bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigation of Underlying Reasons of Factor VIII Deficiency in Haemophilia A Patients with Undetectable Mutations in the Factor VIII Gene.
Authors: El-Maarri Osman , Schröder Jörg , Klein Claudia , Schwaab Rainer , Goodeve Anne , Negrier Claude , Thompson Arthur , Srivastava Alok , Oldenburg Johannes 
Published: October 2019

Abstract
Haemophilia A (HA) is an X-linked bleeding disorder caused by heterogeneous mutations in the coagulation factor VIII (F8) gene. Despite applying sensitive methods for mutation detection, and after excluding the inversions mutations a causative mutation is not identified in F8 gene in about 2,5% of severe HA patients (53 patients out of 2350 German patients). Analysis of mRNA from a small group of such (German) patients has excluded mutations deep in the introns that may affect normal splicing or mechanisms causing some unknown rearrangements of the F8 gene as the cause of HA. Among this group, in one patient no F8 mRNA was detected. Using two common polymorphisms in F8 exon 14, we were able to show that the same allele shared by the patient, his mother and his sister was not detected by reverse transcription PCR (RT-PCR) from total blood mRNA. These findings strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA which points to a novel mechanism leading to HA. To further investigate the cause of Hemophilia A in these patients an international multi-center study was established. The aim of this study to assemble a large collection of such families that could provide clues for novel factors/mechanisims that are important for the F8 molecule biogenesis and protein regulation as well as F8 expression. This multi-center study is therefore suposed to identify novel mechanisms causing HA.
The Bcl-2 Family Inhibitor ABT-263 Shows Significant but Reversible Thrombocytopenia in Mice.
Authors: Shoemaker Alex R. , Mitten Michael J. , Adickes Jessica , Oleksijew Anatol , Zhang Haichao , Bauch Joy , Marsh Kennan , Frost David J. , Madar David , Tse Christin , Fesik Stephen W. , Rosenberg Saul H. , Elmore Steven W. 
Published: October 2019

Abstract
ABT-263 is an orally bioavailable small molecule inhibitor of Bcl-2 family proteins with a Ki of ≤ 1 nM against Bcl-2, Bcl-XL, and Bcl-w. Potent cytotoxic effects have been demonstrated against small cell lung carcinomas and lymphomas (EC50 of less than 500 nM in vitro against numerous cell lines). Tumor growth stasis and regression in xenograft models confirmed this activity. However, a unique thrombocytopenia has been observed in mice, rats, and dogs. Using a mouse model to characterize the extent and duration of decreased circulating platelets, outbred CF-1 or inbred scid-bg mice were treated with increasing doses of ABT-263. Blood samples were collected at various times post dosing and platelet numbers were determined using automated hematology analysis. The results showed a rapid, dose-dependent reduction in circulating platelet counts with a platelet nadir (>80% reduction relative to baseline) occurring approximately 6 hours after a single 100 mg/kg oral dose of ABT-263. Platelet counts returned to normal levels by 72 hrs post-dose. Analysis of bleeding times using a tail-nick method demonstrated a correlation between reduced platelet count and increased bleeding time. At doses of 50 mg/kg and higher, bleed times were increased by more than 5-fold relative to vehicle controls. In multi-dose studies, platelet levels did not continue to decline relative to the first dose of ABT-263. Mice treated with ABT-263 at 100 mg/kg/day for as long as 21 days exhibited no overt signs of toxicity (bleeding, significant weight loss, etc). Because the spleen is a potential site for clearing of damaged or aggregated platelets, a study using splenectomized mice was also conducted. The kinetics of platelet reduction and recovery were similar in splenectomized vs. intact mice with no additional adverse events noted. Evaluations of plasma drug levels associated with preclinical anti-tumor efficacy vs. thrombocytopenic effects indicate that a reasonable therapeutic window exists for ABT-263.
Evidence That the MPL 515L and 515K Mutations Occur in a Lympho-Myeloid Progenitor in Idiopathic Myelofibrosis and Are Associated with TPO Hypersensitivity.
Authors: Chaligne Ronan , Le Couedic Jean-Pierre , Tonetti Carole , Besancenot Rodolphe , Wagner-Ballon Orianne , Debili Najet , Vainchenker William , Giraudier Stephane 
Published: October 2019

