bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nek2 Stabilization By Usp7 Leads to Activation of NF-Kb in Multiple Myeloma
Authors: Franqui Machin Reinaldo , Zhan Xin , Xu Hongwei , Frech Ivana , Tricot Guido J , Zhan Fenghuang 
Published: October 2019

Abstract
NIMA (Never In Mitosis Gene A)-Related Kinase 2 (Nek2), a centrosomal Serine/Threonine kinase, is a key player in numerous malignancies. Overexpression of Nek2 has been related to many cancers including Multiple Myeloma (MM). In MM, Nek2 is one of the chromosomal instability genes associated with drug resistance and disease relapse. However, very little is known about the mechanisms that lead to these Nek2-driven disparities. Here, we show that the Ubiquitin Specific Peptidase 7 (USP7) stabilizes Nek2 leading to activation of NF-kb pathway.
Using gene expression profile (GEP) data from patients and cell lines we discovered that Nek2 overexpression leads to increases of several targets of the NF-kb pathway. We, thus, hypothesize that Nek2 is activating NF-kb. To address this, we overexpressed Nek2 and tested the classic canonical NF-kb hallmarks proteins by western blotting. Nek2 overexpression led to an increase in phosphorylation of IKK, activator of NF-kb, and to decrease levels of IKb-alpha, a negative regulator of the pathway. Nek2 overexpression also increased nuclear and phosphorylated p65 on residue S536, known as active transcriptional site. To further confirm that Nek2 is activating canonical NF-kb luciferase assay was performed. The luciferase reporter is driven by a p65 promoter and in cells overexpressing Nek2 luciferase levels were increased.
To characterize Nek2 interacting partners a tandem affinity purification/mass spectrometry (TAP/MS) approach was performed. We found that Nek2 binds to Usp7, a deubiquitinase overexpressed in numerous cancers. This led to hypothesize that that Nek2, a known target of the ubiquitin proteasome system, is being stabilized by the Usp7 contributing to its overexpression and the increased activation of the NF-kb pathway. To test our hypothesis, we treated different cancer cell lines with the commercially available Usp7 inhibitor, P5091, or silenced the protein using shRNA. In both case, we found a reduction in Nek2 protein level. Additionally, we overexpressed Usp7 and Nek2 increased confirming that Usp7 stabilizes Nek2. To further show that Usp7 stabilizes Nek2 by de-ubiquitination, we overexpressed Usp7 and analyzed Nek2 ubiquitination after immunoprecipitation. When Usp7 was overexpressed no ubiquitination of Nek2 was detected.
Finally, by using GEP data from MM patients, we found that individuals who overexpressed Nek2 along with an active NF-kb signature have worst event free survival as well as overall survival, indicating Nek2 overexpression leading to increased NF-kb signature has clinical significance.

Disclosures
No relevant conflicts of interest to declare.
Ex-Vivo Fucosylation Improves the Anti-Graft-Versus-Host-Disease Effects of Mesenchymal Stem Cells in the NOD/SCID/IL-2R Null Mouse Model
Authors: Galvez Silva Jorge , Mendt Vilchez Mayela , Nobuhiko Imahashi , Blazar Bruce R. , Zweidler-McKay Patrick , Rezvani Katayoun , Shpall Elizabeth J. 
Published: October 2019

Abstract
BACKGROUND: Graft versus host disease (GVHD) remains the major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Due to their immunomodulatory properties, mesenchymal stem cells (MSCs) have produced durable responses in HSCT patients with steroid resistant GVHD, but their positive effects are not seen in all patients. Sackstein et al have shown that MSCs lack an E-selectin ligand on their surface, which impairs their ability to home to the bone marrow and potentially to sites of inflammation associated with GVHD. Our in vitro results confirmed that fucosylation markedly enhanced the E-selectin ligand expression on the MSC cell surface and did not adversely affected MSCs immunomodulatory properties. Hencewe postulated that ex-vivo fucosylation of MSCs would enhance their homing to sites of inflammantion and thus their clinical efficacy in our NOD/ SCID/IL-2R null (NSG) mouse model of GVHD.
METHODS: On day -1 NSG mice were sub-lethally irradiated (300cGy) and infused with 2x106 fucosylated MSCs. Briefly, healthy bone marrow donor derived MSCs were treated for 30 minutes at room temperature with FT-VI (33ng/ml) and GDP-fucose (1mM) (Targazyme, Carlsbad, CA), and then washed prior to tail vein injection. Control mice received 2x106 unfucosylated MSCs or no MSCs. On day 0 all mice received 1x107 normal human donor peripheral blood mononuclear cells (PBMNCs), representing a MSCs:PBMNCs ratio of 1:5. Mice were then followed for weight loss, clinical GVHD score and survival.
RESULTS: We found that a single dose of FT-VI treated MSCs reduced the acute severity of GVHD compared to unfucosylated MSCs and no MSCs (PBMNCs alone) in this xenogeneic mouse model. This was shown by prevention of weight loss (Fig. 1a), and lower GVHD scores (Fig 1b) in the FT-VI treated MSCs mice. Furthermore, when short-term survival was compared among the subgroups, unfucosylated MSCs did not significantly improve short-term survival over PBMNCs group (P=0.3) (Fig. 2a). However, FT-VI treated MSCs mice showed a significantly longer short-term survival compared to PBMNCs group (P=0.002) (Fig. 2b) and untreated MSCs mice (P=0.02) (Fig. 2c).
CONCLUSION. Our data suggest that fucosylated MSCs exert a better anti-GVHD effect at a similar dose than unfucosylated MSCs. Additional data is being generated to confirm these preliminary results and optimize the dose and schedule of fucosylated MSCs.

