bluuebunny/crossref_metadata_2025_split huggingface dataset:
Survivin Is Required for Terminal Differentiation of Erythroid Cells as Well as for the Proliferation and/or Survival of Hematopoietic Progenitors.
Authors: Crispino John D. , Leung Cindy , Xu Yanfei , Mularski Bretton , Liu Hui , Gurbuxani Sandeep 
Published: April 2020

Abstract
Survivin, the smallest member of the Inhibitor of Apoptosis (IAP) family of proteins, is expressed in proliferating cells, including fetal tissues, T-lymphocytes and hematopoietic progenitors. Survivin is also highly overexpressed in a wide spectrum of human cancers. Numerous studies have implicated survivin as a key mediator of the spindle assembly checkpoint and cytokinesis, while others have suggested an additional role as an inhibitor of apoptosis. In order to precisely examine the role of survivin in hematopoiesis in vivo, we induced acute deletion of the survivin gene in mice harboring either heterozygous or homozygous floxed alleles of survivin and the MX1-Cre transgene with polyinosinic-polycytidylic acid (pI-pC). Complete loss of survivin in homozygous floxed, MX1-Cre animals resulted in rapid death, which was associated with near total ablation of the bone marrow and marked hypocellularity of the spleen. The peripheral blood of these mutants displayed pancytopenia, including reticulocytopenia, and the presence of abnormal erythrocytes. Hematopoietic progenitors were markedly decreased, including c-kit+ cells, as well as CD71hiTer119hi erythroid precursors. Analysis of the cell cycle parameters revealed that a significant proportion of erythroid progenitors accumulated in G1, while others became polyploid. These defects could not be rescued by overexpression of the anti-apoptotic protein Bcl-2, consistent with a primary function of survivin in cell cycle progression. Bone marrow transplantation studies further confirmed that there exists a cell autonomous requirement for survivin in hematopoietic progenitors. Surprisingly, acute heterozygous deletion of survivin led to defects in erythropoiesis in approximately 20% of the animals. Upon pI-pC treatment, affected survivin heterozygous floxed mice with the MX1-Cre transgene exhibited a striking decrease in CD71−Ter119hi cells at the terminal stages of maturation. Morphological analysis of erythroid cells from neonatal spleens of these affected mice revealed a dramatic reduction in enucleated erythrocytes in the CD71−Ter119hi population compared to control animals. In addition, the majority of these erythroid cells were megaloblastic, with less condensed nuclear chromatin and nuclear-cytoplasmic dysynchrony. These observations suggest that there is a dose dependence for survivin. We predict that haploinsufficiency of survivin specifically affects “differentiation divisions” that occur concomitant with terminal maturation of red blood cells. Taken together, we show that homozygous deletion of survivin results in a dramatic loss of hematopoietic progenitors and rapid death, while heterozygous deletion leads to a selective defect in red blood cell development.
Transcriptome Classification of B-Cell Non-Hodgkins Lymphoma.
Authors: Aggarwal Mohit , Villuendas Raquel , Al-Shahrour Fatima , Aguilera Abel S. , Martinez Nerea , Ballesteros Elena R. , Camacho Francisca I. , Perez Alberto , Artiga Maria J. , Garcia-Marco Jose A. , Dopazo Joaquin , Piris Miguel A. 
Published: April 2020

Abstract
B-cell lymphomas are presently diagnosed according to the WHO criteria based on morphologic, immunophenotype and cytogenetic findings. However, the precise distinction among common lymphoma types is frequently a difficult task, and there are areas of overlapping and heterogeneity between them. Here we have analyzed whether gene expression profiling (GEP) data, solely considered, could be used to validate the currently used B-cell lymphoma classification, or proposing new lymphoma types, and for identifying functional signatures or genes defining these GEP-based lymphoma classification.
To this aim, we collected Gene Expression Profiling (GEP) for 173 cases of B-cell NHL, including BL (9), DLBCL (36), MALT (3), MCL (20), CLL (38), FL (33), MZL (6) and SMZL (29). The gene expression data for lymphoma cases was normalized against an average gene expression of reactive lymph nodes, except the SMZL which was normalized against normal spleen (3 cases). The analysis of the cases was done using Cluster Accuracy Analysis Tool (CAAT) (Cunningham P., 2005), that enabled us not only to compare gene expression between each node starting from root but also to identify new classes within existing lymphoma diagnosis defined by an internal validation method called Silhouette Width index (Julia Handl. et al, 2005). Using this approach each cluster could be represented by so called silhouette, which is based on the comparison of its tightness and separation. The average silhouette width could be applied for evaluation of clustering validity and can also be used to decide how good the number of selected clusters is.
