bluuebunny/crossref_metadata_2025_split huggingface dataset:
Insights into Disturbed Hemostasis in the Quebec Platelet Disorder Using Thromboelastography, Blood Clots Formed at Low Shear, and Perfusion Studies of Fibrinolysis.
Authors: Diamandis Maria , Adam Frederic , Rivard Georges E. , Kahr Walter H.A. , Hayward Catherine P.M. 
Published: October 2019

Abstract
The Quebec Platelet Disorder (QPD) is an inherited disorder associated with delayed bleeding, increased expression and storage of active urokinase-type plasminogen activator (u-PA) in platelets, normal to increased u-PA in plasma, consumption of platelet plasminogen activator inhibitor-1 (PAI-1), and increased plasmin generation in platelets with proteolysis of stored α-granule proteins, including factor V. Although accelerated fibrinolysis has been proposed to contribute to QPD bleeding, the effects of QPD blood on the lysis of forming and preformed fibrin have not been evaluated. In this study, we modeled fibrinolysis in vitro using thromboelastography, biochemical evaluations of whole blood clots formed at low shear, and perfusion of blood over preformed fibrin. Thromboelastography (TEG) indicated that the clot formation and lysis phases were normal in QPD whole blood clots generated with tissue factor and observed for 180 min, which is the maximum time allowed by the TEG® software. However, when the TEG® were done with limiting amounts of tissue-type plasminogen activator (t-PA), QPD clots showed a shortened lysis phase. The addition of QPD platelets to normal blood hastened t-PA mediated lysis. In QPD whole blood clots, generated at low shear with exogenous thrombin (without t-PA), there was abnormal plasmin generation, with increased fibrinolysis unless fibrinolytic inhibitors were used to inhibit plasmin. These data excluded the defect in platelet factor V as the cause of abnormal lysis. Perfusion studies indicated that QPD platelets adhered to fibrin, although the amount of adhesion was reduced compared to control samples, likely because of the reduced platelet count in QPD blood. When QPD blood was perfused over labeled, preformed fibrin, without a stimulus for thrombin generation, there was accelerated loss of fibrin, and a corresponding increased generation of labeled fibrin degradation products. These results indicate that the QPD is associated with a “gain of function” abnormality that increases the lysis of forming or preformed clots, independent of thrombin generation. Our findings suggest accelerated fibrinolysis is an important contributor to QPD bleeding and support the clinical observation that transfusion of normal platelets does not correct the defect in this bleeding disorder.
Afibrinogenaemia Resulting from a Novel Putative Splicing Mutation, and Evidence for Tissue Specific Expression of FGAα RNA.
Authors: Cutler Jacky , Bourdon Louise , Savidge Geoffrey F. 
Published: October 2019

Abstract
Congenital afibrinogenaemia is a rare autosomal recessive disorder characterised by the complete absence of fibrinogen in plasma. Fibrinogen is predominantly produced in hepatocytes from 3 homologous polypeptide chains (Aα, Bβ, γ) which assemble to form a hexameric structure. The genes that encode the 3 polypeptides (FGAα, FGBβ, FGγ) are clustered in an approximately 50kb region on chromosome 4q28-q31. Defects in each of the 3 genes have been implicated in afibrinogenaemia, and encompass missense and nonsense mutations, insertions, small and gross deletions and splicing defects.
We present a case of congenital afibrinogenaemia, diagnosed in a neonate, the only child of unrelated parents. Sequence analysis of the FGAα, FGBβ and FGγ genes identified a single, novel, homozygous nucleotide substitution (T→A) at IVS1 -19 of the FGAα gene. Both parents genotyped as heterozygous for this variant. Analysis of ~100 normal alleles failed to detect this change, thereby excluding it as a common polymorphism. Using splice prediction software, the IVS1 acceptor splice site is undetected, reflecting poor agreement with acceptor consensus sequences. The mutation presented here, results in the creation of a preferential acceptor splice site (Spliceview score of 77). It has been previously reported (Catia et al, 2003) that intron 1 is preferentially the last intron of the FGAα gene to be spliced, and it may be that the wild type exon 1-intron 1- exon 2- exon 3- exon 4 - exon 5 intermediate presents a better substrate to the spliceosome. If we accept this predicted splicing order the mutation described in this study will not affect expression. Instead, the resulting incorporation of 19 intronic bases will result in a premature termination sequence (TGA) and a truncated protein.
RNA work was undertaken to attempt to demonstrate these findings. However, RNA extracted from peripheral blood leukocytes from both parents failed to amplify for even the normal allele of FGAα (using primers located in exons 1and 5). Integrity of the samples was demonstrated by amplification of F8 RNA. To investigate this further, we tested a panel of commercially available RNAs from various tissues. RNA derived from liver, heart, spleen and kidney cells produced the expected product, with much higher yields being seen with liver cells, while RNA from brain cells and leukocytes failed to yield any product at all. Tissue specific expression has previously been described for FGγ RNA.
In summary: This case study has identified a novel splice site mutation in the FGAα gene which is predicted to result in the introduction of a premature termination sequence at the newly created codon -1. This could not be confirmed due to the previously unreported discovery that FGAα RNA is not expressed in peripheral leukocytes.
FVIII Recovery after a Single Infusion of Recalibrated ReFacto in 14 Severe Hemophilia A Patients.
Authors: Lambert Thierry , Guérois Claude , Gay Valerie , Stieltjes Natalie , Bertrand Marie-Anne , Derlon Annie , Sigaud Marianne , Hassoun Abel , Négrier Claude , Coatmelec Brigitte , Dreyfus Marie , Dubanchet Arnaud 
Published: October 2019