Disclosures
No relevant conflicts of interest to declare.
Intra-Tumor Injection of CAR-Engineered NK Cells Induces Tumor Regression and Protection Against Tumor Re-Challenge
Authors: Boissel Laurent , Klingemann Hans , Khan Junaid , Soon-Shiong Patrick 
Published: October 2019

Abstract
In this era of cancer immunotherapy, the long-term goal of treatment is to eliminate tumor cells anywhere in the body through induction of a specific immune memory response. In addition to spontaneous cytotoxicity, ADCC, and cytokine release, NK cells contribute to an adoptive immune response through crosstalk with dendritic cells and T cells. To further characterize the NK cell-induced adoptive response, aNK cells (formerly NK-92) were transduced with a retrovirus construct coding for a second-generation anti-murine CD19-CAR to create targeted activated NK-92 cells (mCD19.taNK). mCD19.taNK cells were injected intra-tumorally (two injections of 5x106 cells, three days apart) into a murine model of syngeneic subcutaneous lymphoma, created by injecting 1x106 A20 cells into BALB/c mice. Tumor size was monitored over time, and mice showing complete tumor regression were re-challenged with another subcutaneous contralateral injection of A20 cells.
mCD19.taNK cells effectively killed murine cancer cells in vitro (>60% killing at E:T ratio of 5:1). In vivo, intra-tumor injection of mCD19.taNK induced significant tumor regression versus saline (tumor volumes of 342 mm3 and 936 mm3, respectively at day 16, p<0.05) and significantly improved survival, with 75% of the mice showing complete tumor regression at day 32 (p<0.05). In contrast, injection of parental aNK cells did not significantly affect tumor size in mice (815 mm3at day 16). Upon re-challenge with A20 lymphoma cells, >80% mice remained free of tumor after 14 days.
In conclusion, the human aNK cell line expressing an anti-murine CD19-CAR (mCD19.taNK) can effectively kill CD19-positive murine cancer cells. Moreover, intra-tumor injections of mCD19.taNK into a fully immunocompetent mouse model can induce tumor clearance and protection from tumor re-challenge, suggesting induction of a memory ("vaccine") effect after the injection of mCD19.taNK.

Disclosures
Boissel: NantKwest, Inc.: Employment. Klingemann:NantKwest, Inc.: Employment, Equity Ownership, Patents & Royalties. Khan:NantKwest, Inc.: Employment. Soon-Shiong:NantKwest, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Economic, Transfusion, and Efficacy Outcomes with the Addition of IV Iron Sucrose to Oral Iron Therapy in Pregnancy Associated Iron Deficiency Anemia
Authors: Gaspe Mudiyanselage Nilupa , Elrafei Tarek , Lewis Beth , King Mary , Strakhan Marianna , Alexis Karenza , Gralla Richard 
Published: October 2019

