bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human Factor IX Expression and Hemophilia Phenotypic Correction in Mice: A Comparison of AAV Serotypes.
Authors: Chao Hengjun , Chen Wei , Tunstead James R. , Walsh Christopher E. 
Published: October 2019

Abstract
Recombinant adeno-associated virus (rAAV) has been extensively studied as a vector for hemophilia gene transfer. AAV serotype 2 targeting the liver and skeletal muscle has been used in clinical trials for hemophilia B patients. Previously we reported that the use of recombinant non-AAV2 serotype vectors generated persistent expression of supra-normal levels of canine factor IX (cFIX) in immunodeficient mice (Mol Ther ‘2000), and resulted in sustained and complete hemophilia B phenotype correction in immune competent hemophilia B mice (Mol Ther ‘2001). In those studies AAV serotypes 1–5 were tested by intramuscular injection. In this study we tested rAAV serotypes 1–8 for hepatic transduction and FIX production in both C57BL/6 and FIX knockout mice. Animals received intrapotal vein injection of 1x1011 virion particles of rAAV 1 thru 8 carrying a human FIX cDNA linked to a chicken beta actin enhancer-CMV promoter and bovine growth hormone polyA. Animals were followed for 8 months and assessed for plasma levels of human FIX by ELISA. A rapid, sustained and maximal rate of hFIX expression was observed within 1–2 weeks with all vectors except AAV2 where the peak expression occurred at 8–10 weeks. hFIX expression was sustained for all serotyped vectors (n=5 animals tested at each AAV serotype). Animals that received serotypes 7 and 8 maintained hFIX at physiological levels (100% hFIX ~ 5000 ng/ml). A differential hFIX expression pattern emerged with rAAV7 (6213 ng/ml) > 8 (5111 ng/ml) > 5 (2367 ng/ml) > 1 (1090 ng/ml) > 4 (377 ng/ml) > 2 (314 ng/ml) > 3 (232 ng/ml). rAAV7 and rAAV8 generated 20 times more hFIX per virion particle than the rAAV2 vector. We did not detect anti-human FIX antibody in any of the experimental mice. To assess why hFIX production differed between each serotype we performed immunohistochemical staining of the mouse liver using a fluorescent-tagged anti-human FIX antibody. Based on this assay, rAAV7 and AAV8 transduced 30% of hepatocytes while rAAV2 transduced less than 2% of the hepatocytes. We subsequently tested FIX production and effect on hemophilia phenotype of rAAV 7 and 8 (1x1011 virion particles) in hemophilia B mice; the data was similar to that observed using the C57Bl/6 mice. Human FIX levels (>5000 ng/ml) were again sustained during the 6–8 month observation period. No anti-human FIX antibody was detected in these hemophilia B mice. Results of clotting function by aPTT testing demonstrated a normalization of clotting time in all the rAA8/hFIX treated hemophilia mice. Survival of all treated mice by tail clip (lethal for all non-treated knockout animals) confirmed that phenotypic correction had been achieved. Our results again demonstrate that AAV serotypes have differential transduction rates and that new serotypes with greater mouse hepatocyte transduction rates are more efficient for hemophilia gene transfer.
U.S. Factor XI Patients: Is There a Safer Treatment? Yes - Hemoleven.
Authors: Aledort Louis M. , Goudemand Jenny 
Published: October 2019

Abstract
Despite donor screening and newer PCR testing, human-derived fresh frozen plasma (FFP) is not without the potential for viral transmission. Factor XI-deficient patients are plentiful in the U.S. (estimate ~1500–2000). Although there is discordance between phenotype and genotype, with substantially fewer “bleeders” than those with deficiency, and spontaneous bleeding is rare, surgical intervention frequently demands replacement therapy. The English F XI concentrate, virally inactivated, had thrombosis as a complication, and heparin was added, but is no longer available in the U.S. An LFB factor XI concentrate (Hemoleven), doubly viral-inactivated, with heparin and ATIII is available internationally, but not in the U.S. A review of 56 cases treated in France, Spain, Switzerland, Canada, and Israel reveal that for minor and major surgery it is both safe and effective. Between 1992 and 2004, data for the use of Hemoleven for either prophylaxis, minor and major surgery was collected. In all, hemostasis was excellent. In three, concentrate was used because of anaphylaxis with FFP. Three had laboratory evidence of DIC and, of these, one had a PE. In addition, these three cases received excessive dosage. Twelve patients had recovery and half-life studies for this concentrate. T1/2 was 45.5 ± 7.9h and recovery was 1.85 ± 0.38%/u/kg. These data are evidence that a safer and effective therapy for F XI patients exist outside of the U.S. We should not have to wait for a new infectious agent transmitted by FFP before we make it available in the U.S.. We should have learned something from HIV, when DDAVP was used everywhere, but not licensed here.
Experience with the “Off Label” Use of Recombinant Factor VIIa in a University Hospital.
Authors: Escobar Miguel A. , Chong Kenneth , Hoots Keith W. 
Published: October 2019

