bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reversion of the Experimental Hemodilutional Coagulopathy Induced by Crystalloids and Colloids Using Different Coagulation Factor Concentrates
Authors: Caballo Carolina , Galan Ana M , Diaz-Ricart Maribel , Lopez-Vilchez Irene , Lozano Miguel , Cid Joan , Pino Marcos , Beltran Joan , Basora Misericordia , Pereira Arturo , Escolar Gines 
Published: October 2019

Abstract
Abstract 4349

BACKGROUND:
Massive bleeding and subsequent coagulopathy are responsible for 35% of deaths in trauma patients. Hemodilution during resuscitation may worsen the coagulopathy and perpetuate bleeding.

STUDY DESIGN AND METHODS:
Blood samples from healthy donors were diluted (30–60%) using crystalloids (saline, Ringer’s lactate, Plasmalyte™) or colloids (6%hydroxyethylstarch (HES130/0.4), 5% human albumin, and gelatin). The impact of hemodilution on platelet adhesion, thrombin generation (TG), and clot viscoelastic properties by thromboelastometry (TEM) was analyzed. Effects of fibrinogen (Fbn), prothrombin complex concentrates (PCCs), rFVIIa, or cryoprecipates (cryo) on hemodilution were also assessed.

RESULTS:
Hemodilution caused a significant decrease in platelet interaction that was not improved by the addition of any of the plasma derivatives. A decrease in TG and important alterations of TEM were also observed. HES130/0.4 was the expander with the most deleterious action. TG was significantly enhanced by PCCs and their combination with Fbn whereas rFVIIa only slightly accelerated it. Fbn restored the alterations of TEM caused by hemodilution including those more deeply altered by HES 130/0.4. The combination of Fbn with PCC or rFVIIa did not have an additional effect in TEM. Cryo significantly improved the alterations caused by hemodilution on TG and TEM parameters. Effects of cryo on TG disappeared after ultracentrifugation, suggesting that contaminating microvesicular material could account for this effect.

CONCLUSION:
Hemostatic alterations caused by hemodilution are multifactorial and affect both blood cells and coagulation. In our in vitro approach, HES 130/0.4 seemed to exert a more deleterious effect on hemostasis. None of the concentrates improved platelet-mediated hemostasis, although they always showed variable beneficial effects on coagulation parameters. Our data indicate that PCC, rFVIIa and cryo enhance or accelerate thrombin generation. Fbn concentrates could be useful to preserve blood clotting abilities during fluid resuscitation of critically ill patients without exposing them to enhanced thrombin generation.
Grants: PET(2008_0231), FIS(CP04-00112, PS09/00664), SAF2009-10365, RD06/0009

Disclosures:
No relevant conflicts of interest to declare.
2D-DIGE As a Tool to Analyze Lot-to-Lot Consistency of Complex Therapeutic Products Such As BAX 855, a PEGylated Recombinant FVIII
Authors: Rottensteiner Hanspeter , Monetti Christina , Gritsch Herbert , Weber Alfred , Ehrlich Hartmut J. , Scheiflinger Friedrich , Turecek Peter L 
Published: October 2019

Abstract
Abstract 4360
2-Dimensional difference gel electrophoresis (2-D DIGE) is a method that circumvents the gel-to-gel variability inherent in conventional 2-dimensional gel electrophoresis (2-DE). We developed a 2-D DIGE protocol for recombinant factor VIII (rFVIII), a therapeutic protein used for the treatment of hemophilia A. The FVIII heterodimer is composed of heterogenous, strongly glycosylated heavy and light chains that are held together by a divalent cation. 2-DE of rFVIII led to a separation of the various fragments and their identity could be determined by Western blot. A comparison of two rFVIII batches by 2-D DIGE revealed their identical composition, whereas an rFVIII variant lacking its central B domain was congruent with the smallest heavy and light chain fragments of rFVIII only. A simpler pattern was obtained upon removal of the terminal sialic acids of rFVIII’s glycans due to a better focusing in the first dimension. 2-D DIGE was also well suited to structurally evaluate BAX 855, a PEGylated longer-acting variant of recombinant FVIII. 2-D DIGE thus proved an excellent and straightforward method for structural analysis of rFVIII. Our data suggest that the method could serve as a tool to characterize and control quality of very complex pharmaceutically active ingredients such as PEGylated proteins.

