bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of Factors Xa and Iia Results in Synergistic Inhibition of Thrombin Generation with Minimal Clot Time Elevation.
Authors: Welch Kathleen M. , Saiya-Cork Kamlai , Gould Weston R. , Leadley Robert J. 
Published: October 2019

Abstract
Inhibition of either coagulation factor Xa (FXa) or thrombin (FIIa) alters ex vivo biomarkers of coagulation and decreases thrombus size in animal models of experimental thrombosis. The objective of this study was to determine if combining FXa and FIIa inhibitors would synergistically reduce the magnitude of IIa generated without elevating markers of bleeding. A synergistic combination may translate into lower doses, more effective anticoagulation and better safety. To predict the FXa and FIIa inhibitor combinations with the maximum potential for synergy, PD 0313052, a potent, selective FXa inhibitor, and argatroban, a potent FIIa inhibitor, were each tested independently and in combination using an in vitro thrombin generation assay. Individually, PD 0313052 and argatroban reduced total thrombin generation (TG) in a concentration dependent manner with IC50’s of 497±148 and 882±193 nM, respectively. Subsequently, PD 0313052 and argatroban were combined in 96 well plates at concentrations ranging from 0.125x to 8x their respective IC50 concentrations. Analysis using the Bliss Independence Model identified statistically significant synergistic activity, with the greatest increase (33%) over simple additivity at 249 and 441 nM FXa to FIIa, respectively, both below their respective IC50 concentrations. Furthermore, combinations of PD 0313052 and argatroban were evaluated in an assay measuring activated clotting time (ACT). Although both PD 0313052 and argatroban dose-dependently elongated the ACT, the combination of 0.5x the TG IC50 concentrations, which demonstrated the greatest synergy in the TG assay, showed the smallest increase in the ACT, prolonging clotting time by only 15%. These data demonstrate that the combination of a specific factor Xa inhibitor and a specific IIa inhibitor can synergistically reduce thrombin generation without appreciable elevation of ACT, suggesting that dual inhibition of FXa and FIIa, using relatively low doses of each, may provide efficacious and safe treatment for thromboembolic diseases.
Mutations of the Tumor Suppressor Gene SOCS-1 in Classical Hodgkin Lymphoma Are Frequent and Associated with Nuclear Phospho-STAT5 Accumulation.
Authors: Weniger Marc A. , Melzner Ingo , Menz Christiane K. , Wegener Silke , Bucur Alexandra J. , Dorsch Karola , Mattfeldt Torsten , Barth Thomas F.E. , Möller Peter 
Published: October 2019

Abstract
The suppressors of cytokine signaling (SOCS) are critically involved in the regulation of cellular proliferation, survival, and apoptosis via cytokine-induced JAK/STAT signaling. SOCS-1 silencing by aberrant DNA methylation contributes to oncogenesis in various B-cell neoplasias and carcinomas. Recently, we showed an alternative loss of SOCS-1 function due to deleterious SOCS-1 mutations in a major subset of primary mediastinal B-cell lymphoma (PMBL) and in the PMBL line MedB-1, and a biallelic SOCS-1 deletion in PMBL line Karpas1106P (BLOOD, 105, 2535–42, 2005). For both cell lines our previous data demonstrated retarded JAK2 degradation and sustained phospho-JAK2 action leading to enhanced DNA binding of phospho-STAT5. Here we analysed SOCS-1 in laser-microdissected Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). We detected SOCS-1 mutations in HRS cells of eight of 19 cHL samples and in three of five Hodgkin lymphoma (HL)-derived cell lines by sequencing analysis. Moreover, we found a significant association between mutated SOCS-1 of isolated HRS cells and nuclear phospho-STAT5 accumulation in HRS cells of cHL tumor tissue (p<0.01). Collectively, these findings support the concept that PMBL and cHL share many overlapping features, and that defective tumor suppressor gene SOCS-1 triggers an oncogenic pathway operative in both lymphomas.
Optimum Storage Condition of Cord Blood before Cryopreservation.
Authors: Moldenhauer Anja , Wolf Juliane , Habermann Gudrun , Genter Gesche , Selders Lisa , Kiesewetter Holger , Salama Abdulgabar 
Published: October 2019