Abstract
Recombinant factor VIIa (rFVIIa), NovoSeven@ (Denmark), is a bypass agent approved in the United States for patients with hemophilia and inhibitors. The off-label use of this medication (non approved use) is becoming common in large institutions. Its main use seems to be for excessive bleeding due to trauma and surgery. Here we report our 2 year experience with the off-label use of this product and describe a protocol that is followed at our teaching hospital. Administration of rFVIIa was based on the described protocol. The medication is controlled by the hospital pharmacy and authorization for its use has to be given by the hematologists in collaboration with the clinical pharmacist. To be eligible for the use of rFVIIa the patient has to have received at least 10 units of red pack cells and fresh frozen plasma. In addition, the patient should have fibrinogen level greater than 100 mg/dl, platelet count greater than 70,000/mm3 and fibrin split products less than 80 mcg/ml. A total of 25 patients where treated off-label for excessive bleeding and 4 patients where given rFVIIa preoperative for prevention of bleeding. The doses ranged from 35 to 120 micrograms/kg of body weight with an average of 2 doses given per patient. The use of Factor VIIa was more common in the neurosurgical (NS) setting (7), followed by cardiovascular (CV) (6), gastrointestinal (GI) surgery (6), trauma (6) and others (4). Overall 62% of the patients had adequate hemostasis after the administration of rFVIIa. Successful hemostasis with rFVIIa by diagnosis was 100% for preoperative prophylaxis and GI, for NS 57%, for trauma 50% and for CV 17%. All the patients in the CV group where admitted with acute dissecting aortic aneurysms. No evidence of thromboembolic events where described. The “off-label” use of rFVIIa continues to be widely used with variable results. Until further randomized or controlled, double blind, clinical trials are done, no definitive recommendations can be given. Although this report includes a small cohort of patients, we will consider revising our protocol for bleeding following cardiovascular surgery and trauma.
Identification of a Novel Factor V A2 Domain Tyrosine Deletion Mutation in a Patient with Factor V Deficiency and Bleeding.
Authors: Jones Carol D. , Negro Fernando , Darnell Katherine , Zehnder James L. 
Published: October 2019

Abstract
The gene for coagulation Factor V (FV) is located on chromosome 1q23. FV deficiency shows an autosomal recessive mode of inheritance; heterozygotes are generally not clinically affected. The homozygous clinical phenotype occurs in approximately 1 per million individuals with variable severity of bleeding. Thus, genotype-phenotype correlations are likely to shed light on functionally important residues of FV. Here we describe a case of FV deficiency with a severe bleeding phenotype. The proband is a male infant from Argentina. His parents are unrelated. He was born healthy with no bleeding from the umbilical stump or other symptoms. He presented at eight months with a CNS hemorrhage, then suffered a second massive subdural bleed at nine months of age. Both episodes required surgical drainage and treatment with fresh frozen plasma He continues to receive prophylactic FFP infusions and has some residual neurologic impairment. The proband’s FV activity ranges from 2–14%. Two siblings are unaffected. His father’s FV activity is 50% and his mother’s is 70%. We performed DNA sequencing spanning the entire coding region of the proband’s FV gene and found two heterozygous mutations: a heterozygous single base pair deletion, del 2952T in exon 13, located in the B-domain of the FV protein, causing a frameshift mutation followed by a premature termination codon 3 amino acids downstream; and a novel 3-bp deletion in exon 10. This deletion is in-frame and results in the deletion of Y478. The del 2952T frameshift mutation was present in the father, while the del Y478 mutation was present in the mother. Y478 is in the A2 domain of FV and adjacent to another tyrosine, Y477. Evidence suggests that these tyrosine residues are important for co-factor function. Tyrosine residue sulfation has been shown to be required for full activity of the homologous co-factor, FVIII, as well as for hirudin. These sulfated tyrosines and surrounding acidic amino acids have been proposed to be important in interactions with the thrombin anion binding exosite; in the case of hirudin, sulfation of a carboxy-terminal tyrosine increases the affinity for thrombin 10-fold. The homologous tyrosines, Y718 and Y719 appear to be sulfated in FVIII. FV has been shown to be sulfated, but the precise location of the FV sulfation sites has not yet been determined. One of this patient’s FV alleles is nonfunctional due to a frameshift and a premature trancation of translation. With respect to the other allele, we hypothesize that, like FVIII, one or both of FV tyrosines 477 and 478 is sulfated, and that deletion of Y478 may result in disruption of FV co-factor function. In vitro mutagenesis and expression studies to characterize the functional consequences of the del Y478 and/or del Y477 are in progress.
DIC with Ulcerative Colitis: The Combined Effects of Prothrombin G20210A, APC Resistance, and Endotoxemia⇒.
Authors: Aue Georg , Carroll Nancy , Horne McDonald 
Published: October 2019