Disclosures:
Rottensteiner: Baxter Innovations GmbH: Employment. Monetti:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Weber:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.
The Somatic Mutation of PIG-A Gene and the Expressions of EGR-1 and WT1 Genes in Bone Marrow Cells of the Patients with Paroxysmal Nocturnal Hemoglobinuria
Authors: Fu Rong , Li Liyan , Shao Zonghong , Wang Jun , Li Lijuan , Liu Hui 
Published: October 2019

Abstract
Abstract 4379

Objective
To investigate the somatic mutations of PIG-A gene and the expressions of EGR-1 and WT1 genes in the bone marrow cells of the patients with Paroxysmal nocturnal hemoglobinuria, then explore the characteristics of PIG-A gene mutation in Chinese patients with PNH and the possible mechanism of PNH clonal expansion related to EGR-1 and WT1 genes.

Methods
The quantities of CD55− cells and CD59− cells in both peripheral blood and bone marrow were examined with flow cytometric assay(FCM), and then sorting CD59− cells from bone marrow mononuclears (BMMNCs). The mutant segments of PIG-A gene were identified by DNA sequencing; The expressions of EGR-1 and WT1 mRNA in CD59− and CD59+ BMMNC were measured with semiquantitive RT-PCR.

Results
Nine in sixteen patients with PNH and one in four patients with AA-PNH were measured for their PIG-A gene mutation. Five single base substitution were found, including A→G, G→T, A→T, T→G and G→C, resulting in one consense and seven missense mutations. Each of 3 PNH patients had more than two point mutations of PIG-A gene. Insert or deletion mutation of PIG-A gene were not found. The relative mRNA expressions of EGR-1and WT1 genes in CD59− BMMNC, CD59+ BMMNC of PNH or AA-PNH patient and BMMNC of normal controls were (1.00±0.11), (0.86±0.14), (0.85±0.06) and (1.06±0.16), (0.90±0.14), (0.88±0.06), the expression in CD59− BMMNC was significantly higher than that in CD59+ BMMNC or normal controls (p<0.05). There was no significant difference between the expression in CD59+ BMMNC and normal controls(p>0.05). The relative mRNA expressions of EGR-1 and WT1 in BMMNC of PNH and AA-PNH cases were positively correlated with the proportion of CD59− cells(coefficient correlation was 0.509 and 0.481 respectively, p<0.05), but not related to the proportion of CD59+ cells.

Conclusions
The PIG-A gene mutation in some Chinese patients mainly manifested as single base substitution, occurred at random sites and without hot spot, the overexpression of EGR-1 and WT1 genes in PNH clone could promote the abnormal clone expansion.

Disclosures:
No relevant conflicts of interest to declare.
Preliminary Study of the Autoantigens on the Membrane of Erythropoietic Cells of the Patients with BMMNC- Coomb’s Test(+) Hemocytopenia
Authors: Fu Rong , Liu Hui , Shao Zonghong , Wang Jun , Li Lijuan 
Published: October 2019

Abstract
Abstract 4382

Objective
To observe the relationship between EPO receptor(EPOR) and autoantibodies-IgG/IgM(auto-Ab) on the membrane of erythropoietic cells of the patients with IRP and then explore the probable autoantigens of auto-Ab in IRP.

Methods
46 newly diagnosed IRP patients(15 with auto-Ab on erythropoietic cells and 31 without auto-Ab on erythropoietic cells) and 18 healthy controls were enrolled in this study. EPOR expression on their nuclear erythrocytes were tested with FCM to observe the relationship between EPOR and auto-Ab; EPOR mRNA were tested by RT-PCR; Stat5 and P-Stat5 proteins in nucleared erythrocytes were measured by Western blot; EPOR expression on the nucleared erythrocytes membrane were tested again after stripping autoantibodies with glycine buffer.