Abstract
Purpose. Different opinions regarding the storage conditions of cord blood before cryopreservation exist. Most experts recommend the storage at room temperature and the cyropreservation within the first 24 hours after delivery of the baby. However, the optimum storage temperature is unknown.
Methods. Cord blood units (n=46) were stored for 24, 48 and 72 hours at room temperature (RT) and 4°C. After storage, CD34(+) cells were isolated and analyzed for their cell count, flow cytometry profile, apoptosis rate, colony-forming capacity by methylcellulose assays, and transmigration capacity in response to stroma-derived factor 1. If possible, a direct-paired comparison was performed (n=15).
Results. The cell count of peripheral blood mononuclear cells was generally 10fold higher in cord blood units stored at RT than in cord blood units stored at 4°C, leading to a 10fold higher number of isolated CD34(+) cells. There was no difference in the frequency of CD34(+) cells after immunomagnetic isolation (>95%) or in the frequency of CD133(+) and CD45(+) cells. At 48 hours, the apoptosis rate was lower at RT than at 4°C (37.7±7.4 vs 49.9±4.2%). The plating efficiencies (PE) were highest after the units had been stored for 48 hours (RT 14.7±3.6%, 4°C 16.3±4%) independent of the storage temperature. The cells stored at RT for 48 hours had the highest transmigration capacity (54±11.9% vs 4°C 10.7±2%). Storage times of 24 and 72 hours resulted in PEs of on average below 10%. CD34(+)cells stored for 24 hours migrated less than the cells stored at 48 hours. Their transmigration capacity showed no temperature-dependent difference (RT 24.6±7.1 vs 4°C 36.1±10.2%).
Conclusion. Our data imply that CD34(+) cells from cord-blood units stored for 48 hours at room temperature have the highest plating efficiencies and transmigration capacities. A storage time for 48 hours before cryopreservation seems tolerable, since there was no increase of the apoptosis rate.
In Pediatric Mature B-Cell Non Hodgkin Lymphoma (NHL), Complex Karyotype or del(13q) Are Linked Prognostic Factors in Burkitt Lymphoma (BL) While 8q24/c-myc Rearrangement Is Associated with a Strong Adverse Effect in Diffuse Large B-Cell Lymphoma (DLBCL).
Authors: Poirel Hélène A. , Heerema Nyla A. , Swansbury John , Aupérin Anne , Launay Erika , Sanger Warren G. , Talley Polly , Raphael Martine , Perkins Sherrie , McCarthy Keith , Sposto Richard , Weston Claire , Gerrard Mary , Cairo Mitch S. , Bernheim Alain , Patte Catherine 
Published: October 2019

Abstract
Introduction: BL is the main childhood B-cell NHL, characterized by the 8q24 translocation [t(8q24)] leading to a c-myc deregulation. Additional chromosomal alterations have been described in BL as well as in DLBCL, but their prognostic value is not clearly evaluated, especially in children. We have already shown the prognostic value of complex karyotype in childhood B-cell NHL in general (Lugano 2005). We now study the role of c-myc rearrangement and of other chromosomal alterations in each sub entity, BL and DLBCL.
Methods: Karyotypes from 237 children enrolled in the FAB/LMB96 study were centrally reviewed by each participating cooperative group and obtained an international morphological consensus diagnosis: BL 76.8%, BL-like (BLL) 7.6%, DLBCL 12.2%, unclassifiable 3.4%.
Prognosis analyses (event free survival -EFS- and overall survival -OS-) were performed after adjustment on the national cooperative group, the therapeutic stratification group (C vs A & B), the morphological consensus classification (DLBCL vs BL & BLL), the LDH level (>2 vs <=2) & the CNS involvement.
Results: The BL/BLL were usually diploid with a t(8q24) in 92% cases and associated with other chromosomal aberrations in 69%, mainly +1q, del(13q), +7q. The DLBCL were more heterogeneous and more complex than BL/BLL. t(8q24) incidence (34%) was higher than in adult DLBCL and the pattern of chromosomal alterations varied according to the presence of t(8q24) [diploidy, +1q, del(6q), +7q] or not [hyperdiploidy, der(11q) and +12q].
Multivariate analysis identified two different associations of independent cytogenetic factors with worse prognosis on both OS and EFS. Model (A) for EFS: complex karyotype (more than 3 chromosomal alterations) [HR=3, p=0.0008] and t(8q24) [HR=5.1, p=0.04] or model (B) for EFS: del(13q) [HR=2.6, p=0.0008], +7q [HR=2.6, p=0.01] and t(8q24) [HR=5.7, p=0.03]. (+1q had no significant effect). In the BL entity (N=182), only the complexity [HR=2.5, p=0.02] persists in the model A and del(13q) [HR=4.2, p=0.002] in the model B. In the group of 29 DLBCL, the only significant variable (univariate analysis) is the t(8q24): the 3-yr EFS in DLBCL without t(8q24) is 100% while 50% in DLBCL with t(8q24) [p=0.0005].
Conclusion: In BL/BLL, the strong effect of complex karyotype is partly explained by the role of del(13q) and to a lesser extent by +7q. The strong prognosis effect of t(8q24) in B-cell mature childhood NHL is mainly due to its role in DLBCL. This study emphasizes the impact of cytogenetics in both diagnostic characterization and prognostic stratification of childhood mature B-cell NHL.
Constitutive Activation of AKT Contributes to the Pathogenesis and Survival of Blastoid Variants of Mantle Cell Lymphoma.
Authors: Rudelius Martina , Pittaluga Stefania , Pham Trinh H.-T. , Fountaine Thomas , Quintanilla-Martinez Leticia , Fend Falko , Jaffe Elaine S. , Raffeld Mark 
Published: October 2019