Abstract
Background: Prior studies have indicated that transfusion is unusual (2%) in pregnant women with iron deficiency anemia. Nonetheless, compliance with oral iron replacement can be an issue and physicians may wish to use IV iron therapy in markedly anemic pregnant women.
Objectives: to evaluate the effectiveness of adding intravenous iron sucrose concentrate (ISC) to pregnant patients already taking oral iron in terms of effect on hemoglobin, effect on ferritin levels, rates of transfusion, and cost.
Methods: We analyzed all referrals from Obstetrics to Hematology clinic and Obstetrics consultation (Internal medicine) clinic from January 2014 to June 2016. Of the 176 pregnant patients, 98 were referred for anemia, including 81 patients with Hgb < 12 g/dl and ferritin < 20 ug/L. All had previously been given oral ferrous sulfate prescriptions. Patients with hemoglobinopathy were excluded. All 81 patients were advised to continue on the oral iron, and 40 were given IV iron sucrose (ISC group).
Results: The average cumulative dose of iron sucrose was 700 mg, a mean of 5.575 doses (initiated in the third trimester in 38 of 40 patients). The lowest antepartum Hgb was 8.18 g/dl in the ISC group and 9.58 in the oral only group; there was an average Hgb increase of 2.17 vs 1.76 g/dl respectively (p=.107 NS and the 0.41 g/dl difference was considered to be of no clinical consequence). 89% in the ISC group vs 30% in the oral achieved a ferritin >20 (p=0.000015). No adverse events in the IV iron group were reported. There was 1 transfusion in the oral iron group attributable to iron deficiency (2.4%) vs none in the IV iron group (p = 0.107 NS). Two patients were transfused in the antenatal period before IV iron was started and 1 transfused because of post-partum hemorrhage. The total cost of the IV iron therapy would add an average of $1,500 per patient. Thus, and additional cost of $60,000 in IV iron would be required to prevent 1 transfusion [40:1].
Conclusions: ISC corrects ferritin in more patients than oral iron replacement, but did not significantly increase Hgb levels or have a meaningful impact on the transfusion rate. The additional cost and lack of clinically improved outcomes with IV iron argue against its use and in favor of strategies to ensure compliance with oral iron.

Disclosures
No relevant conflicts of interest to declare.
Endogenous Adamts-13 Regulates Angiogenesis in Cultured Human Endothelial Cells
Authors: Tang Huiyuan , Lee Manfai , Ho Kim Eun , Bishop Daniel Allen , Rodgers George M. 
Published: October 2019

Abstract
ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is a zinc-containing metalloprotease enzyme that cleaves von Willebrand factor (vWf). Previous publications by our laboratory have shown that ADAMTS-13 may also be involved in angiogenesis; for example; addition of exogenous ADAMTS-13 enhances angiogenesis in cell migration and proliferation assays. For this study, we report the successful transient knockdown of endogenous ADAMTS-13 in human umbilical vein endothelial cell (HUVEC) via siRNA. 100pmols of ADAMTS-13 siRNA inhibited HUVEC ADAMTS-13 expression levels by 60% after 7hr incubation, whereas control siRNA did not reduce endogenous ADAMTS-13. The effects of reduced endogenous ADAMTS-13 on HUVEC function were studied in proliferation and scratch wound assays. Transfection of HUVEC with 100pmols of ADAMTS-13 siRNA resulted in a 47% decrease in proliferation after 19hr incubation. The effects of ADAMTS-13 knockdown on migration of HUVEC across a scratch wound were also studied. 9hr after transfection with control siRNA, cells initiated migration across the scratch wound. This migration did not occur in ADAMTS-13 knockdown cells. The activity of the AKT pathway, one of the angiogenesis downstream signaling pathways was down regulated by ADAMTS-13 siRNA while ERK, a component of the MAP kinase pathway was not affected upon knockdown of ADAMTS-13. These data indicate that one role of endogenous ADAMTS-13 in HUVEC is regulation of angiogenesis, mediated through the AKT signaling pathway. Overall our data suggest an additional model of endogenousADAMTS-13 functionality, beyond that of cleaving von Willebrand factor.

Disclosures
No relevant conflicts of interest to declare.
Prospective, Non-Randomized, Open-Label, Single-Arm, Multi-Center Clinical Trial to Evaluate the Efficacy and Safety of Intravenous Immunoglobulin 10% Formulation in Patients with Immune Thrombocytopenia (ITP)
Authors: Bang Soo-Mee , Mun Yeung-Chul , Shin Ho-Jin , Jung Chul Won , Bae Sung Hwa , Sohn Sang Kyun , Kim Kyoung Ha , Kim Hawk , Yhim Ho-Young , Hong Junshik , Kim Hye Joo , Lee Jae-hoon , Oh Doyeun 
Published: October 2019