Using this approach, we obtained the following categorization of lymphoma cases:
Figure Figure
Using T-Rex (Herrero J & Dopazo J., 2005) to compare differential expression between the categories obtained by CAAT, and FatiScan analysis (Al-Shahrour, F., 2006), we identified genes that were differentially expressed between molecular categories of lymphoma types, assigning them to Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes)-defined pathways. The functional signatures that were identified as distinguishing between these lymphoma types were defining cell cycle, cytokine-cytokine receptor interaction, T-cell receptor, B-cell receptor, cell adhesion, NF-kB activation, and other significant interactions. Comparison between these lymphoma clusters following this definition yielded large number of genes distinguishing them, this list including already known genes and a large number of new potential markers.
Expression Levels of Glutathione Peroxidase 1 (GPx1) and Multi-Drug Resistance 1 (MDR1; ABCB1) Identify Patients with Diffuse Large B-Cell Lymphoma (DLBCL) That Have Superior Clinical Outcomes.
Authors: Andreadis Charalambos , Gimotty Phyllis , Hammond Rachel , Wahl Peter , Houldsworth Jane , Schuster Stephen , Rebbeck Timothy 
Published: April 2020

Abstract
Background: Standard chemotherapy fails in 40–50% of patients with DLBCL. Some patients can be salvaged with high-dose regimens, suggesting a role for drug resistance in this disease. While genes in the glutathione (GSH) pathway and ATP-dependent transporter (ABC) family have been implicated in drug resistance in other malignancies, their role in lymphoma, and DLBCL in particular, remains unclear.
Methods: We examined expression of 21 key members of these families in two tissue-based expression-microarray datasets of DLBCL patients, prior to CHOP chemotherapy. Expression signals derived from Affymetrix MAS 5.0 software (SF/NF=1) were publicly available for the primary dataset (Monti et al, Blood2005; 105: 1851; http://www.broad.mit.edu/cgi-bin/cancer/datasets.cgi) and were independently generated for the validation dataset (Houldsworth et al, Blood, 2004; 103: 1862). Univariable analysis was performed for freedom from progression (FFP) using a linear-effects Cox proportional-hazards model. Recursive partitioning with CART (Salford Systems, San Diego, CA) was used to develop a classification tree for the 2-year freedom-from-progression rate (FFP2) based on median-adjusted gene expression.
Results: In univariable analysis, expression of GPx1 had the most significant adverse association with FFP in the primary dataset (n=130), hazard ratio (HR) 1.68 (95% CI: 1.26 – 2.22, p&lt;0.001). This effect remained highly statistically significant after controlling for the Monti biological signature, the LLMPP cell of origin signature, and the IPI score, and was confirmed in the validation dataset (n=39) (HR 1.5, 95% CI: 1.01 – 2.3, p=0.048). None of the ABC transporters examined were associated with FFP in the univariable analysis. Recursive partitioning identified a group of patients (n=34) with low-level expression of GPx1 and ABCB1 that had no early failures (FFP2: 100%, 95% CI: 90% – 100%). Notably, this group was comprised of patients in all IPI and LLMPP signature subgroups and achieved superior long-term FFP compared to the entire cohort (p&lt;0.001).
Conclusions: Overall, our findings suggest an important role for redox-stress defense and drug elimination in the treatment failure of DLBCL and identify GPx1 and ABCB1 as potentially powerful biomarkers of early response and response duration. Coordinate expression of these genes delineates a subgroup of patients with superior clinical outcomes regardless of other clinical and biological risk stratification in current use. Prospective, treatment-based studies with uniform inclusion and response criteria are needed to validate these markers in clinical practice.
T Cell Lymphopenia in Rhoh−/− Mice Results from Combined Defects of T Cell Migration and IL-7-Regulated Naïve T Cell Homeostasis.
