bluuebunny/crossref_metadata_2025_split huggingface dataset:
Factor VII Deficiency Remains Post-Puberty Due to Point Mutation in the HNF4 Binding Site in the Coagulant Factor VII Gene.
Authors: Zheng XingWu , Tidd Theresa M. , DiMichele Donna , Pollak Eleanor S. 
Published: October 2019

Abstract
A novel T to C point mutation at −60 in the gene for coagulant Factor VII results in life-long severe coagulant Factor VII deficiency in post-pubertal twin males. The clinical course of these patients provides an informative in vivo example of the regulation of expression of vitamin K-dependent clotting protein Factor VII. An analogous point mutation in the HNF4 binding site in the Factor IX gene results in the clinical phenotype Hemophilia B Leyden, a sex-linked antigen-negative Factor IX deficiency that resolves post-puberty. The affected Factor VII deficient patients have prolonged prothrombin times (46 and 52 secs), normal aPTTs and decreased FVII levels of FVII:Coagulant activity: < 1% and FVII:Antigen: < 3%. The −60 mutation, ACTTTG → ACTCTG occurs 9 base pairs before the start site of transcription and 59 bps before the before the start site of translation. The twins are compound heterozygotes and also possess a mutation in exon 8 at amino acid 348, a mutation that has previously been reported to cause FVII deficiency. Both affected individuals have recurrent target joint hemorrhage (shoulder, elbow, ankle) requiring replacement therapy 6–12 times/year.
Results: Gel mobility shift assays using a radio-labeled probe spanning from −76 to −46 in the FVII promoter region demonstrate the loss of binding of transcription factor HNF-4. Transient transfection assays in HepG2 cells using 186 bps of the mutant and the wildtype promoters (−185 to +1) revealed a loss of expression with the mutant allele. Co-transfection with an HNF4 expression plasmid resulted in an increase in expression of the wildetype construct in HeLa cells, a non-hepatic cell line. However, co-transfection of the HNF4 expression plasmid failed to increase expression with the construct containing the mutant allele sequence.
Conclusion: The lack of phenotypic change of the FVII:C in 19 yo twin boys provides dynamic support of the necessity of an overlapping androgen binding site in the homologous Factor IX gene as responsible for the phenotypic resolution of Factor IX deficiency (Hemophilia B Leyden) post-puberty. It is of interest that an increase in FVII:C did not occur with advancing age in FVII deficiency due to this HNF4 binding site mutation.
Human Factor VIIai(K62E) Is a More Effective Inhibitor of Coagulation Than Wild-Type FVIIai.
Authors: Clarke Bryan J. , Sridhara Sampath , Blajchman Morris A. 
Published: October 2019

Abstract
Background: In previous work we provided evidence for a linked conformational change of the recombinant factor VII(rFVII) first epidermal growth factor-like(EGF1) domain associated with chloromethylketone(ck) covalent modification of the wild-type(WT) rFVIIa active site (J Biol Chem275:34894,2000). All naturally-occurring mutations of the human FVII gene hitherto described encode plasma FVII with both reduced activity and affinity for their cell-surface receptor tissue factor (TF). We have recently characterized a mutant rFVII protein, rFVIIa(K62E), which has both increased enzymatic activity and a 5-fold greater affinity for TF than wild-type rFVIIa (J Thromb Haemost3:1250, 2005). In the present report we provide data about the TF-binding characteristics and conformational state of rFVIIa(K62E) before and after inactivation with a variety of chloromethylketones.
Methods: Purification of both the rFVIIa(K62E) mutant protein and rFVIIa(WT) and their ck-inactivated derivatives was achieved by anion-exchange chromatography. FVII antigen concentration was determined by ELISA and biological activity by modified prothrombin time (PT) assay. Affinity of inactivated rFVIIa(rFVIIai) for both TF and a FVII EGF1 domain conformation-specific monoclonal antibody(Moab 231–7) was determined by competitive ELISA (IC50).
Results: The affinity of rFVIIai(K62E)DEGR and rFVIIai(K62E)FFR for TF were increased 24.4-fold and 7.7-fold respectively versus rFVIIa(WT)[shown below]. Notably, the enhanced affinity of both rFVIIai(K62E) and unmodified rFVIIa(K62E) for TF was associated with substantially increased affinity for Moab 231–7 as compared to rFVIIa(WT). The clotting time of diluted human plasma was significantly more prolonged by rFVIIai(K62E)DEGR and rFVIIai(K62E)FFR than rFVIIai(WT).