Abstract
This study investigated the safety and efficacy of IV-Globulin SN, a 10% intravenous immunoglobulin (IVIg), in patients with severe ITP (platelet count ¡Â20 x 109/L). Among 81 eligible patients, 31 patients were newly diagnosed, 7 patients had persistent ITP, and 43 patients had chronic ITP. Five patients had received splenectomy. Patients received IV-Globulin SN 1 g/kg/day on two consecutive days; infusion rate was initially 1 mg/kg/minute and then doubled every 30 minutes to a maximum of 8 mg/kg/minute.
Fifty-eight patients (72%) attained the primary efficacy endpoint of clinical response (platelet count ¡Ã 50 x 109/L within 7 days). Their median time to response was 2 days and the median duration of response was 10 days (range from 1 to 28 days). Complete response (platelet count ¡Ã 100 x 109/L over 7 days) was obtained in 14 patients (17%). Response rates were not significantly different when compared the patients with newly diagnosed, persistent or chronic ITP; previous treatment with immunosuppressant or not; splenectomized or not. IV-Globulin SN 10% was well tolerated and the frequency of mucocutaneous bleeding was decreased during the study period (figure 1). There was no unexpected adverse event. The mean half-life and Cmax of study drug were 28.9 days and 34.6 g/L in 25 tested patients. There were no unexpected adverse events. In conclusion, IV-Globulin SN was efficacious in adult ITP patients regardless of their disease status as well as safe given that the resolution of bleeding and minimal infusion-related adverse events. (NCT02063789)
Bleeding rate according the anatomic sites during the study period in 81 patients. *Visit 2 means day 1; visit 3, day 2; visit 4, day 4¡¾1; visit 5, day 6¡¾1; visit 6, day 8¡¾1, visit 7, day 11¡¾1; visit 8, day 15¡¾1; visit 9, day 22¡¾3; visit 10, day 29¡¾3) Bleeding rate according the anatomic sites during the study period in 81 patients. / *Visit 2 means day 1; visit 3, day 2; visit 4, day 4¡¾1; visit 5, day 6¡¾1; visit 6, day 8¡¾1, visit 7, day 11¡¾1; visit 8, day 15¡¾1; visit 9, day 22¡¾3; visit 10, day 29¡¾3)

Disclosures
Kim: Green Cross Corp.: Employment. Lee:Green Cross Corp.: Employment.
Assessment of Intracranial Hemorrhage Demographics in Children with Hemophilia: A Multicentre Study in China
Authors: Haque Qareen , Feng Xiaoqin , Li Chunfu 
Published: October 2019

Abstract
Objective: Intracranial hemorrhage (ICH) is a severe complication in hemophilia patients with high mortality and disabling sequelae. This study is to provide key insights on the demographics of hemophilic patients with ICH in China.
Method: The database and medical notes ofHemophilia patients who were diagnosed between 1995 and 2014 are reviewed with a goal to find the effective factors which relate to ICH such as age of onset, type, severity, cause, and ICH position, presence of inhibitor and neurological sequelae. A total of 145 hemophilic boys who are diagnosed with ICH and come from multiple hemophilia centers in China, are included.
Result: The median age of ICH is 18 months. 123/145 (85%) are Hemophilia A and 22/145 (15%) are Hemophilia B. Intracranial hemorrhages most commonly occur in patients with severe disease 79/145 (54%), while 58/145 (40%) and 8/145 (5%) in moderate and mild disease respectively. There is obvious trauma history in 53/145 (37%) children. Neurological disabilities are seen only in 34/145 (23%). No deaths from ICH have been reported in our study. Data about the position of ICH is available only in 30% of patients; including involvement of multiple areas is seen in 50%. ICH in epidural space is 14%, brain parenchyma is 14%, subdural is 11% and subarachnoid is 5%.Out of 30 patients inhibitor is found only in 9 cases while in other 115 patients inhibitor data is unavailable. After the onset of ICH only 53/145 (37%) children are put on regular prophylaxis to prevent further episode of ICH.
Conclusion: Risk of ICH is high in patients with severe hemophilia. In most cases of ICH, there is no trauma history and relatively less neurological consequences. About half of children with ICH multiple areas of brain are involved. We need more data to conclude the other risk factors in the incidence of ICH.

