bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanisms of Erythropoietin-Mediated Cardioprotection during Ischemia-Reperfusion Injury: Role of Protein Kinase C Signaling.
Authors: Arcasoy Murat O. , Hanlon Paul , Fu Ping , Steenbergen Charles , Murphy Elizabeth 
Published: October 2019

Abstract
The biologic effects of erythropoietin (EPO) are mediated by its cellular receptor EPOR, a member of the cytokine receptor superfamily. EPOR expression in non-hematopoietic cells is associated with novel biologic effects for EPO in diverse organ systems. We recently demonstrated functional EPOR expression in adult rat cardiac myocytes and found that recombinant EPO exerts a rapid cardioprotective effect during ischemia-reperfusion injury of the isolated, perfused heart. Here we investigated the mechanisms of the cardioprotective effect of EPO using Langendorff-perfused rat hearts while left-ventricular-developed pressure (LVDP) was measured continuously to assess contractile function. Hearts were treated directly with EPO in the presence or absence of inhibitors of specific signal transduction pathways prior to normothermic global ischemia followed by reperfusion. Post-ischemic recovery of contractile function was determined by measuring LVDP at the end of reperfusion and expressed as a percentage of the baseline pre-treatment measurement. We investigated EPO-mediated activation of signal transduction pathways in the isolated, perfused heart and observed phosphorylation of p44/p42 MAP kinases ERK 1/2 (Thr202/Tyr204) and protein kinase B/Akt (Ser473), a downstream target of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Furthermore, EPO treatment of the isolated, perfused heart was associated with translocation of protein kinase C (PKC) ε and δ isoforms to the membrane fraction. We investigated the role of specific signaling pathways in EPO-mediated cardioprotection by employing inhibitors targeting PI3K, PKC and MAP kinase kinase (MEK1). PI3K inhibitors LY294002 and wortmannin attenuated EPO-induced phosphorylation of Akt but had no effect on EPO-mediated cardioprotection. MEK1 inhibitor U0126 had no effect on EPO-mediated cardioprotection. The PKC catalytic inhibitor chelerythrine (chel) significantly inhibited EPO-mediated improvement in post-ischemic recovery of LVDP (figure 1). Hearts pre-treated with EPO exhibited significantly improved post-ischemic recovery of LVDP compared to control hearts (mean±SE: 72±3 in EPO-treated versus 35±3% in control hearts, P<0.05 by ANOVA and Bonferroni post-hoc test, n=10 experiments each group) and the protective effect of EPO was significantly inhibited in chel-treated hearts (52±4% in EPO+chel versus 72±3% in EPO-treated hearts, P<0.05, n=10). As a control, treatment of the hearts with chelerythrine alone had no significant effect on LVDP (49±4%) compared to control hearts. These data demonstrate that EPO-mediated activation of the PKC signaling pathway is required for the cardioprotective effect of EPO during ischemia-reperfusion injury.
Figure Figure
Autologous Stem Cell Transplantation (ASCT) as Consolidation Therapy for Patients with Low-Intermediate (LI) Risk Diffuse Large B-Cell Lymphoma (DLBCL) and Overexpression of Bcl2 Protein. Results of the First Interim Analysis of the GELA Trial LNH98-B2.
Authors: Morel Pierre , Mounier Nicolas , Briere Josette , Ferme Christophe , Coiffier Bertrand , Tilly Herve , Gaulard Philippe , Lederlin Pierre , Reyes Felix , Gisselbrecht Christian 
Published: October 2019

Abstract
The anti-apoptotic protein bcl2 expression is associated with treatment failure in elderly patients (pts) with DLBCL (Blood 101:4279,2003). The ACVBP regimen with conventional consolidation therapy (JCO 18:3025,2000) yield a 2-year (yr) EFS at 71% in DLBCL pts aged 60 yrs or less with only 1 adverse age-adjusted International-Prognostic-Index factor (LI risk pts). To assess if ASCT reduces treatment failure in these pts, we initiated a trial stratified by bcl2 expression. Centralized staining for bcl2 was prospectively performed within 6 weeks after inclusion. Induction regimen consisted of 4 courses of ACVBP given every 2 weeks. In case of response, bcl2 negative pts received the conventional consolidation regimen, whereas bcl2 positive pts underwent peripheral blood stem cell collection after the 4th cycle, and ASCT (mitoxantrone 45 mg/m2, cyclophosphamide 1.5 g/m2 x 4d, etoposide 250 mg/m2 x 4d and carmustine 300 mg/m2) between d80 and d90. From 1999 to 2002, 272 LI-risk pts aged 18 to 59 (median 46, M/F=1.4) presented with bcl2 overexpression (151) or without (121). There was no difference in initial characteristics, toxicity of induction and complete response (CR+CRu) rates between bcl2 positive and bcl2 negative pts (78% and 82%, respectively, p = 0.38). ASCT was not performed in 35 pts, mainly because of early failure (37%). Although bcl2 negative pts retained a better 2-yr survival than bcl2 positive pts (95% vs. 86%, p=0.01) with a median follow-up of 21 months, 2-yr EFS and 2-yr DFS did not differ significantly (84% vs. 79%, p=0.33, and 89% vs. 87%, p=0.94 respectively). This analysis demonstrates the feasibility of taking bcl2 expression into account for the design of multicentre protocol. The lack of difference in EFS and DFS between bcl2 positive pts who received ASCT and bcl2 negative pts treated with conventional consolidation suggested that ASCT may overcome the adverse prognostic value of bcl2 expression in LI-risk pts who achieve CR or Cru.