Comparison of rFVIIai(K62E) and rFVIIai(WT) rFVIIa Preparation Affinity for TF(IC50) Relative Increase in TF Affinity Relative Increase in Affinity for Moab 231–7 Prolongation of Plasma Clotting time(sec) Reduction of Coagulant Activity(%) Affinity of rFVII for TF (IC50 expressed as molar excess of rFVII) was determined by competitive ELISA. Plasma clotting times were determined at 2.5-fold molar excess of each rFVIIai inhibitor. Control plasma clotting time was 81 sec. rFVIIai(K62E)DEGR 0.13 24.4 3.1 68 90 rFVIIai(K62E)FFR 0.41 7.7 4.9 57 86 rFVIIa(K62E) 0.46 6.9 4.1 ND ND rFVIIai(WT)DEGR 0.98 3.2 3.5 1 4 rFVIIai(WT)FFR 0.62 5.1 1.2 27 65 rFVIIa(WT) 3.17 1 1 ND ND
Conclusions: We provide evidence that both active-site modification and the substitution K→E in human rFVIIa(K62E) causes conformational change(s) within the FVII EGF1 domain associated with a substantially increased affinity of rFVIIa(K62E) and rFVIIai(K62E) for TF. Both rFVIIai(K62E)DEGR and rFVIIai(K62E)FFR show considerable promise as more effective inhibitors of coagulation than rFVIIai(WT).
Factor IX Substrate Binding Exosite on the Light Chain of Factor Xia.
Authors: Sinha Dipali , Marcinkiewicz Mariola , Navaneetham Duraiswamy , Walsh Peter N. 
Published: October 2019

Abstract
Binding of factor IX (FIX) to an exosite on the heavy chain of factor XIa (FXIa) is essential for the optimal activation of FIX (Biochemistry26:3768–3775, 1987). We now provide evidence that a second substrate binding exosite resides on the light chain (LC) of FXIa. To understand the mechanism of activation of FIX by LC we have investigated 1) the kinetics of S-2366 hydrolysis by FXIa and its isolated LC in the presence and absence of a reversible inhibitor of serine proteases (p-aminobenzamidine, PAB); 2) the kinetics of activation of the macromolecular substrate FIX by FXIa and its isolated light chain in the presence and absence of either PAB or the alternative substrate S-2366; and 3) the effect of active site inhibited FXIa LC (LCi, i.e., FXIa LC treated with phenylmethylsulfonyl fluoride) on the activation of FIX by FXIa. Kinetic parameters for the hydrolysis of S-2366 by FXIa and its isolated LC are comparable. However kcat for the activation of the macromolecular substrate FIX by LC compared to full-length FXIa is reduced by ~300-fold whereas the Km was only slightly higher (2- to 3-fold). PAB inhibited the hydrolysis of S-2366 both by FXIa and LC in a classical competitive fashion. If an exosite on the heavy chain of FXIa is solely responsible for formation of the Michaelis complex then PAB should have inhibited the activation of FIX by LC in a competitive fashion. However, PAB and S-2366 were found to be noncompetitive inhibitors of FIX activation by FXIa or LC demonstrating the presence of an exosite for FIX binding on the LC remote from its active site. The presence of the second exosite on the light chain of FXIa was further confirmed by the fact that the V vs. S as well as the V vs. I plots of the activation of FIX by FXIa in the presence of LCi were sigmoidal suggesting formation of a nonproductive SI complex. We conclude that for FIX-activation, two macromolecular substrate-binding exosites, one on the heavy chain and one on the light chain of FXIa are required to mediate the formation of the Michaelis complex.
Myeloid Expansion after Insertional Activation of MDS1/EVI1, PRDM16 and SETBP1 in a Successful Chronic Granulomatous Gene Therapy Trial.