Abstract
Thrombosis is a recognized complication of inflammatory bowel disease (IBD). In contrast disseminated intravascular coagulation (DIC) has rarely been described with IBD, and then only during periods of intense disease activity or during colectomy. However, we recently encountered a young woman with a 3-year history of ulcerative colitis (UC) and a 10-year history of recurrent thrombosis who presented with DIC when her disease appeared to be well controlled. For the previous two months she had been forced to discontinue her usual warfarin because of extreme over anticoagulation and extensive ecchymoses. Otherwise she felt well and was working full time. When she presented to us, the following laboratory data were obtained: pro time 18.7s, aPTT 43.6s, fibrinogen 56 mg/dL, D-dimer 8–16 μg/mL, platelet count 123K/μL, thrombin-antithrombin complexes 47 μg/mL (nl, 1.3–4.0), plasma endotoxin 6.3 EU/mL (control 0.1), positive prothrombin G20210A heterozygosity, negative FVLeiden, but positive APC resistance. Antithrombin, protein C, protein S, and homocysteine were within normal limits. Extensive imaging studies failed to reveal evidence of malignancy or venous malformations. The patient was treated with 1 mg/kg/d enoxaparin with resolution of her ecchymoses and normalization of her laboratory values. Subsequently we exposed cultured human umbilical vein endothelial cells (HUVECs) to our patient’s serum and assayed HUVEC lysates for tissue factor. After a 2 hour incubation a 20% dilution of her serum increased the tissue factor in the HUVECs to a level twice that observed with serum from a normal donor. Furthermore, the effect of her serum was negated by the addition of 1 μg/mL polymyxin-B, which specifically inhibits endotoxin.
Discussion: Prothrombin G20210A, APC resistance, and UC are individually associated with an increase risk of thrombosis. However, the combination of all 3 factors in our patient apparently led to an intense degree of hypercoagulability that provoked not only recurrent thrombosis but DIC. Furthermore, the contribution of UC to her hypercoagulability may have been mediated by endotoxin diffusing from her inflamed bowel and upregulating tissue factor in her endothelium and/or monocytes. Because this patient’s recurrent thromboses predated her diagnosis of UC by 7 years, we suggest that occult IBD should be considered in the evaluation of idiopathic thrombophilia.
Elevated Factor VIII: C Level Is a Risk Factor for Cerebral Infarction.
Authors: Hua Baolai , Pan Jiraqi , Zhu Yicheng , Liu Xiaoli , Fan Liankai , Cun Liying , Zhao Yongqiang , Shen Ti 
Published: October 2019

