bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tissue Factor-Dependent Cellular Responses of Factor VII/VIIa in the Absence and Presence of Factor X.
Authors: Petersen Lars C. , Albrektsen Tatjana , Hjortø Gertrud M. , Kjalke Marianne , Rojkjaer Rasmus , Bjørn Søren E. , Sørensen Brit B. 
Published: October 2019

Abstract
Factor VIIa (FVIIa) when bound to tissue factor (TF) triggers both coagulation and intra-cellular signaling. The dose-response of TF/VIIa-mediated factor X (FX) activation and gene regulation by TF-expressing cells were measured and interpreted in relation to plasma in vivo conditions. EC50s in the range 10–30 pM for FVIIa-mediated FX activation were measured with LPS-stimulated monocytes, vascular smooth muscle cells and MDA-MB-231 cells indicating that saturation of FX activation required less than 100 pM FVIIa. Compared to a plasma concentration for FVII/FVIIa of 10 nM this ensures that TF exposure is the determining factor for the coagulation response. This notion was further substantiated by data showing that FVII is readily activated to FVIIa on the TF-expressing cell surface, and that the FX activation kinetics mediated by FVII was similar to that of FVIIa. In MDA-MB-231 cells FVIIa induced TF-dependent signal transduction as measured by IL-8, CXCL-1 and CSF-2 mRNA. Stimulation reached saturation at about 10 nM FVIIa in the presence or absence of 100 nM FX. Furthermore, the maximal level of FVIIa-induced mRNA was similar with or without FX. FX enhanced TF/FVIIa-stimulated mRNA induction at low FVIIa concentrations, whereas a physiological level of FX or FXa (100 nM) alone was insufficient to induce a significant cellular response. In conclusion our results indicate that optimal conditions for triggering both coagulation and signaling prevail already at normal plasma concentration of FVII/FVIIa and FX, and that TF exposure is the determining factor for both responses.
Age Related Expression Patterns of Mouse Liver Nuclear Proteins.
Authors: Kurachi Sumiko , Yasui Manabu , Kasama Emi , Kurachi Kotoku 
Published: October 2019

Abstract
Previously we reported the discovery of the ASE/AIE-type age related regulatory mechanism of gene expression, the first molecular mechanism for age dimension homeostasis (Kurachi et al, Science 1999; Zhang et al, J Biol Chem 2002; Kurachi & Kurachi J Thromb Haemost 2005). Here we report a comprehensive analysis of the age associated changes of mouse liver nuclear proteins, an experimental model for studying the dynamic regulatory mechanisms of the age-dimension homeostasis. Liver nuclei obtained from mice (C57BL/6xSJL, male) at 1, 3, 6, 12, 18 and 21 months of age (n=10–20/age) were used for preparing protein extracts. The protein extracts were then subjected to 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) (pH range of 4–11) and protein identification by MALDI-TOF/MS. As analyzed by the PDQuest program combined with Mascot Protein Identifier, out of over 5000 protein spots identified by 2D-PAGE, approximately 2800 protein spots were found unique including isomers. Intensity changes of these protein spots along the age axis were determined and analyzed in details with the PDQuest software. Twelve proteins were found to show no significant changes in their intensity throughout the life-span (smaller than 20% fluctuation). Besides some complex patterns, at least 6 unique age-related patterns were found, including those of puberty-onset increase, puberty-onset decrease, puberty-onset increase up to 6 months of age followed by decrease, unique increase only at around 12 months of age, ageing associated increase or decrease. Age related pattern changes were observed for some isomers among many, which were likely generated by differential posttranslational modifications or conformational changes. These findings support that in addition to the ASE/AIE type mechanism, there exist multiple novel age-dimension regulatory mechanisms, and their various combinations are responsible for producing complex and subtle age related regulatory patterns for a large number of genes. This is the first comprehensive analysis of the liver nuclear proteins in relation to age, and would provide a valuable basis for studying many age related phenomena, diseases as well as epigenetic effects.
NMR Structural Analysis and Folding Studies of the Factor XI Apple 4 Domain as a Model for Factor XI Homodimerization.
Authors: Riley Paul W. , Samuel Dharmaraj , Cheng Hong , Roder Heinrich , Walsh Peter N. 
Published: October 2019

