bluuebunny/crossref_metadata_2025_split huggingface dataset:
Non-Classical Anti-Factor VIII C2 Domain Antibodies Are Pathogenic in a Murine in Vivo Bleeding Model.
Authors: Meeks Shannon , Healey John F. , Parker Ernest T. , Barrow Rachel T. , Lollar John (Pete) 
Published: April 2020

Abstract
Approximately 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). In addition, autoimmune antibodies to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. Patients with congenital hemophilia who develop inhibitors usually have a polyclonal antibody response directed against the A2 and C2 domains of fVIII. Patients with acquired hemophilia typically have a more limited B-cell epitope response with antibodies directed against the A2 or C2 domain not both. Classical anti-C2 antibodies inhibit the binding of fVIII to phospholipid membranes and to von Willebrand factor. We recently have identified anti-C2 antibodies that inhibit the activation of fVIII, but do not inhibit the binding of fVIII to phospholipid membranes or to von Willebrand factor. These non-classical inhibitors are found in the plasmas of most inhibitor patients (Meeks, S.L. et al. Blood112, 1151-1153, 2008). The pathogenicity of classical and non-classical murine anti-human fVIII monoclonal antibodies (MAbs) was tested in a murine in vivo bleeding model. MAbs were injected into the tail veins of –hemophilia A mice to a peak plasma concentration of 60 nM followed by an injection human B domain-deleted fVIII to a concentration of 2 nM. At 2 hours the mice were anesthetized and a 4 mm tail snip was made. The amount of blood lost into a collection tube of normal saline over 40 minutes was measured. 4A4 is a type I anti-A2 inhibitor with an inhibitory titer of 40,000 Bethesda units (BU)/mg IgG. I54 and 1B5 are classical type I anti-C2 inhibitors with inhibitory titers of 1300 and 930 BU/mg IgG, respectively. 2-77 is a non-classical type II anti-C2 inhibitor that produces a residual fVIII level of 40% at saturating concentrations and whose titer is 21,000 BU/mg IgG. 2-117 is a non-classical anti-C2 MAb with inhibitory activity less than 0.4 BU/mg IgG. All of these MAbs except 2-117 significantly increased blood loss over control mice injected with fVIII alone (p= 0.01-0.02, Mann-Whitney Test) (Fig .1). The amount of blood loss was similar at these saturating concentrations of antibody despite inhibitory titers ranging from 930-40,000 BU/mg IgG. Increasing the dose of fVIII to 4 nM could overcome the bleeding diathesis produced by the non-classical MAb 2-77, but not the type I antibodies, 4A4 and I54. Similar results were seen in the in vitro Bethesda assay where 4A4 completely inhibited both 1 U/ml and 3 U/ml fVIII at saturating concentrations, while 2-77 had 40% residual activity with either 1 or 3 U/ml fVIII (0.4 U and 1.2 U respectively) (Fig. 2). These results suggest that high-dose fVIII rather than bypassing agents may be warranted in patients with an inhibitor response dominated by non-classical anti-C2 antibodies.
FIGURE 1. FIGURE 1. FIGURE 2. FIGURE 2.
A Unique Recombinant Plasmin Molecule Lacking Kringles 2-5 Possesses Equivalent Hemostatic Safety as Full-Length Human Plasmin.
Authors: Marder Victor J. , Novokhatny Valery , Manyak Steve , Gruber Theresa , Hunt Jennifer , Bromirski John , Goyal Abha , Scuderi Philip , Petteway Steve 
Published: April 2020

Abstract
Background: All thrombolytic agents in current clinical use are plasminogen activators (PA). While effective, PA have a downside because of unavoidable and potentially lifethreatening bleeding complications in vulnerable patients. We previously demonstrated a striking margin of hemostatic safety for the “direct-acting” thrombolytic enzyme, plasmin. Now, a novel recombinant derivative of plasmin has been synthesized which lacks kringles 2-5, consisting only of kringle 1 linked to the serine protease domain.
Methods: We evaluated escalating dosages of recombinant D (K2-K5) plasmin in a randomized and blinded manner in comparison with molar equivalent dosages of full-length plasmin in an animal model of fibrinolytic hemorrhage. Hemostatic safety was measured by primary bleeding time (PBT) and plasma concentrations of a-2 antiplasmin, factor VIII and fibrinogen.
