bluuebunny/crossref_metadata_2025_split huggingface dataset:
ADAMTS13 Reduces Vascular Inflammation and Early Development of Atherosclerosis Via VWF-Dependent Mechanism.
Authors: Gandhi Chintan , Lentz Steven R. , Chauhan Anil K 
Published: October 2019

Abstract
Abstract 2178

Background and objective:
The metalloprotease ADAMTS13 prevents spontaneous thrombosis in the microvasculature by cleaving hyperactive ultra large von Willebrand factor (ULVWF) multimers into smaller and less active forms. Recently, we and others have demonstrated that ADAMTS13-deficiency aggravates vascular inflammation and early atherosclerosis in apolipoprotein E-deficient (ApoE−/−) mice fed a high-fat Western diet. Although VWF is the only known substrate for ADAMTS13, it is not known if the effects of ADAMTS13 on vascular inflammation and atherosclerosis are mediated through its proteolytic effects on VWF or possibly another ADAMTS13 substrate. In this study, we determined whether the exacerbated atherosclerosis observed in the Adamts13−/−/ApoE−/− mice is dependent or independent of VWF.

Model and methods:
ApoE−/−, Adamts13−/−/ApoE−/−, Adamts13−/−/Vwf−/−/ApoE−/− and Vwf−/−/ApoE−/− male mice were fed a high-fat Western diet (20% fat, 0.2% cholesterol) beginning at 6 weeks of age until they were sacrificed at 4 months. We compared the extent of atherosclerosis in the cross section area of the aortic sinus using the VerHoeffs/Van Gieson stain. Inflammatory cells (neutrophils and macrophages) in the aortic lesions were quantitated by immunohistochemistry.

Results:
Similar to previous published reports by us and others, we found that the mean lesion area in the aortic sinus of the Adamts13−/−/ApoE−/− mice were significantly larger (mean ± SEM: 26.6 % ± 1.9 %, P<0.01) compared with ApoE−/− mice (mean ± SEM: 20.4 % ± 1.5 %). Next, we quantitated macrophage and neutrophil infiltration into lesions in the aortic sinus by immunohistochemistry. We observed significantly increased macrophages (mac-3 positive) and neutrophils (Ly6 B.2 positive) recruitment in the aortic sinus of Adamts13−/−/ApoE−/− mice compared with ApoE−/− mice. The mean lesion area in the aortic sinus of Adamts13−/−/Vwf−/−/ApoE−/− mice (mean ± SEM: 15.2 % ± 1.5 %) were similar to Vwf−/−/ApoE−/− mice (mean ± SEM: 15.6 % ± 0.9 %), suggesting that the accelerated atherosclerosis observed in ADAMTS13-deficient mice is VWF-dependent. Finally, macrophages and neutrophils recruitment in the aortic sinus of Adamts13−/−/Vwf−/−/ApoE−/− mice were similar to Vwf−/−/ApoE−/− mice, suggesting that increased vascular inflammation observed in ADAMTS13-deficient mice is also VWF-dependent. Total cholesterol and triglyceride levels were similar among groups fed a high-fat Western diet.

Conclusion:
These findings reveal that VWF-deficiency abrogates accelerated early atherosclerosis in ADAMTS13-deficient mice, suggesting that VWF the only relevant substrate for ADAMTS13 in murine atherosclerosis.

Disclosures:
Lentz: Novo Nordisk A/S: Consultancy, Investigator Other.
Vascular Disrupting Combretastatins Impair Bone Marrow Endothelial Cells by Depolymerizing the Microtubule Cytoskeleton.
Authors: Bosse Raphael C , Rohatgi Sunil , Meacham Amy , Wise Elizabeth , Scott Edward W , Cogle Christopher R 
Published: October 2019

Abstract
Abstract 2183

Background:
The vascular disrupting combretastatins CA1P (OXi4503) and CA4P (Zybrestat) are known to target solid tumor neovasculature by altering endothelial cell morphology and occluding cancer neovessels. In prior work, we and others have demonstrated that combretastatins target acute myeloid leukemia (AML) cells by generating reactive oxygen species within the malignant myeloid cells. We are currently conducting a phase 1 clinical trial of OXi4503 in patients with AML (NCT01085656). However, the vascular targeting mechanisms by which combretastatins regress AML are ill defined. We hypothesized that combretastatins also target bone marrow endothelial cells (BMECs).