Abstract
Factor XI (FXI) is a 160kD homodimeric component of the intrinsic pathway of the blood coagulation system. The FXI Apple 4 domain (A4) mediates homodimer formation even when the cysteine responsible for the intermolecular disulfide is mutated to serine (C321S). Preliminary NMR structural studies of the isolated wild type (wt) A4 domain showed a flattened, planar structure (Samuel D, Cheng H, Riley PW, Walsh PN, Roder H. Blood104, 481a, 2004), compared to a more globular structure consisting of two layers of β-sheet and a flanking α-helix that was observed on further analysis of wt and C321S A4. Hydrogen exchange NMR and surface NMR analysis (SEAHSQC), as well as temperature dependent hydrogen exchange NMR showed that in C321S and wt A4, the loop surrounding C321 or S321 is flexible and exposed to the solvent. Thus, the globular structure is the major species in both wt and C321S A4, but the planar structure is present as well. Fluorescence anisotropy studies of C321S A4 yielded a Kd value of ~64 nM (monomeric protein concentration), compared with ~72 nM for a dissociable mutant full-length FXI (G326C) obtained by analytical ultracentrifugation (Sinha D, Marcinkiewicz M, Lear JD, Walsh PN. Biochemistry, in press, 2005), suggesting that only the A4 domain mediates dimer formation in the intact protein. In addition, fluorescence and circular dichroism (CD) studies of the folding equilibrium of C321S A4 indicate that starting from the unfolded monomeric state, partial folding precedes dimerization. This folding mechanism for FXI A4 is consistent with the observed failure of a naturally occurring mutant (FXI F283L) to dimerize intracellularly (Meijers JC, Davie EW, Chung DW. Blood79, 1435–1440, 1992). To determine the structural consequences of the mutation, F283L was introduced into the noncovalent dimer C321S A4, which increased the Kd of the homodimer interaction to ~5 μM, but did not change the secondary structure (from comparison of the CD spectra), or the high degree of thermostability observed for C321S A4. Since the F283 sidechain has been tentatively assigned to the surface of FXI A4 near the interface, the mutation to Leu may significantly alter the interface, thereby raising the Kd but not changing the overall secondary structure significantly. In summary, there appears to be a mechanism by which a conformational change or productive folding process occurs in the monomer state that activates it for native dimer formation. Also, inserting the F283L (type III) patient mutation does affect the dimer stability, but not the overall secondary structure.
Hematopoietic Repopulation, In Vivo, with Genetically Defined Clones Derived from HOXB4 Expressing ES-Cells.
Authors: Pilat Sandra , Carotta Sebastian , Schiedlmeier Bernhard , Kamino Kenji , Mairhofer Andreas , Schmidt Manfred , von Kalle Christoph , Steinlein Peter , Ostertag Wolfram , Baum Christopher , Beug Hartmut , Klump Hannes 
Published: October 2019

Abstract
In the context of somatic gene therapy of the hematopoietic system, transplantation of molecularly defined and, hence, “safe” clones would be highly desirable. However, techniques which allow gene targeting, subsequent in vitro selection and clonal expansion are only available for embryonic stem (ES) cells. After in vitro differentiation, some of their progeny cells are capable of mediating long term hematopoietic repopulation after transplantation into immunodeficient recipient mice, in vivo. This is especially efficient when the homeodomain transcription factor HOXB4 is ectopically expressed (1). We have recently shown that HOXB4-ES-cell derivatives behave similar to bone marrow cells also expressing this transcription factor ectopically, both in vitro and in vivo (2). Here we demonstrate that long term repopulation (>6 months) in Rag2(−/−)γ C(−/−) mice can be achieved with ES-cell derived hematopoietic cells (ES-HCs) obtained from single, molecularly characterized ES-clones, in which the insertion sites of the retroviral expression vector had been defined. Clones expressing HOXB4 above a certain level showed a high extent of chimerism in the bone marrow of transplanted mice (average 75%; range 45–95%, n=4) whereas ES-HC clones expressing lower levels only repopulated with very low efficiency (average 2.5% chimerism, range 1–4%, n=6 mice). These results suggest that the capability of long-term repopulation, in vivo, is highly dependent on the expression levels of HOXB4 in the transplanted clones. Only mice reconstituted with ES-HC clones expressing high amounts of HOXB4 and thus showing substantial chimerism, recapitulated the morphohistological phenotype observed in polyclonally reconstituted mice. This included the bias towards myelopoiesis, “benign” myeloid proliferation in spleen and the incompatibility of HOXB4 expression with T-cell poiesis (2). In summary, we demonstrate that repopulation of the hematopoietic system can be achieved with preselected clones of genetically manipulated stem cells in which a) the insertion site of the retroviral (gene therapy) vector has been characterized prior to transplantation and b) in which ectopic HOXB4 has to be expressed above a certain threshold level. Thus, ES cells carry the potential for performing safe somatic gene therapy when using integrating gene therapy vectors. Nevertheless, advanced cell therapy will certainly require the expression of HOXB4 in a regulated manner to avoid unwanted effects such as disturbed lineage differentiation.
BCR/ABL Modifies the Kinetics and Fidelity of DNA Double-Strand Breaks Repair in Leukemia Cells.
Authors: Slupianek Artur , Nowicki Michal O. , Skorski Tomasz 
Published: October 2019

