bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhanced Synthesis of Functional Recombinant Factors IX and VII by BHK Cells Engineered to Overexpress VKORC1 Combined with siRNA Silencing of the γ-Carboxylation Inhibitor Calumenin.
Authors: Wallin Reidar , Wajih Nadeem , Owen John 
Published: October 2019

Abstract
Because of the importance and demand for recombinant factors IX, VII and protein C in prophylactic medicine, our laboratory is focused on engineering eukaryotic cells with enhanced capacity to produce fully γ-carboxylated functional forms of these recombinant vitamin K-dependent proteins. Here we report our work on recombinant human factor IX (r-hFIX) and recombinant human factor VII (r-hFVII). Successful engineering was accomplished by overexpressing the vitamin K cofactor producing enzyme VKORC1 of the vitamin K cycle in BHK cells stably overexpressing r-hFIX and r-hFVII respectively. VKORC1 provides the γ-carboxylase with reduced vitamin K1 (vit.K1H2) cofactor and is the rate limiting step in vitamin K-dependent post translational γ-carboxylation of proteins. We have identified the ER chaperone protein calumenin as an inhibitor of vitamin K-dependent γ-carboxylation. Here we show that siRNA silencing of calumenin in BHK cells engineered to overexpress VKORC1 and r-hFIX and r-hFVII respectively significantly increases production of both coagulation proteins as functional fully γ-carboxylated proteins. Engineering of the γ-carboxylation system and silencing of calumenin do not affect total synthesis of r-hFIX and r-hFVII by BHK cells. However the fraction of functional r-hFIX and r-hFVII isolated from the total pool of each recombinant coagulation factor pool was found to be 80 and 85% compared to 12 and 14 % from the pools isolated from non engineered BHK cells stably overexpressing r-hFIX and r-hFVII respectively. This new recombinant technology enforced on the vitamin K-dependent γ-carboxylation system demonstrates that r-hFIX and r-hFVII used as pharmaceuticals can be produced at a much higher yield than currently is accomplished from r-hFIX and r-hFVII overexpressing cells not modified by the new technology.
Plasmin-Mediated Coagulation Factor Xa Derivative, Xa33/13, Enhances Plasminogen Activation and Fibrin Clot Lysis.
Authors: Pryzdial Ed L.G. , Talbot Kimberley , Meixner Scott C. 
Published: October 2019

Abstract
Previous reports showed that coagulation factor Xa (FXa) treated with the fibrinolysis protease, plasmin, loses procoagulant activity and can accelerate tissue plasminogen activator (tPA). The resulting FXa derivative, Xa33/13, acquires binding sites for plasminogen and was consequently presumed to be the form of FXa involved in generating plasmin. By evaluating the function of purified Xa33/13, the current work addresses the hypothesis that conversion of FXa to Xa33/13 is the basis of enhanced plasmin generation by tPA and that this newly acquired FXa function can also contribute to clot lysis. Using purified proteins in a chromogenic assay, both Xa33/13 and FXa were found to increase the generation of plasmin activity by tPA at least 10-fold. To explain the involvement of FXa reported previously, Western blots demonstrated a correlation between the disappearance of FXa, appearance of Xa33 antigen and enhanced plasmin production. The Western blots also revealed that detection of Xa33 antigen is lost at later time points, which explains the observation that plasmin generation is eventually shut down during the chromogenic assay. Despite fibrin being recognized as the principal accelerator of tPA, at concentrations as low as 10 nM, either Xa33/13 or FXa reduced the lysis time of thrombin-mediated fibrin clot in a turbidity assay. The Xa33/13 or FXa could be added during clot formation before the tPA or after clot formation with the tPA to facilitate a 50–70% reduction in the time required to achieve half maximal lysis at 0.1 μM. The time-dependent formation of Xa33 antigen in plasma that was induced to clot with tissue factor, anionic phospholipid vesicles and Ca2+, demonstrated that the plasmin-mediated fragmentation pattern of purified FXa represents the complex physiological mixture. However, the use of specific inhibitors and immuno-depleted plasma indicated that plasmin is not required for production of Xa33 antigen in plasma. Cumulatively the data presented here support a role for coagulation FXa in fibrinolysis. These diametrically opposing functions derived from the same molecule, may contribute to the balance between coagulation and fibrinolysis, thereby facilitating tolerance for a wide normal physiological range of FX and possibly other coagulation proteins.
Morphological and Functional Studies of Ultra Long Von Willebrand Factor (ULVWF) Anchored on Endothelial Surface.
Authors: Huang Jing , McEver Rodger P. , Heuser John , Sadler J. Evan 
Published: October 2019