Methods:
BMECs were isolated and cultured from bone marrow of healthy subjects and patients with AML. The cells were treated with various doses of the combretastatins CA1 and CA4 for 24 and 48 hours. Cell viability was measured by XTT assay. BMEC function in response to combretastatin treatments was quantified using a modified in vitro scratch assay and 2D capillary formation using Matrigel. Analysis of microtubule cytoskeleton was performed by staining treated BMECs with anti-β-tubulin antibodies and fluorescence microscopy. To ascertain the effects of combretastatin treatment on the ability of BMECs to support and protect AML cells, co-culture experiments were performed.

Results:
The combretastatins CA1 and CA4 did not decrease BMEC viability or metabolic activity. However, scratch assay showed impaired BMEC migration, with only 15.6% and 7.5% of the wound covered by BMECs after CA1 and CA4 respectively, compared to untreated control (80%) (p=0.0173). Matrigel assay also showed impaired BMEC function after combretastatin treatment, with complete lack of capillary tube formation compared to untreated BMECs which generated an average of 27.3 capillary tubes per 15 BMECs (p<0.001). Immunohistochemistry showed a distinct degradation of microtubule cytoskeleton in BMECs treated with CA1 and CA4. Finally, when treated with a vascular targeting combretastatin, AML cells co-cultured with BMECs showed a lower survival rate than AML cells alone (64.1% vs. 75.3%, p = 0.0267).

Conclusion:
Combretastatins directly impair BMEC function and eliminate BMEC protection of AML cells. The dual targeting of combretastatins on BMECs and AML cells leads to enhanced AML regression. These data highlight the role of the leukemia microenvironment as a protective reservoir of disease and provide methods of tracking biologic activity of combretastatins in early phase clinical studies.

Disclosures:
Cogle: OXiGENE: Research Funding; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding.
Glycosylphosphatidylinositol Anchored TFPI As Main Regulator of Factor X Activation On the Surface of Endothelial Cells.
Authors: Dockal Michael , Pachlinger Robert , Baldin-Stoyanova Angelina , Knofl Fabian , Ullrich Nadja , Koehn Jadranka , Ehrlich Hartmut J. , Scheiflinger Friedrich 
Published: October 2019

Abstract
Abstract 2210
Tissue factor pathway inhibitor (TFPI) is a key regulator of factor X (FX) activation in the extrinsic pathway of blood coagulation. TFPI inhibits FXa generation by formation of a quaternary complex consisting of factor VIIa (FVIIa), tissue factor (TF), FXa and TFPI. The main portion (∼80%) of TFPI in humans is reportedly associated with endothelial cells. We used human umbilical vein endothelial cells (HUVECs) as a model to obtain further insight into the function of TFPIα and the glycosylphosphatidylinositol (GPI) anchored TFPI form, which represents TFPIα bound to GPI-anchored surface proteins and/or TFPIβ. In contrast to TFPIα, which consists of 3 Kunitz domains (KD) and a basic C-terminal part, GPI-anchored TFPIβ lacks the third Kunitz domain (KD3) and the basic C–terminal region due to alternative splicing. In TFPIβ these two domains are replaced by a sequence that adds a GPI anchor to the protein linking it to the cell membrane.
Treatment of HUVECs with phosphatidylinositol phospholipase C (PI-PLC) that cleaves GPI-anchors and subsequent fluorescence activated cell sorting (FACS) on living cells showed that GPI-anchored TFPI represents about 70–80% of cell surface TFPI. Staining of TFPI on and in fixed and permeabilized cells (total TFPI) demonstrated that GPI-anchored cell surface TFPI contributes to ∼20% of total cellular TFPI. Enzyme-linked immunosorbent assay (ELISA) showed that PI-PLC treatment released a TFPI protein lacking the KD3 and basic C-terminus. These findings strongly suggest that TFPIβ is the predominant GPI-anchored form of TFPI on HUVECs. FX activation assays performed on the cell surface of PI-PLC treated living HUVECs showed the importance of GPI-anchored TFPI on extrinsic Xase complex activity. PI-PLC treatment resulted in increased FX activation. Although GPI-anchored TFPI displays ∼70–80% of cell surface TFPI, overall FXa generation was increased only by ∼50%. In conclusion, HUVEC surface TFPI is predominantly TFPIβ, and GPI-anchored TFPI is the main but not sole regulator of FX activation on the surface of HUVECs.