Results
(1)EPOR of auto-Ab(+) arm(1.59±0.87)% was significantly lower than that of auto-Ab (−) arm(4.58±4.09)%(P<0.01)and the latter was significantly higher than that of normal controls(2.27±1.76)%(P<0.05); GEPOR of IRP patients was inversely correlated with their auto-Ab (r=−0.543,P=0.000).(2) EPOR mRNA of auto-Ab(+) arm(0.685±0.136)was significiantly higher than that of auto-Ab (−) arm(0.554±0.116)(P<0.01)and normal controls(0.580±0.119)(P<0.05);(3) Protein Stat5 of auto-Ab(+) arm(1.45±0.94) was significantly higher than that of normal controls(0.54±0.36)(P<0.05). While P-Stat5 of auto-Ab(+) arm(0.42±0.18) was significantly lower than that of normal controls(0.85±0.38)(P<0.05). (4) EPOR expression increased significantly after auto-Ab stripped from nucleared erythrocytes with glycine buffer.

Conclusion
The auto-Ab of some IRP patients might block or competitively inhibit the EPOR on the membrane of erythropoietic cells. EPOR might be one of autoantigens in IRP.

Disclosures:
No relevant conflicts of interest to declare.
Off-the-Shelf Cord Blood Mesenchymal Stem Cell Bank
Authors: Montemurro, Tiziana , Lavazza Cristiana , Andriolo Gabriella , Viganò Mariele , Baluce Barbara , Rebulla Paolo , Giordano Rosaria , Lazzari Lorenza 
Published: October 2019

Abstract
Abstract 4403
Our hospital-based GMP facility, named Cell Factory, built in Milano (Italy) and the first one authorized by the National Drug Agency (AIFA) in 2007, has pioneered innovative manufacturing technologies in order to supply therapeutic clinical development programs.
By our group and by others, cord blood (CB) mesenchymal stem cells (MSC) have been already tested both in vitro and in extensive pre-clinical disease models, demonstrating that CBMSC are safe and effective. Therefore, CBMSC are the best candidate product for several clinical trials including neurologic and renal repair (1–3).
In this context, our group developed the first off-the-shelf (OTS) bank where CBMSC are expanded and banked in GMP conditions, and now they are available as “a drug”, to be distributed for patient doses, much like an off-the-shelf pharmaceutical product. From informed consent CB donations, CBMSC were grown and expanded in CellSTACK kit that includes devices for a closed-system cell culture (Macopharma) with EMEA-grade Australian FBS and they were manufactured through a multi-step controlled and well-validated process. Cell Factory established this innovative culture approach through sufficient passages to produce hundreds of thousands of potential doses from an individual donor. After culture, our quality control approach has been designed to ensure that controls are implemented and completed satisfactorily during manufacturing operations, and that developed procedures and specifications are appropriate and followed, including those performed by contract testing laboratories. In fact, at each step and at the end of the production process before cryopreservation, CBMSC were analyzed and qualified according to pre-established criteria (cell count, viability, extensive immunophenotype, CFU-F, karyotype, CGHArray, telomerase activity, sterility, mycoplasma and endotoxin assays) to ensure that a consistent, well characterized product candidate is produced. The same CBMSC batches can be provided for pre-clinical testing and for further validations to prepare the investigational medicinal product dossier (IMPD) for each clinical trial approvals.
Now these GMP batches of CBMSC are cryopreserved and available in our OTS CBMSC bank in different cell doses to comply any kind of medical needs. Products are provided under specific contract conditions developed with the clinical centers. (Contact: cellfactorypoliclinico@gmail.com)

Disclosures:
No relevant conflicts of interest to declare.
K562-R As a Model to Study Jak2 in a Non Bcr-Abl Addicted Cell Line
Authors: Perazzona Bastianella , Lin Yu-Hsi , Arlinghaus Ralph B. 
Published: October 2019