Abstract
Von Willebrand factor multimers that are tethered on endothelial cells are ultra large, hyperactive and susceptible to ADAMTS13 cleavage. However, the morphology of acutely secreted ULVWF that are deposited on endothelial surface and the mechanism by which they persist under fluid shear stress remain elusive. In this study, we use immunofluorescence and electron microscopy to characterized HUVEC surface associated VWF. By conducting perfusion assays in a parallel plate flow chamber in the presence of an agonist and a fluorescent anti-VWF antibody, we directly visualized VWF secretion and dynamics in real time. Upon histamine or forskolin stimulation, VWF multimers secreted by confluent HUVEC formed extended strings parallel to flow direction. While strings formed independent of platelet adhesion, they bound platelets without requiring platelet activation. Quick-freeze, deep-etch electron microscopy of immuno-gold labeled VWF showed that multiple single strings can merge and form parallel or twisted bundles, suggesting lateral association between individual VWF multimers. These “strings” or “bundles of strings” were tethered to the plasma membrane by a limited number of anchoring points, remained stable for over 30 minutes of continuous laminar flow at the shear stress of 2.5dyn/cm2, and about 50% of these strings were disrupted (bent or washed away) by a reversal of flow direction. Static adhesion assays indicated that both “RGDS” binding integrin and P-selectin were involved in binding of CHO-P cells to ULVWF containing HUVEC supernatant. However, “RGDS” peptide, but not soluble P-selectin or a polyclonal P-selectin antibody by itself, could interfere with the formation of fluorescent VWF strings under flow. These observations suggest that “RGDS” binding integrins are implicated in the presentation of ultra-long VWF strings on endothelial lumen, and that multiple protein-protein interactions could contribute to this process.
Altered Von Willebrand Factor Subunit Proteolysis and Multimer Processing Associated with the Cys1599(2362)Phe Mutation in the B2 Domain.
Authors: De Marco Luigi , Casonato Alessandra , Gallinaro Lisa , Sztukowska Marita , Mazzucato Mario , Battiston Monica , Pagnan Antonio , Ruggeri Zaverio M. 
Published: October 2019

Abstract
Cysteine residues clustered in carboxyl- and amino-terminal domains mediate the assembly of von Willebrand factor (VWF) subunits into dimers and multimers, respectively. We have identified a patient with bleeding symptoms and very low plasma VWF antigen and ristocetin cofactor activity, compatible with clinically severe von Willebrand disease (VWD), whose abnormal plasma VWF multimers show a distinctly altered proteolytic processing. Analysis of plasma VWF multimers in low resolution agarose gels demonstrated a smearing extending to the origin of the separating gel in both the proband and her parents, suggestive of the presence of unusually large species with low electrophoretic mobility. In high resolution gels, these large multimers were stacked at the origin of the gel, clearly seen in a sample of proband’s plasma obtained after DDAVP infusion even though the treatment caused only a modest increase in VWF:Ag and VWF:RCo (from 4.7 to 7.5 U/dL and from 5.2 to 8 U/dL, respectively). Individual VWF multimers in the proband’s plasma comprised only one clearly visible band with different mobility as compared to the main band of normal multimers, and lacked the satellite bands that form the normal triplet structure. Analysis of the proband’s plasma VWF subunit composition using anti M7 antibodies (which probe the aminoterminal region of VWF) revealed the apparent 225 kDa mature form and a single 205 kDa fragment, but notably absent was the 140 kDa aminoterminal fragment generated by ADAMTS-13 cleavage. Subunit analysis using anti-M31 antibodies (which probe the carboxyl-terminal region of VWF) revealed the 225 kDa intact subunit and the 205 kDa fragment, but notably absent were the fragments generated by ADAMTS-13 cleavage. Analysis with anti-22K antibodies, which react with one or more epitope(s) located in the extreme carboxyl terminal region of VWF downstream of Val1927, revealed the presence of only the intact subunit of 225kDa. Concentration of ADAMTS-13 antigen and VWF-cleaving activity in the patient plasma were within normal limits.The patient carried a homozygous Cys to Phe mutation at position 1599 in the mature VWF subunit B2 domain (Cys2362 of pre-pro-VWF). In her heterozygous parents, the 205 kDa fragment was distinctly visible along with the normal VWF cleavage products. Our finding indicate that a proper conformation of the B2 domain, dependent on critical Cys residues, may be required for normal proteolytic processing of VWF multimers. The association of these structural and functional abnormalities with a missense mutation in the B2 domain provides insights into the mechanism that may regulate the post-secretion processing of VWF multimers, and, thus, their prothrombotic potential.
Role of Tissue Factor in Platelet Adhesion: Real-Time Perfusion Studies.
Authors: Tonda Raul , Galan Ana M. , Navalon Fulgencio , Munday Adam D. , Chen Junmei , Lopez-Vilchez Irene , Lopez Jose A. , Escolar Gines 
Published: October 2019