Disclosures:
No relevant conflicts of interest to declare.
Thrombosis and Inflammation Are Coupled by a Common Thioredoxin Reductase Pathway Downstream of the P2×7 Receptor.
Authors: Rothmeier Andrea S , Furlan-Freguia Christian , Petrich Brian G. , Marchese Patrizia , Ruggeri Zaverio M. , Ginsberg Mark H. , Ruf Wolfram 
Published: October 2019

Abstract
Abstract 2218
The purinergic P2X7 receptor contributes to thrombosis by promoting tissue factor (TF) activation and the release of prothrombotic microparticles (MP). On primed macrophages, P2X7 stimulation induces the procoagulant activity of cell surface TF and the release of MP that carry TF, integrin β1 and protein disulfide isomerase (PDI). The generation of TF+ MP is dependent on extracellular thiol-disulfide exchange, but intermediates leading to MP generation downstream of P2X7 signaling are incompletely defined. Tracking of cell surface TF by confocal microscopy shows that constitutive internalization of TF is prevented by P2X7 activation. In non-stimulated cells, inhibition of dynamin-dependent endocytosis retains TF on the cell surface, rapidly upregulates TF activity, and releases procoagulant MP carrying TF and integrin β1. Integrin recycling is dependent on the small GTPase ARF6 that is found incorporated into MP released when internalization is blocked. In contrast, activation of P2X7 releases ARF6 into the MP-depleted supernatant rather than associated with MP. Decreased ARF6 GTP loading in P2X7-stimulated cells and additional data in integrin α4 S988A mutant mice with reduced ARF6 activity support the conclusion that P2X7 interrupts constitutive ARF6 recycling to increase cell surface availability of TF-integrin complexes for incorporation into MP. Confocal imaging shows that P2X7 stimulation promotes filopodia formation and the peripheral transport of TF to the tips of filipodia. Proteomics identifies γ-actin as a thiol-regulated MP protein that is released by P2X7 signaling. We show that specific inhibition of thioredoxin reductase attenuates P2X7-induced actin remodeling and filopodia formation, the generation of highly procoagulant MP carrying γ-actin and PDI, and the increased availability of extracellular thioredoxin and free thiols. Importantly, inhibition of thioredoxin reductase also prevented the processing and release of the pro-inflammatory cytokine IL1β. Thus, the thioredoxin reductase system is a crucial intermediate downstream of P2X7 activation that may couple coagulation and inflammation in cardiovascular diseases.

Disclosures:
No relevant conflicts of interest to declare.
Efficiency of RBC Antibody Detection Methods: Comparison of Manual Indirect Antiglobulin Test (IAT) to a Method Using Very Low Ionic Strength Conditions (vLIS/IAT).
Authors: Szymanski Irma O. 
Published: October 2019

