bluuebunny/crossref_metadata_2025_split huggingface dataset:
Localization of Stereo-Specific Phosphatidylserine Binding Sites within the Factor V C2 Domain.
Authors: Liang Xiaoe , Quinn-Allen Mary Ann , Kane William H. 
Published: October 2019

Abstract
Binding of activated factor V (Fva) to phosphatidylserine (PS) is required for formation of the prothrombinase complex on the surface of activated platelets. The interaction between Fva and PS has been shown to be stereo-specific with bovine Fva favoring L-PS over D-PS. Our previous low resolution alanine scan data localized PS binding sites to the FV-C2 domain and identified two tryptophan residues, W2063 and W2064, which are required for high affinity binding. Two putative PS binding sites were also predicted based on modeling studies using the FV-C2 structure. In this study, the stereo-specific interaction of Fva with PS was evaluated with plasma derived human Fva (pd-HFVa), recombinant human Fva (rHFVa), rHFVa-C2 domain and rHFVa mutants. Quantitative fluorescence binding assays and functional prothrombinase assays were conducted using phospholipid vesicles containing 20 mol% dioleoyl phosphatidyl-L-serine (L-PS) or dioleoyl phosphatidyl-D-serine (D-PS). Direct binding experiments demonstrated that pd-HFVa and rHFVa bound to membranes containing L-PS with 6 to 9 fold higher affinity than to membranes containing D-PS. Analysis of prothrombinase activity on these membranes demonstrated the difference in Vmax/K1/2Va between L-PS and D-PS was 15-fold and 23-fold higher for pd-HFVa and rHFVa, respectively. These data confirm that HFVa, similar to bovine FVa, prefers L-PS over D-PS. The rHFV-C2 domain bound to L-PS membranes with an affinity of 5.3 ± 1.7 nM. In contrast, the binding of C2 domain to D-PS membranes was undetectable. This indicates that the rHFV-C2 domain contains a stereo-specific binding site for PS. We next analyzed the membrane binding of four rHFVa mutants containing alanine substitutions within the FV-C2 domain. rHFVa mutants W2063A and W2064A both bound to membranes containing 20% L-PS with high affinity similar to native rHFVa with Kd’s of 0.97 ± 0.30 nM and 0.38 ± 0.18 nM, respectively. In contrast no detectable binding of W2063A or W2064A could be detected on membranes containing 20% D-PS. Mutants were also prepared containing alanine substitutions for amino acid side chains predicted to participate in two stereospecific PS binding sites based on modeling studies using the rHFV-C2 structure. The binding of (K2060, Q2085, S2115)A and (Q2078, N2082, R2187)A to L-PS and D-PS were characterized as described above. These mutants bound to both L-PS and D-PS membranes with relatively high affinity. The Kd values for binding of (K2060, Q2085, S2115)A and (Q2078, N2082, R2187)A to 20% D-PS membranes were 1.19 ± 0.31 nM and 1.11 ± 0.32 nM, respectively. These results suggest that these two sites play little or no role in the binding of FVa to PS membranes. In conclusion, the HFV-C2 domain contains stereo-specific binding sites for L-PS which require the indole side chains of W2063 and W2064. These findings provide new insights into the regulation of prothrombinase activity by PS.
Molecular Background of “Smeary” von Willebrand Factor Multimers.
Authors: Schneppenheim Reinhard , Marggraf Olivier , Eckert Heike , Obser Tobias , Oyen Florian , Pieconka Antje , Schneppenheim Sonja , Will Kerstin , Budde Ulrich 
Published: October 2019

Abstract
Background: Multimer analysis of von Willebrand factor (VWF) is a most important technique to classify patients with von Willebrand disease (VWD) type 2. Besides “classical” multimer patterns a “smeary” appearance of individual VWF oligomers is increasingly observed and has previously been regarded as a pre-analytical artifact.
Objective: To phenotypically and genotypically assess the molecular background of “smeary” VWF multimers.
