bluuebunny/crossref_metadata_2025_split huggingface dataset:
The MDM2 Antagonist Nutlin-3 Is Lethal to Mantle Cell Lymphoma with Wild Type p53.
Authors: Tabe Yoko , Sebasigari Denise , Rudelius Martina , Pittaluga Stefania , Raffel Mark 
Published: October 2019

Abstract
Mantle cell lymphoma (MCL) has one of the poorest overall prognoses of the non-Hodgkin lymphomas, and is resistant to standard chemotherapy. P53 mutations are rare in typical mantle cell lymphoma and occur in only about 1/3 of the less common blastoid variant. The relatively low overall rate of p53 mutation in MCL suggests that this lymphoma may be a good candidate for biologic therapies that upregulate p53 and lead to cell death. Nutlin-3 is a novel small-molecule antagonist of MDM2 that efficiently activates p53 by blocking the MDM2-p53 interaction. In this study, we investigated the effects of nutlin-3 in 5 MCL cell lines with known p53 mutation status (wt-p53: REC-1, Z138, Granta-519, JVM-2; mt-p53: Jeko-1). IC50s for all cell lines were determined, and the activation of p53 and multiple p53 target genes were studied by western blot analysis. Cell proliferation and apoptosis were assessed using the MTT test and flow cytometry. Treatment with Nutlin-3 resulted in a marked reduction in cell proliferation/viability, and an increase in the apoptotic fraction, in a time and concentration-dependent manner in wt-p53 cells (IC50 at 48 hrs; 7.5 uM for Granta-519, 9.2 uM for REC-1, 0.5 uM for Z138, and 5.7 uM for JVM-2), while mt-p53 Jeko was resistant to nutlin-3 treatment (IC50>60 uM), and showed no increase in the apoptotic cell fraction. In the wt-p53 MCL cell lines, nutlin-3 treatment increased the cellular levels of p53, and several p53 dependent proteins including p21, MDM2 itself and the proapoptotic BH3-only protein Puma, while there was no change in the levels of these proteins in Jeko-1. Recently, attention has been focused on novel p53 target genes that function to inhibit cell growth and proliferation, rather than by inducing apoptosis. These include the AKT-mTOR regulators PTEN, TSC2 and AMPKβ1. We saw no increase in these novel p53 targets in any of the cell lines analyzed, suggesting that nutlin-3-activated p53 activates only a subset of its possible targets. These findings demonstrate that nutlin-3 successfully activates wt-p53 in mantle cell lymphoma leading to the upregulation of traditional targets such as p21 and proapoptotic proteins including Puma, and result in apoptotic cell death. The data suggest that p53 activators such as nutlin-3 may be effective agents in the treatment of mantle cell lymphoma.
Localization and Functional Characterization of the Deoxyhemoglobin Binding Site on Human Erythrocyte Band 3.
Authors: Chu Haiyan , Breite Andrew , Ciraolo Peter , Franco Robert S. , Low Philip S. 
Published: October 2019

Abstract
Band 3, the major protein of the human erythrocyte membrane, associates with multiple metabolic, ion transport, and structural proteins of the cell. Functional studies demonstrate that the oxygenation state of the erythrocyte regulates metabolic, ion transport and mechanical properties of the cell. Because deoxyhemoglobin, but not oxyhemoglobin, binds band 3 with high affinity, these observations raise the hypothesis that hemoglobin (Hb) might regulate erythrocyte properties through its oxygenation-dependent association with band 3. To explore how reversible association of deoxyHb with band 3 might modulate erythrocyte properties, we have characterized the binding site of deoxyHb on human erythrocyte band 3. We report that:
the deoxyHb binding site on the erythrocyte membrane comprises residues 12–23 of band 3; mutation of residues on either side of this sequence greatly enhances affinity of deoxyHb for band 3, suggesting that evolution of a higher affinity interaction was possible had it been beneficial; Hb does not bind to two similar sequences in band 3 despite their high sequence homology to residues 12–23, and the Hb binding site on band 3 lies proximal to binding sites for glycolytic enzymes, band 4.1 and ankyrin, suggesting possible mechanisms through which multifarious erythrocyte properties might be regulated by Hb oxygenation.
We conclude that nature has evolved a deoxyHb binding site on band 3 whose affinity is regulated by O2, enabling the reversible association of deoxyHb with band 3 to serve as a regulator of the oxygenation-dependent properties of the cell.