Identification of Genes Potentially Involved in Disease Transformation of CML.
Authors: Janssen Jeroen J. , Klaver S. Marjolein , Waisfisz Quinten , Pasterkamp Gerard , de Kleijn Dominique P. , Schuurhuis Gerrit J. , Ossenkoppele Gerrit J. 
Published: October 2019

Abstract
In patients with chronic myeloid leukemia who do not reach a (near) complete cytogenetic response upon treatment, the disease progresses over months to several years from an indolent, chronic phase into a rapidly fatal blast crisis. Events that are responsible for this transformation process are largely unknown. To identify changes occurring in the course of the disease, we performed cDNA subtraction on sequentially stored peripheral blood mononuclear cell pellets, collected from a single CML patient throughout the course of disease. Thirty-three differentially expressed sequences were identified, of which 28 corresponded to known genes. Quantitative PCR confirmed cDNA subtraction results for 24 of 25 tested genes. Differential expression levels ranged from <2- to 106-fold. The sequences that were identified include genes encoding for several apoptosis related proteins, cell cycle proteins, interleukins/chemokines, heat shock proteins and a DNA repair enzyme (see table). Thus far, expression levels of eight genes have been tested in sequential samples of three to six additional CML patients, gathered throughout their disease course. PBCEF, IL8, IL6, CCL4 and YWHAZ show comparable patterns as the index patient; expression levels of APEX, NXF1 and PRG1 were consistent with those of the index patient in only a part of the additional patients. More patients are being tested on these and remaining genes. We conclude that this set of genes can be considered as a valuable starting point for further research on the causes of disease transformation in CML.
Name of gene Accession No. Fold difference (Q-PCR) * Genes hypothetically encoding proteins, ** Reverse to subtraction result, ***orf: open reading frame diagnosis > blast crisis: PRG1 NM_002727 14 IVNS1ABP NM_006469 <2 MPO NM_000250 ND blast crisis > diagnosis: EMR1 NM_001974 <2 CD96 NM_198196 3 GAS2 NM_005256 12 CHI3L2 NM_004000 11 late chronic phase > blast crisis: IL8 NM_000584 2 S100A8 NM_002964 16 FPR1 NM_002029 51 LYZ NM_000239 ND MEN1 AF001893 ** blast crisis > late chronic phase: MMRN NM_007351 6 NACSIN NM_015252 24 LARS NM_020117 7 FLJ25286* NM_152546 ND C5orf13*** NM_004772 ND C15orf15*** NM_016304 ND FLJ10849* NM_018243 ND diagnosis > late chronic phase: DNAJB1 NM_006145 35 HSPA8 NM_006597 5 NXF1 NM_006362 2 DC2 NM_021227 <2 APEX1 NM_080648 4 RPS6 NM_001010 <2 EEF1A1 NM_001402 3 FLJ25286* NM_152546 ND late chronic phase > diagnosis: SAT NM_002970 7 CCL4 NM_002984 106 YWHAZ NM_145690 3 PBEF1 NM_182790 7 IL6 NM_000600 42 IL8 NM_000584 9
STAT5 and STAT5A Signaling Play an Important Role in the Pathophysiology of the CML-Like Myeloproliferative Disorder in Mice.
Authors: Ye Dan , Li Li , Ilaria Robert L. 