Abstract
Abstract 4412
Chronic myeloid leukemia (CML) is a hematological disease caused by the fusion protein Bcr-Abl tyrosine kinase. Development of the tyrosine kinase inhibitor Imatinib Mesylate (IM) has significantly improved the long-term survival of early stage CML patients. However, occurrence of drug resistance, permanence of residual disease and recurrence of active leukemia if IM is discontinued, remain problems awaiting solution. Therefore, new therapeutic strategies aimed at targeting alternative signaling pathways or CML progenitor cells that survive IM treatment are needed. We have previously shown that Janus kinase 2 (Jak2) is activated in Bcr-Abl+ cells. We have demonstrated that reduction of Jak2 activity by the Jak2-specific inhibitor TG101209 (TG) or by genetic knock down (Jak2 shRNA and siRNA) in Bcr-Abl+ cell lines, IM-resistant cells and CML blast crisis cell lines resulted in reduced levels of phosphorylation of Tyr177 and of total Bcr-Abl protein. Jak2 inhibition results in diminished activation of the Ras, PI-3 kinase pathways and reduced levels of pTyrSTAT5 (Samanta et al., Leukemia 2011). During these studies we observed that K562 cells and IM-resistant cell line K562-R had different susceptibility to the effect of TG, with K562-R showing increase sensitivity to lower concentrations of TG resulting in faster destabilization of the Bcr-Abl protein. Based on these observations, we hypothesized that increased sensitivity of the K562-R cells was due to the different state of activation of Jak2. In addition, based on recent studies by (Dawson et al., Nat 2009) and by (Rinaldi et al., Blood 2010) we also hypothesized that different levels of Jak2 activation may influence the localization of Jak2 in the cell. We used cell fractionation and western blotting analysis to show that in K562-R cells, active Jak2 is mostly localized in the nucleus with a minor pool found in the cytoplasm. In K562 cells, active Jak2 is equally distributed in both cytoplasmic and nuclear compartment. In addition, immuno-fluorescence confocal analysis of total Jak2 distribution in K562 shows a very organized localization of Jak2 at one pole of the cells but this organization is lost in K562-R and total Jak2 appears uniformly distributed within the cell. K562-R cells were isolated as a Bcr-Abl independent IM resistant cell line that expressed high levels of activated Lyn kinase (Donato et al., Blood 2003). We used K562-R as a model to study the role of Jak2 in a non Bcr-Abl addicted cell line. Since we have previously published that Jak2 up-regulates Lyn kinase activity (Samanta et al., Oncogene 2009), we propose that the higher activation of Jak2 in K562-R is the main driver of oncogenicity and IM resistance and that this cell line may be used to model the role of Jak2 in a cell that is not Bcr-Abl “addicted.”

Disclosures:
No relevant conflicts of interest to declare.
5 Year Experience in Chronic Phase-Chronic Myeloid Leukemia Treated with Imatinib Copy Drugs in a Peruvian Hospital
Authors: Torres Aimee , Navarro Juan Ramon , Untama Jose , Moreno Mariela , Murillo Sergio 
Published: October 2019

Abstract
Abstract 4449

BACKGROUND AND OBJECTIVES:
In our Hospital patients with Chronic Myeloid Leukemia (CML) in chronic phase (CP) were treated with imatinib (Glivec) 400 mg qd from june 2001 to june 2006. Since july 2006, only imatinib copy drugs (brand names: Zeite, Imatec, Imatib, Millatus and Celonib, all of Indian origin) are being used to treat this disease at the Rebagliati Hospital in Lima Peru. We present here the efficacy, security, event-free survival, progression free survival, overall survival of patients treated with imatinib copy drugs.

METHODS
Retrospective and descriptive analysis of CP-CML patients treated only with imatinib since july 2006. They all initiated the treatment with 400 mg qd. Five year experience is presented here.