CD133+ Purified Hematopoietic Stem Cells in Co-Culture with Mesenchymal Stromal Cells - The Cell to Cell Contact Matters.
Authors: Alakel Nael , Jing Douhui , Bornhaeuser Martin , Ehninger Gerhard , Ordemann Rainer 
Published: October 2019

Abstract
Hematopoietic stem cells (HSC) are defined by their capacity of self-renewal and differentiation. In recent years it became aware that cell to cell contact mediated communication between mesenchymal stromal cells (MSC) and HSC is important for homeostasis of hematopoiesis. MSC play a crucial role in the so called bone marrow niche giving rise to the majority of marrow stromal cell lineages. In vitro we investigated the impact of MSC on CD133 purified HSC expansion and differentiation in terms of phenotype, (CXCR4) chemokine receptor expression, migration capacity and clonogenicity. After one week of co-culture with MSC adherent and non-adherent HSC were isolated and analyzed separately. As expected in co-culture experiments with MSC a significant higher expansion of TNC was observed in comparison to the expansion culture without MSC (cytokine driven), 37±6 fold vs. 19±3 fold, p<0.001. However the co-culture expansion was accompanied by a significant increase of differentiation and maturation of the progenitor cells. Therefore we were focusing on the adherent cell fraction of the co-culture system. According to FACS analysis results, adherent HSC in the co-culture display a more immature phenotype than non adherent cells (CD34+CD38−). Next colony assays and LTC-IC were performed showing that the adherent cell fraction contained a significant higher proportion of primary and secondary CFU-C, indicating that this cell fraction contains the majority of clonogenic HSC. The CXCR4 - SDF-1 axis plays an important rule in homing, mobilization and engraftment of the hematopoietic stem cells. Therefore we looked for CXCR4 expression in the two cell fractions using FACS analysis. The adherent cells expressed consistently and significantly more CXCR4 than the non adherent cells; 43±4% vs 15±2%, p<0.001. Data could be confirmed by performing western blot. Due to this data we evaluated the capacity of HSC migration toward SDF-1 gradient using transwell migration assay. The migration capacity of the adherent cells was significantly higher than that of the non adherent cell fraction correlating with the CXCR4 expression; 13±1% vs. 6±1%, p<0.001. We checked the expression of different cell adhesion molecules by FACS analysis. CD44 and CD49e (integrin α5) were significantly higher expressed in adherent cells comparing with non-adherent cells. Our data indicate that the cell-to-cell interaction mediated impact on CD133+ selected HSC leads to an enhanced clonogenic capacity and migratory potential. Further experiments are needed to investigate engraftment and NOD/SCID repopulation potential of the adherent and non-adherent cells after ex-vivo expansion.
Pharmacodynamic Analysis of the Anticoagulant Effects of Idraparinuxparinux after Termination of 6 and 12 Months Therapy for Prevention of Recurrent Venous Thromboembolism: Observations after the vanGogh Trials.
Authors: Harenberg Job , Joerg Ingrid , Hagedorn Antje , Giese Christina , Weiss Christel , Mikus Gerd 
Published: October 2019

Abstract
Idraparinux is a synthetic, polymethylated O-methyl, O-sulphate pentasaccharide with a higher affinity to antithrombin and has been investigated in patients with acute venous thromboembolism (VTE) for prophylaxis of recurrent events (vanGogh studies). 2.5 mg Idraparinux was given once weekly subcutaneously in patients with a creatinine clearance > 30 ml/min and continoued with 1.5 mg Idraparinux from the second injection in patients with lower creatinine clearance. This treatemnt was randomly compared to bodyweight adjusted enoxaparin followed by INR-adjusted warfarin for 3 to 6 months to prevent symptomatic recurrent VTE. The vanGogh extension trial randomised patients of the vanGogh trials or other patients with a 3 to 6 months course of warfarin after an initial first event of VTE for an additional double blind 6 months therapy of Idraparinux compared to placebo. The pharmacodynamic parameters of factor Xa (aXa) inhibition using the S2222 chromogenic substrate and Heptest assay were determined from plasma samples of 23 patients obtained after termination of a 6 or 12 months (Idraparinux for 6 months each in the DVT/PE and extension studies) therapy up to 15 