Abstract
The MPL 515L mutation has recently been described in 10% of Idiopathic Myelofibrosis (MMM) as an essential oncogenic event. Here we report another MPL mutation in the same codon of MPL (MPL515K). The MPL515L mutation has been detected in granulocytes but has not been yet detected in patient hematopietic stem cells. This raises the question whether this molecular event occurs in a true stem cell (ie: lymphoid/myeloid progenitor cell), as it has already been shown for JAK2 V617F occurrence in MMM and PV.
The aim of this work was to study the presence of the mutation and another MPL mutation (MPL 515K) in both myeloid and lymphoid lineages in JAK2 V617F negative MMM. We therefore looked for the mutation first in mature myeloid and lymphoid cells and second in lymphoid/myeloid progenitors progeny after CD34+ and CD34+CD38- cell isolation from peripheral blood. Three IMF, patients harbouring MPL mutations were enrolled in this study after informed consent. Peripheral blood granulocytes and platelets were purified by standard methods and B, T, NK cells and monocytes were solated by combined immunomagnetic and flow cytometric procedures. The same techniques were used to sort CD34+ and CD34+CD38- subpopulations from peripheral blood. Clonal B/NK/Myeloid differentiation from CD34+CD38- cells and T cell differentiation from CD34+ cells were performed respectively onto a MS5 layer in the presence of SCF, FLT3L, IL2, IL3, IL7, IL15, TPO and in Fetal Thymic Organ Cultures (FTOC). Genotyping of mature cell populations, B/NK/Myeloid clones and CD34+ derived T cells were performed by direct sequencing. Moreover, CD34+ cells were cultured in a one cell per well experiment to determine the sensitivilty of these patient’s cells to low dose TPO.
The MPL mutations were present in granulocytes and platelets from all patients, and in monocytes. We detected the mutation in B and NK cells in all three cases. The MPL 515L and MPL 515K mutations could be subsequently detected in CD34+ cells and in B/NK/Myeloid and/or NK/myeloid CD34+CD38- derived clones from all IMF patients. Interestingly, MPL 515L homozygous clones were detected in B/NK/Myeloid and/or NK/Myeloid clones from 1 MMM patient. Using the FTOC assay, the mutation was also detected in all T cell fractions derived from CD34+ cells from 2 MMM patients. At least we found that MPL mutations induce a spontaneous megakaryocytic growth in cell culture but also a hypersensitivity to low dose TPO.
These results demonstrate that the mutation occur in a lymphoid/myeloid progenitor cell in MPL 515L or K MMM and give rise to a hypersensitivity to TPO. Thus, these MPD take their origin in a true lymphoid/myeloid progenitor cell. In accordance with mouse model, this represent a good argument for a causative role of MPL mutations in MMM.
Effect of Valproate on In Vitro Steroid Responsiveness in 2-Phase Erythroid Culture in Diamond Blackfan Anemia (DBA): Potential Role for Histone Deacetylation in the Mechanism of Action of Steroids in DBA.
Authors: Ohene-Abuakwa Yaw , Marius Carine , White David , Sarah Ball 
Published: October 2019

Abstract
We have previously adapted a 2-phase liquid erythroid culture system to demonstrate a consistent and profound failure in erythroid expansion in DBA cultures, which localizes to early in the second erythropoietin-dependent phase of culture. We have shown a partial corrective effect of steroids, which both enhance the clonogenicity of early erythroid colony forming cells, and increase erythropoietin sensitivity by a direct effect on erythroid progenitors at the later stage of CFU-E expansion. Following a report of the induction of remission by valproic acid in a patient with refractory DBA, we have extended our study to include the effect of added valproate at concentrations equivalent to therapeutic plasma levels. In preliminary studies of cultures from normal controls, sodium valproate at 50ng/ml markedly increased erythroid expansion in cultures without added dexamethasone (p&lt;0.001, n=13). A similar effect was observed in DBA cultures, although this was not statistically significant (p=0.07, n=7). However, the enhancing effect of 10−7M dexamethasone in control cultures was inhibited by valproate 100 ng/ml, (n=13, p&lt;0.05). A similar inhibition of the steroid effect was observed in both of 2 DBA cultures showing in vitro steroid responsiveness. In contrast, at low dose valproate (25ng/ml) and dexamethasone (10−8M), there was evidence of synergistic enhancement of erythroid expansion. Valproate is a known histone deacetylase (HDAC) inhibitor, and its interactions with dexamethasone in this culture system strongly suggest that dexamethasone influences erythroid histone acetylation status. In support of this, a similar dose-dependent interaction with dexamethasone was observed with trichostatin A, another HDAC inhibitor. This represents a potential molecular basis for the mechanism of action of the stimulatory effect of steroids in erythropoiesis, although the complex dose-dependent interaction between valproate and dexamethasone would be consistent with differential effects of steroids on histone deacetylation at different stages in erythropoiesis. Interestingly, the inhibition of the effect of dexamethasone at higher concentrations of valproate resembles our previously reported effect of prolactin in this culture system, suggesting that modification of histone acetylation also underlies the effect of prolactin. We will address this further by analysing the histone acetylation status of primary erythroid cells throughout the 2-phase culture system, especially at the time of divergence of DBA and control cultures, and the effect of different concentrations of steroids. This may elucidate the individual variation in steroid responsiveness in DBA, and possibly suggest steroid sparing agents for clinical use.
pak2a Mutations Cause Cerebral Hemorrhage in Redhead Zebrafish.
Authors: Buchner David A. , Shavit Jordan A. , Su Fengyun , Yamaoka Jennifer S. , Kamei Makoto , Mcgee Beth , Hanosh Andrew W. , Weinstein Brant M. , Ginsburg David , Lyons Susan E. 
Published: October 2019