Abstract
Clinical observations and experimental findings indicated that BCR/ABL stimulates genomic instability leading to mutations and chromosomal abnormalities. The accumulation of genetic errors is believed to be responsible for the transition from a relatively benign CML chronic phase (CML-CP) to the aggressive blast crisis phase (CML-BC) and the resistance to imatinib mesylate. BCR/ABL- positive leukemia cells accumulate an excess of potentially lethal DNA double-strand breaks (DSBs) caused by reactive oxygen species (ROS) or genotoxic treatment. However, BCR/ABL tyrosine kinase facilitates the repair of DSBs and promotes survival. Therefore, the infidelity of DSBs repair processes may contribute to genomic instability in leukemia cells exposed to elevated numbers of spontaneous and/or induced DSBs. To test this hypothesis DSBs repair efficiency and fidelity was examined and compared in parental and BCR/ABL-transformed cells. Nuclear foci detected by γ-H2AX (the form of H2AX histone that is quickly phosphorylated on Serine 139 by ATM, ATR and/or DNA-PKcs kinases on megabase-length fragments near DSB sites) immunofluorescence served as indicators of DSBs. We found that BCR/ABL-positive leukemia cells acquire more DSBs after γ-irradiation in comparison to normal cells. Homologous recombination (HR) and non-homologous end-joining (NHEJ) represent two major mechanisms of DSBs repair in mammalian cells. HR and NHEJ reaction sites in the nuclei can be visualized by double-immunofluorescence detecting co-localization of γ-H2AX foci with RAD51 or Ku70, respectively. NHEJ and HR appear to work in a time-dependent fashion (NHEJ followed by HR) and be more active in BCR/ABL-transformed cells in comparison to normal counterparts. Time-dependent engagements of NHEJ and HR mechanisms in repair of DSBs after γ-irradiation are accompanied by elevated accumulation of Ku70 and RAD51 proteins in cell lysates obtained from BCR/ABL cells. Specific DSBs repair assays confirmed that BCR/ABL leukemia cells in comparison to normal cells displayed enhanced capability of HR and NHEJ. However, analysis of DSBs repair products revealed that the repair mechanisms were less faithful in former cells generating large deletions and point mutations during NHEJ and HR, respectively. In summary, BCR/ABL leukemia cells display facilitated, but unfaithful HR and NHEJ, which may contribute to accumulation of genetic errors in surviving leukemia cells leading to malignant disease progression.
Graft-Versus-Tumor Effects after Allogeneic Hematopoietic Cell Transplantation for the Treatment of Hematologic Malignancies.
Authors: Usui Eiji , Fukuda Takahiro , Kato Ruri , Yamasaki Satoshi , Maruyama Dai , Morita Yuriko , Kim Sung-Won , Mori Shin-ichiro , Tanosaki Ryuji , Tajima Kinuko , Heike Yuji , Makimoto Atsushi , Tobinai Kensei , Takaue Yoichi 
Published: October 2019