Patients and methods: Samples of 8 VWD patients were analyzed in our reference lab (UB) for further classification and molecular testing. Multimer profiles were assessed by intermediate resolution gels. VWF:CB and VWF:GpIb binding were used as functional assays. VWF gene mutation analysis was performed in all index cases (IC). The causal relationship between genotype and phenotype was studied by analyzing recombinant mutants in comparison to wildtype VWF.
Results: In all IC the phenotype correlated with particular mutations in the VWF D3 domain (G1172D), the A1 domain (R1315C, R1374S, R1374C, R1399C), the D4 domain (C2257R), the C1 domain (R2464C) and in the region close to the CK domain (C2671Y), respectively. The multimer patterns of recombinant mutant VWF was of a “smeary” appearance and closely resembled those of mutant plasma VWF. Mutations in the A1 domain additionally correlated with severe GpIb binding deficiency.
Conclusions: Our data suggest a molecular cause of the “smeary” multimer structure rather than pre-analytical artifacts. Most of the mutations identified involved cysteine residues suggesting an influence on the VWF secondary structure which is determined by intra- and intermolecular disulfide bonds. This could explain the peculiar multimer appearance. The functional deficits, however, seem to depend on the location of the mutations with a significant impact on GpIb binding of mutants in the A1 domain.
Bortezomib Retreatment in Relapsed Multiple Mycelia - A Retrospective Multicenter Survey.
Authors: Hrusovsky Irena , Emmerich Berthold , Engelhardt Monika , Kornacker Martin , Gann Claudia-Nanette , Pliskat Harold , Hess Georg 
Published: October 2019

Abstract
Bortezomib (Velcade) has demonstrated highest single agent response rates in anti-myeloma therapy. There is no data from preclinical studies to suggest that resistance develops from repeated treatment with bortezomib. It was therefore of interest to investigate, whether repeated bortezomib treatment is safe, feasible and efficient. Here, we have retrospectively collected data from multiple myeloma patients who had previously responded to bortezomib (previous bortezomib treatment), presented with relapsed disease and who received bortezomib for a second time (retreatment). Treatment and retreatment had been on discretion of the physician according to prescribing information, and no diagnostic or therapeutic instructions were given during the course of this multicenter non-interventional survey (26866138MMY4014). Data from from a total of 15 centers and 65 patients were obtained: these patients had all received bortezomib and were eligible for safety analyses. Sixteen patients had to be excluded due to major protocol violations, leaving 49 patients for the modified intention to treat population. Patients had a median age of 66 years and had been treated with a median of 4 prior therapies before receiving bortezomib for the first time. Median cycle number for previous bortezomib therapy and retreatment was 5 and 4, respectively. The majority of patients (85.7%) received doses of 1.3 mg/m2 body surface area. Concomitant dexamethasone was given in 38.8% of patients with previous bortezomib treatment, and in 62.2% with retreatment. Six patients (12.2%) received various anti-myeloma therapies between bortezomib treatment and retreatment. Efficacy data is summarized below, revealing very encouraging responses and similar response durations for previous bortezomib therapy and retreatment.
Thirty-three (50.8%) patients experienced a total of 109 adverse drug reactions (ADRs). Three (4.6%) patients experienced a total of 12 life-threatening or disabling ADRs and six (9.2%) experienced 13 severe ADRs (related to bortezomib = 6 SADRs). By the end of the documentation period, 21 patients had died (32.8%). This retrospective survey suggests that the safety profile of bortezomib retreatment is in line with the current SmPC of Velcade and that high remission rates can be achieved. It remains to be shown if these bortezomib retreatment results can be repeated in prospective trials and larger patient cohorts.
Previous bortezomib treatment Retreatment Overall response rate 100% (per protocol) 63.3% Complete remission rate 12.2% 10.2% Time to response (median) 3.2 months 3.0 months Duration of response (median) 6.3 months 4.5 months Treatment free interval (median) 6.6 months 4.1 months Time to progression (median) 10.9 months 6.7 months
Longer Duration of Thalidomide Monotherapy Results in Improved Outcome in Relapsed/Refractory Multiple Myeloma.