Abstract
The serine/threonine protein kinase AKT [protein kinase B (PKB)] plays a pivotal role in the tumorigenesis of many human malignancies, including hematopoietic neoplasms. AKT mediates cancer cell survival and cell growth through the phosphorylation of key regulatory proteins such as FRKHL-1, MDM2 and p27. We wished to determine whether the AKT signaling pathway might be involved in the pathogenesis of mantle cell lymphoma (MCL).The activated form of AKT (pAKT) and phosphorylation of several AKT targets, including FRKHL-1, MDM2 and p27 as well as the upstream modulators PTEN and TCL-1 were analyzed by immunoblotting in 31 MCL cases (16 classic MCL and 15 MCL of the blastoid variant) and in four MCL cell lines (Granta 519, NCEB, REC-1 and Z138). PI3KCA mutation analysis at two hotspot regions (Exons 9 and 20) were completed for all cases and cell lines. Subsequently, inhibition of the PIK3/AKT-pathway with LY294002, Wortmannin and AKT-inhibitor was performed in the four cell lines. pAKT was expressed in all 15 blastoid variants of MCL and in the 4 cell lines, with high expression in 13/15 cases and low expression in 2/15 cases. In contrast, only 2/16 typical MCL expressed pAKT, at low levels. Furthermore, pAKT expression in the blastoid variants and cell lines was accompanied by the phosphorylation of multiple downstream targets of activated AKT, including the cell cycle regulatory proteins p27, FRKHL-1, MDM-2 and the apoptosis associated protein bad. 42% of the blastoid MCL cases showed loss of PTEN, while only 2 (12%) of the typical cases showed diminished PTEN expression.TCL-1 expression levels were not correlated with AKT activation and no mutations of PI3KCA were detected. Functional studies of all four MCL lines demonstrated that the phosphorylation of the downstream effectors were regulated by activated AKT:Inhibition of the PI3K/AKT pathway by both 20 μM LY294002 and 8 μM Wortmannin or 40 μM AKT-inhibitor abrogated the phosphorylation of AKT, p27, FRKHL-1, bad and MDM2 after 24 to 48 hours of treatment, and resulted in G1 arrest after 24 hours of treatment and measurable apoptosis by 48 hours. We conclude that constitutive activation of AKT contributes to the pathogenesis of the blastoid variants of MCL. One possible mechanism of AKT activation is the loss of PTEN expression. The inhibition of the PIK3/AKT pathway in MCL cell lines leads to induction of cell cycle arrest and apoptosis, raising the possibility that PI3K/AKT inhibitors may be effective in the treatment of aggressive (blastoid) variants of MCLs.
Hormonal Regulation of the Anticoagulant Protein S.
Authors: Hughes Quintin , Staton Janelle , Watson Mark , Harvey Jennet , Baker Ross 
Published: October 2019

