combination with activated mutants of the GM-CSF receptor common beta subunit (hβc) to dissect receptor pathways and gene expression changes that can contribute to myeloid cell growth and differentiation. Of particular interest are the mechanisms by which signalling from the GM-CSF receptor can promote simultaneous granulocyte and macrophage differentiation. In this context we have characterised differentiation of FDB1 cells in response to an activated hβc mutant (FIΔ). For this mutant, which confers growth factor-independent granulocyte-macrophage differentiation in the FDB1 cell line, the presence of a single intracellular tyrosine residue (Y577) is a requirement for granulocyte differentiation. In the absence of this tyrosine residue FDB1 cells expressing FIΔ undergo uni-lineage macrophage differentiation (Brown et al., 2004). Comparative transcriptional profiling of these FDB1 populations has allowed us to identify gene expression changes associated with receptor-induced granulocyte or macrophage differentiation. An examination of the genes selectively associated with macrophage differentiation revealed a potential role for the transcription factor TCF4/TCF7L2, which is a central mediator of the canonical Wnt signalling pathway through its role as the DNA binding co-factor for β-catenin. Over-expression of TCF4 in FDB1 cells could initiate growth arrest and differentiation, under growth-promoting conditions. In FDB1 cells expressing the FIΔ mutant forced TCF4 expression resulted in an increase in the proportion of cells differentiating to macrophages. Further examination of signalling in the FDB1 FIΔ populations demonstrated that stabilisation of β-catenin is associated with the switch to macrophage differentiation. GM-CSF signalling in FDB1 cells also induces transient β-catenin stabilisation during bi-lineage GM differentiation. In FDB1 cells stabilisation of β-catenin and myeloid differentiation is also induced using the GSK3β inhibitor BIO (6-bromoindirubin-3′-oxime). The results using this unique model of receptor-induced GM differentiation are consistent with a role for the β-catenin pathway in regulating the macrophage lineage.
In Vitro Myelo-Lymphoid Colony Formation by Mouse Hematopoietic Stem Cells
Authors: Ooehara Jun , Takano Hina , Takayanagi Shin-ichiro , Nakauchi Hiromitsu , Ema Hideo
Published: October 2019

Hematopoietic stem cells (HSCs) clonally differentiate into all myeloid, B-lymphoid, and T-lymphoid lineages. Mouse HSCs are known to form in vitro colonies comprised of morphologically identifiable myeloid cells such as neutrophils, macrophages, erythroblasts, and megakaryocytes. Whether HSCs are able to differentiate along B-and T-lymphoid lineages in such colonies remains obscure. The co-culture systems with stromal cells such as S17, OP9, OP9/Delta cells have been shown to support B- and T-cell development. These systems have been used to identify subclasses of progenitors with lymphoid potentials. However, neither B cells nor T cells have been successfully generated from HSCs in vitro. This is most likely due to the lack of culture conditions which support HSCs to differentiate into a certain stage of lymphoid progenitors. In this study, we attempted to use serum-free single-cell culture to identify cytokines which fill the developmental gap between HSCs and lymphoid progenitors. Here we show that myelo-lymphoid colonies are formed by HSCs in the presence of thrombopoietin (TPO), interleukin (IL)-11, or IL-12 together with stem cell factor (SCF). CD34-negative/low, c-Kit-positive, Sca-1-positive, lineage marker-negative (CD34-KSL) bone marrow cells were individually cultured with a combination of cytokines for 7 days. All cells in each colony were transplanted into each from a group of lethally irradiated mice, along with compromised bone marrow cells. The recipient mice were periodically analyzed after transplantation to detect transient myeloid and lymphoid reconstitution. All myeloid, B-, and T-lymphoid progenitor activities were detected in single colonies formed in the presence of SCF+TPO, SCF+IL-11, SCF+IL-12. Only myeloid progenitor activity was predominantly detected in single colonies formed in the presence of SCF+IL-3, consistent with previous observations in blast colony assays. All these combinations of cytokines support self-renewal in HSCs to varying degrees. We conclude that TPO, IL-11, and IL-12 directly act on HSCs and support them to differentiate into progenitors with lymphoid differentiation potential. Early differentiation pathways in HSCs are likely to be used in common by myeloid and lymphoid lineages and be supported in common by multiple cytokines.
CSF-1 Promoter-Driven IL-34 Expression Can Rescue Phenotypes of CSF-1-Deficient Mice
Authors: Wei Suwen , Lin Haishan , Stanley E. Richard
Published: October 2019