Abstract
To explore the association between the risk of cerebral infarction (CI) and markers of coagulation and fibrinolytic systems. A prospective case-control study was carried out in PUMCH, in which 124 unselected patients with CI as well as 91 control patients were studied. Blood samples in acute phase of CI were obtained within 10 days from onset, and the convalescent smples were obtained at least 6 months after onset. Factor VII, FVIII:C, FX, vWF, AT-III, PC, PS, FPS,protein Z( PZ), D-dimer and TAT levels were measured. Associtions were assessed by multiple logistic regression analyses. Compared with control group, levels of FVIII:C, vWF, FVII, DD, TAT in patients with acute CI were marked elevated (FVIII:C194.6±48.6% vs 91.1±26.9%, p=0.000; vWF 123.2±28.8% vs 90.0±20.5%, p=0.003; FVII 98.7±25.2% vs 82.3±25.3%, p=0.002; DD 0.41±0.61 mg/l vs 0.12±0.10 mg/l, p=0.008; TAT 40.9±73.3 ng/ml vs 12.3±11.5 ng/ml, p=0.028, whereas level of FX was not changed significantly. Levels of anticoagulation proteins were decreased. Levels of PZ was 0.41μg/ml lower than cortrol (1.32±0.83μg/ml vs 1.73 ±0.55μg/ml, p=0.004) The result of logistic regression analysis showed levels of FVIII:C and vWF in acute phase were related with CI, with OR 1.143, 95%CI 1.099~1.189, P<0.01 and OR 1.040, 95%CI 1.003~1.082, P<0.05 for FVIII:C and vWF respectively. Compared with acute phase, levels of FVIII:C, DD and TAT in convalescent phase were reduced (FVIII:C 144.3±37.2% vs 193.5±46.2%, P=0.000; DD 0.21±0.20 mg/l vs 0.41±0.78 mg/l,p=0.025; TAT 15.2±10.3 ng/ml vs 42.9±72.6 ng/ml, p=0.030). However, the levels of FVIII:C, vWF and FVII were still higer than that of control(FVIII: C 144.3±47.2%, vs 91.1±26.9%, vWF 110.7±23.2% vs 90.0±20.5%, FVII 93.6±25.5% vs 82.3±25.3%, P<0.05). Level of PZ was elevated by 0.33μg/ml. The result of multiple logistic regression analyses indicated FVIII:C, vWF were the independent risk factors for CI
A Novel Flow Cytometric Annexin A5 Competition Assay for the Diagnosis of Antiphospholipid Syndrome.
Authors: Senzel Lisa , Wu Xiao-Xuan , Rand Jacob 
Published: October 2019

Abstract
Annexin A5 (A5) is a potent anticoagulant protein that crystallizes over phospholipid surfaces, shielding them from availability for coagulation enzyme reactions. The antiphospholipid antibody (aPL) syndrome (APS) is an autoimmune thrombophilia that is marked by the presence of antibodies against phospholipid-binding proteins and the paradoxical lupus anticoagulant phenomenon. aPL antibodies can promote coagulation by interfering with the crystallization of A5, thereby increasing the exposure of blood to thrombogenic anionic phospholipids. Since a flow cytometric assay for aPL measuring A5 binding to frozen thawed platelets was previously reported, we investigated whether phosphatidylserine-bound polystyrene beads could provide a robust platform for the assay. Beads were incubated with sera from patients with the aPL syndrome and from non-aPL controls. Fluorescence-tagged A5 was added and samples were analyzed by flow cytometry. Similarly treated beads were also used in coagulation assays to determine whether A5 anticoagulant function was also inhibited. Control sera permitted fluorescent labeling of nearly all beads.
In contrast, sera from aPL syndrome patients produced a major population of unlabeled beads, sometimes accompanied by a minor population of labeled beads.
7 of 13 confirmed aPL syndrome patients, and 7 of 12 anticardiolipin antibody positive patients, demonstrated reduced A5 binding in this assay, while 10 of 10 healthy blood donor controls did not. Dilution of the aPL sera resulted in progressive increase of A5 binding. Reduction of A5 binding appeared to correlate with reduced A5 anticoagulant activity (r=0.42, n=33, p=.01). This assay shows promise for investigating the effects of aPL antibodies on A5 binding and for the clinical diagnosis of the aPL syndrome.
Figure Figure Figure Figure
Cyp33 Immortalizes Mouse Bone Marrow Progenitor Cells.
Authors: Chen Jing , Diaz Manuel O. 
Published: October 2019