RESULTS
We analyzed data from 51 patients (pts) with CP-CML. Median age at diagnosis was 45.1 years (15–90), 49% female and 51% male, all patients were Chromosome Philadelphia positive. Low, Intermediate and High Hasford Score were 32%, 48% and 20%, respectively. Median time from diagnosis to start of imatinib was 7.6 months (0–123). Five patients (9.8%) began treatment more than six months after diagnosis. All patients had at least a 3 month cytogenetic monitoring.
Thirty five (68.6%) and 39 patients (76.4%) achieved Complete Cytogenetic Response (CCyR) and Mayor Cytogenetic Response (MCyR), respectively. Median time to CCyR was 7.6 months and 71.4% of these patients achieved it during the first six months of treatment. CCyR and MCyR were 38% and 67% at 3 months, 57% and 70% at 6 months, 63% and 83% at 12 monhts, 78% and 91% at 18 months, respectively. Quantitative RT-PCR was performed in 22 patients with CCyR and 18 pts (81.8%) had Mayor Molecular Response (MMR) in five years of follow up.
Four pts (7.8%) progressed to Blastic Phase and all of them had Low Hasford Risk at diagnosis. Eight pts (15.6%) changed to 2nd line treatment. Two pts (3.9%) abandoned treatment.
Only 1 pt (1.9%) had both anemias 3–4. Eighteen pts (35.2%) had neutropenia-3, 8 pts (15.6%) had neutropenia-4, 7 pts (13.7%) had thrombocytopenia-3 and 4 pts (7.8%) had thrombocytopenia-4. Neutropenias 3–4 were managed with filgrastim, as treatment and as prophylaxis.
At 5 years, Event Free Survival (EFS), Progression Free Survival (PFS) and Overall Survival (OS) were 82%, 89% and 91%, respectively (Figure 1, 2 and 3).

CONCLUSIONS
In our hospital, CP-CML patients treated with imatinib copy drugs obtained important cytogenetic and molecular responses with prolonged EFS, PFS and OS and an acceptable safety profile. Hasford Score was not a good prognosis predictor. Treatment with Imatinib copy drugs has proven to be an acceptable treatment in CML patients in our hospital.

Disclosures:
Torres: Bristol Myers Squibb: Speakers Bureau.
Frontline Autologous Stem Cell Transplantation As Intensive Consolidation in Patients with Peripheral T Cell Lymphomas: Multicenter Phase II Trial in Korea
Authors: Ahn Jae-Sook , Yang Deok-Hwan , Chae Yee Soo , Sohn Sang Kyun , Kwak Jae-Yong , Yhim Ho-Young , Lee Se-Ryeon , Jung Sung-Hoon , Bae Soo-Young , Kim Yeo-Kyeoung , Kim Hyeoung-Joon , Lee Je-Jung 
Published: October 2019

Abstract
Abstract 4477
Peripheral T cell lymphomas (PTCLs) are an aggressive subtype of non-Hodgkins lymphoma and they have shown the shorter survival compared with B cell lymphoma. High-dose chemotherapy (HDT) followed by autologous stem cell transplantation (HDT/ASCT) for PTCLs has a potential meaning of consolidating remission for PTCLs. However, the effectiveness of ASCT on distinct conditioning regimens, the optimal transplant timing in the frontline or relapsed are still unclear.
We investigated the clinical outcomes of HDT/ASCT as frontline intensification in 46 patients with newly diagnosed PTCLs except ALK(+) anaplastic large cell lymphoma. Patients underwent ASCT with a uniform conditioning regimen (busulfex, cyclophosphamide and etoposide). The median age was 47 years (17–65). The histological subtypes were 47.9% PTCL-NOS (n=23), 18.8% anaplastic large cell lymphoma (n=9), 4.2% angioimmunoblastic T cell lymphoma (n=2), 25% extranodal NK/T cell, nasal type (n=12), 2.1% hepatosplenic T cell lymphoma (n=1) and 2.1% enteropathy-associated T cell lymphoma. Thirty patients (62.6%) presented with advanced stage disease (III/IV) and 16 (33.3%) had B symptoms. At diagnosis, 21 patients (43.8%) were classified as high-intermediate/high risk by the age-adjusted IPI (aaIPI) and 10 (20.9%) were classified as high-risk (more than 2 factors) by the prognostic index for PTCL (PIT). Thirty-one patients (67%) could undergo HDT/HSCT and disease status at pretransplant consisted of 23 patients (50 %) with CR and 8 patients (17.4%) with PR. 6 out of 8 patients with PR at pretransplantation improved the response to CR after HDT/ASCT. There was no significant difference of the response rate between CHOP alone or CHOP-like chemotherapy and non-anthracycline-based chemotherapeutic regimen. At a median follow-up of 32.9 months, 23 patients (50%) are alive. The 5-year probability of overall and progression-free survival (PFS) was 48.2 ± 8.1 % and 47.4 ± 8.1%, respectively. However, the 5-year OS and PFS rate in transplanted patients was 57.3± 10.2 % and 55.3 ± 11.3 %, respectively.