Abstract
Adjustment of immune reaction, balancing between graft-versus-tumor (GVT) effect and graft-versus-host disease (GVHD), is of primary importance for improved therapeutic outcome after allogeneic hematopoietic cell transplantation (HCT). We retrospectively reviewed the medical records of 315 patients with hematologic malignancies who underwent allogeneic HCT at our center between 2000 and 2004. The median age of the patients was 46 (range, 1–68) years. The diagnoses included acute myeloid leukemia or myelodysplastic syndrome (n=153), chronic myelogeneous leukemia (n=30), acute lymphoblastic leukemia (n=36), lymphoma (n=90), and other hematologic malignancies (n=6). Seventy-two patients had low-risk and 243 had high-risk disease. Patients were conditioned with fludarabine-based reduced-intensity (n=160) or conventional myeloablative regimen (n=155). Donors included HLA-matched (n=120) or mismatched (n=53) relatives and unrelated volunteers (n=142). Stem cell source was G-CSF-mobilized peripheral blood stem cell (n=169), bone marrow (n=117), or cord blood (n=29). Of the 187 patients with measurable disease before HCT, 139 (75%) achieved complete remission. In a multivariate analysis, myeloid disease and the development of acute or chronic extensive GVHD were associated with a higher probability of achieving complete remission. Currently, 155 patients (49%) are alive with a median follow-up of 931 (range, 88–1968) days. Multivariate analyses revealed that a higher HCT-specific comorbidity index (HCT-CI) score, disease risk, graft rejection, and transplant from donors other than HLA-matched relatives were associated with increased risks of poor overall survival (OS) and progression-free survival (PFS). A higher HCT-CI score, greater patient age, graft rejection, and transplant from donors other than HLA-matched relatives were associated with an increased risk for non-relapse mortality (NRM). Patients with high-risk disease were associated with an increased risk of relapse or progressive disease (PD). After adjusting for these significant variables, grade III–IV acute GVHD was associated with increased risks of high NRM and poor OS and PFS. Chronic extensive GVHD was associated with an increased risk of NRM, without any significance for PD, OS, and PFS. In a subset analysis of 160 patients who received reduced-intensity conditioning, chronic extensive GVHD was associated with a decreased risk of PD. In conclusion, marginally intensified GVT effect with severe acute or chronic extensive GVHD appears to be offset by increased toxicities and mortality. Future study should focus on identification of narrow window for better control of immune reaction to induce tolerable GVHD with clinically significant GVT effect.
Large Granular Lymphocytosis Following Transplantation: Institutional Experience over a 10-Year Period at City of Hope.
Authors: Popplewell Leslie L. , Chang Karen , Palmer Joycelynn , Forman Stephen J. 
Published: October 2019

Abstract
Large Granular lymphocyte (LGL) expansion and LGL leukemia have been described in patients following both ablative and non-myeloablative conditioning regimens for hematopoietic cell transplantation (HCT), and have been associated with viral infection and graft-vs.-host disease (GVHD). We describe here an analysis of 41 cases of clonal LGL expansion identified at our institution over a 10-year period. Of these, 18 occurred in patients post autologous HCT (autoHCT), 23 in patients post allogeneic HCT (alloHCT). In the same time period, an additional 23 diagnoses were made in patients who had never undergone HCT. LGL expansion appeared an average of 485 days (range 30 – 1935 d) from alloHCT. In most patients, this occurred as an isolated finding, while in 5 patients, the clonal expansion was detectable on serial evaluations over a period of months or years (75 – 1662 d). LGL expansion was detected an average of 133 days from auto HCT, and only one patient had detectable clonal expansion over a prolonged period of time (70d). AutoHCT patients ranged from 23 to 72 years, average age 48.5 years. AlloHCT patients ranged from 26 to 62 years, average age 47.8 years. Incidence of reactivation of CMV postHCT was 56.5% in the alloHCT and 11% in the autoHCT. Relapse rates were 39% in the alloHCT and 28% in the autoHCT groups. At a median follow-up of 1372 days, 14 alloHCT patients remain in remission, with 9 relapses, and 5 deaths. At a median follow-up of 887 days, autoHCT patients remain in remission, with 5 relapses, and 3 deaths. Deaths were primarily attributed to relapse of disease and infectious complications, while no deaths were attributable to LGL clonal expansion. No patients with LGL clonal expansion have required treatment specifically for this condition.
Detectable Minimal Residual Disease before Haematopoietic Stem Cell Transplantation Predicts Extremely Poor Prognosis in Children with Acute Lymphoblastic Leukaemia.
Authors: Muzikova Katerina , Fronkova Eva , Sramkova Lucie , Reznickova Leona , Formankova Renata , Sedlacek Petr , Stary Jan , Trka Jan 
Published: October 2019