Authors: von Lilienfeld-Toal Marie , Hahn-Ast Corinna , Bertolini Francesco , Bila Jelena , Boulin Mathieu , Oakervee Heather , Cibeira Teresa , Cook Gordon , Dmoszynska Anna , Fenk Roland , Guglielmelli Tommasina , Goldschmidt Hartmut , Hattori Yukata , Myers Bethan , Offidani Massimo , Patriarca Francesca , Petrucci Maria Teresa , Pini Massimo , Prince Miles , Schey Steve , Sonneveld Pieter , Yakoub-Agha Ibrahim , Waage Anders , Glasmacher Axel 
Published: October 2019

Abstract
Although efficacy of thalidomide (thal) monotherapy in relapsed/refractory Multiple Myeloma (MM) is proven, the optimal duration of treatment is unknown. Here, we analysed individual patient data of 852 patients (pts.) with relapsed/refractory MM from 21 centres who received thal monotherapy in a trial or in a compassionate use program. To assess the influence of treatment duration, pts. who discontinued thal because of lack of efficacy, progression or death were excluded, so we analysed only pts. who continued on thal as long as they tolerated it or until the study they were enrolled in was complete. 162 pts. were evaluable; 32 completed the trial, 57 had severe neurotoxicity, 2 severe somnolence, 72 other adverse events leading to discontinuation of thal. The median age was 65 years (interquartile range 58–72), 92 (56%) were male, 96 (59%) had relapsed MM. Sixteen (10%) achieved complete remission (CR), 64 (39%) partial remission (PR), 29 (18%) minimal response, 34 (21%) no change, 9 (6%) had progressive disease, 9 (6%) were unevaluable for response. The median daily dose at 3 months was 200 mg/d (100–400), the cumulative dose after 3 months 23200 mg (9025–34150). Median time to response was 3.6 months (3–7), median treatment duration 5 months (2.3–12). Median event-free survival (EFS) was 16 months (95%CI 12–21), median overall survival (OS) 33 months (95%CI 22–44). In univariate analysis, prognostic factors for EFS/OS were leukocyte/platelet count, haemoglobin, cumulative dose at 3 months, duration of treatment and CR/PR. In multivariate analysis, duration of treatment, CR/PR and platelet count remained independent prognostic factors for EFS/OS. If pts. took thal for ≥ 10 months (n=51), median EFS was 31 (95%CI 14–48) vs 12 (9–14, p<0.001) months and median OS 83 (95%CI 49–116) vs 24 (95%CI 17–31, figure 1, p<0.001) months. We analysed responding pts. (n=80) separately to assess the role of treatment duration in maintenance. Patients in CR/PR who took thal ≥ 10 months (n=41) had a longer EFS (39 (95%CI 19–59) vs 18 (95%CI 9–28, p<0.001) and OS (83 (95%CI 49–116) vs 32 (95%CI 20–44, p<0.001) months). This analysis of patients whose duration of treatment was determined by factors independent of disease demonstrates a clear survival benefit of longer duration of thal treatment for relapsed/refractory MM and supports its value as maintenance therapy.
Figure Figure
Recruitment of Rad50 and MCM Complexes on the Human Beta Globin Locus Control Region (LCR): A Novel Role for LCR Hypersensitive Site 4 (HS4).
Authors: Karmakar Subhradip , Mahajan Milind C. , Weissman Sherman M. 