months. The elimination half life of Idraparinux was 60±14 days (d, mean+SD) after termination of therapy (S2222) and 107±34 d (Heptest, p<0.0001) and did not differ between 6 and 12 months of treatment. The mean residence time differed between S2222 (75±31 d) and Heptest (121±49 d, p<0.0001) and not between 6 and 12 months. The Cmax was lower after 6 months of therapy compared to 12 months (S2222: 0.26±0.10 vs 0.44±0.08 μg/ml, p=0.0013, Heptest: 0.35±0.13 vs 0.54±0.11 μg/ml, p=0.0085). The volume of distribution was higher after 6 months of therapy compared to12 months (S2222: 8.5±3.5 vs 4.1±0.6 liter, p=0.0052, Heptest: 9.8±4.5 vs 4.7±1.3 liter, p=0.0145) without differences between the assays. The clearance differed between assays (S2222: 0.09±0.06. Heptest: 0.06±0.03 ml/min, p<0.0001) and was higher after 6 than after 12 months of treatment (S2222; 0.11±0.06 vs 0.05±0.02 ml/min, p=0.0088, Heptest: 0.07±0.03 vs 0.03±0.01 ml/min, p=0,0145). The data show a very long pharmacodynamic elimination half-life of 60 days or longer after termination of therapy after once weekly injection of Idraparinux. The half live is longer when determined by Heptest indicating also non-anti-factor Xa activities of Idraparinux. The steady state of Idraparinuxparinux is reached between 6 and 12 months of therapy or later. The low VTE-recurrence rate of 0.9% of patients randomized to placebo in the vanGogh Extension trial and after an initial course of 6 months of Idraparinux in the DVT/PE trial can be explained by the long half life of Idraparinux.
The Cytofluorimetric/FISH Diagnostic Approach Define a B-Cell Variant-CLL with Peculiar Clinico-Biological Features.
Authors: Cro Lilla , Baldini Luca , Nobili Lucia , Neri Antonino , Zucal Nadia , Ferrario Andrea , Lionetti Marta , Bertoni Francesco , Morabito Fortunato , Guffanti Andrea , Goldaniga Maria , Cutrona Giovanna , Cortelezzi Agostino , Luminari8 Stefano , Ferrarini Manlio , Deliliers Giorgio Lambertenghi 
Published: October 2019

Abstract
In the present study, we describe the clinico-biological features of 63 cases of variant B-CLL (v-B-CLL) defined according to a previously described diagnostic algorithm for CD5+ mature B-cell leukemias, based on the combined use of Cytofluorimetric (CFM) analysis and t(11;14)(q13;q32) detection by means of fluorescence in situ hybridisation (FISH). These leukemias were characterized by SIgbright/CD23+ or CD23+/− , CD79b/CD20 bright, and negativity for t(11;14)(q13;q32). A historical series of 112 classical B-CLL was used as comparison. The mean age was 61 years (33–85) and M/F ratio 1.9. The v-B-CLL cases were significantly different from the B-CLL cases in terms of the following clinico-hematological variables: age <70 yrs, lymphocytosis <20 ×109/L (P <.0001), lymphocyte doubling time ≤ 12 months (P=.04), high serum ß2-microglobulin levels (P=.003) and presence of splenomegaly (P=.007). No differences were found in terms of sex, Hb levels, platelets count, presence of serum monoclonal component, serum LDH levels and HCV-Ab positivity. In v-B-CLL and B-CLL pts the median follow-up was respectively 56 and 84 months. The median overall survival was 80 months in v-B-CLL pts while it was not reached in B-CLL pts; 37 pts (58.7%) in the first group started chemotherapy vs 84 pts (56.4%) in the latter. The analysis of leukemic cell biological characteristics related to the prognosis was also made in part of the cases. A classic or mixed-CLL cytomorphology was seen only in 22/61 pts (36.1%). The percentage of CD38 positive cases was higher in v-B-CLL (60.3) than in B-CLL (44.6; P=.028); the IgVH mutation status, evaluated in 30/63 cases, was more frequently hypermutated in v-B-CLL group (23/30) than B-CLL (P=.03); the percentage of CFM Zap-70 positivity, evaluated in 42/63 cases, was similar in two groups (47.6 vs 57.4%, respectively). Interestingly, significant differences were found about the frequence of the recurrent chromosome alterations, evaluated in 48/63 cases, by means of FISH analysis: trisomy 12 was more frequent in v-B-CLL (37.5 vs 18.4%, p=0.02), while del13q14 was more frequent in B-CLL (14.5 vs 37.8%, P=0.007). In both groups, del 11q22.3 and del 17p13.1 were relatively rare (4 vs 8%, and 2 vs 7.7%, respectively). In conclusion, our study identifies, on the basis of a defined CFM-FISH diagnostic approach, a variant form of B-CLL that shows significant differences in terms of genetic and clinical features.