Authors: Schwarzwaelder Kerstin , Schmidt Manfred , Ott Marion G. , Stein Stefan , Glimm Hanno , Deichmann Annette , Fischer Marlene , Ball Claudia , Pilz Ingo , Schmidt Sonja , Prinz Claudia , Fessler Sylvia , Wissler Manuela , Klingenberg Silke , Hoelzer Dieter , Seger Reinhard , Grez Manuel , von Kalle Christof 
Published: October 2019

Abstract
Successful gene therapy trials of ADA-SCID and SCID-X1 demonstrated the curative potential of oncoretroviral gene transfer. Integration of the retroviral vectors used in these studies has been thought to be a random process but severe side effects in gene therapy and in vitro studies revealed preferred insertion of these vectors mainly around transcription start sites. In SCID patients proliferation advantage of gene corrected cells was one reason for the success of the trials, whereas in the most recent chronic granulomatous disease (CGD) gene therapy trial corrected cells do not have any selective advantage therefore the two patients received mild busulfan treatment before transplantation. High efficiency transduction and conditioning have helped in the successful correction of the patients. Peripheral blood granulocytes show a stable expression (>10%) of the transgene (gp91phox) in patient 1 (15 months post treatment) as well as in patient 2 (11 months post treatment). We reasoned that, unlike T cells, which have the capability to proliferate independent of their bone marrow progenitors, granulocytes more directly reflect the influence of retrovirus insertion, and should therefore allow to closely monitor clonal fate in vivo and its potential relation to vector insertion. To study the clonality of the corrected myelopoiesis, the long term activity of individual cell clones, and the distribution of integration sites in active cells we carried out high sensitive LAM-PCR. The highly polyclonal composition of transduced cells forming myelopoiesis caused the sustained expression of gp91phox. Individual clones carrying the transgene could be detected at multiple time points. To define whether corrected cells have a proliferation advantage due to their vector integration we started large-scale sequencing and mapping of involved insertion sites. We here present >700 unique mappable integration sites of the two treated patients. The distribution of the SFFV based retroviral vector integration sites in this trial turned non random 5 months after transplantation. Corrected long-term myelopoiesis expanded 3- to 5- fold in the two patients due to activating common integration sites (CIS) in the zinc finger transcription factor homologs MDS1/EVI1, PRDM16, or in SETBP1, suggesting that these genes influence regulation of normal long-term hematopoiesis in humans. Our data indicate that the therapeutic benefit in this trial was activated through insertional side effects, therefore our findings have important implications in novel gene therapy approaches.
Variables Predicting for Poor Mobilization of Peripheral Blood Stem Cells (PBSC) in Newly Diagnosed Myeloma Patients, Treated on TT-2 Protocol.
Authors: Cavallo Frederica , Bolejak Vanessa , Cottler-Fox Michele , Hoering Antje , van Rhee Frits , Zangari Maurizio , Tricot Guido 
Published: October 2019

Abstract
Autotransplantation is now considered the standard of care for myeloma patients ≤ 65 years. Tandem autotransplants result in doubling of the percentage of patients alive and event-free at 7 years when compared to a single transplant (IFM-94). A minimum number of 2 x 106/kg CD34 cells is required per transplant for reliable engraftment. The aim of this study was to identify variables associated with the inability to collect sufficient numbers of PBSC for tandem transplants. 668 recently diagnosed patients were enrolled in our TT-2 protocol consisting of intensive induction with VAD, DCEP, CAD and DCEP followed by tandem transplants. PBSC were collected after CAD plus hematopoietic growth factors (either G-CSF or GM-CSF). 49 patients (7.5%) failed to collect ≥ 4 x 106/kg CD34 cells (poor mobilizers) and the characteristics of these patients were compared to those of 77 patients who collected > 30 x 106/kg CD34 cells (excellent mobilizers) using chi-square tests. A multivariate analysis was performed using a stepwise logistic regression. The median number of CD34 cells collected in the group of poor mobilizers was 2.1 x 106/kg (range 0–3.9) in a median of 3 days (range 1–9). In the group of excellent mobilizers the median number of CD34 cells was 38.7 x 106/kg (range 30.1–86) in a median of 2 days (range 1–4). Baseline and mobilization variables significantly different between these 2 groups are listed in Table 1. On multivariate analysis duration of cytopenia (platelets < 50,000/μl and/or neutrophils < 1,000/μl) > 7 days (OR: 27; p<0.0001), randomization to thalidomide (OR 4.5; p=0.007), age ≥ 65 years (OR: 4.96; p=0.01), female gender (OR: 3.62; p=0.02), and neutropenic infection requiring intraveneous antibiotics (OR 3.8; p=0.03) were independently associated with poor collection. No differences in event-free (p= .68) or overall survival (p=.73) were observed between the excellent and poor mobilizers. These data confirm our previous observations of inferior collection in older patients and in those receiving thalidomide as part of upfront therapy. The duration of cytopenia after chemotherapy is a good reflection of patient’s stem cell reserve, while administration of broad spectrum antibiotics prior to collection has been shown to be myelosuppressive. The negative impact of female gender on the ability to collect well is new.