Published: October 2019

Abstract
Although numerous studies have implicated signal transducers and activators of transcription (STAT)5 in Bcr/Abl-dependent transformation and survival, the role of STAT5 in chronic myelogenous leukemia (CML) has not been fully elucidated. In mice, the CML-like myeloproliferative disorder can occur in the absence of STAT5A/5B, but the repopulating and colony-formation defects of STAT5A/5B double knockout (−/−) bone marrow (BM) cells have made it difficult to separate Bcr/Abl-specific and Bcr/Abl-non-specific effects. To clarify the role of STAT5 signaling in P210BCR/ABL leukemogenesis, we have expressed Bcr/Abl in primary murine STAT5A-deficient (−/−) BM cells, which have no demonstrable intrinsic hematopoietic defects. Interestingly, the absence of STAT5A profoundly decreased the incidence of the pure CML-like disease. Only 24% of wild-type mice reconstituted with P210-transduced STAT5A(−/−) BM cells developed CML, compared to 93% of P210-STAT5A(+/+)- and 83% of P210(+/−)-reconstituted mice. The remainder of P210-STAT5A(−/−)-reconstituted mice developed an acute lymphoblastic leukemia (ALL)-like disease (20%) or a CML/ALL mix (56%), underscoring the relative importance of STAT5 signaling in P210-induced myeloid leukemia compared to P210-induced lymphoid leukemia. There was also a trend for increased latency of the CML-like disease in P210-STAT5A(−/−)-reconstituted mice, with P210-STAT5A(−/−)-reconstituted mice having a median survival of 38 days, compared to only 22 days for P210-STAT5A(+/+) animals. Compared to P210-control cells, primary leukemia cells from P210-STAT5A(−/−) mice had decreased constitutive expression of the STAT5 target genes Bcl-XL and oncostatin M, but preserved expression of pim-1 and cis. Of mice that developed CML, P210-STAT5A(−/−)-reconstituted animals had greater splenomegaly and tended to have higher Bcr/Abl expression than P210-control animals, suggesting that enhanced expression of Bcr/Abl might compensate for the absence of STAT5A in these mice. Importantly, there was no difference in the number of unique P210-STAT5A(−/−) or P210-STAT5A(+/+) leukemic clones by Southern blot, indicating that the reduced incidence of CML in STAT5A (−/−) mice was not due to the decreased availability of CML progenitors. Altogether, these findings indicate that STAT5, particularly STAT5A, plays an important role in the efficient induction of the CML-like disease in mice. A greater understanding of the STAT5 target genes important in CML will provide important insights into the pathophysiology of CML, and may provide additional molecular targets for CML therapy.
Single Nucleotide Polymorphisms Influence the Phenotype of Mutations P618A and R1336W found in the ADAMTS13 Gene of Congenital TTP Patients.
Authors: Plaimauer Barbara , Mohr Gabriele , Wernhart Waltraud , Bruno Katharina , Antoine Gerhard , Rieger Manfred , Scheiflinger Friedrich 
Published: October 2019

Abstract
ADAMTS13 cleaves plasmatic von Willebrand factor (VWF) between Tyr1605 and Met1606 and regulates thereby the hemostatic activity of VWF. Mutations in the ADAMTS13 gene leading to severe ADAMTS13 deficiency have been found in patients with congenital thrombotic thrombocytopenic purpura (TTP). We have analyzed the ADAMTS13 gene defects in two brothers with hereditary TTP [Antoine et al, Brit. J. Hematol., 2003] where we detected a total of six nucleotide exchanges causing point mutations. On the maternal allele we found an accumulation of five amino acid substitutions (R7W, Q448E, P618A, A732V, R1336W) and on the paternal allele a stop mutation (Q44X) leading to premature protein termination in the propeptide region. Both brothers were double heterozygotes with < 3% of ADAMTS13 activity, whereas their asymptomatic parents have ~ 50% activity. Four (R7W, Q448E, P618A, A732V) of the five maternal mutations constitute single nucleotide polymorphisms (SNP) but R1336W was identified as novel rare mutation in the second cub domain. To evaluate the biologic phenotype of a given haplotype, e.g. the functional significance of the presence of the various SNPs, we analyzed the functional impact of the individual mutations on ADAMTS13 antigen levels and ADAMTS13 activity. A series of mutant ADAMTS13 molecules was expressed which contained either single amino acid substitutions or combinations of mutations with each other. We found that the common SNPs R7W, Q448E and A732V, as single mutations, had either no or only a minor impact on ADAMTS13 secretion and ADAMTS13 activity, whereas P618A and R1336W conferred a dominant adverse effect on ADAMTS13 secretion levels. Co-expression of SNPs R7W or Q448E with SNP P618A lead to improved ADAMTS13 secretion levels and could therefore partly attenuate the detrimental effect of P618A. Concomitant expression of all four SNPs reconstituted secretion levels similar to wild-type implicating a complex synergistically interaction of SNPs located in different ADAMTS13 domain regions, however, functional activity was impaired to 50%. Mutation R1336W was shown to be, as a single amino acid exchange, responsible for reduced ADAMTS13 antigen levels, but in contrast to P618A, the negative effect of R1336W was rather enhanced by the co-expression of R7W and Q448E, than rescued, leading to the total absence of ADAMTS13 secretion from the maternal allele. Our findings provide for the first time evidence that fairly common SNPs, dependent on the presence or absence of other mutations, may differently modulate functional ADAMTS13 protease levels.