Colony Stimulating Factor-1 (CSF-1) is the primary regulator of the survival, proliferation and differentiation of mononuclear phagocytes. All of the effects of CSF-1 are mediated by the high affinity CSF-1 receptor (CSF-1R), a protein tyrosine kinase encoded by the c-fms proto-oncogene. Previous studies have shown that the phenotype of CSF-1 receptor-deficient (Csf1r−/−) mice is more severe than the phenotype of CSF-1-deficient (Csf1op/op) mice. Csf1r−/− mice have a more severe osteopetrosis, fewer osteoclasts, lower survival and fewer tissue macrophages and these differences are more pronounced on single strain backgrounds. This led us to speculate that there may be another ligand for the CSF-1R (Dai et al., Blood99:111–120, 2002; Dai et al., J. Bone Min. Res.19:1441–1451, 2004). This was recently confirmed by the discovery of the novel cytokine Interleukin 34 (IL-34), which specifically binds to the CSF-1R (Lin et al., Science320:807-11, 2008). Like the secreted glycoprotein isoform of CSF-1 (sgpCSF-1), with which it shares no sequence similarity, IL-34 is a dimeric glycoprotein. Like CSF-1, it is broadly expressed in embryonic and adult tissues and it stimulates macrophage colony formation. We hypothesized that there is an incomplete overlap in the expression pattern of IL-34 and CSF-1 in the various cell types in which they are expressed and/or that expression of IL-34 and CSF-1 are differently regulated during mouse development. In transgenes, the CSF-1 promoter and first intron drives normal tissue specific and developmental expression of CSF-1 and such transgenes expressing sgpCSF-1 on the Csf1 op/op background partially rescue defects associated with CSF-1 deficiency (Nandi et al., Blood107:786–795, 2006). Thus one test of this hypothesis is to determine whether expression of IL-34 under the control of the same driver rescues those aspects of the CSF-1-deficient phenotype rescued by sgpCSF-1. We therefore generated the transgenic lines in which the expression of mouse IL-34 is under the control of CSF-1 promoter and first intron. One of these transgenic mouse lines rescued the Csf1op/op deficiences, like similarly-driven sgpCSF-1. These studies indicate that IL-34 can function like sgpCSF-1 in vivo when expressed in a CSF-1-dependent manner.
Effect of Iron Chelators on Cardiac Iron Assessed by MR T2* in Thalassemia Major
Authors: Galanello Renzo , Piga Antonio , Lai Maria- Eliana , Forni Gianluca , Danjou Fabrice , Longo Filomena , Agus Annalisa , Origa Raffaella
Published: October 2019

Introduction: Cardiac iron overload secondary to red blood cell transfusion is a common complication of thalassemia major despite the wide use of chelation therapy and cardiac disease still account for up to 70% of deaths in these patients. Furthermore, myocardial siderosis can be accurately assessed by cardiovascular magnetic resonance (CMR) using T2* sequence. Available chelators seem to have unique profiles of organ iron removal. We report a retrospective analysis of the effect of three available chelators on cardiac iron in patients with thalassemia major.