Results: A dose-related nadir in a-2 antiplasmin, factor VIII and fibrinogen followed administration of 4, 6, 8 and 10 mg/kg of D (K2-K5) plasmin, with residual fibrinogen of 65%, 40%, 30% and 0% of initial pre-treatment concentrations. Hemostasis was undisturbed with 4 mg/kg of D (K2-K5) plasmin (mean PBT 2.2 +/- 0.7 min), minimally so at the 6 or 8 mg/kg dosages (5 +/- 2.9 and 4.4 +/- 2.2 min). Hemostasis was markedly abnormal at the purposefully toxic 10 mg/kg dose (12.8 +/- 18.8 min), in association with complete depletion of plasma fibrinogen. Comparison of agents at 10 mg/kg showed no difference, but at 4 mg/kg (2-4-fold more than needed for efficacy), D (K2-K5) plasmin showed slightly less prolongation of PBT (2.2 +/- 0.7 vs 3.3 +/- 2.1 min). Both agents were equally potent for in vitro whole blood clot lysis, indicating that the safety advantage of D (K2-K5) plasmin was not due to decreased fibrinolytic activity.
Conclusions: Recombinant D (K2-K5) plasmin has equivalent hemostatic safety as full-length plasmin in an animal model of fibrinolytic hemorrhage and warrants evaluation in clinical thrombotic disease.
Mutations of the SBDS Gene Result in Nuclear Mislocalization.
Authors: Yamaguchi Masafumi , Fujimura Kingo , Toga-Yamaguchi Hanae , Svetic Valentina , Okamura Naoki , Chopra Rajesh 
Published: April 2020

Abstract
Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported that Shwachman-Bodian- Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of granulocyte precursor cells. (Exp Hematol35; 579, 2007). A wide variety of mutations in SBDS gene has been identified, and almost of all patients show truncated immature proteins, p.K62X (c.183_184TA&gt;CT) or p.C84fsX3 (c.258+2T&gt;C). However, it is not yet clear how these truncated proteins affect cellular processes that result in the SDS phenotype. The SBDS protein is localized to the nucleoli but does not have the canonical nuclear localization signal. In order to clarify the molecular basis of pathogenicity of mutated SBDS proteins, we explored the subcellular distribution of normal and mutant SBDS proteins in Hela and 32Dcl3 cells. Using various N-terminal and C-terminal deletion constructs, we found N-terminal region, domain I (1-87 amino acid residue) in particular, was necessary to localize to the nucleus. The disease related mutations (C31W, K33E, N34I, L71P) and the mutations which are conserved among the species in the domain I (E44K, K62E, D70N, E82K) were generated. C31W and N34I mutants failed to localize SBDS to the nuclei. The SV40 derived nuclear localization signal was fused to these mutated SBDS protein, and these proteins were clearly localized to the nuclei. In addition to the mislocalization, the protein expression level of these mutants showed a dramatic decrease compared to the wild type. We also established SBDS wild type and domain I overexpressed 32Dcl3 cell. SBDS wild type overexpressed cells could differentiate to normal neutrophils in the presence of mG-CSF, however domain I overexpressed cells did not differentiate. Almost of all cells showed apoptosis in this domain I overexpressed cells in the presence of mG-CSF, and this was very similar like SBDS RNAi knockdown cells. The localization of endogenous SBDS protein was also analyzed in this domain I overexpressed cells. The domain I was concentrated to nuclei, however endogenous SBDS protein was diffused to cytosol.
Conclusions: The present findings enable us to document the nuclear localization signals in SBDS domain I, and that the shuttling protein would promote SBDS to nuclei. These results also showed that mislocalization and/or low expression level of mutated SBDS protein would cause SDS.
Reactive Oxygen Species Regulate Spontaneous and Fas-Mediated Apoptosis in SBDS-Deficient Cells.