Abstract
Abstract 2276
Is has long been known that RBC antibody detection is enhanced following a decrease in the ionic concentration. In the past we added into our routine pre-transfusion antibody screen a new test, which employs ionic strength conditions between 15 to 17% of the ionic strength of 0.9% NaCl (vLIS/IAT): 2 drops of patient's plasma and one drop of 3% RBC are incubated for 15 minutes in 0.5 mL of 5% mannitol, pH 6.0 with phosphate buffer, containing 8mM EDTA. The RBCs are washed with normal saline and tested for agglutination by an anti-IgG reagent. Other published low ionic strength methods decrease the ionic concentration less, up to about 50%.
In the process of pre-transfusion testing we found 711 patients with one, two or more blood group antibodies. The following table shows the specificities and quantities of clinically significant antibodies detected by these two methods. The following antibody specificities were not included: those that were present only in small quantities, directly agglutinating IgM antibodies and anti-Lea and Lebantibodies which are usually considered to be clinically insignificant.
Antibody specificity Total Number detected % Detected by IAT % Detected by vLIS/IAT C 71 71.8% 93.0% c 36 80.5% 92.0% D 147 68.7% 97.3% E 220 40.3% 95.5% Fya 44 70.5% 95.5% Jka 60 60.0% 96.7% K 154 89.0% 76.0% M 23 34.8% 87.0% S 18 50.0% 100% Total 773 Avg. 62.8% Avg. 92.6%
Overall, vLIS/IAT detected an increase in clinically relevant antibodies compared to IAT. This is clearly seen in the case of anti-E, anti-D, anti-Jka, anti-M and anti-S antibodies. Anti-K antibodies, however, were detected preferably by IAT. It should be noted that more anti-Le antibodies, although not shown here, were detected either by IAT or by direct agglutination (IgM antibodies).
In summary, these data show that vLIS/IAT improved antibody detection when incorporated into routine pre-transfusion tests. They also indicate that one method does not detect all clinically significant antibodies; therefore it is beneficial to use two methods which in combination cover the varying spectra of RBC antibodies.

Disclosures:
No relevant conflicts of interest to declare.
Validation of a Non-Invasive Hemoglobin Estimation in Whole Blood Donors.
Authors: Al-Khabori Murtadha K. , Farsi Khalil Al , Al-Huneini Mohammed , Daar Shahina , Al-Hashim Abdulhakeem , Al-Kemyani Nasser , Al-Riyami Arwa Z. 
Published: October 2019

Abstract
Abstract 2280

Introduction:
Pre-donation screening of blood donors are currently based on venous or capillary blood sampling. Adoption of a non-invasive hemoglobin estimation may increase blood donor recruitment. Masimo Pronto-7 Pulse CO-oximetry device is a spectrophotometry based device used to estimate the hemoglobin (Hb) level non-invasively, waiving the need of blood sampling. It has not been validated in normal blood donors. The primary objective of our study was to validate the pulse CO-oximetry based hemoglobin estimation in normal blood donors.

Methods:
We conducted a prospective observational study on 106 whole blood donors attending the blood bank of a tertiary care center over 4 weeks. We estimated a Spot Hemoglobin (Sp Hb) concentration using Masimo Pronto-7 Pulse CO-oximetry device (two measurements per donor) and compared it to a venous sample Hb (Reference Hemoglobin; Ref Hb) measured using Abbott CELL-DYN Sapphire hematology analyzer. We calculated Pearson correlation coefficient and coefficient of determination (R2). The multivariable linear regression model of predicting the estimation differences included age, gender, weight, height, blood pressure and reference hemoglobin.

Results:
We enrolled 106 donors (98 males, 8 females) with a mean age of 27 years (SD 6.2; 18–49) and a mean Ref Hb of 14.2 g/dL (SD 1.2; 11.5–17). The mean Sp Hb was 14.4 g/dL (SD 1.2; 11.3–16.7). The correlation coefficient between the Sp Hb and Ref Hb was 0.46 (R2 = 21%) with a mean difference of 0.2 g/dL (SD 1.2; −4.5 to 3). In the multivariable model, height (p =0.015) and Ref Hb level (p <0.001) were statistically significant predictors of the difference in measurement. There was a strong correlation between the two CO-oximetry Hb measurements (correlation coefficient 0.78, R2 = 60%).

Conclusions:
Our study demonstrated the validity of the CO-oximetry Hb measurement in normal blood donors and with good reproducibility. Larger prospective studies are needed to confirm our findings.