Methods: Fifty-two patients on subcutaneous desferrioxamine (DFO), 28 on deferiprone (DFP) and 28 on deferasirox (DFX) are included in the study. DFO was administered at a mean dosage of 36 ± 8 mg/kg/d for 10 to 14 hours per day for 21 ± 13 months, DFP at 86 ± 10 mg/kg/d for 23 ± 12 months and DFX at 27 ± 6mg/kg/d for 15 ± 5 months. T2* was measured according to Westwood et al (2005). Entry criteria included in each patient a value of T2* lower than 20 ms at baseline or at final assessment. Left ventricular ejection fraction (LVEF) was measured by echocardiography.

Results: At baseline, the 3 treatment groups did not show any significant difference in age blood consumption and cardiac T2* (DFO group: 13.4 ± 4.5 ms, DFP 13.9 ± 3.7 and DFX 12.8 ± 3.7; p=0.573).. At the last evaluation mean cardiac T2* was slightly increased in patients on DFO (13.9 ± 8.6 ms, p=0.8) and in those on DFX (13.8 ± 4.4 ms, p=0.04), while was substantially increased in patients on DFP (21.7 ± 9.2 ms, p=0.001). To correct for the different duration of treatment we calculated the percentage monthly cardiac T2* changes that were significantly higher in patients on DFP (1,84 ± 1.94), as compared to patients on DFO (0.2 3 ± 2.15) or DFX (0.45 ± 1.49) (p<0.001). No differences were detected between mean LVEF at baseline and at last assessment in all 3 groups.

Conclusions: In this retrospective study monotherapy with deferiprone was significantly more effective than desferrioxamine and deferasirox in alleviating myocardial siderosis in patients with beta-thalassemia major. Further studies are needed to understand if the cardiac T2* changes are influenced by the chelator dosages.
Prolylcarboxypeptidase Murine Hypomorphs Are Hypertensive and Prothrombotic
Authors: Adams Gregory N , Zhou Yihua , Larusch Gretchen , Nieman Marvin T , Hoit Brian , Jacobs Gretta , Yu Xin , Mahdi Fakhri , Shariat-Madar Zia , D’Alecy Louis , Okada Yoshio , Schmaier Alvin H.
Published: October 2019

Prolylcarboxypeptidase (PRCP) is a serine protease that degrades bradykinin and angiotensin II. It also activates prekallikrein (PK) (K =9 nM) to plasma kallikrein when bound to high molecular weight kininogen on cells and matrix independent of factor XIIa. PRCP polymorphisms are a risk factor for metabolic syndrome and pre-eclampsia. PRCP murine hypomorphs (PRCPgt/gt) were created from ES cells where the Prcp gene was interrupted by gene trap with a LacZ insertion in intron 4 (KST302gt/+). PRCPgt/+ mice were created and backcrossed 10 generations in a C57BL6 background. PRCPgt/gt mice contain <10% PRCP mRNA than littermate wild type (WT) mice. LacZ staining and immunostaining indicate PRCP is highly expressed in renal proximal tubules where it co-localizes with lotus lectin and arterial vessels (small, medium, carotid, aorta) where it co-localizes with alpha-SMA, a vascular smooth muscle cell marker. Studies show that PRCPgt/gt mice are hypertensive. Using carotid sensors and continuous telemetry BP monitoring for 7 days, the mean systolic and diastolic BP in PRCPgt/gt mice were ~138, 125, and 113 mm Hg, versus ~125, 113, and 111 mg Hg in WT mice. Transthoracic ultrasound studies show significantly reduced anterior and posterior cardiac muscle velocity and strain at 6 and 12 months in PRCPgt/gt mice versus WT. Murine micro-MRI show reduced ejection fraction (p=0.038) and an enlarged aortic root (p=0.048) in PRCPgt/gt mice. Additionally, PRCPgt/gt mice have shortened carotid artery closure times using the Rose Bengal (25±0.8 min PRCPgt/gt vs 51±4 min WT, p<0.0001) and ferric chloride (20.6±3.4 min PRCPgt/gt vs >60 min WT, p<0.0001) models of carotid artery thrombosis. Pharmacological inhibition of PRCP with Z-Pro-Prolinal or plasma kallikrein with PSKI-571, SBTI, or Pro-Phe-Arg- CK in WT C57BL/6 or 129/SvJ mice resulted in shortened Rose Bengal carotid artery closure times. PRCPgt/gt murine hypomorphs also have reduced plasma factor XII (69±12% vs 114±15% WT, p<0.03), increased PK levels (110±4% vs 94±3% WT, p<0.006) with normal angiotensin II and bradykinin levels. PRCPgt/gt mice also have normal contact activation- or tissue factor-induced thrombin generation times, indicating that arterial thrombosis risk is independent of thrombin formation. These combined investigations indicate the PRCP is an important modulator of constitutive arterial blood pressure and thrombosis risk by mechanisms independent of thrombin generation. Further, reduced PK activation or pharmacologic inhibition of plasma kallikrein may increase arterial thrombotic risk.
Platelet Interaction with Bone Marrow Derived CD34+ Stem Cells: Potential Role of Chemoattractant Chemerin
Authors: Du Xiao-Yan , Leung Lawrence L.
Published: October 2019