Abstract
Exposure of Tissue Factor (TF) to circulating blood and excessive thrombin generation are major contributors to acute thrombosis. Platelets also play a central role in the pathogenesis of occlusive events. Despite the importance of both components, convincing evidence of direct adherence of platelets onto TF immobilized on a surface is lacking. Here, we studied the interactions of platelets with different TF-rich substrata. Real-time perfusion techniques were applied to visualize thrombus formation and to evaluate the time necessary for platelets to both attach to the surface and to form thrombi. We used three different sources of TF as substrates:Thromborel S®, which is TF purified from human placenta (pTF) (at 0.4 ng/cm2);Innovin®, recombinant TF that has been relipidated (rTF) (at 0.4 ng/cm2); andTF expressed on the membranes of TF-transfected CHO cells (CHO-TF).
As negative controls, we used equivalent surfaces sprayed with 0.5% bovine serum albumin or cultured sham-transfected CHO cells (CHO-C-), respectively. Platelet-rich plasma (PRP) from blood anticoagulated with low-molecular-weight heparin was perfused over these substrata at a shear rate of 300s−1 for 5 min. Images were captured with a digital camera at high resolution and streamed to a computer. Continuous real-time photographs were taken during the full 5 min. Single frames were captured at predetermined times and the mean area of the platelet aggregates at these times was measured. Every experiment was replicated at least 4 times. Results are expressed as mean ± S.E.M. Under these conditions, platelets bound and formed thrombi on all substrata containing TF, although the time to aggregate formation and aggregate area varied depending on the substratum. Platelet deposition was minimal on the BSA-coated surfaces or on CHO-C- cells.
Initial platelet deposits on pTF were observed after 120 sec of perfusion. After 5 min, thrombi reached an area of 122±41 μm2. Relipidated rTF was less effective in supporting platelet deposition, requiring 135 seconds on average for the initiation of platelet aggregates. After 5 min, the average area of platelet aggregates was 34.3±9.6 μm2. Formation of platelet aggregates on CHO-TF cells was noticeable after 80 seconds. Aggregates formed at 5 min reached an area of 213±58 μm2. In conclusion, our studies demonstrate that platelets adhere to and aggregate on different TF substrata. The presence of other adhesive molecules in cell-derived TF preparations (pTF, CHO-TF) appears to accelerate the interaction of platelets with TF. These observations suggest that in addition to its role in blood coagulation, TF also has role in platelet adhesion by providing an adhesive surface at sites of vessel injury.
SAF2003-05780, SGR2005-00952, FIS PI040887, FIS CP04-0011, NHLBI 5R01HL064796.
Plasminogen Activator Inhibitor 1 Deficiency Reduces the Progression of Atherosclerosis in a Murine Model of Human Familial Hypercholesterolemia.
Authors: Iwaki Takayuki , Victoria Ploplis A. , Castellino Francis J. 
Published: October 2019