Epigenetic Profiling Identifies EGLN3 as a Frequent Target for Transcriptional Silencing in Plasma Cell Neoplasias.
Authors: Shah Reshma , Hatzimichael Eleftheria , Syed Nelofer , Bourantas Konstantinos L. , Crook Tim 
Published: October 2019

Abstract
Background: Multiple myeloma (MM) results from an excess of monoclonal plasma cells in the bone marrow. MM cells are interleukin (IL)-6 and stromal cell dependent. Although the molecular signature of MM cells has been identified, the signature of the stromal component of the BM microenviroment is not yet fully determined and holds important clues to the understanding of disease pathobiology and progression. It has been suggested that bone marrow hypoxia is lessened during myeloma progression and that myeloma-associated angiogenesis is functional. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor and a master regulator of hypoxia responsive genes in cancer, which promote angiogenesis, vasodilation and glycolysis. As part of a programme to seek novel genes subject to epigenetic regulation in B cell neoplasia, we have identified the egl9 homologue 3 (EGLN3) as a frequent target for methylation-dependent transcriptional silencing in multiple myeloma (MM) and Waldenström’s macroglobulinemia (WM). EGLN3 is a prolyl hydroxylase (POH) that functions as a cellular oxygen sensor that targets HIF-1 alpha for proteasomal degradation via the von Hippel-Lindau (VHL) complex.
Patients and Methods: Thirty six MM patients (18 male, 18 female, age range 50–87 years), 4 patients with WM (2 male and 2 female, age range 47–75 years) and two patients with monoclonal gammopathy of undetermined significance (MGUS; 2 male age 56 and 67 years old) were included in the study. Twenty out of 36 MM patients had advanced stage disease (Durie Salmon stage≥II, IPI≥II). All samples were taken at diagnosis except for 5 that were taken when progression occurred from plateau phase. The Durie-Salmon staging system and the International Prognostic Index (IPI) were used for clinical and prognostic discrimination of patients. Bone marrow aspiration was performed with aseptic technique. Genomic DNA was isolated with a commercially available kit (QIAmp DNA mini kit, Qiagen).
Results: We identified a CpG island in the 5′ sequences of the EGLN3 gene. Using bisulphite sequencing and methylation specific PCR, we observed aberrant CpG methylation in 17/36 cases of MM and 1/4 cases of WM. Expression of EGLN3, assessed by quantitative PCR (qPCR) correlated inversely with methylation. In contrast, we detected no methylation in the related EGLN1 or EGLN2 in either MM or WM. Cases with methylated EGLN3 were more likely to have bone lytic lesions (r=0.39, p≤0.05), but there was no correlation with extramedullary disease, hemoglobin<10 mg/dl or advanced disease stage.
Conlusions: EGLN3 is frequently methylated in patients with multiple myeloma. We suggest that methylation of EGLN3 leading to decreased HIF-1a degradation could be a possible mechanism of increased angiogenesis and altered bone marrow microenviroment that is more supportive for survival and growth of MM cells. The role of POH in MM merits additional study.
GA101, a Novel Humanized Type II CD20 Antibody with Glycoengineered Fc and Enhanced Cell Death Induction, Mediates Superior Efficacy in a Variety of NHL Xenograft Models in Comparison to Rituximab.