Abstract
The anticoagulant Protein S (PS) is coded for by the PROS1 gene and serves as a co-factor to APC in the inactivation of FVa and FVIIIa. It is produced predominately by the liver. Low PS levels are associated with an increased thrombotic risk. Clinically it is well documented that circulating PS levels are reduced in response to heightened estrogen (E2) levels (oral contraceptive use, pregnancy, etc.). However, an explanation as to how this is occurring at the molecular level has not yet been elucidated.
Two approaches have been utilized. Firstly, a liver carcinoma cell line, HepG2, was exposed to 5 and 10nM concentrations of testosterone (T) and E2 for 6, 12 and 24h at which point’s total RNA was extracted and quantitated using real-time PCR. Secondly, a reporter plasmid pQH5′[2]DsRed2 was constructed that has 948bp of the PROS1 promoter region (up to but not including the ATG start codon) fused to an enhanced green fluorescent protein (EGFP) reporter gene sequence. A second reporter gene, DsRed2, was incorporated into pQH5′[2]DsRed2, and is constitutively expressed by a cytomegalovirus (CMV) promoter. This provides a means of standardizing any change in expression of the EGFP reporter.
pQH5′[2]DsRed2 was transiently transfected into a breast cell carcinoma line MCF7 [chosen for its high levels of oestrogen receptor alpha (ERα)] and subsequently exposed to 5 and 10nM concentrations of E2 and T for 24h. Expression levels were assessed using a COULTER EPICS flow cytometer.
The HepG2 6, 12 and 24h PROS1 mRNA hormone response experiment showed an erratic response to E2. However there were consistently higher levels of PROS1 mRNA over time in response to T compared to the vehicle alone. Higher levels still were evident for the 10nM concentration.
The transient transfection of pQH5′[2]DsRed2 into MCF7s followed by a 24h exposure of either T or E2 at 5 and 10nM concentrations, showed a significant change in response to both the 5 and 10nM concentrations of E2, but little response to either of the T concentrations
These results suggest that E2 is exerting a direct effect on the promoter of PROS1, down regulating transcription, reflecting clinical observations. However, T may be affecting PROS1 mRNA stability via a yet undetermined mechanism. Therefore, hormonal regulation of PS may be occurring at several levels of the overall production process.
The Cytoplasmic Domain of Tissue Factor Inhibits Migration and Enhances Adhesion of Human Breast Cancer Cells.
Authors: Jiang Xiaofeng , Panetti Tracee S. , Bromberg Michael E. 
Published: October 2019

Abstract
Tissue factor (TF) is a 47 kDa transmembrane glycoprotein that when complexed with its cofactor, factor VIIa (FVIIa), initiates blood coagulation. Apart from hemostasis, TF has been shown to have roles in cellular signaling, development, inflammation, metastasis and angiogenesis. We showed previously that both the cytoplasmic and extracellular domains of TF are required for the full metastatic effect of TF. Recently, we showed that TF-FVIIa-FXa complex induces cellular signaling in human breast cancer cells and is associated with enhanced cell migration and prevention of apoptosis. However, the role of the cytoplasmic domain of TF in tumor cell function is not fully known. In the present study, the role of the cytoplasmic domain of TF in cell migration and adhesion was investigated using the Adr-MCF-7 cell line, a multidrug resistant subline of the human breast cancer cell line, MCF-7. The Adr-MCF-7 cell line has high endogenous expression of TF and expression of PAR1 and PAR2. Adr-MCF-7 cells were retrovirally transfected with either a cDNA construct encoding a FLAG epitope tag fused to the transmembrane and cytoplasmic domains of TF (known as FLAG-TFCD) or vector (LXSN) alone as a control, and stable, polyclonal cell lines selected using G418. Expression of the FLAG-TFCD construct was verified by RT-PCR, Western blot analysis and flow cytometry. To test the effect of overexpression of the FLAG-TFCD construct on cell motility a modified Boyden chamber chemotaxis assay was used. The control LXSN cell line had a nearly 9 fold increase in cell migration [33.5± 3.2 cells/hpf (mean± SEM)]using the combination of rFVIIa (10 nM) and FX (150 nM) as the chemoattractant compared with 0.1% bovine serum albumin (BSA) [3.9± 1.2 cells/hpf]. In contrast, the FLAG-TFCD cell line had no increase in migration of using the combination of rFVIIa and FX [6.6± 0.57 cells/hpf] compared with BSA [5.4± 0.67 cells/hpf]. We then examined the ability of the transfected cell lines to adhere to type IV collagen. The number of adherent cells for the transfected cell line, FLAG-TFCD, was nearly 3 fold higher than that for the LXSN line using a colorimetric MTS assay (0.295± 0.041 vs 0.119± 0.011). Moreover, treatment of the FLAG-TFCD cells with the combination of rFVIIa and FX increased the adhesion by nearly 2 fold compared with untreated FLAG-TFCD cells (0.561±0.055 vs 0.296±0.044). In summary, overexpression of TF cytoplasmic domain leads to inhibition of tumor cell migration and enhancement of cell adhesion and potentially acts as a dominant negative in these cellular processes. These data suggest that a function of the cytoplasmic domain of TF in metastasis is to regulate tumor cell migration and adhesion.
Enhancement of Tissue Factor Dependent Procoagulant Activity and Factor Xa Generation by Alpha2-Macroglobulin.
Authors: Bhattacharjee Gourab , Arandjelovic Sanja , Mackman Nigel , Gonias Steven L. , Edgington Thomas S. 
Published: October 2019