Chemerin is a 16 kDa chemoattractant circulating in blood in an inactive form (prochemerin). Its chemotactic activity is released following proteolytic cleavage of its labile carboxyl-terminal amino acids by proteases of the coagulation, fibrinolytic and inflammatory cascades. Chemokine-like receptor 1 (CMKLR1) is a chemerin receptor expressed on plasmacytoid dendritic cells, natural killer cells and tissue macrophages. We identified the expression of CMKLR1 on mouse CD34 positive (CD34+) bone marrow-derived stem cells (BMSC) using anti-mouse CMKLR1 antibody by FACS. Fluorescein-labeled CD34+/CMKLR1+ BMSC adhered to immobilized chemerin dose-dependently. The EC50 of BMSC adhesion to human and mouse chemerin is 2.5 and 5 ug/mL respectively. BMSCs also adhered to SDF-1 (stromal cell-derived factor-1) with EC50 of 0.3 ug/mL, but did not bind to immobilized fibronectin. We have previously reported that platelets express chemerin and release it upon activation. CMKLR1 is also expressed on platelet surface as demonstrated by FACS. Immobilized chemerin could induce platelet adhesion and CD62P (p-selectin) expression as detected by fluorescein-labeled anti-CD62P antibody. BMSC formed complexes with both resting and activated platelets as demonstrated by fluorescein-labeled washed platelets incubated with unlabeled mouse CD34+/CMKLR1+ BMSCs using FACS analysis. Both anti-chemerin and anti-CD62P antibodies could partially block BMSCs binding to immobilized platelets at 50% and 35% respectively. In conclusion, chemerin directly mediates platelets interaction with BMSCs. Chemerin also facilitates platelet/BMSC complex formation by inducing platelet p-selectin (CD62P) expression. Platelet-derived chemerin, similar to SDF-1, may be involved in tissue regeneration by inducing progenitor cells mobilization and differentiation.
Inhibition of the ADP/P2Y12 Pathway Confers Additional Protection against Arterial Thrombosis in PAR-4 Deficient Mice
Authors: Cornelissen Ivo , De Candia Erica , Palmer Daniel , Coughlin Shaun R
Published: October 2019

Clopidogrel, a potent anti-thrombotic drug widely used for the secondary prevention of myocardial infarction and ischemic stroke, inhibits the ADP/P2Y12 pathway by inducing irreversible alteration of the platelet receptor P2Y12, ultimately uncoupling ADP from adenylyl cyclase inhibition. Clinical trials have demonstrated additional benefits of dual antiplatelet therapy, such as combined treatment with clopidogrel and aspirin, in the prevention of ischemic cardiovascular disease. We previously established that protease-activated receptor (PAR) signaling is necessary for platelet activation by thrombin and plays an important role in mouse models of hemostasis and thrombosis. Indeed a PAR antagonist is currently being used as an anti-thrombotic agent in clinical trials.