Published: October 2019

Abstract
The human beta globin locus control region (LCR) aids in the establishment of an open chromatin configuration thereby facilitating the expression of linked downstream globin genes in a temporal and spatial manner along the developmental ontogeny. The LCR also functions as a classical enhancer of the beta like globin genes. The four DNAase-I hypersensitive regions (HS 1–4) on the LCR are believed to act as nucleation centers for the assembly of multi-protein transcriptional complexes that interact with the distant gene promoters. Present work is focused on characterizing the protein complex assemblies on the HS4 region and delineating their function in the context of goblin gene regulation. Our finding shows an assembly of a 2–5 MDa complex on the HS4 locus that is both erythroid restricted and sequence specific. Electrophoretic mobility shift assays (EMSA) were performed using K562 and HeLa nuclear extracts and ∼35mer double stranded oligonucleotides with 7 bp overlaps tiling the entire core HS4 sequence. We detected prominent K562 specific bands formed with an AT rich 39 bp HS4 sequence. Sequence specificity of DNA-protein complex formation on this AT rich sequence was determined by competitive EMSA assays. Gel supershift assays with a wide range of antibodies and subsequent partial purification of this multiprotein complex followed by the LC-MS/MS analysis reveal the presence of a mini chromosome maintenance (MCM) complex that is associated with the DNA replication, DNA repair associated Rad50-Mre-11-NBS-1 complex, histone acetyl transferase p300 and DNA binding transcription factors ILF2 (NF45) and ILF3 (NF90). The association of these proteins was determined by co-immunoprecipitation experiments. Chromatin immunoprecipitation (ChIP) experiments demonstrate the in vivo recruitment of these proteins on the HS4 region of the LCR. We are currently working on further characterization and functional studies of this complex and its significance on the role of the LCR in DNA replication and repair.
Bortezomib, Doxorubicin and Dexamethasone (PAD) Combination Therapy Followed by Thalidomide and Dexamethasone (TD) as a Salvage Treatment for Relapsed Multiple Myeloma (MM): Preliminary Analysis of Efficacy and Safety.
Authors: Lee Sung Sook , Suh Cheolwon , Kim Bong-Seog , Chung Jooseop , Joo Young-Don , Ryoo Hun-Mo , Do Young Rok , Jin Jong-Youl , Kang Hye Jin , Lee Gyeong-Won , Lee Moon Hee , Shim Hyeok , Kim Kihyun , Yoon Sung-Soo , Kim Ho Young , Lee Dong Soon , Lee Jae-Hoon 
Published: October 2019

Abstract
Introduction: PAD was reported to be highly effective regimen as an induction therapy before high dose therapy. TD is an another effective regimen with no cross resistance. We conducted a phase II study with PAD followed by TD in relapsed MM to test effectiveness of this combination.
Method: Patients were planned to receive 6 cycles of PAD, (bortezomib 1.3 mg/m2 days 1, 4, 8 and 11, doxorubicin 4.5 mg/m2 days 1–4, dexamethasone 40 mg days 1–4, every 21 days). Responders following 6 cylces of PAD received 12 cycles of TD (thalidomide 100 mg days 1–28 and dexamethasone 40 mg days 1–4, every 28 days). In patients with progression during PAD therapy, regimen was changed to 12 cycles of thalidomide 200 mg days 1–28 and dexamethasone 40 mg days 1–4, every 28 days.
Result: This study aimed to enroll 40 patients till Oct 2007 and we are reporting preliminary result with 35 patients. Efficacy could be assessed in 29 patients. After two cycles of PAD, 29 patients showed response with 5 CR. Overall response rate to 6 cylces of PAD was 96.4% with 39.3% CR. Six of total 13 patients with TD showed further improvement of response status with 5 additional CR. Overall response to PAD followed by TD was 92.8%: CR 57.1%, nCR 10.7%, VGPR 7.1%, PR 17.9%, SD 3.6%, PD 3.6%. There was no prognostic factor for CR+nCR achieving in the univariate analysis. The median follow-up was 8.4 months with 1 year PFS 66.8% and 1 year OS 59.4%. One hundred fourty-six PAD cycles (median 5, range 1–6) in 35 patients were assessable for safety. The most common hematologic toxicity was thrombocytopenia, with grade 3/4 in 37.1%. Grade 3/4 neutropenia was observed in 5.8%. Sensory neuropathy occurred with grade 2 in 31.4% and grade 3 in 8.6%. The median dose intensity was 1.44 mg/m2/week for bortezomib and 5.25 mg/m2/week for doxorubicin, which correspond 83.2% and 87.5% of the planned dose intensities, respectively. A total of 112 TD treatment cycles (median 0, range 0–12 cycles) was administered. Two patients developed grade 3 neutropenia. One patient die by the pneumonia.