Authors: Ambekar Chhaya , Das Bikul , Yeger Herman , Dror Yigal 
Published: April 2020

Abstract
Background and hypotheses: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, pancreatic insufficiency, and a marked propensity for myelodysplastic syndrome and leukemia. Approximately 90% of the patients have mutations in the SBDS gene, but the function of the gene is unknown. We previously showed that marrow cells from SDS patients and SBDS-deficient HeLa Cells are characterized by accelerated apoptosis, overexpression of Fas and hypersensitive to Fas stimulation. Involvement of reactive oxygen species (ROS; oxidative stress) have been shown to be related to Fas hypersensitivity and overexpression in a variety of cell types. Therefore, we hypothesized that functional deficiency in SBDS in cells that express Fas could lead to impaired ROS generation and a subsequent increase in spontaneous and Fas-mediated apoptosis and decrease in cell growth.
Methods: We used shRNA-mediated SBDS-knockdown HeLa cells as a model. We investigated whether SBDS-deficiency increases ROS levels and if antioxidants can rescue the cell growth and apoptosis phenotype. To measure ROS formation cells were incubated with DCFH-DA and fluorescence measured in Gemini Spectra MAX microplate reader. Staining with annexin V and propidium iodide was done to determine apoptosis and necrotic cell death. MTT assay was used to measure cell viability.
Results: ROS levels in SBDS knockdown cells were significantly increased compared to control. Apoptosis analysis by annexin V and propidium iodide showed a marked decrease in cell viability in the SBDS-knockdown cells. NAC treatment decreased ROS levels, enhanced ERK phosphorylation (pERK), improved cell viability, and decreased apoptotic and necrotic cell death. Stimulation of the Fas signaling pathway by CH-11 (activating anti-Fas antibody) and Fas ligand showed increased ROS production in SBDS Knockdown cells. CH-11 treatment showed a marked increase in apoptotic and necrotic cell death after 24 and 48hrs incubation. Cell viability decreased by 40% and 80% after 24 and 48hrs incubation with CH-11. Treatment with NAC lowered ROS levels, enhanced pERK expression, protected the cells from Fas-mediated early apoptosis and improved cell survival.
Conclusion: We have demonstrated that stable loss of SBDS results in increased ROS levels, leading to apoptotic and necrotic cell death. Thus, increased baseline and Fas-stimulated ROS could result in increased sensitivity to apoptosis and necrotic cell death. NAC appeared to reverse the ROS-mediated decrease in cell survival and apoptotic cell death. Our data support the novel concept that SBDS may be a homeostatic regulator of oxidative stress
Recurrent Chromosomal Intermingling Interactions at the BCL2 Locus in T(14;18) +Ve and −Ve Cell Lines.
Authors: Yang Youwen , Wrench David J , Lister T. Andrew , Fitzgibbon Jude 
Published: April 2020

Abstract
Introduction Chromosomes locate non-randomly within the nucleus where they physically associate via intermingling. This physical association leads to co-ordinated regulation of gene expression but also makes these regions prone to translocations. The loci involved in many of the common translocations in lymphoma co-localise together and include IGH and BCL2 which are juxtaposed by t(14,18)(q32q21) in ~90% of Follicular Lymphoma (FL). This frequency suggests the BCL2 locus may be prone to intermingling, with the major breakpoint region (MBR) representing a potential interactive hotspot as it flanks a matrix attachment region (MAR) which tethers chromatin.
Methods-Results To investigate this possibility, intermingling associations with the BCL2 locus were determined using the Chromosome Conformation Capture technique (3C) in B-lympho-blastoid (HRC57 and NcNc), t(14;18) +ve (RL, SuDHL4, SuDHL6, DoHH2) and -ve (HT, SuDHL8, SuDHL5, RAJI) B-NHL cell lines. The 3C technique allows cross-linking of associating chromosomal segments, generating a library of DNADNA hybrid molecules. Intermingling profiles were established for the following three BCL2 fragments contained within this library:
a 10kb section containing the BCL2 promoter region including exons 1 and 2; a 7kb segment within intron 2 containing a highly Conserved Non-coding Sequence (CNS) (associated with gene regulation) and overlapping MAR; and a 6kb section containing part of exon 3 and the MBR with flanking MAR.