Authors: Li ShuShun , Hildeman David A. , Grimes H. Leighton , Williams David A. , Gu Yi 
Published: April 2020

Abstract
The Rho family GTPases are increasingly implicated for their important roles in T cell development and function. We have found that RhoH, a hematopoietic-specific member of this family is essential for thymocyte development (Gu et al, Nat Immunol, in press). Further, Rhoh−/− mice showed T cell lymphopenia. In particular, naïve T cells were reduced in secondary lymphoid organs and blood both in unchallenged and LCMV-challenged Rhoh−/− mice compared to WT controls. These findings suggest a possible defect in T cell emigration from thymus and peripheral T cell homeostasis in Rhoh−/− mice. To study the role(s) of RhoH in T cell migration and homeostasis, purified splenic T cells were adoptively transferred into common γ−/−; Rag2−/− mice. T cells were then recovered from spleens 3d and 8d post-transplantation. The recovery of Rhoh−/− T cells was decreased by 60% compared with WT cells. In vitro migration of Rhoh−/− T cells towards SDF-1α, a homeostatic chemokine for T cells, in a transwell migration assay was significantly reduced compared to WT cells. Flow analysis showed decreased number of CXCR4 expressing and reduced expression levels of CXCR4 on Rhoh−/− T cells. Furthermore, apoptotic T cells were increased twofold in Rhoh−/− mice compared to controls. CFSE staining of adoptively transferred T cells demonstrated a comparable proliferation rate between Rhoh−/− and WT cells in the recipient mice. Our data suggest that RhoH plays an important role in T cell homeostasis via regulating cell survival and SDF-1α-mediated migration. To further investigate how RhoH regulates T cell survival, we focused on IL-7, an essential factor for prolonged survival of naïve T cells. One way IL-7 exerts its effect is through upregulating the Bcl-2 family of antiapoptotic proteins. Our data showed that in vitro survival of Rhoh−/− T cells in response to IL-7 was impaired, and expression of IL-7Rα and Bcl-2 were both decreased in Rhoh−/− T cells. To further study a potential role of RhoH in regulation of IL-7R expression, FACsvantage sorted naïve T cells (CD4+CD44low, or CD8+CD44low) were cytokine starved overnight, then cultured with or without the addition of IL-7 for 6 hrs, and analyzed for IL-7Rα expression by flow cytometry. In WT cells, during cytokine starvation CD4 and a subset of CD8 naïve cells upregulated IL-7Rα expression, but this upregulation was reduced followed IL-7 stimulation. In contrast, Rhoh−/− T cells failed to show either up- or subsequent down-regulation of IL-7Rα in response to cytokine starvation and IL-7 exposure. These data may indicate a role for RhoH in regulating IL-7R expression in naïve CD4 T cells. Taken together, these findings suggest that RhoH is required for IL-7-mediated T cell survival and SDF-1α-mediated homing and/or emigration from thymus. Thus, deficiency of naïve T cells in Rhoh−/− mice likely results from combined defects in T cell migration and homeostasis.
Monitoring of Clopidogrel Using Multiple Electrode Aggregometry.
Authors: Calatzis Andreas , Theisen Franz , Reininger Armin J. , Spannagl Michael 
Published: April 2020

Abstract
A control of clopidogrel response is one proposed strategy for an improvement of anti-platelet therapy. Different methods have been evaluated for this indication. We assessed ADP induced aggregation in whole blood in healthy blood donors and patients treated with clopidogrel 75 mg qd using a new monitoring method.
Methods: Platelet function was determined using multiple electrode aggregometry (MEA) on the Multiplate analyzer (Dynabyte, Munich, Germany). This device uses a single use test cell with two separate impedance sensors, consisting of a total of 4 electrodes and has 5 channels for parallel tests. Aggregation was triggered using ADP (6.4 μM, ADPtest, Dynabyte) and using a combination of ADP (6.4 μM) and prostaglandin E1 (20 nM) (ADPtest high sensitivity = ADPtest HS, Dynabyte). The combination of ADP + prostaglandin E1 (PGE1) is used to enhance sensitivity for the effects of clopidogrel onto ADP induced platelet activation. PGE1 reduces intracellular calcium mobilisation and therefore platelet activation and acts thus synergistical to the effect of clopidogrel. For the analysis 300 μl of blood are analyzed with the addition of 300 μl of saline. Aggregation was quantified by the area under the curve in arbitrary U (1 U corresponds to 10 AU*min). Following IRB approval venous blood was collected from 120 blood donors without anamnestic intake of platelet inhibitors in the 2 weeks before the analysis and 160 patients taking clopidogrel 75 mg qd using the direct thrombin inhibitor Melagatran in a final concentration of 15 μg/ml as the anticoagulant.