Abstract
Zebrafish are a powerful vertebrate model system for the study of human disease as they share many molecular pathways with mammals. Of note, nearly all of the mammalian coagulation factors are also highly conserved in fish. As part of a whole genome ENU mutagenesis screen, we identified a mutant zebrafish which displayed intraventricular hemorrhage between 2–3 days post fertilization (dpf), which we named redhead. Using an F2 intercross strategy, we mapped this recessive mutant to a 100 kilobase interval on chromosome 2 and identified a splice site mutation in the gene for an ortholog of the p21-activated kinase Pak2. This IVS9 T+2A mutation renders greater than 90% of transcripts nonfunctional, resulting in a hypomorphic allele. Surveying zebrafish expressed sequence tag databases, we identified two Pak2 orthologs in zebrafish, designated Pak2a and Pak2b, with the redhead mutation in pak2a. Central nervous system (CNS) hemorrhage in redhead embryos was rescued by injection of wild type pak2a mRNA. Morpholino knockdown of pak2a in wild type fish phenocopies the redhead mutant, but with an increase in penetrance and severity, including hydrocephalus and pericardial edema secondary to severe hemorrhage. Injection of either pak2a or pak2b mRNA was able to rescue this phenotype. In addition, pak2b knockdown worsened the bleeding phenotype in redhead embryos, with no effect on wild type fish. These results suggest a partial overlap in function between Pak2a and Pak2b. Confocal microscopy was performed at 2.5 dpf in pak2a knockdown embryos transgenic for Gata1-dsRED and Flk1-eGFP, distinctively labeling erythrocytes and endothelial cells, respectively. The sites and magnitude of hemorrhage were variable between individual embryos, but there were no obvious abnormalities in vessel patterning. In summary, we have identified a critical role for Pak2 in maintaining CNS vessel integrity in zebrafish, without apparent effects on other vascular beds. Further analysis of this signaling pathway in the vessel wall may provide novel insight into the mechanisms underlying vascular heterogeneity in mammals, and the tissue specific function of Pak2 in the CNS vasculature.
Notch1 Provides a Proliferative Signal in Mouse T Cell Leukemia by Directly Targeting c-Myc.
Authors: Draheim Kyle M. , Sharma Vishva M. , Calvo Jennifer A. , Cunningham Leslie A. , Hermance Nicole , Krishnamoorthy Veena , Beverly Levi , Bhasin Manoj , Capobianco Anthony , Kelliher Michelle A. 
Published: October 2019