Conclusion: PAD followed by TD in patients with relapsed multiple myeloma is very active and tolerable. Updated results will be presented at the meeting.
*Protocol Number: KMM55-NCT00319865
Effect of Disease Stage and Time Since Diagnosis on Time to Progression for Pegylated Liposomal Doxorubicin + Bortezomib vs Bortezomib Alone in Relapsed or Refractory Multiple Myeloma.
Authors: Sutherland Heather J. , Blade Joan , Miguel Jesus San , Nagler Arnon , Sonneveld Pieter , Spencer Andrew , Robak Tadeusz , O’Neal Wane , Mundle Suneel D. , Zhuang Sen H. , Harousseau Jean-Luc , Orlowski Robert Z. 
Published: October 2019

Abstract
A recent report of a phase III randomized trial with pegylated liposomal doxorubicin (PLD)+bortezomib vs. bortezomib alone in relapsed or refractory multiple myeloma (RRMM) demonstrated improved time to progression (TTP) with PLD+bortezomib (Orlowski, JCO 2007). The present post-hoc analyses evaluated the efficacy of PLD+bortezomib according to International Staging System (ISS) disease stage, and time since initial myeloma diagnosis (TSD). Patients with ≥1 prior therapy were randomized to receive PLD at 30 mg/m2 on day 4 and bortezomib at 1.3 mg/m2 on days 1, 4, 8, and 11, or bortezomib alone for up to eight 21-day cycles, or at least 2 cycles beyond CR, or optimal response unless disease progression or unacceptable toxicities occurred. The improved TTP reported previously with PLD+bortezomib over bortezomib alone in the total study population was also observed in the higher risk groups based on disease stage (ISS 2 & 3; Table). TTP was also significantly longer with PLD+bortezomib vs. bortezomib for TSD >2 yrs despite more protracted disease. The therapeutic advantage of prolonged TTP with the PLD+bortezomib combination was maintained consistently across subgroups (heterogeneity tests: ISS 1 vs. 2 vs. 3, p=0.407; TSD ≤2 yrs. vs. >2 yrs., p=0.946). Incidence of grade 3/4 neuropathies was low (<10%) in the two treatment arms in all subgroups. PLD-related hand-foot syndrome was also <10% in all PLD+bortezomib subgroups. Despite high-grade disease or protracted disease history, the PLD+bortezomib combination shows significantly improved TTP as compared to bortezomib alone. Also, the combination therapy was well-tolerated.
Use of Gene-Modified T-Cells as Antigen Presenting Cells (T-APC) for Vaccination Against Myeloma Antigens.
Authors: Anderson Larry D. , Cook Danielle R. , Berger Carolina , Maloney David G. , Riddell Stanley R. 