Disclosures
No relevant conflicts of interest to declare.
Evaluation of Direct Oral Anticoagulants in the Treatment of Venous Thromboembolism for Inherited Thrombophilia Disorders
Authors: McBride Ali , Diri Reem , Krishnadasan Ravitharan , Chalasani Pavani , Abraham Ivo , Campen Chris 
Published: October 2019

Abstract
Background
Venous thromboembolism can be classified according to the presence of either environmental or genetic risk factors. Risk factors for thrombosis can include activated protein C resistance, and heritable including deficiencies of antithrombin, protein C or protein S. Factor V Leiden deficiency and prothrombin gene mutations are some of the more common thrombophilias, with a slight increased risk for venous thromboembolism (VTE). Current guidelines suggest the use of low-molecular weight heparins for secondary prophylaxis in patients with VTE. However, there is a lack of data on the use of Direct Oral Anticoagulant (DOACs) in patients with inherited thrombophilia. We evaluated our use of rivaroxaban in patients with thrombophilia disorders treated for secondary DVT prophylaxis.
Method
We performed a retrospective evaluation of patients in our institution with inherited thrombophilia with an active VTE diagnosis who received DOACs for secondary prophylaxis from November 2013 until April 2016. Data collected included patient demographics, inherited thrombophilia mutation, previous history of VTE, prior treatments, and efficacy and safety of anticoagulation with DOACs.
Results
We had 13 patients with inherited thrombophilia mutation and 4 patients diagnosed with concomitant cancer (non-Hodgkin lymphoma, melanoma, and 2 with breast cancer) (Table 1). Out of 13 patients 3 failed warfarin, and one failed fondaparinux prior to switching to a DOAC. Mutation with heterozygous Factor V Leiden deficiency was reported in 7 patients, while mutations with Protein C and/or S deficiency were found in 4 patients. One patient had both Factor V Leiden and Protein C deficiency mutations. The prothrombin gene mutation was identified in one patient. The median of length of therapy was 2 years with 8/13 still on rivaroxaban in April 2016. The shortest treatment duration was 41 days for a patient who failed rivaroxaban with a second clot and was switched to apixaban without subsequent treatment failure. Two patients experienced 4 non-major episodes of gastrointestinal bleeding, nose bleeding and dark stool. One patient developed rash with noted bruising during their rivaroxaban therapy.
Conclusion:
This is the first report on outcomes for secondary DVT prophylaxis with DOACs in patients with underlying thrombophilia mutations. Safety and efficacy of DOACs for secondary VTE prophylaxis yielded favorable results; however, future prospective studies in the setting of thrombophilia are warranted.
Table 1 Summary of baseline characteristics and outcomes. Table 1. Summary of baseline characteristics and outcomes.

Disclosures
McBride: Sanofi: Research Funding.
The Effectiveness and Safety of Direct Oral Anticoagulants (DOACs) Vs. Traditional Anticoagulants in Patients with Cirrhosis
Authors: Hum Justin , Jou Janice , Deloughery Thomas G. , Shatzel Joseph 
Published: October 2019

Abstract
Introduction: The coagulopathy associated with cirrhosis is complex and places patients at risk for both bleeding and thrombosis. Direct oral anticoagulants (DOACs) have been shown to have superior efficacy and safety compared to vitamin K antagonists; however their efficacy and safety in cirrhotic patients is not clear. The aim of this study is to retrospectively compare the effectiveness and bleeding complications of DOACs as compared to traditional anticoagulants in cirrhotic patients.
Methods: This study was a retrospective review of patients treated at a single academic center between 2012-2015 who were prescribed a DOAC (apixaban or rivaroxaban), or a traditional anticoagulant (warfarin or low molecular weight heparin), with an ICD-9 code for the diagnosis of cirrhosis. The primary outcomes of interest are recurrent thrombosis or stroke (efficacy failure), or bleeding events (safety failure). Major bleeds were characterized as fatal bleeding, symptomatic bleeding in critical organ area, or bleeding causing a fall in hemoglobin level >2 or leading to transfusion of 2+ units of packed red blood cells.
Results: During the study period, 27 cirrhotic patients were prescribed a DOAC and 18 were prescribed a traditional anticoagulant (either LMWH or warfarin). Both groups had similar total bleeding events (8 DOAC vs. 10 traditional anticoagulation, p = 0.12). There were significantly less major bleeding episodes in the DOAC group, (1 (4%) vs. 5 (28%), p = 0.03) and less intracranial bleeding (3 (17% ) vs. 0 (0%) p=0.06). Recurrent thrombosis or stroke occurred in 1 (4%) patient in the DOAC group and 1 (6%) patient in the traditional group (p = 1.0).
Conclusions: Anticoagulation with DOACs in cirrhotic patients may be as safe as traditional anticoagulants with respect to bleeding events. Patients with cirrhosis at our center prescribed DOACs had less major bleeding events, while maintaining efficacy at preventing stroke or recurrent thrombosis.
Table Table.

Disclosures
No relevant conflicts of interest to declare.