In order to evaluate the possible interaction between the thrombin/PAR and ADP/P2Y12 pathways, we orally administered clopidogrel (30mg/kg) or vehicle to PAR-4 null, heterozygous and wild type littermates, and evaluated arterial thrombus formation and bleeding during surgical challenge. In a FeCl3-induced carotid thrombosis model, PAR-4 deficient mice treated with clopidogrel were completely protected from thrombosis compared to their wild type treated littermates. Interestingly, clopidogrel treated PAR-4 heterozygotes showed an intermediate level of protection compared to treated wild type and null mice that was similar to vehicle-treated PAR-4 deficient mice. Additionally, we scored bleeding during the surgical procedure while blinded to genotype and treatment. The bleeding score was increased in clopidogrel treated mice compared to vehicle treated mice and was exacerbated with increasing haplotype deficiency of Par-4.

To better define the relationship between these two activation pathways, we generated mice lacking both PAR-4 and P2Y12. All genetic combinations were born at the expected Mendelian distribution. Females lacking both receptors carried offspring to term, but signs of bleeding were observed during parturition. Preliminary data from PAR-4/P2Y12 double null mice, in both the tail bleeding assay and in a FeCl3-induced carotid thrombosis model, is in agreement with the results obtained in clopidogrel treated PAR-4 deficient mice. This study provides useful information to evaluate the efficacy and safety of dual antiplatelet therapy inhibiting the thrombin/PAR and ADP/P2Y12 pathways and explores the relationship between these two critical pro-thrombotic pathways in an in vivo setting.
Use of Recombinant Activated Factor VII in Severe Postpartum Haemorrhage: Data from the South European Registry. A Multicentric Observational Retrospective Study
Authors: Barillari Giovanni ,   On behalf of the rFVIIa - PPH South European Registry Study Group
Published: October 2019

Introduction: Major Post Partum Haemorrhage (PPH) is a life threatening complication of labor, which mainly occurs without warning, predictive signs and symptoms and often in absence of predisposing conditions. A retrospective survey of cases of severe primary PPH treated with Recombinant Activated Factor VII (rFVIIa) in Italy, Greece, Spain and Portugal was performed. The aim of our study was to report the south european real experience in clinical practice about the use rFVIIa in PPH treatment.

Methods: anamnestic, clinical and haemostatic data about fortyfive patients, treated between 2005 and 2007, were collected. Coagulative parameters and transfusion requirements before and after rFVIIa treatment were compared.

Results: INR significantly decreased, while levels of fibrinogen markedly increased after rFVIIa administration. The median need of packed red blood cells units, platelets units, fresh frozen plasma and crystalloids and colloids, before and after rFVIIa administration, dramatically reduced being respectively 6 and 2 units (p<1.2exp-6), 1.5 and 0 units (p = 0.001), 1250 and 0 mL (p<4.4exp-5), 3000 and 1250mL (p<0,0042). No cases of adverse effects or thromboembolic complications were observed.

Conclusions: Our clinical and haemostatic data suggest that recombinant activated factor VII may be a safe and helpful adjunctive therapy in the management of postpartum haemorrhage.
Sickle Cell Disease Is Associated with Reduced Adenosine Deaminase Catalytic Activity, Resulting in Altered Adenosine Metabolism
Authors: Eiymo Mwa Mpollo Marthe-Sandrine , Malik Punam
Published: October 2019