Disclosures:
No relevant conflicts of interest to declare.
Immune Complexes Enhance Uptake of Blood Coagulation Factor VIII by Antigen Presenting Cells
Authors: Wroblewska Aleksandra , Herczenik Eszter , ten Brinke Anja , Voorberg Jan 
Published: October 2019

Abstract
Abstract 37
The X-chromosome-linked bleeding disorder hemophilia A is caused by the absence or dysfunction of blood coagulation factor VIII (FVIII) which can be corrected by regular intravenous administration of FVIII. Development of antibodies directed against FVIII (referred to as FVIII inhibitors) is common complication in hemophilia care. Immune tolerance induction (ITI) comprising frequent administration of high doses of FVIII is the treatment of choice for hemophilia A patients with inhibitors. Thus far, the molecular mechanisms contributing to tolerance induction have not been defined. Here we sought to study the role of immune complex (IC) formation in endocytosis and functional presentation of FVIII by antigen presenting cells. Bone marrow-derived dendritic cells (DC) were used as model antigen-presenting cells, as DCs are known for their ability to take up and process immune complexes via FcγR. Purified recombinant FVIII was labeled using the Microscale Alexa Fluor 488 protein-labeling kit. For T-cell proliferation assays, FVIII−/− mice were injected intravenously 5× weekly with 1 μg of recombinant FVIII. Subsequently, spleens collected after weekly injections were processed into single-cell suspensions and used as a source of FVIII-specific T cells. Bone marrow-derived murine DCs were able to efficiently take up FVIII pre-complexed with anti-FVIII antibodies (FVIII-IC) in a dose-dependent manner. Endocytosis of FVIII-IC was 3–6 fold more efficient when compared to equimolar concentrations of soluble FVIII. Uptake of FVIII-IC, but not FVIII alone, could be inhibited with 2.4G2 antibody indicating functional involvement of FcγRII/III in this process. These results were confirmed using murine DCs isolated from FcγR-deficient mice. Moreover, antibodies used for IC formation were analysed based on isotype – IgG2a antibodies were most efficient in mediating IC endocytosis. Experiments performed with FVIII-specific T cells showed that in the low concentration range (<1 nM), FVIII-IC induced stronger T cell proliferation when compared to soluble FVIII. Collectively, these data provide further insight and understanding of modulation of anti-FVIII immune response in hemophilia A patients with pre-existing FVIII inhibitors.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition of Histone Deacetylase 6 (HDAC6) Disrupts the Tolerogenic STAT3 Signaling Pathway and Augments Antitumor Immune Responses in Mantle Cell Lymphoma (MCL)
Authors: Wang Hongwei , Cheng Fengdong , Wang Zi , Lienlaf Maritza , Perez-Villarroel Patricio , Knox Tessa , Sahakian Eva , Pinilla-Ibarz Javier , Horna Pedro , Chen-Kiang Selina , Martin Peter , Bergman Joel , Kozikowski Alan , Villagra Alejandro , Tao Jianguo , Shah Bijal D. , Sotomayor Eduardo M. 
Published: October 2019