Abstract
As demonstrated in mouse models and in primary human T cell leukemic samples, gain of function mutation(s) in Notch1 is a common genetic event in T cell acute lymphoblastic leukemia (T-ALL). The Notch1 receptor signals through a γ-secretase-dependent process that releases intracellular Notch1 from the membrane to the nucleus where it forms part of a transcriptional activator complex. We have demonstrated that mouse leukemic growth is Notch1-dependent, since treatment with γ-secretase inhibitors (GSI) results in rapid cell cycle arrest and/or apoptosis. To specifically identify Notch1 target gene(s) in leukemia, we developed mouse T cell leukemic cell lines that express intracellular Notch1 in a doxycycline-dependent manner. Using gene expression profiling and chromatin immunoprecipitation, we identified c-myc as a novel and direct Notch1 target gene. Consistent with these findings, retroviral insertional mutagenesis screening of our tal1 leukemic mouse model reveal common insertions in either notch1 or c-myc. Retroviral expression of c-myc, like intracellular Notch1, rescues the growth arrest and apoptosis associated with GSI treatment or Notch1 inhibition in 83% mouse tal1 leukemic cell lines tested. Yet in a subset of leukemic cell lines, retroviral expression of c-myc fails to rescue leukemic growth, whereas expression of intracellular Notch1 in these lines remains capable of restoring growth. These data suggest that additional Notch1 target genes other than c-myc contribute to leukemogenesis. Other Notch1 target genes in thymocyte developement and their potential role in leukemogenesis will be discussed.
Platelet Microparticles, Platelet Activation and Platelet-Leukocyte Conjugates in HIV Infected Patients with Optimal Response to Highly Active Antiretroviral Therapy (HAART).
Authors: Corrales-Medina Vicente F. , Simkins Jacques , Chirinos Julio A. , Lopez-Sanabria Bernardo , Symes Stephen , Jayaweera Dushyantha T. , Jy Wenche , Jimenez Joaquin J. , Horstman Lawrence L. , Ahn Yeon-Soong 
Published: October 2019

Abstract
Background: HIV infection and its treatment have been associated with an increased risk of atherothrombotic cardiovascular disease. Little is known about the status of platelets in optimally controlled HIV infection. We aimed to assess the status of platelets using flow cytometry markers of platelet activation/apoptosis in HIV-infected patients who demonstrated an optimal response to highly active antiretroviral therapy (HAART).
Methods: In the current study we measured P-selectin expression in platelets, platelet microparticles (PMP) and platelet-leukocyte conjugates (PLC) by flow cytometry in 29 patients (mean age=42.1±7 years) with HIV infection on HAART who demonstrated an optimal viral and immunological response (CD4+ cell count > 200 /mm3 and undetectable viral load by ultrasensitive PCR analysis). Patients with a history of diabetes, smoking, illicit drug use, thrombotic, cardiovascular or malignant disease were excluded. We used age- and gender-matched healthy individuals as controls. PMP were measured by flow cytometry based in the co-expression of CD31 and CD42b. Platelet activation was also assessed by expression of activation marker P-selectin (expressed in fluorescence intensity units, FIU). PLC were measured based on the co-expression of CD45 and CD41 (expressed as percentage of leukocytes positive for CD41).
Results: Platelet expression of P-selectin was not significantly different between HIV infected patients (7.35 FIU; IQR=4.00–15.43) and controls (13.32 FIU; IQR=4.91–23.02; p=0.11). However, the levels of PMP were markedly elevated in HIV-infected patients (24,951 (counts/μl; IQR=17,887–36,175) compared to the control group (13,862 counts/μl; IQR=9,984–20,736; p=0.001). The levels of PLC were lower in HIV-infected patients (52.2%; IQR=42.65–65.75) compared to controls (63.8%; IQR=56.9–78.7; p=0.0007). There was a trend for a negative correlation between PMP levels and the CD4 count (Pearson R=−0.34; p=0.07).
Conclusions: HIV infected patients with optimal response to HAART demonstrate an increased number of circulating free PMP, normal levels of platelet expression of activation marker P-selectin and decreased levels of PLC. A decreased number of PLC indicates decreased binding of platelets and PMP to leukocytes in HIV-infected patients, which could be partially responsible for increased levels of free PMP. In addition, PMP (unlike P-selectin expression) may be a sensitive marker indicating low-grade platelet activation in HIV infection. Increased PMP levels are likely to contribute to the atherothrombotic risk HIV+ infected patients on HAART.
Utility of Optical Density Values from Heparin-Platelet Factor 4 Antibody Testing and Probability Scoring Models To Diagnose Patients with Heparin Induced Thrombocytopenia.
Authors: Janatpour Kim A. , Gosselin Robert C. , Dager William E. , Lee Andrew , Owings John T. , Wun Ted 
Published: October 2019