Abstract
MLL is a gene involved in translocations associated with leukemia. It encodes a protein with PHD fingers and SET domains that are conserved in the related Drosophila protein trithorax. Cyp33, a nuclear cyclophilin with an RNA binding domain, interacts with the MLL PHD finger domain. Overexpression of Cyp33 represses the Hoxc8 gene in human leukemia cells in an MLL dependent manner. This close relationship between Cyp33 and the genes involved in hematopoiesis and leukemogenesis inspires us to further clarify the function of Cyp33 in primary hematopoietic cells. Murine bone marrow progenitor cells (BMPC) were retrovirally transduced to express Cyp33 and their replicative potential was then investigated by repeatedly plating on methylcellulose medium supplemented with GM-CSF. Upon over-expression of Cyp33, p27 is down-regulated and the progenitor cells keep proliferating after the fourth plating while the control cells (infected with empty retroviral vector only) differentiate and stop forming colonies after the third plating. Furthermore, after the third plating, Cyp33 over-expressing cells exhibit primitive cell morphology after Wright staining, while the control cells show mature macrophage/granulocyte characteristics. We also generated a Cyp33-BMPC line by growing the Cyp33 over-expressing cells in liquid culture containing GM-CSF, after the fourth plating. Regardless of the presence of GM-CSF, the Cyp33-BMPC cell line cycles and grows exponentially maintaining stem cell surface markers: Sca-1+, c-Kit+, CD34+ and Lin− (B220−, CD3e−, Mac-1−, Gr-1−). Morphologically, Cyp33-BMPCs are like immature progenitor cells. In addition, these cells can again form compact colonies on the same methylcellulose media as described before. These data suggest that Cyp33 may immortalize mouse bone marrow progenitor cells and arrest them at the primitive cell stage.
Induction of Transient Cytopenia to Detect Hematopoietic Defects in the Mouse.
Authors: Anderson Nicole M. , Adamson Lee , Barber Dwayne L. , Stanford William L. 
Published: October 2019

Abstract
In an attempt to generate new mouse models of human hematopoietic diseases and increase our knowledge of the genetic networks that orchestrate hematopoiesis, we are developing a sensitized screen that will more effectively identify hematopoietic in heterozygous mutants. The chemical mutagen ethylnitrososurea (ENU) is a highly efficient mutagen in the mouse and other model organisms, generating primarily point mutations. As a part of the Centre for Modeling Human Disease (CMHD), we have used simple clinical assays such as blood morphology and automated blood cell counts to test saphenous vein collected peripheral blood from approximately 6500 offspring of ENU mutagenized mice to identify clinically relevant dominantly inherited hematopoietic disease models. Phenodeviant G1 animals are bred to wild type mice to generate G2 mice, and G2 strains demonstrating high penetrance undergo a genome scan. This strategy has yielded 14 heritable dominant mutants to date. However, many genes and pathways will not express phenotypes in a dominant manner due to the homeostatic balance maintained by complex genetic networks, possessing excess capacity or redundancy, a problem exacerbated by using a viable peripheral blood assay. For example, only one of the 14 heritable hematopoietic mutants demonstrates anemia. Because environmental sensitization can exacerbate latent phenotypes, such as the effect of a high salt diet in an individual with prehypertension, sensitized screens should identify subtle and additional phenotypes of dominant mutations. Thus, we are developing a sensitized screen to uncover subtle dominant phenotypes by inducing transient cytopenia and following the kinetics and magnitude of the recovery. We are using a variety of drugs (5-fluorouracil, phenylhydrazine, and hydroxyurea) to induce cytopenia and recruit hematopoietic stem cells and progenitors to repopulate the blood with differentiated cells. We hypothesize that animals with defects in blood development will demonstrate altered recovery kinetics. This strategy will allow us to determine if there is a defect in blood cell progenitors, while keeping the animals viable for further screening and breeding. We are testing these drugs on known dominant and recessive mutants to validate the assay, determine the best dosages, and compare the effects of each drug. However, we have already begun to use 5-fluoruracil on a cohort of G1 animals and have identified two G1s that display latent recovery in at least 1 blood lineage. The two G1s are confirmed heritable and are currently undergoing a genome scan to map position the mutation. We believe this strategy will further our understanding of the molecular mechanisms that regulate hematopoietic development, as well as generate new mouse models of human hematopoietic diseases and identify the underlying molecular defects of diseases whose etiological origins are currently unknown.