Authors: Friess Thomas , Gerdes Christian , Nopora Adam , Patre Monika , Preiss Susanne , van Puijenbroek Erwin , Schuell Christine , Bauer Sabine , Umana Pablo , Klein Christian 
Published: October 2019

Abstract
GA101 is a novel humanized and glycoengineered CD20 antibody that was derived by humanization of the parental B-Ly1 mouse antibody and subsequent glycoengineering leading to the following characteristics: high affinity binding to the CD20 type II epitope; low complement dependent cytotoxicity (CDC) activity; high direct cell death induction; and high antibody-dependent cellular cytotoxicity (ADCC) potency. In vivo studies examined the consequences of GA101 on the growth of aggressive NHL xenograft models in SCID beige mice and the subsequent effects on survival. These studies demonstrated an outstanding dose-dependent anti-tumor activity of GA101. In particular, GA101 mediated superior anti-tumor efficacy in staged s.c. Z138 mantle cell lymphoma (MCL) and SU-DHL4 diffuse large B cell lymphoma (DLBCL) xenograft models in direct comparison to rituximab. Weekly dosing of GA101 at 10 mg/kg (Z138 model) or 30 mg/kg (SU-DHL4) was able to induce complete tumor remission and long-term survival (cures) in all animals. Consistent anti-tumor efficacy against established tumors was also observed in a number of additional NHL models including Raji Burkitt’s lymphoma or OCI-LY18 DLBCL models. In addition GA101 was evaluated in an orthotopic disseminated Z138 model. After i.v. injection of Z138 cells SCID beige mice developed large intraperitoneal lymphoid tumors in the ovary. Several experiments showed that GA101 mediated increased overall and median survival in this model in direct comparison to rituximab. All xenograft studies were performed in SCID beige mice that are incompetent for NK-mediated ADCC. Indeed, control experiments with non-glycoengineered GA101 in s.c. xenograft models suggest that the superiority of GA101 does not depend on enhanced interactions of the engineered Fc region with murine effector cells such as macrophages/monocytes or granulocytes. Thus, the anti-tumor effects of GA101 in s.c. xenograft models can be primarily attributed to non-effector cell mediated activities. Currently, experiments are ongoing to evaluate the efficacy of GA101 in combination with classical chemotherapy regimens and novel targeted agents. First results from these studies will be presented during the 2007 ASH Annual Meeting. In summary it can be assumed that the combination of the recognition of a type II epitope together with improved ADCC potency might translate into superior efficacy in the clinical treatment of CD20 positive malignant diseases.
Structural Determinants for Heparin Binding in Human Coagulation Factor XI.
Authors: Shikov Sergei , Wu Wenman , Walsh Peter N. 
Published: October 2019

Abstract
Previous studies from our laboratory and others have demonstrated that zymogen factor XI (FXI) binds to heparin with moderate (KD ∼110 nM) affinity via residues (K252, K253 and K255) located in the Apple 3 (A3) domain. In contrast, the enzyme, FXIa, was shown to bind to heparin (Biochemistry40: 7569–7580, 2001) with significantly higher affinity (KD ∼9 nM by SPR and ∼1.5 nM by ELISA) via residues (K529, R530 and R532) within the catalytic domain (CD). This interaction potentiates by ∼10-fold the inhibition of FXIa by protease nexin-2. Also, polyanions heparin and dextran sulfate inhibit the catalytic activity of the enzyme factor XIa. The present study was designed to determine the relative contributions of positively charged residues as well as the dimeric structure of FXI to heparin binding. Mutational analysis of full-length FXI expressed in HEK293 cells was based on the following criteria:
Conservation of the positively charged residues in FXI among various species; Surface exposure of the residues based on the X-ray crystal structure of FXI (Papagrigoriou E, McEwan P, Walsh PN, Emsley J,Nat. Struct. & Mol. Biol. 2006; 13:557–558); and comparison with human plasma prekallikrein (PK), which does not bind heparin.
Two positively charged residues Arg507 (147, chymotrypsin numbering) and Arg532 (173) are conserved in FXI genes of all species for which sequences are available. In human PK, Arg507 is replaced by lysine, while Arg532 is replaced by a neutral glutamine. We have expressed and purified wtFXI, R507A, R532A as well as monomeric C321S/K331A and C321A/I290A. While wtFXI, R507A and R532A demonstrated normal activity in APTT assays; monomeric FXI mutants retained 60-70% activity. The R532A and R507A mutants demonstrated ∼75% decrease in total number of heparin binding sites based on the solid phase ELISA assay using 5F7 monoclonal antibody. Also, the apparent dissociation constants for R507 (11 nM) and R532A (22 nM) were 7 and 11-fold increased respectively compared with 1.6 nM for the wtFXI. We also characterized monomeric FXI C321S/K331A and C321A/I290A proteins for their ability to bind to heparin compared with wtFXI using surface plasmon resonance (SPR). Surprisingly, the monomeric FXI mutants, C321S/K331A and C321A/I290A, which had no mutations in any heparin-binding regions, displayed major defects in binding to heparin by SPR. Although kinetic analysis is challenging due to complex binding kinetics, while Rmax is about 10-fold lower, the off-rate for the binding of the monomeric FXI mutants is drastically increased when compared to that of wtFXI. These results suggest the possibility that the unique dimeric structure of FXI is required for cooperative binding to heparin. Thus, the dimeric structure of FXI and basic residues R507 and R532 in the catalytic domain of factor XI are both necessary for high-affinity heparin binding.