The Stress-Induced PAI-1 Expression Is Enhanced in Obese Mice: A Possible Mechanism of Stress-Associated Thrombosis in Obese Subjects.
Authors: Yamamoto Koji , Kojima Tetsuhito , Adachi Tatsuya , Hayashi Mutsuharu , Matsushita Tadashi , Takamatsu Junki , Loskutoff David J. , Saito Hidehiko 
Published: October 2019

Abstract
Cardiovascular/thrombotic diseases are frequently induced by a variety of stressors. Obese patients are susceptible to thrombotic diseases associated with stress, but the underlying mechanisms are unknown. We previously reported that restraint stress to mice caused a significant induction of a primary inhibitor of fibrinolysis, plasminogen activator inhibitor-1 (PAI-1), in association with tissue microthrombosis. We have investigated the effect of obesity on the stress-induced PAI-1 expression using genetically obese mice. Male obese mice (C57BL/6J ob/ob) of 6 weeks old and their lean counterparts (C57BL/6J +/?) were placed into 50-100 ml conical centrifuge tubes fitted with multiple punctures so as to allow ventilation. The tubes were placed in horizontal holders and the animals thus maintained for a continuous period of restraint. After 2 or 20 hours of restraint, the mice (n=8) were sacrificed, and the plasma and tissues were harvested. PAI-1 antigen in plasma was measured by ELISA and PAI-1 mRNA in tissues was quantitated by competitive RT-PCR assay. Obese mice were hyperresponsive to short- and long-duration of restraint stress in the induction of PAI-1 antigen in plasma and PAI-1 mRNA in tissues, especially in their hearts and adipose tissues (Fig. 1).
Figure Figure
In situ hybridization analysis of the stressed mice revealed that strong signals for PAI-1 mRNA were localized to adipocytes, cardiovascular endothelial cells, and renal glomerular cells in obese animals. Immunohistochemical analysis for fibrin revealed that renal glomerular fibrin deposition was induced by 2 hour-restraint stress in obese mice (arrow in Fig. 2B), but rare in lean mice (Fig. 2A). Quantitative evaluation of fibrin was also achieved by counting the number of fibrin-positive glomeruli in each kidney section in a blinded fashion (Fig. 2C).
Figure Figure
Elevation of tumor necrosis factor-alpha (TNF-alpha) level in plasma after stress was pronounced in obese mice and pre-treatment of mice with anti-TNF-alpha antibody partially attenuated the stress-mediated induction of PAI-1 gene in adipose tissues. These results suggest that larger induction of PAI-1 may be relevant to the increased risk of stress-induced thrombosis in obese subjects and that TNF-alpha may be involved. This study presents a novel finding regarding the molecular process of the stress-induced thrombosis in obesity, and suggests that PAI-1, stress, and obesity, may be closely associated with the increased risk for cardiovascular and thrombotic diseases.
Monitoring of FVIII Replacement Therapy with Cell-Based Monitoring Tests.
Authors: Bassus Steffen , Wegert Wolfgang , Krause Manuela , Scharrer Inge , Scholz Thomas , Escuriola-Ettinghausen Carmen , Siegemund Annelie , Kreuz Wolfhart , Engelmann Lothar , Kirchmaier Carl M. 