Conclusion:
Frontline HDT/ASCT in patients with PTCL could be performed with a high response rates and a substantial impact on improving outcome for PFS. Our findings also indicate that busulfex, cyclophosphamide and etoposide is a feasible conditioning regimen in ASCT for PTCLs.

Disclosures:
No relevant conflicts of interest to declare.
NVP-BEZ235, A Dual Inhibitor of Phosphoinositol-3-Kinase (PI3K) and Mammalian Target of Rapamycin (mTOR), Is a Potent Inhibitor of Lymphoma Cell Growth and Survival
Authors: Buglio Daniela , Lemoine Manuela , Neelapu Sattva S. , Vega Francisco , Berry Donald , Younes Anas 
Published: October 2019

Abstract
Abstract 4965
The Phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR pathway is frequently deregulated in Hodgkin (HL) and non-Hodgkin lymphoma (NHL), and has been linked with tumor cell growth and survival. Although several proteins/enzymes in this pathway can be targeted by a variety of small molecules in vitro and in vivo, it remains unclear which protein target is the ideal for clinical testing. Previous studies demonstrated that the clinical activity of mTOR inhibitors may be attenuated by a negative feedback loop that involves activation of AKT, suggesting that a dual inhibition of AKT and mTOR activation may produce a better therapeutic outcome. To test this hypothesis, we evaluated the in vitro activity of NVP-BEZ235, a dual inhibitor of PI3K and mTOR, in a panel of 13 HL and NHL cell lines. NVP-BEZ235 inhibited cell growth and induced apoptosis in lymphoma cell lines in a time and dose dependent manner. After 48 hours of incubation, the IC50 ranged between 50 and 100 nM, and it was equally effective in ABC and GCB-derived DLBCL cell lines. NVP-BEZ235 induced cell death was primarily due to induction of apoptosis, as evident by the annexin-V and PI dual staining method, and the induction of caspase 3 and PARP cleavage. NVP-BEZ235 effectively inhibited the activation of the PI3K pathway at several steps, including decreasing the phosphorylation level of p-Akt (Ser473), p-Akt (Thr308), p-mTOR, p-4-EBPI and pP70S6K. Because lymphoma cells frequently depend on multiple activated signaling pathways to promote their survival, including the JAK/STAT pathway, we investigated the potential synergy between PI3K and JAK/STAT pathway inhibitors. Lymphoma cells were variably sensitive to the JAK1/2 inhibitor INCB16562 in vitro. Submaximal concentrations of NVP-BEZ235 demonstrated a synergistic activity with INCB16562. Collectively, our data show that the PI3K/mTOR inhibitor NVP-BEZ235 is highly effective against a wide range of lymphoma cell lines, and warrants evaluating it alone and in combination with JAK/STAT inhibitors in phase I/II clinical trials in patients with relapsed lymphoma.

Disclosures:
No relevant conflicts of interest to declare.