Abstract 1078

Sickle cell disease (SCD), a chronic hemolytic anemia due to a mutant beta-globin, is associated with prominent inflammatory features with elevated levels of inflammation mediating molecules and an oxidative state, with elevated levels of reactive oxygen species (ROS). Additionally, elevated levels of adenosine (Ado) have recently been shown to play a role in exacerbating sickle cell pathophysiology. However, the etiology of increased Ado is unclear; and presumed to occur secondary to increased ATP release from cellular damage (Xu et al, Nature Medicine, 2010). Herein, we show that increased levels of Ado in the plasma of patients with SCD and the mouse model of SCD [Berkeley sickle (SS) mice] were accompanied by significantly reduced catalytic activity of the Ado degrading enzyme, adenosine deaminase (ADA) in sickle red blood cell (RBCs). ADA activity in Berkeley sickle (SS) RBCs was 246 ± 50 U/L as compared to 570 ± 36 U/L in normal mouse RBCs (derived from Berkeley AA/WT mice), as measured by a color-based enzymatic assay. Notably, despite lower enzyme activity of ADA, immunoblotting assays showed that the levels of ADA protein in sickle RBC were similar, or even slightly higher compared to that in wild type (WT) RBC, suggesting that ADA catalytic activity was impaired in sickle RBCs. In vitro plasma swap experiments, where WT RBC are incubated with sickle plasma and vice versa for an hour, revealed that sickle plasma induced a significant decrease in ADA activity in WT RBC, suggesting that plasma from sickle mice contains a soluble factor that is capable of impairing ADA enzyme activity. Interestingly enough, the decrease in ADA activity correlated inversely with the levels of intracellular RBC ROS in sickle mice. Next we directly assess in vivo the effect of induced oxidative stress on RBC ADA activity in sickle mice by injecting sickle mice with 35umoles/Kg body weight of hemin and saline respectively. Preliminary data suggests a decrease in RBC ADA activity in hemin–injected mice (294 ± 197 U/L) compared to the saline-injected group (346 ± 185.8 U/L), and this data was consistent with a concomitant increase in intracellular RBC and white blood cells ROS levels. We conclude that increase in oxidative stress levels mediates a post translational alteration of ADA catalytic activity and alters Ado metabolism in SCD.

Disclosures:

No relevant conflicts of interest to declare.
Batf3-Dependent CD11blow/− Peripheral Dendritic Cells Are GM-CSF-Independent and Are Not Required for Th Cell Priming After Subcutaneous Immunization
Authors: Edelson Brian T. , Murphy Kenneth M.
Published: October 2019

Abstract 1113

Granulocyte/macrophage colony stimulating factor (GM-CSF) regulates the development and activity of several myeloid cell types, including dendritic cells (DCs), thereby influencing the initiation and maintenance of adaptive immune responses. Mice lacking GM-CSF or its receptor show decreased antigen specific T cell priming against the encephalitogenic peptide of myelin oligodendrocyte glycoprotein (MOG35–55), and are resistant to experimental autoimmune encephalitis (EAE), a mouse model of multiple sclerosis. Beyond this role in priming adaptive immune responses, GM-CSF acts to sustain ongoing T cell effector responses, both in EAE initiated by MOG35–55 peptide and in other models of autoimmunity.

A recent report has argued that part of the role for GM-CSF in EAE is based on its requirement for the development of a subset of dermal DCs, termed CD11blow/−Langerin+CD103+ DCs (J Exp Med. 207:953–61, 2010). These dermal DCs represent one anatomic subtype of peripheral tissue CD11blow/−CD103+ DCs, and share the properties of efficient antigen cross-presentation and IL-12 production with lymphoid tissue-resident CD8α+ DCs. Peripheral tissue CD11blow/−CD103+ DCs and lymphoid tissue-resident CD8α+ DCs are developmentally related, each being dependent on FMS-like tyrosine kinase 3 ligand (Flt3L), and requiring the transcription factors Batf3, Irf8, and Id2 for development. Mice deficient in any one of these transcription factors selectively lack these DC subsets, with Batf3−/− mice representing the best model for studying the role of these DCs in vivo, as Irf8−/− and Id2−/− mice display more widespread immune defects.