Published: October 2019

Abstract
Although FVIII replacement therapy is clinically safe and effective, there is still a debate about the individually sufficient efficacy of treatment. With routinely performed one-stage and chromogenic assays individual haemostatic effects of FVIII therapy are poorly detectable due to artificial test conditions, e.g. strong dilution and adding contact activators, phospholipids or activated coagulation factors to patients plasma. Moreover, the crucial relevance of platelets for coagulation characteristics can not be investigated. For our study cell-based coagulation assays (thrombin generation in platelet rich plasma and thrombelastography in whole blood) as well as an analytic strategy were adapted to make them suitable for measuring individual haemostatic effects of FVIII therapy. 40 patients (age 7–79) with haemophilia A replacements therapies were investigated. We measured FVIII activity resp. efficacy before and after substitution with one-stage, chromogenic, thrombin generation and thrombelastography assay. 2/3 of patients received a recombinant, 1/3 of patients a plasma-derived concentrate. The results showed an average FVIII activity, measured with chromogenic and one-stage assays, of 8% before and 76% after substitution of an average of 27,3 U/kg body weight. Thrombin generation and clot firmness showed almost maximum values for relatively low FVIIIactivities (20 to 30%). Moreover, the results of the cell-based tests showed large interindividual variations in pre-substitution residual activities and in haemostatic effects after substitution for same FVIII plasma activities. This indicates the need for different interindividual FVIII activities for achieving sufficient thrombin activity and clot firmness. Therefore it seems to be advantageous to measure patients FVIII efficacy profiles before treatment and use them as their own standard to assess FVIII efficacy after treatment instead of using standard curves from pooled plasma. According to this we measured thrombin generation and clot firmness before treatment. Than we added in vitro one unit FVIII to the same sample to determine patients individual maximum of thrombin generation and clot firmness (100%). After FVIII replacement therapy we determined the same parameters and calculated the relative haemostatic effect of FVIII therapy compared to in vitro results in relative per cent of the maximum FVIII efficacy. With thrombin generation and thrombelastography assays and our revised strategy of analysis we are able to determine the individual haemostatic effect of FVIII replacement therapy and, moreover, to predict the required FVIII-amount for successful treatment with pre-substitutional in vitro spiking of patients’ own blood.
HIV-1 Nef Alleles Show Differential Activation of Hematopoietic Cell Kinase in a Yeast Model System.
Authors: Trible Ronald P. , Smithgall Thomas E. 
Published: October 2019

Abstract
Hematopoietic cell kinase (Hck) is a member of the Src family of non-receptor protein-tyrosine kinases that is expressed strongly in macrophages, an important target cell type for human immunodeficiency virus (HIV). The HIV Nef protein, a critical AIDS progression factor, binds Hck with unusually high affinity and induces Hck activation. Nef-mediated Hck activation has been proposed to trigger signaling pathways important for the establishment of HIV infection and subsequent progression to AIDS. Previous studies have utilized sequence alignments and binding assays to predict the functionality of Hck-Nef interactions, but these studies do not directly investigate the ability of Nef alleles to activate Hck. To better address this issue, we have created a model system in Saccharomyces cerevisiae based on previous findings that ectopic expression of c-Src arrests yeast growth in a kinase-dependent fashion. To establish the utility of this system for Hck and Nef, we first created a Hck variant containing a C-terminal sequence modified to encode the high-affinity SH2-domain binding sequence Tyr-Glu-Glu-Ile (HckYEEI). This modified sequence allows for autophosphorylation of the new tail and the subsequent intramolecular binding to the Hck SH2 domain. The resulting HckYEEI molecule models the physiologically down-regulated (inactive) form of Hck in structure and function. Unlike wild-type Hck, HckYEEI showed little kinase activity and failed to induce growth suppression in yeast. Importantly, co-expression with the Nef-SF2 allele activated HckYEEI, resulting in growth arrest. This effect was dependent on the conserved Nef proline-rich motif essential for Hck binding through its SH3 domain. However, Nef-SF2 did not activate FynYEEI, consistent with our previous data that the Fyn tyrosine kinase is not activated by Nef in mammalian cells. We then tested several additional laboratory HIV-1 Nef alleles for their ability to activate HckYEEI. Consensus, Lai, NL4.3, SF2, and YU-2 Nef alleles activated HckYEEI to varying degrees, as reflected by yeast growth inhibition. In contrast, Nef-Eli failed to suppress growth in the presence of HckYEEI, consistent with its inability to bind the Hck SH3 domain in vitro. Growth inhibition paralleled tyrosine phosphorylation of yeast proteins on anti-phosphotyrosine immunoblots. The differential effects of these Nef alleles on Hck activation correlated closely with previous results in mammalian cells. Taken together, these studies validate the use of yeast to reconstitute Nef-Hck interactions in a system amenable to screens of clinical Nef isolates, which may have predictive value in terms of AIDS progression.