Abstract
Background: A field study among 6 reference laboratories in the EU using chromogenic substrate assays revealed in some laboratories a FVIII level lower than expected when measuring the potency of ReFacto, a B-domain deleted recombinant FVIII concentrate. In an attempt to resolve these discrepancies, the standard used for establishing the potency of ReFacto was recalibrated in 2003. Indeed after this recalibration, the amount of ReFacto protein in each International Unit (IU) has increased by approximately 20 percent without change in the labeled dosage strength.
Objectives: The primary objective of this prospective study was to assess the FVIII recovery in severe hemophilia A patients receiving recalibrated ReFacto.
Methods: The study was conducted in 10 French Hemophilia Treatment Centers in Previously Treated Patients (> 150 exposure days to any FVIII concentrate). A series of 4 blood samples per patient were collected in a non-bleeding state, respectively before, 15mn, 30mn and 60mn after intravenous bolus infusion of a single dose of 50 ±5 IU/kg of ReFacto. Plasma FVIII activity was determined in a central lab using a chromogenic substrate assay (Coamatic FVIIITM).
Results: Fourteen severe hemophilia A patients (FVIII: C < 1%) were evaluable for intention to treat analysis. Median age was 25.5 years (range: 12–48). Median injected dose was 53.3 UI/kg (range 48.4–56.8). Maximal plasma FVIII activity level was obtained 15mn (n=10) or 30 mn (n=4) after the end of infusion. Mean incremental recovery (K value) was 2.20 ±0.27 UI/dL per UI/kg infused (range: 1.89–2.75) with a mean in vivo recovery of 105.2% (range: 87.6–133.8).
Conclusions: In most cases the peak of FVIII activity was obtained 15 mn after the end of infusion. Recovery of recalibrated ReFacto was similar to the expected recovery with full-length FVIII concentrates.
Treatment of Diffuse Alveolar Hemorrhage with Recombinant Factor VIIa in Patients with Hematologic Malignancies.
Authors: Jha Asit , Balasubramanian Manjula , Charania Roseleen , Sandilya Vijay , Topolsy David , Styler Michael 
Published: October 2019