Abstract
Alpha2-macroglobulin (α2M) is a broad-spectrum proteinase inhibitor (M~718,000) which is unique in its ability to inhibit proteinases from all four mechanistic classes. α2M inhibits coagulation proteinases, such as thrombin and factor Xa, and fibrinolytic proteinases, such as plasmin. α2M is also a major carrier of growth factors and may regulate growth factor activity. The primary receptors for α2M are the low density lipoprotein receptor related protein (LRP, KD~5 nM) and a higher affinity receptor recently identified as the 78 kDa glucose regulated protein (Grp78, KD~50 pM). In its native conformation α2M is present in plasma at very high concentrations (up to 5 μM). Upon binding to serum proteinases or small primary amines α2M is converted to the receptor-recognized form (α2M*) and then binds to LRP. LRP-associated α2M-proteinase complexes are rapidly internalized and degraded by phagocytic cells such as macrophages. Previous work in our laboratory has demonstrated that Grp78 binds to tissue factor (TF) and inhibits its functional activity. We now demonstrate that α2M* enhances TF-dependent procoagulant activity and factor Xa generation and this function is dependent on α2M* binding to LRP, but not Grp78, since receptor-associated protein (RAP) blocks the response. The effects of α2M on TF-dependent procoagulant activity are most pronounced in RAW 264.7 macrophage-like cells, but also observed in THP-1 monocytic cells. We are performing additional studies, using both α2M mutants and LRP-deficient cells, to further elucidate this novel mechanism by which α2M* may regulate hemostasis.
Gene Therapy for Severe Combined Immunodeficiency X1.
Authors: Hacein-Bey-Abina S. , Schmidt M. , Le Deist F. , Garrigue A. , Borkhardt A. , Wulffraat N. , Alexander I.E. , Wintergerst U. , Leiva L.E. , Delabesse E. , Macintyre E. , Gougeon M.L. , Gyapay G. , Weinssenbach J. , Fraser C. , de Saint-Basile G. , Casanova J.L. , Von Kalle C. , Fischer A. , Cavazzana-Calvo M. 
Published: October 2019

Abstract
We have previously reported that ex vivo retroviraly-mediated gc gene transfer into CD34 (+) bone marrow precursor cells led to the correction of the immunodeficiency in 9 out of 10 patients with X-linked severe combined immunodeficiency. Follow-up now reaches more that 6 years for the first 2 treated patients. Patients’immune function has been restored. The distribution of both TCR Vb family usage and TCR Vb CDR3 length still reveals a broadly diversified T cell repertoire. Moreover 6 years after treatment the thymus is still seeded by transduced progenitor cells as attested by the presence of TRECS in peripheral blood RTE. Among these patients, three (P4, P5 and P10) developed at 30 to 34 months after gene therapy a monoclonal T cell proliferation requiring a chemotherapy. P4 received also an allogenic HSCT from a MUD but died 26 months after the occurence of the lymphoproliferation. For P5 and P10, chemotherapy has led to an overall control of the clonal proliferation. These two patients are doing well and P5 is off treatment with a good immunological recovery. Genetic analysis of the blastic cells showed that in the two first cases the vector had integrated within or upstream of the LMO2 locus causing an insertional activation of LMO2 transcription. The last case revealed the involvement of several targeted sites, but their exact contribution to the lymphoproliferation is still under investigation. The repeated involvment of LMO2 as a site of vector integration in the proliferating T-cells points to an insertional activation of this gene as at least one of the causes of the oncogenic process. However, the long latency observed in all cases (> 30 months) suggests that additional “hits” have been required for overt desease. Synergy with gc expression and thereby induced proliferative signals (explaining occurrence in SCID-X1 patients only) is the most obvious hypothesis which we are trying to analyse in a mouse model. A deep analysis of retroviral integration patterns has been performed on patients’PBMCS by LAM-PCR to estimate the frequency of potentially harmful integration events and to assess the risk factors associated with the LTR’s strong enhancer effect of the MLV-based retroviral vector. 708 unique integration sites (IS) have been obtained from all analysed patients post-gene therapy and among them, 577 could be mapped unequivocally to the human genome. *Most of these insertions (63%) are located in the vicinity of 10kb or within the coding sequence of a known gene*. A significant peak of insertion frequency is related closely to the transcription strart site *among the 577 IS, 43 are common integration sites. Among the latter, we found out a high selection of genes involved in human oncogenic process.