Abstract
The level of minimal residual disease (MRD) prior to allogeneic haematopoietic stem cell transplantation (HSCT) was shown to be an independent prognostic factor for the outcome of paediatric patients with high-risk acute lymphoblastic leukemia (ALL). Retrospective studies which used (semi-)quantitation of clone-specific immunoglobulin/T-cell receptor (Ig/TCR) rearrangements documented feasibility and practicality of such an approach. Recently, this approach was disputed by Imashuku et al (BMT 2003) due to great occurrence of the clonal evolution and generally high MRD levels prior HSCT in their cohort. In our prospective study, MRD before and after HSCT was monitored in a cohort of 36 children with ALL consecutively transplanted in our centre between VIII/2000 and VII/2004. We used a quantitative real-time PCR approach introduced and standardised by European Study Group on MRD in ALL. In 25 of 36 patients MRD level prior HSCT was assessed (9 patients lacked adequately sensitive Ig/TCR target; two lacked analysable DNA prior HSCT). Seventeen patients were MRD-negative prior HSCT (including two with MRD level below the quantitative range 10(−4)) and 8 were MRD-positive up to 9x10(−2). In the MRD-positive subgroup, 7 events (6 relapses) occurred post-transplant in striking contrast to only one relapse in MRD-negative subgroup (EFS log-rank p<0.0001). MRD proved to be the only significant prognostic factor in a multivariate analysis (p<0.0001). Adoptive immunotherapy including donor lymphocyte infusions in patients with adverse dynamics of MRD after HSCT had only limited and/or temporary effect. Clonal evolution did not present a problem precluding MRD monitoring in any of patients suffering a post-transplant relapse. We show that MRD quantitation using clonal Ig/TCR rearrangements represents a feasible approach for the risk assessment in paediatric ALL patients undergoing allogeneic HSCT. However, our ability to respond to detectable MRD levels after HSCT and to avert an impending relapse is very limited. The change of the approach to MRD-positive patients prior HSCT is necessary because of very questionable benefit of HSCT in these children. Supported by grants MSM0021620813, FNM 9735 and GAUK 62/2004.
Incompatible Inhibitory KIR Ligands Contribute To Lower Survival Rates in Leukemia Patients Receiving T-Replete Hematopoietic Cell Transplants (HCTs).
Authors: Sun J.Y. , Dagis A. , Miller M.M. , Longmate J. , Rodriguez R. , Gaidulis L. , Forman S.J. , David D. 
Published: October 2019

Abstract
Natural killer (NK) cells are becoming increasingly recognized as an important part of the immune system. Killer Ig-like receptors (KIRs) are the major form of receptors used by human NK cells. KIRs transmit either inhibitory or activating signals regulating NK cell activity. Some inhibitory KIRs specifically recognize HLA-A, B or -Cw allotypes on target cells. Thus differences in inhibitory ligand (iKIRL) phenotypes between donors and patients could result in NK cell alloreactivity that further results in different HCT outcomes. To test this hypothesis, the present study evaluated 362 patients with either ALL (99), AML/MDS (141), or CML (122). The patients were transplanted (1996~2003) with T-replete bone marrow, or peripheral blood stem cells from unrelated donors. High resolution HLA typing with DNA-based methods had been prospectively performed for donor selection. The KIR genes of patients and donors were retrospectively typed by a multiplex PCR-SSP method. The cohort was divided into three groups by the HLA and KIR profiles: 247 cases with matched HLA at the antigen level of HLA-A, B, Cw, DRB1, and DQB1 (MH), 64 with mismatched HLA (mMH) and 51 with mMH plus mismatched iKIRL (mMH+miKIRL). A significantly different rate (P=0.009) of estimated one year overall survival was found between the MH (61%, 95%CI 55–67%), the mMH (46%, 34–59%), and the mMH+miKIRL (29%, 18–43%) groups. When analyzing the event free survival, a similar difference (P=0.003) was observed between the MH (57%, 51–63%), the mMH (43%, 32–56%) and the mMH+miKIRL (25%, 15–39%) groups. Further analysis demonstrated a different rate (P=0.02) of relapse between the MH (two-year estimation: 21%, 16–28%), the mMH (16%, 7–31%), and the mMH+miKIRL (36% 18–58%) groups. That the MH group experienced more relapses than the mMH is consistent with the GvL effect. The mMH+miKIRL group experienced the most relapses implied miKIRL undermine the GvL effect. We also tested the influence of lacking iKIRL in patients within the MH group, and found a significant survival difference (P=0.05) among the patients lacking two iKIRLs (n=67, 54% 42%–65%), one iKIRL (n=104, 59% 49%–68%), or none (n=76, 70% 58%–79%). This finding contrasts with the previously reported beneficial role of potential NK cell alloreactivity in T-depleted HCT. Additional studies are required to resolve these issues.