Baseline variables Poor mobilizers (%) Excellent mobilizers (%) P-value Female gender 59 26 <0.001 Age ≥ 65 35 13 0.004 Hb<10g/dl 45 21 0.005 B2M ≥4mg/L 39 17 0.006 P1<150,000/ μl 20 8 0.04 Mobilization variables Cytopenia > 7 days 59 5 <0.001
Low-Level Expression of the Tumor Suppressor Bim in CML Cells: Role of BCR/ABL, Delineation of Underlying Signaling Pathways, and Re-Expression by Imatinib, AMN107, and Proteasome Inhibitors.
Authors: Aichberger Karl J. , Mayerhofer Matthias , Krauth Maria-Theresa , Vales Anna , Derdak Sophia , Pickl Winfried F. , Selzer Edgar , Deininger Michael , Druker Brian J. , Sillaber Christian , Valent Peter 
Published: October 2019

Abstract
Chronic myeloid leukemia (CML) is a myeloproliferative disease in which BCR/ABL enhances survival of leukemic cells through modulation of pro- and anti-apoptotic molecules. Recent data suggest that pro-apoptotic Bim plays a role as a tumor-suppressor in myeloid cells, and that leukemic cells express only low amounts of this death activator. In the current study, we have investigated expression of Bim in primary CML cells and in the CML cell lines K562 and KU812, and in Ba/F3 cells inducibly expressing BCR/ABL on exposure to doxycycline (TonB.210-X). As assessed by Northern- and Western blotting, primary CML cells were found to express significantly lower amounts of Bim mRNA and Bim protein compared to normal bone marrow cells. The BCR/ABL-inhibitors imatinib (Novartis Pharma AG) and AMN107 (Novartis Pharma AG) were found to promote Bim expression in CML cells at pharmacologic concentrations. Correspondingly, BCR/ABL was found to down-regulate expression of Bim in TonB.210-X cells. The BCR/ABL-induced decrease in expression of Bim in leukemic cells was found to be a post-transcriptional event that depended on signaling through MEK, and was abrogated by the proteasome-inhibitor MG132. Interestingly, MG132 was found to up-regulate Bim-expression and to suppress the growth of Ba/F3 cells containing either wild-type BCR/ABL or various imatinib-resistant mutants of BCR/ABL including the T315I mutant that is resistant to all currently available ATP-competitive tyrosine kinase inhibitors (IC50: 30–100 nM). To confirm the role of Bim as a tumor suppressor in CML, a Bim specific siRNA was transfected into K562 cells. This siRNA was found to counteract imatinib- and MG132-induced cell death. In conclusion, our data identify BCR/ABL as a Bim-suppressor in CML cells and suggest, that re-expression of Bim by proteasome inhibition or by targeting of signaling pathways downstream of BCR/ABL may be an attractive therapeutic approach in imatinib-resistant CML.
Evaluation of Ph+/CD34+ Residual Cells in Chronic Myeloid Leukemia Patients after Long Lasting Treatment with Imatinib Mesylate.