Abstract
Diffuse alveolar hemorrhage (DAH) is a potentially life-threatening pulmonary complication in patients following hematopoietic stem-cell transplantation (HSCT) or acute leukemia induction therapy. This syndrome is associated with a high mortality rate. Current management strategies include high-dose steroids, platelet transfusions and mechanical ventilatory support to treat acute respiratory failure. Recombinant factor VIIa (rFVIIa) is approved for treatment of bleeding in patients with hemophilia A/B with inhibitors. A greater understanding of the mechanism by which rFVIIa restores hemostasis has become available; with the in-vitro evidence supporting that the thrombin burst achieved by rFVIIa is independent of the presence or binding to tissue factor. This insight has suggested a range of other potential clinical uses for the drug. We report our experience of using rFVIIa for the treatment of DAH in the setting of HSCT and acute leukemia induction therapy. We treated 5 episodes of DAH in 4 adult patients with hematologic malignancies. Two patients had undergone matched unrelated donor HSCT. Clinically, DAH was suspected because of progressive respiratory failure. DAH was confirmed by radiologic and bronchoscopic exams. All patients received high-dose steroids, broad-spectrum antibiotics and respiratory support, as indicated. Packed red cells and platelets were transfused to keep the hemoglobin above 8gm/dL and platelets above 50,000/μL. Patients received boluses of rFVIIa at 90mcg/kg, every 3-12 hrs. The dose of rFVIIa ranged from 7,200–28,000 mcg/day. All five episodes of DAH achieved clinicoradiologic improvement. No toxicity or adverse events were observed with rFVIIa use. Our experience indicates that rFVIIa might be an effective treatment option for patients with DAH, post-HSCT or acute leukemia induction therapy. The optimal dosing schedule in this clinical situation needs to be studied.
Patient demographics and outcomes Patient Diagnosis Pre-intervention rFVIIa used (microgms) PRBC/Platelet transfused (units) Clinical outcome OW ALL-MUD, BMT, day +75 8.8/12,000 24000/d X 4days 3/10 100 Non-rebreather to 6L nasal cannula O2 OW ALL-MUD, BMT, day +80 8/8,000 28000/d X 4 days 6/14 Ventilatory support to successful weaning CS Relapsed AML, S/P induction 8.8/19,000 8400/d X 2 days 2/4 O2 requirement decreased LW CML blast crisis, S/P induction 8.6/101,000 10800/d X 2 days 4/1 Clinically no bleeding, decreased O2 requirement RB Relapsed ALL 8.7/7,000 7200/d X 1 day 2/4 Ventilatory support to successful weaning
c-mpl Mutations in Congenital Amegakaryocytic Thrombocytopenia: Residual TPO Receptor Activity in Patients of Group CAMT II.
Authors: Ballmaier Matthias , Welte Karl , Germeshausen Manuela 
Published: October 2019

Abstract
Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare inherited bone marrow failure syndrome. Mutations in the gene for the thrombopoietin receptor c-mpl were defined as the molecular cause of CAMT. Extending our sequence analyses from 8 to a total of now 23 CAMT patients we could confirm our hypothesis of genotype-phenotype correlation in CAMT. Seven different nonsense, frame-shift or splice-site mutations, all predicted to lead to a complete loss of function of the thrombopoietin receptor were found in 13 patients belonging to group CAMT I with persistently low platelet counts and a fast progression into pancytopenia. Nine different missense mutations were detected in ten patients of group CAMT II, characterized by a transient increase in platelet counts over 50 nl−1 during the first years of life. Mutations spread nearly over the entire gene with a clear accumulation in exons 2 and 3. Except for two missense mutations in exon 8 and exon 12 coding for the second cytokine receptor homology domain and for the distal half of the intracytoplasmic domain, respectively, all mutations were found in exons 2–5 coding for the first cytokine receptor homology domain of the thrombopoietin receptor. The mutations found in group CAMT I patients were located in exons 2, 3 and in the splice sites between exons 1–3. Missense mutations found in patients of group CAMT II spreaded over exons 3, 4, 5, 8 and 12. Using in vitro assays with hematopoietic progenitors from patients of both patient groups we could demonstrate a residual activity of the thrombopoietin receptor in CAMT II patients: In samples from patients of group CAMT II the addition of TPO led to a significant higher number of total colonies. No difference was found in the number of megakaryocytic colonies. In contrast, TPO did show neither an effect on the total colony number nor on megakaryocytic colony number in samples from patients of group CAMT I. This is a first experimental confirmation of our hypothesis that the type of mutation has a strong influence on the course of the disease and that the transient increase of platelet counts and the delayed development of pancytopenia in CAMT II patients is due to a residual activity of the TPO receptor.
Rapid, Exponential Extra-Splenic Platelet Consumption in WASP Deficient Mice.
Authors: Strom Ted S. , Wan Jim Y. , Du Haiming , Jackson Carl W. 
Published: October 2019