Here, we directly compared the development of peripheral tissue DCs and susceptibility to EAE in GM-CSF receptor deficient (Csf2rb−/−) and Batf3−/− mice. We confirmed that Csf2rb−/− mice displayed poor priming of MOG35-55 peptide-specific T cell responses and resisted induction of EAE. Importantly, however, we found that Batf3-dependent dermal CD11blow/−Langerin+ DCs did, in fact, develop in Csf2rb−/− mice, but that these DCs expressed reduced, but not absent, levels of the surface marker CD103. In contrast, Batf3−/− mice, lacking all peripheral CD11blow/− DCs and lymphoid tissue-resident CD8α+ DCs, showed robust Th cell priming after subcutaneous immunization, and were fully susceptible to EAE. These results exclude the hypothesis that defective T effector cell priming and resistance to EAE exhibited by Csf2rb−/− mice result from the absence of dermal CD11blow/−Langerin+CD103+ DCs, and instead suggest that GM-CSF is acting via different mechanisms to promote autoimmunity.

Disclosures:

No relevant conflicts of interest to declare.
Ewing Sarcoma Cells Are Sensitized to NK Cell Mediated Killing by SNDX-275, An HDAC Inhibitor
Authors: Ray Anish K. , Lee Dean A.
Published: October 2019

Abstract 1126

Natural Killer (NK) cells are an important a part of the innate immune system response to infection and malignancy. Tumors may escape NK cell surveillance by upregulating expression of inhibitory ligands (HLA), or downregulating expression of activating ligands (MICA/B, ULBP). Histone Deacetylase (HDAC) inhibitors promote cell cycle arrest, differentiation, and apoptosis in tumor cells by decompacting chromatin to a more transcriptionally active state. In previous studies, we and others have shown that HDACi can sensitize tumor cells to NK cell mediated cytotoxicity by modulating expression of activating or inhibitory receptors. We investigated whether HDACi could modulate sensitivity of Ewing Sarcoma (EWS) cell lines to lysis by NK cells. EWS cells were cultured in vitro in the presence of SNDX-275, a Class I HDAC inhibitor, for 24, 48 and 72 hours, and then assessed for viability, cell proliferation, expression of activating and inhibitory receptors, and susceptibility to NK cell mediated cytolysis. We found a dose and time dependent inhibition of EWS cell proliferation mediated by SNDX-275. We also found a dose and time dependent decrease in the expression of HLA on tumor cell surface, which correlated with drug treated cells being more susceptible to NK cell mediated killing. SNDX-275 not only has a direct toxicity on Ewing sarcoma cells but it also appears to improve NK cell mediated cytolysis of these cells by down regulation of HLA, an inhibitory receptor of NK cells.

Disclosures:

No relevant conflicts of interest to declare.
Bypassing Agents in Children with Inhibitors: How Much Do We Use and How Much Is Too Much?
Authors: Gordon Deana , Wang Michael , Ruegg Katherine , Villalobos-Menuey Elizabeth , Manco-Johnson Marilyn J.
Published: October 2019

Abstract 1217

Background:

Children with hemophilia A and high-titer inhibitors suffer frequent severe bleeding events and often respond to bypassing agents more poorly than adults resulting in increased dosing and cost. Aims: This study was conducted to determine the amount and variability of bypass agent therapy currently given to children with inhibitors at a large combined adult and pediatric hemophilia treatment center in comparison to treatment of adult inhibitor patients. Methods: All subjects were enrolled onto a single institution prospective inceptional cohort study of bleeding disorders with appropriate IRB-approved consent and assent. All children who developed high-titer inhibitors were treated with immune tolerance induction, and children treated with bypassing agents primarily represent difficult inhibitors. Data regarding treatment agents, dose and duration were extracted for 24 bleeding events in 6 children in comparison to comparable data on 26 bleeding events in 5 adults with high-titer inhibitors. Treatment decisions were made clinically by academic hematologists in consultation with hemophilia nurses, patients and parents based on perceived severity of bleeding events and therapeutic response. Treatment was given as recombinant factor VIIa (NovoSeven, N, n=28), prothrombin complex concentrates (FEIBA, F, n=10) or alternating doses of both (A, n=12). Median results for individual children and aggregated adult data are shown on Table 1. Children received seven times the treatment doses and duration compared with adults. In addition, children received both N and F for half of the bleeding events, while this was used for only one adult event. Treatment duration in children was not affected by initial dose or dose frequency of N (p > 0.05 for both). Bleeding events were severe and difficult to manage; significant long-term morbidity in these 6 children include ankle arthropathy in 3, elbow contracture following compartment syndrome in 2 and quadrucep pseudotumor in 1.