Authors: Bocchia Monica , Abruzzese Elisabetta , Ippoliti Micaela , Calabrese Simona , Gozzetti Alessandro , Pirrotta Maria Teresa , Crupi Rosaria , Tozzuoli Daniela , Lauria Francesco 
Published: October 2019

Abstract
Although the success of imatinib mesylate therapy represents an exciting advance in targeted cancer therapy, it has still to be determined whether responses to this p210 inhibitor in chronic myeloid leukemia (CML) patients will be durable. In fact most of clinical studies agree on the evidence of a persistent molecular disease in the majority of treated patients and altough the absolute level of bcr-abl transcript may vary over the treatment, yet a molecular complete response is of rare observation. In addition, discontinuation of imatinib exerts always in rapid loss of response. In accordance to this the persistence of malignant progenitors in patients in complete cytogenetic response (CCR) after short term imatinib treatment, has been recently demonstrated. In particular, Bathia et al. showed in 12/15 patients studied after a median time of 10 months of imatinib treatment a median of 11% of residual CML CD34+ progenitors in the bone marrow (by FISH Dual Fusion bcr/abl analysis)while only 3/15 patients had no detectable residual CD34+ cells. Less is known about residual Ph+/CD34+ cells surviving after a prolonged therapy with this targeting drug. Thus, we evaluated the amount of bone marrow residual CD34+ cells in 17 CML patients in stable CCR after a long lasting treatment with imatinib. At the time of evaluation, the patients were on conventional dose (400mg) Imatinib for a median time of 48 months (range 36–58 months) having achieved a CCR status (conventionally defined as the complete absence of t(9;22) on caryotypic analysis) within 3 to 6 months of treatment. However all of them still showed molecular disease as detected by nested RT-PCR. Bone marrow CD34+ cell-enriched populations were selected from mononuclear cells using immunomagnetic column separation and were evaluated after cytospin by FISH using a bcr-abl Dual Color Extra Signal Probe(LSI bcr-abl ES, Vysis), that is able to detect bcr-abl fusion in interphase nuclei with a false positive signal rate close to 0. A minimum of 100 CD34+ nuclei per each sample were evaluated. Interestingly, in 8/17 patients no Ph+/CD34+ cells were detected, while in the remaining 9/17 patients a median of 2% (range 0.5–11%) of bcr-abl positive progenitors were still observed. In this small selected serie of patients prolonged treatment with imatinib appears to be correlated with a lower, yet detectable, amount of residual bone marrow Ph+/CD34+ cells when compared to previously published data. This result could be partly explained with the different specificity and sensitivity of the probe used (bcr/abl ES<1% false positive; bcr-abl Dual Fusion 8–10% false positive) The clinical significance of these data as well as the role of this cell target to monitor minimal residual disease in CML needs to be evaluated on a larger serie of patients.
Pathologic Co-Expression and Physical Interaction of c-MYC and BCL6 in B-Cell Lymphomas.
Authors: Saito Masumichi , Phan Ryan T. , Morse Herbert C. , Pasqualucci Laura , Dalla-Favera Riccardo 
Published: October 2019

Abstract
Deregulated expression of the proto-oncogenes BCL6 and c-MYC caused by chromosomal translocation or somatic hypermutation is common in non-Hodgkin B cell lymphoma derived from germinal center (GC) B cells, including diffuse large cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Normal GC B cells express BCL6, whereas, surprisingly, they do not express c-MYC, suggesting that the expression of this oncogene in BL and DLBCL (20% of cases) is ectopic (Klein, U. et al. Proc Natl Acad Sci U S A100, 2639–2644, 2003). Here we report that c-MYC is absent in proliferating GC B cells because it is transcriptionally suppressed by BCL6, as demonstrated by the presence of specific BCL6 binding sites in the c-MYC promoter region and by chromatin immunoprecipitation experiments showing that BCL6 is bound to these sites in vivo. Thus, c-MYC escapes BCL6-mediated suppression in lymphoma leading to the co-expression of the two transcription factors, an event never observed in immunohistochemical and gene expression profile analysis of normal GC B cells. Surprisingly, co-immunoprecipitation experiments and in vitro binding experiments indicate that, when co-expressed, BCL6 and c-MYC are physically bound in a novel complex detectable in DLBCL and BL cell lines as well as in primary lymphoma cases. The formation of the BCL6/c-MYC complex has several significant functional consequences on the function of both c-MYC and BCL6: 1) a two fold, BCL6-binding dependent increase in c-MYC half-life, an event that has been shown to contribute to its oncogenic activation; 2) a synergistic increase in the ability of both BCL6 and c-MYC to suppress MIZ1-activated transcription of the p21CIP cell cycle arrest gene; 3) MYC-dependent inhibition of BCL6 acetylation by p300, an event that physiologically inactivates BCL6 via c-MYC-mediated recruitment of HDAC. Notably, the pathologic co-expression of c-MYC and BCL6 was shown to have pathologic consequences in vivo, since double transgenic BCL6/c-MYC mice display accelerated lymphoma development and the appearance of a novel GC-derived tumor phenotype not recognizable in single transgenic animals and containing the pathologic c-MYC/BCL6 complex. Thus, the pathologic co-expression and illegitimate physical interaction of BCL6 and c-MYC leads to an increase in the constitutive activity of both oncogenes. These results identify a novel mechanism of oncogenic function for BCL6 and c-MYC and a novel tumor-specific protein complex of potential therapeutic interest.