Abstract
The thrombocytopenia seen in patients with the Wiskott-Aldrich Syndrome (WAS) is thought to be due primarily to rapid platelet consumption, and is markedly improved by splenectomy. While a murine model of WAS shows only mild thrombocytopenia, we have previously demonstrated rapid platelet turnover in this model; that splenectomy improves the platelet count in WASP-(C57Bl/6J) mice; and that the latter mice do not achieve the same platelet count found in splenectomized WT mice. Here we show that CMFDA-labeled WASP- platelets are consumed rapidly and exponentially in WT recipients, with an estimated lifespan of 18 hours (vs. 87 hours for WT platelets). WASP- platelets are consumed only slightly more slowly in splenectomized WT recipients (lifespan 28 hours, vs. 102 hr for WT platelets). On the C3H background, WASP- mice have normal platelet counts but show a similar rapid, exponential platelet consumption rate (lifespan 25 hours in either C3H or C57Bl/6J recipients, vs 85 hours for WT platelets). In vivo platelet biotinylation studies demonstrate less effective labeling of WASP- (C57Bl/6J) platelets than of WT platelets. After a plateau phase, in vivo labeled WASP- platelets show the same kind of rapid platelet turnover seen with ex vivo labeling (figure 1). Our results imply a three-fold increase in platelet production rate in WASP- mice on either background, consistent with the markedly increased splenic extramedullary hematopoiesis seen in WASP- mice. WASP- mice also show an increased number of bone marrow megkaryocytes, the ploidy distribution of which is normal. We conclude that WASP- mice demonstrate a significantly increased rate of extra-splenic platelet consumption that is largely (on the C57Bl/6 background) or completely (on the C3H background) compensated by increased platelet production. The ability of WASP- mice to compensate for their rapid platelet consumption, and the normal ploidy of their megakaryocytes, suggests that platelet production is not impaired by WASP deficiency in this model.
In vivo biotinylation of WASP-and WT platelets In vivo biotinylation of WASP-and WT platelets
Progressive Emergence of a Dominant Unit during Dual Unit Umbilical Cord Blood (UCB) Culture.
Authors: Liu Min , Reese Jane S. , Jaroscak Jennifer J. , Gerson Stanton L. 
Published: October 2019

Abstract
Umbilical cord blood (UCB) as a stem cell source for allogeneic transplantation is limited by the cell dose a single unit can provide to adult recipients. One approach to overcoming the cell dose limitation is the use of two UCB units. However, murine and human clinical studies have shown that after infusion of two UCB units, one unit dominates as early as 3 weeks post engraftment. To understand the biology that determines the engraftment of each donor, we investigated the effect of dual donor competition on hematopoietic progenitors from 3 unrelated units and 2 units from dizygotic twins during methylcellulose and LTC-IC co-culture of equal numbers of mononuclear cells from each unit pair. Donor chimerism was evaluated in primary CFU (t=0) and secondary CFU from LTC-IC cultures (weeks 4 and 5) by PCR of informative polymorphic short tandem repeats (STR analysis). In 10 units analyzed, the CFU frequency per 100,000 mononuclear cells was 152 ± 18 and the LTC-IC frequency was 1 in 20,000 ± 5.81. The mononuclear cell composition was CD34: 1.07 ±.29%; CD3: 56.3 ± 4.1%; CD33: 17.4 ± 3.4%; and CD19: 8.42 ± 1.1%. In 4 UCB pairs, the unit with the higher primary CFU frequency dominated by an average ratio of 2:1 based on STR analysis of the primary CFU co-culture. In secondary CFU derived from the LTC-IC cultures, the skewing progressively became more pronounced with the same unit identified in primary culture predominating by an average ratio of 7:1. In mixed cultures, the absolute number of primary CFU from the dominant units increased by 15 ± 2% over the number from individual units while CFU from the non-dominant units decreased by 37 ± 4%. Interestingly, there was no dominant unit in primary CFU cultures from one set of dizygotic twins and the CFU number from each unit increased by 30% in the mixed culture. This set of twins emerged with a dominant unit during LTC-IC culture, yielding a donor ratio of 2:1. We found no correlation between the predominating unit and the CD34 percentage or LTC-IC frequency. However, in all 5 dual UCB experiments, the unit with the higher CD3 percentage dominated in primary CFU cultures and was intensified during LTC-IC culture. These results demonstrate an early and progressive dominance of one donor based on CFU analysis during mixed UCB culture. The correlation with CD3 content suggests both an immediate and protracted T-cell effector mechanism even though the LTC-IC culture does not promote T-cell proliferation. Additionally, the increase in the absolute cell number of the dominant unit may suggest a graft vs. graft progenitor cell growth-stimulatory effect which may explain the observed more rapid engraftment of the dominant unit after dual UCB transplantation. Thus, immediate UCB competition predicts the dominant unit during in vitro culture and presumably mimics the process occurring after clinical dual UCB transplantation.
Quantitative Real Time PCR Analysis of MDR1 Transduced Rhesus Macaque Hematopoietic Stem Cells Shows No Indication for Proliferative Advantage after Long-Term Follow-Up.
Authors: Bozorgmehr Farastuk , Laufs Stephanie , Buss Eike C. , Nagy Katalina Z. , Sellers Stephanie E. , Zeller W. Jens , Dunbar Cynthia E. , Fruehauf Stefan 
Published: October 2019