Abstract
Background: Heparin induced thrombocytopenia (HIT) is a potentially life threatening complication of heparin administration caused by antibodies directed to the heparin-platelet factor 4 (PF4) complex and characterized by thrombocytopenia with a seemingly paradoxical high risk of thrombosis. Diagnosis is challenging, and is based on both clinical suspicion and laboratory detection of heparin-PF4 antibodies. The Warkentin 4 T’s “pre-test” probability and Chong’s “post-test” probability models have been developed to aid the diagnosis of HIT. Enzyme-linked immunoabosorbant assay (ELISA) laboratory measurement of heparin-PF4 antibodies is commonly used but has a low positive predictive value for thrombosis. Recent reports suggest that using an ELISA optical density (OD) value of ≥ 1 may improve the predictive value for thrombosis.
Methods: We performed a retrospective analysis of 105 patients with suspected HIT who were treated with a direct thrombin inhibitor and evaluated the anti-heparin-PF4 ELISA OD values and Warkentin 4 T’s scores for sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) using the Chong’s score to define HITBoth the manufacturer’s OD threshold of 0.4 and ≥ 1 were evaluated.
Table 1. Comparison of sensitivity, specificity, and predictive values for 4 T’s and OD levels, alone and in combination Sensitivity (%) Specificity (%) NPV (%) PPV (%) For calculations, Chong’s categories of definite/probable and unlikely were used to define presence or absence of HIT. 4T’s score (high and low) 81 100 80 100 OD level 0.4 69 75 65 78 OD level ≥1 38 85 52 77 4T’s + OD 0.4 94 100 94 100 4T’s +OD ≥1 69 85 81 75
Results:
The sensitivity, specificity and predictive value of ELISA alone were inferior to the 4 T’s clinical scorings system. The sensitivity and negative predictive values of the 4T’s score were improved by considering positive or negative ELISA test results.
Conclusions: Consistent with previous reports, the PPV of a high probability 4T score alone was 100%. Thus, ELISA results might not change clinical decisions in this situation. Alternative anticoagulation might be inappropriately withheld in 20% of patients if a low probability 4T score alone were to be used for decision-making. The addition of ELISA data using an OD level of 0.4 increased the NPV to a threshold that might be considered clinically acceptable (94%) to withhold therapy. Addition of an OD level of ≥ 1 did not improve the PV of the 4 T’s. However, in contrast to other studies that found better predictive value to using this higher OD threshold, our study group was limited to people considered high risk for HIT. Validation of this strategy in a prospective trial with clinical endpoints should be considered.
Cause of Death among Patients with Local or Regional Stage Cancer of the Breast, Colon and Lung Who Develop Venous Thromboembolism (VTE).
Authors: Wun Ted , Chew Helen K. , Zhou Hong , Harvey Danielle , White Richard H. 
Published: October 2019

Abstract
Background: Previous data has shown that cancer-related VTE is associated with reduced two-year survival for patients with several tumor types. The reason for the survival difference is not clear.
Methods: Using the California cancer registry and hospital discharge data, we identified all cases diagnosed with colon, breast and lung cancer over a 6-year period. Patients with local or regional stage cancer who developed incident VTE within 2 years after the diagnosis of cancer were identified. The cause of death was determined using a master death registry. Each VTE case was compared to 4 case-controls without VTE after matching for age, race, sex, stage and number of medical co-morbidities. The odds ratio (OR) for dying from any cause and dying due to the underlying cancer in the subsequent 2 years was calculated.
Results (Table): Approximately 40% of all deaths occurred < 30 days of the VTE diagnosis for all cancers. The longer the time interval between cancer diagnosis and VTE, the greater the relative risk of dying of cancer.
Conclusions: Patients with lung, breast and colon cancer who developed VTE < 2 years after cancer diagnosis were significantly more likely to die, and death was attributed to the underlying cancer in a sizable percentage of these cases. This indicates that among patients initially diagnosed with local or regional stage cancer of the breast, colon or lung, VTE is frequently a harbinger or consequence of metastatic stage.
Odds Ratio (OR) for Death and Death Due to Cancer VTE Cases < 2 Years From Cancer Dx N Died < 2 Years From Date of VTE (%) OR (CI) For Death in VTE Cases < 2 Years Cancer As the Cause of Death N (%) OR (CI) for VTE Cases to Die of Cancer % Deaths Due to Cancer CI = Confidence Interval Local Stage     Breast 545 85 (16) 4.1 (3.0 – 5.6) 30 (6) 4.5 (2.7 – 7.7) 53     Colon 377 130 (35) 3.9 (3.0 – 5.1) 58 (15) 5.0 (3.3 – 7.5) 45     Lung 234 150 (64) 3.7 (2.6 – 5.5) 110 (45) 3.0 (2.0 – 4.4) 73 Regional Disease     Breast 493 116 (24) 3.4 (2.6 – 4.4) 73 (15) 2.8 (2.1 – 3.9) 63     Colon 887 375 (42) 2.1 (1.9 – 2.6) 273 (31) 2.1 (1.8 – 2.5) 73     Lung 485 386 (80) 3.7 (2.8 – 5.0) 320 (66) 2.5 (2.0 – 3.3) 66