This selection contained 81% of the BCL2 coding sequence. The intermingling partners of these BCL2 fragments were amplified using either BCL2- “unknown DNA walking primer” or BCL2-IGH enhancer primer combinations and their identity determined by direct sequencing. Nine intermingling partners were identified from the 3C libraries of at least two cell lines. Of these, IGH enhancer–BCL2 promoter associations were identified in RL, SuDHL4 and DoHH2 and appear restricted to t(14;18)+ve cell lines. MYCBP2 (chr13q22), which functions as a transcriptional activator of MYC, associated with the CNS+MAR sequence within intron 2 and this association was detected solely in malignant cell lines. In comparison, OPCML (chr11q25), a tumour-related gene, associated with the BCL2 MBR+MAR region in all cell lines examined.
Conclusions These preliminary studies suggest that the BCL2 locus has multiple recurring intermingling partners. These may be specific to BCL2, as demonstrated for OPCML which was a consistent partner in all lines investigated, or may be restricted to cells containing the translocated allele as shown for the IGH enhancer.
High-Resolution Array-Based Comparative Genome Hybridization (CGH) Identifies Novel and Recurrent Regions in CLL.
Authors: Grubor Vladimir , Krasnitz Alex , Troge Jennifer E. , Meth Jennifer L. , Lakshmi B. , Kendall Jude T. , Yamrom Boris , Alex Garrick , Pai Deepa , Navin Nicholas , Hufnagel Lisa A. , Lee Yoon-ha , Cook Kerry , Allen Steven L. , Rai Kanti R. , Damle Rajendra , Calissano Carlo , Chiorazzi Nicholas , Wigler Michael H , Esposito Diane 
Published: April 2020

Abstract
Although we have some understanding of the genetic abnormalities occurring in B-cell chronic lymphocytic leukemia (CLL) and their association with clinical outcomes, there is an incomplete comprehension of all of the mutations contributing to disease development and progression. In most abnormalities previously observed, it has been difficult to pinpoint specific candidate genes, reflecting the inadequacy of present tools for assessing chromosomal damage.
We examined copy number changes in the genomes of B cells from 58 patients with CLL using a 390,000-probe microarray, enabling us to perform comparative genomic hybridization (CGH), at a resolution exceeding previously published studies. We observed at least one genomic lesion in each CLL sample and considerable variation in the number of abnormalities from case to case. Virtually all abnormalities previously reported were also observed, most of which were highly recurrent. We observed the boundaries of known events with greater clarity and identified lesions previously not described, some of which were also recurrent.
A newly identified 3.6 Mb deletion at 8p21.2-p12 includes the gene TRIM 35. A second novel deletion at 2q37.1 (587kb) encompasses the gene SP100/110/140. Of the refined regions, a 249kb region at 9p21.3 spanning the CDKN2A (p16-INK4) and a 156kb region at 18q23 containing NFATC1 are particularly interesting. In the case of NFATC1, the minimal region of overlap spans that single gene.
Furthermore, we tilized our arrays to examine the clonal heterogeneity of CLL within the same patient from mixed sub-populations. The presence of greater than 30% of B-cells with the CD38 cell surface marker has been associated with poor outcome in CLL. It is an open question whether this reflects genetic heterogeneity and possibly clonal evolution. To investigate this possibility, we analyzed CD38+ and CD38− fractions from individual patients and demonstrated that three out of the four patients examined had undergone clonal diversification leading to new subclones of appreciable size.
Additionally, we have utilized a 2.1 million probe ultra-high density (HD2) array from NimbleGen (Madison, WI), providing us with the capability to scan the human genome for copy number changes at a resolution of a probe every 1500 bp. Utilizing the HD2 array, we are presently re-analyzing all the CLL samples in our study, and in the process are discovering many more previously unpublished lesions, several of which pinpoint single genes. We will present the results of our findings and discuss the potential applications of high resolution CGH analysis in a clinical setting.
A High Number of Losses in 13q14 Chromosome Is Associated with a Worse Outcome and Biological Differences in Patients with B Chronic Lymphoid Leukemia.
Authors: Hernández José-Ángel , Rodríguez Ana-Eugenia , González Marcos , Benito Rocío , Fontanillo Celia , Sandoval Virgilio , Romero Mercedes , Martín-Núñez Guillermo , de Coca Alfonso García , Fisac Rosa , Galende Josefina , Recio Isabel , Ortuño Francisco , García Juan-Luis , de las Rivas Javier , Gutiérrez Norma-Carmen , Hernández Jesús-Maria , Miguel J.F. San 
Published: April 2020

Abstract
Background: Cytogenetic abnormalities in B chronic lymphoid leukemia (B-CLL) define groups of patients with different prognosis. Thus, patients with B-CLL and 13q14 deletion display a favourable outcome. However, whether the percentage of cells showing 13q-, as it has been demonstrated in other genetic aberrations such as loss of P53, could influence the prognosis or the biological characteristics of the disease remains unknown.