Published: October 2019

Abstract
Multiple myeloma is incurable with currently available standard treatments, and novel therapies with lower toxicity, including immunological therapies, are needed. Myeloma cells express class I MHC molecules and several potentially immunogenic proteins. Candidate target antigens, other than the patient-specific immunoglobulin idiotype, include cancer-testis antigens, such as MAGE-C1 (CT-7), and tissue differentiation antigens, such as B-Cell Maturation Antigen (BCMA). We have isolated, from normal donors, CD8+ T-cells that are CT-7 and BCMA peptide-specific, suggesting that immunologic tolerance to these proteins is incomplete. A subset of these CD8+ T-cells with the highest avidity are capable of recognizing myeloma cells. However, the frequency of T-cells recognizing these peptides is very low. Vaccination offers a potential approach for augmenting the frequency of tumor-reactive T-cells, provided that a vaccine strategy can be developed that elicits high-avidity T-cells. We have previously shown that adoptive transfer of autologous gene-modified T-cells leads to the rapid development of potent CD8+ T-cell immunity against transgene products expressed in the transferred T-cells. The aims of this study were to determine if vaccination with T-cells modified to express the CT-7 or BCMA genes (T-APC) could induce anti-myeloma immunity in an HLA-A2 transgenic mouse model, and to map additional immunogenic epitopes of these proteins presented by HLA-A2. To address this we genetically modified HLA-A2 transgenic mouse T-cells with retroviral vectors encoding the candidate myeloma antigens CT-7 and BCMA, or with a control vector encoding green fluorescent protein (GFP). The gene-modified T-APC were injected intravenously into HLA-A2 transgenic mice, and ELISPOT and cytotoxicity assays were performed on splenocytes from immunized mice to evaluate T-cell recognition of target cells expressing CT-7 or BCMA (including human myeloma cell lines). T-cell responses specific for CT-7, BCMA, and control antigens were elicited following gene-modified T-APC immunization, and in some cases these T-cells were capable of recognizing human myeloma cells. Furthermore, responses to CT-7 have been mapped to two different dominant CT-7 peptides. CD8+ T-cells from some normal A2+ donors also recognized these two epitopes after in vitro peptide stimulation, suggesting that these epitopes may be applicable to human myeloma therapy. Characterization of the BCMA target peptide(s) is ongoing. The ease with which T-cells can be obtained, genetically-modified, and expanded in vitro, together with data showing potent immunogenicity, suggests that T-APC vaccination should be evaluated as an alternative cell based vaccine strategy for human cancer therapy.
Inhibition of KSP by ARRY-520 Induces Cell Cycle Block and Cell Death Via the Mitochondrial Pathway in AML Cells.
Authors: Carter Bing Z. , Mak Duncan H. , Schober Wendy D. , Woessner Richard , Gross Stefan , Harris David , Estrov Zeev , Andreeff Michael 
Published: October 2019

Abstract
Kinesin spindle protein (KSP or Eg5), a microtubule-associated motor protein, plays an important role in establishing a bipolar spindle during mitosis and is essential for cell cycle progression. It has been demonstrated that inhibition of KSP prevents bipolar spindle formation leading to mitotic arrest and cell death. As such, a KSP inhibitor may have anti-tumor potential without toxicities associated with anti-microtubule agents such as taxanes. We found that KSP is highly expressed in various acute leukemic cells. Inhibition of KSP by a specific inhibitor, ARRY-520, at low nanomolar concentrations, blocked cell cycle progression and led to subsequent cell death in OCI-AML3, Molm13, HL-60, U937, and Jurkat cells. Knocking down p53 by p53shRNA in OCI-AML3 cells did not alter the effectiveness of ARRY-520. U937 cells overexpressing XIAP (U937XIAP) and Jurkat cells lacking caspase 8 (JurkatI2.1) showed unchanged sensitivities to ARRY-520 compared with their respective control cells, suggesting that cell cycle block and cell death induced by KSP inhibition is independent of p53 status, XIAP levels, and the activation of the extrinsic apoptotic pathway. However, although ARRY-520 blocked cell growth and induced mitotic arrest in both HL-60 cells and HL-60 cells overexpressing Bcl-2 (HL-60Bcl-2), cell death (determined by annexin V staining and changes in mitochondrial potential) was significantly abolished in HL-60Bcl-2 cells. These results suggest that cell death following cell cycle blockade by KSP inhibition is mediated through the intrinsic mitochondrial pathway. Furthermore, ARRY-520 induced the protein level of Bim, a proapoptotic BH3-only Bcl-2 family protein, prior to the activation of caspases in HL-60 cells. Although KSP inhibition by ARRY-520 had no effect on the survival of non-dividing AML blasts in vitro, ARRY-520 significantly inhibited the colony formation capacities of AML blasts, further supporting the critical role of KSP in cell proliferation. Our studies demonstrated that inhibition of KSP by ARRY-520 potently induces blockage of cell cycle progression which leads to cell death of various leukemic cells via the mitochondrial pathway and has the potential to eradicate AML progenitor cells.