Abstract
Atherosclerosis has been described as a “self-perpetuating” inflammatory disease which progresses in discrete stages and involves a number of cell types and effector molecules. Lipid metabolic disorders are heavily involved in the genesis and progression of atherosclerosis in humans, and thrombotic complications substantially contribute to the coronary artery disease that frequently accompanies atherosclerosis. A relevant mouse model of human atherosclerosis with elevated LDL-cholesterol (C) is one in which both the Ldlr and Apobec1 genes are deleted (Ldlr−/−/Apobec1−/−), since these mice lack the ability to convert apoB-100 to apoB-48 in liver and also are defective in LDL clearance. We have generated Ldlr−/−/Apobec1−/−/PAI-1−/−and characterized atherosclerosis progression in these mice. VLDL, LDL, HDL, and total C levels were identical between Ldlr−/−/Apobec1−/−and Ldlr−/−/Apobec1−/−/PAI-1−/−mice (VLDL-C; 30.3±1.8 vs 26.0±1.7 (mg/dl), LDL-C; 268.7±6.3 vs 274.1±13.1 (mg/dl), HDL-C; Total-C; 353.8±8.1 vs 356.8±14.5 (mg/dl), respectively). However, both the plaque size in the aortic sinus and the extent of plaque area in aorta were significantly smaller in Ldlr−/−/Apobec1−/−/PAI-1−/−mice relative to Ldlr−/−/Apobec1−/−mice (plaque in aortic sinus; 158792.9±11958.2 vs 227243.5±15291.1 (μm2), plaque area in aorta; 3.90±0.34 vs 5.10±0.27 (%), respectively). To elucidate the cellular functions, especially macrophages, in the plaque, bone marrow-derived macrophages (BMMs) were isolated and utilized for Dil-oxidized (Ox) LDL uptake assays and migration assays using 8 μm pore culture inserts. There were no significant differences in Dil-OxLDL uptake by BMMs between the two genotypes. The kinetics of migration of BMMs was more rapid in Ldlr−/−/Apobec1−/−/PAI-1−/−cells than Ldlr−/−/Apobec1−/−cells. Furthermore, thioglycollate-induced intraperitoneal migration of macrophages was increased in Ldlr−/−/Apobec1−/−/PAI-1−/−mice vs Ldlr−/−/Apobec1−/−mice. Results from these studies indicate that cell migratory differences may contribute to attenuating atheroma formation in a PAI-1 deficient state.
Calcineurin/NFAT Pathway Is a Novel Target of Therapeutic Interest in Lymphoid Malignancies.
Authors: Medyouf Hind , Alcade Helene , Berthier Caroline , Janin Anne , de Thé Hugues , Ghysdael Jacques 
Published: October 2019

Abstract
Calcineurin (PP2B) is an ubiquitously expressed serine/threonine protein phosphatase dependent on calcium calmodulin. Calcineurin plays a pivotal role in many important biological processes, including the development and function of the immune system. Sustained signalling through calcium/calcineurin results in the activation of transcription factors of the NFAT family (Nuclear Factor of Activated T cells). Mouse genetic studies have demonstrated a strong epistatic relationship between calcineurin and NFAT activation and function in many developmental processes. In lymphoid cells, the calcineurin/NFAT signalling pathway is essential to specific aspects of T cell development and plays a central role in the activation of the immune response and in its homeostatic control.
Despite their central role in T cell development, immune function and homeostasis, deregulation of calcineurin/NFAT pathway have not been shown so far to have a pro-oncogenic role in lymphomagenesis. Using our TEL-JAK2 transgenic mouse model of T-ALL, we investigated the activation status and importance of the calcineurin/NFAT signalling pathway in leukemogenesis. We found that the calcineurin/NFAT pathway is activated in leukemic cells of TEL-JAK2 transgenic mice. Western blot and EMSA experiments showed that all NFAT members expressed in the lymphoid lineage (NFAT1, 2 and 4) are found in an active hypophosphorylated form, they are located in the nucleus, and exhibit an increase in their DNA binding activity. In order to investigate whether constitutive calcineurin/NFAT activity was specific to TEL-JAK2 leukemia or whether it is a more general property of leukemic cells, we analyzed NFAT activation in leukemic cells of transgenic mice aberrantly expressing oncoproteins involved in lymphoma in human. This showed that calcineurin/NFATpathway is not only aberantly active in leukemic cells of TEL-JAK2 transgenic animals, but also in other mouse models of human leukemia. We also found that the activation of the calcineurin/NFAT pathway is only observed when cells are rapidly analyzed following their explantation and dissociation from diseased animals. Indeed, if cells are incubated in tissue culture for a short period of time (60–120min.), essentially stochiometric rephosphorylation, meaning inactivation, of NFAT is observed. This indicates that activation of the calcineurin/NFAT pathway depends upon signals generated in the in vivo tumor cell environment and is not -or not solely- under the control of the initiating oncogene. Using these mouse models we conducted pre-clinical studies that indicated that calcineurin/NFAT pathway is an attractive novel target of therapeutic interest in lymphoid malignancies.
Cerebrospinal Fluid (CSF) Proteomics in Children with Acute Lymphoblastic Leukemia (ALL).
Authors: Trueworthy Robert C. , Stork Linda , Zhong Yanping , Pine Sharon , Matloub Yousif , Laderas Ted , McWeeney Shannon , Fan Guang 
Published: October 2019