Aim: To analyze the clinical and biological characteristics of B-CLL displaying, at diagnosis, loss of 13q as the sole cytogenetic aberration.
Patients, material and methods: A total of 350 patients diagnosed of B-CLL were included. All the clinical data as well as FISH information (13q14, 12, 11q21, 14q32 and 17p13 probes) and molecular studies were carried out. In addition, a gene expression profile from a microarray-based analysis was also performed.
Results: One hundred and sixty-two patients (46.3 %) had 13q14 deletion. In 109 out of the 350 cases (31.1 %) loss of 13q was the sole cytogenetic aberration at diagnosis. Interestingly, in the subgroup of patients with ³ 80 % of cells with loss of 13q (18 cases, 15.5%), the OS was 143 months (95 % CI: 104 m-154 m), while in the group with &lt; 80 % of losses in 13q, the OS has not been reached (95 % CI: 175 m-253 m). In the multivariate analysis for OS in patients with 13q-, the variables included in the final model were the percentage of losses of 13q14 (P = 0.001) and the presence of B symptoms (P = 0.007). Regarding the time to the first therapy (TFT), in the group with more than 80 % of losses in 13q the median TFT was 134 months while in the group of patients with &lt; 80 % of losses it was 38 months (P = 0.05). In the multivariate analysis for TFT in patients with 13q- the variables selected were unmutated status of IgVH (P = 0.001) and a high level of b2microglobulin (P = 0.003). In addition, the gene expression profile showed that patients with a high number of losses in 13q14 had a high proliferation rate, downregulation of genes apoptosis-related, and deregulation of genes related to mitochondrial functions compared to B-CLL patients with low numbers of 13q- cells.
Conclusion: B-CLL patients with a high number of losses in 13q14 as the sole cytogenetic aberration display different clinical and biological features: short OS and TFT as well as more proliferation and less apoptosis. Therefore, a quantification of the number of cells showing a genetic abnormality should be included in the study of the prognostic factors of B-CLL.
Invariant Deletion of One Mir-34b/C Locus in CLL with del11q.
Authors: Malek Sami , Ouillette Peter 
Published: April 2020

Abstract
CLL with del11q constitutes a clinical syndrome characterized by a relatively aggressive disease course, short response duration to therapies and compromised overall survival. CLL with del11q carries mutations in ATM in about one third of cases, which may further negatively affect prognosis. Little is known about additional molecular changes within or due to del11q in CLL and it remains unclear what the selective advantage of acquisition of del11q is for CLL cells.
To gain insights into the physiology of CLL with del11q, we have mapped this lesion using the Affymetrix 50K SNP array platform in 178 CLL cases using DNA from sorted CD19+ cells versus paired buccal DNA. Focusing on the region that spans ATM, a minimal deleted region (MDR) of approximately 8 Mb was identified, spanning approximately 106.6–114.6 Mb physical position.
Scanning the MDR for genes with the potential to affect the physiology of cancerous cells uncovered invariant monoallelic deletion of the miR-34b/c gene, the only known human microRNA gene within the del11q MDR. miR-34a-c have been implicated as direct p53 target genes by a number of groups and these microRNAs have been shown to be direct regulators of pro-proliferative and anti-apoptotic genes, including E2F3, Cyclin E2, Cdk6 and Bcl2. Given that ATM acts upstream of p53 in the ds-DNA damage response pathway, it appears that del11q lesions in CLL affect p53 apoptotic effector functions through two independent processes (ATM loss or mutation and miR-34b/c loss).
Work is in progress measuring miR-34 b/c expression levels in a large CLL cohort and correlating miR-34b/c levels with expression levels of critical miR-34 targets in CLL. In summary, our data implicate miR-34b/c loss as a potential contributor to the physiology of del11q in CLL and should stimulate investigations into miR-34 function as a response predictor of therapy for CLL.