Abstract
Abstract 3724
MCL is an aggressive and incurable subtype of B-cell Non-Hodgkin's lymphomas. Although patients often respond to first-line chemotherapy plus monoclonal antibodies, relapse and decrease response to further lines of treatment eventually occurs. Manipulation of the immune system to unleash its specificity and long-lasting protective effect might provide a unique opportunity to induce more durable responses or prevent relapse in MCL.
Previous studies by our group have shown that HDAC6 is required for production of the immunosuppressive cytokine, IL-10 in antigen-presenting cells (APCs) and that genetic or pharmacologic disruption of HDAC6 in these cells triggers potent antigen-specific T-cell responses. Given the role of IL-10 in suppressing the immunogenicity of B-cells, we asked therefore whether inhibition of HDAC6 with the isotype-selective inhibitor, Tubastatin-A (Tub-A) could reverse the tolerogenic properties of malignant B-cells in a murine model of MCL1. First, in vitro treatment of FC-muMCL1 cells with Tub-A resulted in increased acetylation of a-tubulin, a known HDAC6 target. Treated B-cells also displayed an enhanced expression of MHC class II and the co-stimulatory molecules B7.1, B7.2 and CD40 relative to untreated cells. Such changes resulted in more immunogenic MCL cells able to effectively activate naïve antigen-specific CD4+ T-cells and more importantly, capable of restoring the responsiveness of anergic T-cells from lymphoma-bearing mice. Second, in vivo treatment of FC-muMCL1-bearing C57BL/6 mice with Tub-A resulted in lymphoma rejection. This antitumor effect was not observed in immunodeficient (SCID) C57BL/6 mice treated with Tub-A, pointing to the immunological effects triggered by HDAC6 inhibition in B-cells as playing a dominant role in Tub-A induced anti-MCL activity. Third, mechanistically we have found that HDAC6 physically interacts with STAT3 and it is required for STAT3 phosphorylation and recruitment to the IL-10 gene promoter. Furthermore, Tub-A treated MCL cells displayed a diminished STAT3 phosphorylation and abrogation of IL-10 gene transcriptional activity.
Taken together, a concerted regulatory mechanism involving HDAC6 and STAT3 influence the immunogenicity of MCL cells. Targeting HDAC6 with specific inhibitors to disrupt the tolerogenic STAT3/IL-10 axis represents a novel strategy to trigger effective anti-MCL immunity.

Disclosures:
Martin: Millennium Pharmaceuticals, Inc.: Speakers Bureau.
Human ROR1 Activates AKT and Accelerates Leukemia Cell Proliferation
Authors: Zhang Suping , Chen Liguang , Zhang Ling , Yu Jian , Rassenti Laura Z. , Kipps Thomas J. 
Published: October 2019

Abstract
Abstract 3872
ROR1 is an orphan-receptor-tyrosine-kinase-like developmental surface-antigen that is expressed by embryonic cells, chronic lymphocytic leukemia (CLL) cells, and neoplastic cells of many other types of cancer, but not by virtually all normal adult tissues. ROR1 can serve as a receptor for Wnt5a and potentially complex with other surface receptors for growth/survival factors elaborated by the tumor microenvironment or expressed by the tumor itself. In prior studies, we and others found that silencing ROR1 could impair tumor-cell growth and/or survival in vitro and in vivo. Although the CLL cells of ≥96% of all examined patients (N= 800) expressed ROR1, we found that a B-cell line derived from CLL cells, namely MEC1 (Leuk Res 23:127, 1999), lacked expression of this protein. This provided us with an opportunity to assess how introduction of ROR1 could affect the biology of this CLL-cell line, which has relatively slow doubling-time of ≈40 hours. We transfected MEC1 cells with either an expression vector encoding human ROR1 or a control vector, and then selected for stable transfectants in selection medium. MEC1 cells transfected with the ROR1 vector expressed high-levels of ROR1, but not MEC1 cells transfected with the control vector. MEC1 cells made to express ROR1 had higher levels of phosphorylated and activated-AKT and activated cAMP response-element-binding (CREB) protein than non-transfected MEC1 cells or cells transfected with the control vector. Furthermore, MEC1 made to express ROR1 had significantly greater proportions of cells in S/G2/M phase than did control-transfected cells 16 hours after transfer from serum-free medium to complete growth medium, indicating that MEC1 cells made to express ROR1 had increased relative proportions of cells undergoing cell division. Consistent with this, we found that ROR1+ MEC1 cells had significantly shorter doubling times in culture than did comparably cultured parental MEC1 cells or MEC1 cells transfected with the control vector. This study indicates that expression of ROR1 in the MEC1 CLL cell line can activate AKT/CREB and accelerate leukemia cell growth, providing us with a model system with which to interrogate the function of ROR1 in vitro.

Disclosures:
No relevant conflicts of interest to declare.