Abstract
Introduction: It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral MDR1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analyses on the clonal level and a long-term follow-up in a model with more direct relevance to human HSC biology were lacking.
Methods: In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were transduced either with a MDR1 gene-retroviral vector (HaMDR1-vector) or a NeoR-retroviral vector (G1Na-vector). After autologous transplantation granulocytes and mononuclear cells of each animal were obtained at different time points and analysed by using a highly sensitive and specific ligation-mediated PCR (LM-PCR) and by quantitative real time PCR.
Results: In monkey M120 73 different cell populations (clones) were detected between 8 weeks and 4 years after transplantation; in monkey M038 49 clones could be identified between 16 weeks and 4 years after transplantation. Remarkably, 99 clones descend from the G1Na-vector, whereas only 23 clones descend from the HaMDR1-vector. Quantitative real time PCR analyses support these data: in both animals, the mean proportion of the G1Na-vector and the HaMDR1-vector, respectively, is 2,46%±0,014% and 0,455%±0,003%. Furthermore, single G1Na clones identified with LM-PCR 4 years after transplantation were quantified, showing a copy number between 1.43±0,52 and 7.1±0.34, according to 0.001%±0.0003% and 0.004%±0.0003% of the granulocytes fraction. In the MNC fraction, the G1Na clones were not detectable. At the same time point, 2 HaMDR1 clones were quantified, one in the granulocyte and one in the MNC fraction, respectively. Here, the copy numbers were 2.87±0.56 and 3.52±1.11, according to 0.002%±0.0003% and 0.002%±0.0008% of the granulocyte and MNC fraction, respectively.
Conclusions: We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.
Impaired Alloantigen Presenting Activity of Cord Blood Nucleated Cells.
Authors: Chunduri Sandeep , Mahmud Dolores , Abbasian Javaneh , Rondelli Damiano 
Published: October 2019

Abstract
Transplantation of HLA-mismatched cord blood (CB) nucleated cells has limited risk of severe acute graft-versus-host disease and graft rejection. This may depend on naïve T cells not yet exposed to many antigens and on immature antigen-presenting cells (APC) not delivering appropriate signals to allogeneic T cells. In order to test the APC activity of human circulating CB cells in-vitro, we initially used irradiated CB mononuclear cells (MNC) or immunomagnetically selected CD34+ cells, CD133+ cells, or CD14+ monocytes to stimulate the proliferative response of incompatible blood T cells in mixed leukocyte culture (MLC). CB MNC failed to induce allogeneic T cell proliferation, while CD34+ and CD133+ progenitors or CD14+ monocytes induced potent T cell alloresponses. Nevertheless, since allogeneic T cell response was not restored after depletion of CD3+ cells in the CB, nor the add-back of irradiated CB MNC to CD34+ or CD14+ stimulators inhibited allo-T cells, a direct suppressive effect of CB MNC was excluded. Allogeneic peripheral blood cytotoxic T-lymphocyte (CTL) responses were not induced after 7 days of stimulation with irradiated CB MNC, although after 4 weekly rechallenges with CB MNC, on average a 23% lysis of antigen-specific CB PHA-blasts was observed at the highest effector:target ratio (50:1). To test the tolerogenic potential of CB MNC, T cells initially exposed to CB MNC were rechallenged in secondary MLC with CB MNC, or CD34+ cells, or monocyte-derived dendritic cells (Mo-DC) generated in liquid culture with GM-CSF and IL-4. Allogeneic T cells were still unresponsive upon rechallenge with CB MNC, but proliferated upon 3 days of restimulation with CD34+ cells or Mo-DC from the same CB. Surprisingly, the supernatant of these latter MLCs did inhibit completely a 3rd party MLC. Instead, the supernatant of blood T cells that had been activated by CB CD34+ cells or Mo-DC both in primary and secondary MLC did not. These results show an impaired allo-APC activity of CB MNC but not CB CD34+ cells, and suggest that T cells releasing immunosuppressive cytokines may be activated by CB MNC and then expanded by a second more potent stimulation with professional APC. This hypothesis could explain the sustained engraftment of HLA-mismatched CB stem cell transplants in humans. Based on these results, the in-vivo or ex-vivo downregulation of T cell alloreactivity induced by CB MNC will be tested in experimental models of stem cell, as well as solid organ transplantation.