Abstract
Relapse within the central nervous system (CNS) remains a major challenge for the treatment and cure of childhood ALL. With current intensive treatment, at least one-third of relapses occur within the CNS. Evaluation of CSF is essential to the management of childhood ALL and relies on morphologic examination of cytospin smears. CSF has been re-discovered in the post genomic era as a source of potential protein biomarkers for various diseases. Such proteins may help establish a diagnosis, provide insight into pathogenesis, or identify therapeutic toxicity. In the present study, acellular CSF supernatants from newly diagnosed children with standard risk ALL treated on CCG-1991 were used for protein profiling.
The morphologically negative CSF samples had previously been classified as minimal residual disease (MRD) negative (−) or positive (+) using real-time PCR techniques on centrifuged cell lysates. Three MRD −, 3 MRD +, and 2 morphologically + CSF samples were concentrated and desalted using Microcon filter device. Albumin was removed using a Human Albumin Kit, Albuminomics™. The MRD − and MRD + samples, each containing 15 mcg of protein, were pooled separately and processed for protein identification and quantitation using a multidimensional LC/MS/MS method. Custom software was developed (TandTRAQ) for database searching and peptide quantitation. Protein annotation was primarily derived from ENSEMBL and NCBI sources. Rank order summarization was used to identify putative candidates. In the comparison between MRD + and MRD− samples, we identified 226 unique genome identifiers, with 89 having functional annotation. Of these 89, 49 have high quality protein-level annotation, 35 have mRNA-level annotation, and 5 are predicted (have functional protein evidence). Using a 1.5 fold cut-off, there were 18 up-regulated and 12 down-regulated proteins among the 89 proteins in MRD + compared to MRD − supernatants. Focusing on the 49 with high quality protein-level annotation, 8 proteins show higher-level expression and 2 show lower level expression in MRD + CSF samples (Table). Eight highly expressed proteins are involved in either DNA binding interactions, cell migration, apoptosis, or immune response. Two under-expressed proteins are involved in cell adhesion. The 2 morphologically + CSF samples confirmed 4 over expressed and 1 under-expressed proteins.
This research is the first attempt at protein profiling of CSF from patients with ALL. The proteins identified in this study may further our insight into the pathogenesis of CNS leukemia. Furthermore, future research may find that protein profiling of supernatants is a sensitive way to detect leukemia in CSF and to monitor treatment-related CNS toxicities.
Immunogenicity of Childhood T-ALL.
Authors: Panzer-Grümayer Renate , Inthal Andrea , Krapf Gerd , Felzmann Thomas , Gadner Helmut , Dohnal Alexander 
Published: October 2019

Abstract
Background: Acute lymphoblastic leukemia (ALL) with a T cell immunophenotype accounts for about 15% of childhood ALL. While contemporary treatment protocols have improved the relapse free survival for children with T-ALL, it has for a long time been considered a malignancy with a dismal prognosis. In fact, this type of leukemia is still at a higher risk for treatment failure and early relapses than B cell precursor ALLs. Further intensification of conventional treatment or stem cell transplantation may increase the cure rate for only some subtypes of ALL but is also associated with considerable treatment-related mortality and morbidity. Thus, new treatment strategies that are less toxic and, at the same time, more effective for patients with a resistant disease, are needed. The aim of the study was to evaluate whether a specific immune reaction is triggered in children with T-ALL and to identify the respective targets. We chose SEREX (serological identification of leukemia derived antigens by recombinant expression cloning) to screen a T-ALL expression library with plasma from 18 children with T-ALL.
Results: Thirteen antigens with homology to known genes that are involved in critical cellular processes were detected. Some of them have already been implicated in the induction of an immune response in a variety of cancer types. From four of these genes novel isoforms were identified and further analyzed. mRNA expression of three isoforms (HECTD1Δ, CX-ORF-15Δ and hCAP-EΔ) was restricted to more than 70% of T-ALLs (n=22) and specific antibodies against these isoforms were detected in up to 30% of patients (n=16) with the highest frequency for HECTD1Δ. The alternative splicing leads to the deletion of one glutamic acid located in a putative PEST domain thereby decreasing the PEST score suggesting a higher stability of the protein. Interestingly, the HECTD1 protein was present at high abundance in T-ALLs while is was not detectable in normal hematopoietic tissues. Since the leukemia-associated antigens detected in this study have an intracellular localization, a feature that is shared by the majority of SEREX defined antigens, the generation of immune effector responses most likely requires antigen presentation. To test this assumption, dendritic cells were loaded with HECTD1Δ protein and used for T cell stimulation. A specific T cell response was induced in vitro in all three donors, including a former T-ALL patient.
Conclusion: Leukemia-associated antigens, identified by SEREX, appear to be capable of inducing both a humoral and cellular immune response in children with T-ALL. Thus, these data support further studies to establish new approaches for immunotherapy.