Discussion:

Children with hemophilia A and inhibitors experience bleeding episodes that are poorly responsive to both N and F, resulting in higher dosing, longer duration and enormous costs. Children require better inhibitor therapies. Clinical trials of new bypassing agents designed specifically for children are urgently needed.

Disclosures:

No relevant conflicts of interest to declare.
SAR1 Is Critical to the HbF-Inducing Effect of HU and Other Therapeutic Cytotoxic Agents
Authors: Zhu Jianqiong , Chin Kyung , Aerbajinai Wulin , Kumkhaek Chutima , Liu Wenli , Rodgers Griffin P.
Published: October 2019

Abstract 1306

Hydroxyurea (HU) is a drug that is effectively used in the management of the β-hemoglobinopathies sickle cell disease and β-thalassemia by augmenting the production of fetal hemoglobin (HbF). However, the molecular mechanisms underlying HU-mediated HbF regulation remain unclear, as does the reason why some proportion of the patients failed to show a beneficial therapeutic response. A better understanding of how hydroxyurea acts at the molecule levels and induces fetal hemoglobin production would offer targeted molecular therapy with higher specificity, and less toxicity and may provide alternative effective therapies to current non-responders. Secretion-associated and ras-related protein (SAR1) is a small guanosine triphosphate (GTP)-binding protein that is induced by HU and closely mimics the known effects of HU on erythroid cells, including gamma-globin induction and cell-cycle regulation. Here we report of our further investigation on whether SAR1 is an obligatory mediator to the HbF-inducing effect of HU and other therapeutic cytotoxic agents. Our results indicate that blocking SAR1 expression not only significantly lowered the basal and HU-elicited HbF production in CD34+ and K562 cells (see table), it also abolished HU-mediated S-phase cell-cycle arrest (see table) and cell apoptosis (P<0.001).Transcriptional factor BCL11A, which controls globin switching and suppresses HbF, is involved in SAR1 and HU regulated gamma-globin expression, and SAR1 is required for HU, 5-AZA and Ara-C mediated BCL11A reduction. We also found HU acts through the interaction of NFκB with its conjugated binding site on SAR1 promoter to regulate its transcriptional expression. Our data show that SAR1 is not only sufficient, but indeed necessary and central, to HU's beneficial effects. Our data suggests that SAR1 is not only sufficient, but indeed necessary and central, to HU's erythroid effects. Further exploration of the mechanistic details underlying SAR1 effects may reveal novel therapeutic targets for the beta-globin disorders.MockControl shRNA or microRNASAR1 shRNA or microRNAHU−HU+HU−HU+HU−†pHU+‡pHbF positive CD34 + cells27.00 ± 1.01%50.70 ± 1.88%20.59 ± 0.76%0.00331.77 ± 1.17%0.00001HbF positive K562 cells18.21 ± 0.21%41.00 ± 1.41%19.72 ± 2.11%43.37 ± 0.2%14.29 ± 1.2%0.00426.63 ± 1.23%0.00002Fraction of S-phase cells24.94 ± 0.84%32.38 ± 0.59%24.08 ± 0.59%34.26 ± 0.54%24.88 ± 0.4%0.09727.64 ± 0.25%0.00192†p compared with control HU-‡p compared with control HU+

Disclosures:

No relevant conflicts of interest to declare.