Results: The distribution of the aggregation values is shown in Fig. 1. The median (min-max) was 83 (36–143) for the blood donors in the ADPtest and 68 (1–130) in the ADPtest HS. In the patient group the median (mix–max) was 30 (2–147) in the ADPtest and 10 (0–108) in the ADPtest HS. If a non-response rate of 25% is assumed, the cut-off between clopidogrel responders and non-responders would be 51 for the ADPtest and 21 for ADPtest HS.
Discussion: The comparison of the results of clopidogrel-treated patients and healthy controls (blood donors) reveals a higher sensitivity of ADPtest HS vs. ADPtest for the effects of clopidogrel onto ADP induced aggregation in whole blood. Due to the use of a direct thrombin inhibitor as the anticoagulant in our study platelet function was determined under physiological levels of ionized calcium.
For both test methods a significant proportion of patients does not show adequate reductions in their vitro platelet aggregation. Prospective trials are required to show whether a clopidogrel-non-response in this particular method is associated with an increased occurrence of arterial thromboembolism.
Fig. 1 Fig. 1.
Use of Therapeutic Dose Low Molecular Weight Heparin in Patients with Renal Insufficiency: Analysis of Anti-Xa Levels and Creatinine Clearance.
Authors: Lim Wendy , Sridhara Manasa , Wang Luqi , Kinnon Krystyna , Douketis James , Crowther Mark 
Published: April 2020

Abstract
Low molecular weight heparin (LMWH) is predominantly eliminated by the kidneys. In patients with severe renal impairment, use of therapeutic dose LMWH may be associated with accumulation and a resultant bleeding risk. Tinzaparin may be less dependent on renal clearance due to its higher molecular mass and greater negative charge compared to other LMWHs. The objective of this prospective cohort study was to serially measure the anti-Xa anticoagulant effect of therapeutic dose tinzaparin over 5–7 days, used for the initial treatment of venous thromboembolism (VTE) in patients with varying degrees of renal insufficiency. We present the anti-Xa results from the first 78 patients enrolled in the study, correlated with renal function. In this study, consecutive in- and outpatients with objectively confirmed VTE requiring anticoagulation were enrolled and stratified into 4 groups based on the calculated Cockcroft-Gault creatinine clearance (CrCl): &gt; 60 mL/min, 30–60 mL/min, ≤ 30 mL/min and hemodialysis-dependent. Tinzaparin 175 IU/kg was administered subcutaneously once daily for 5–7 days or until the INR ≥ 2.0 with warfarin therapy. Trough anti-Xa levels were measured prior to the 3rd, 5th and/or 7th tinzaparin doses. Patients with anti-Xa level &gt; 0.5 IU/mL received tinzaparin dose adjustment using a standardized nomogram. Bleeding and recurrent VTE events were recorded. The relationship between anti-Xa levels and CrCl is shown in Figure 1. Based on our predefined anti-Xa threshold of 0.5 IU/mL, 5 of 78 patients (6.4%) required dose adjustment; 1 hemodialysis dependent patient, 2 patients with CrCl &lt; 30 mL/min, 1 patient each with CrCl 30–60 and &gt; 60 mL/min, respectively. None of these patients developed bleeding or recurrent VTE. Among all 78 patients, 1 hemodialysis-dependent patient developed a hematoma following a traumatic line insertion, and no patients developed recurrent VTE. In conclusion, in a cohort of 78 patients with differing degrees of renal function including patients requiring hemodialysis, use of therapeutic-dose tinzaparin for the initial treatment of VTE resulted in accumulation (defined by trough anti-Xa &gt; 0.5 IU/mL) in 6% of patients. There appears to be a weak inverse relationship between renal function and trough anti-Xa levels, but does not result in clinically significant accumulation when tinzaparin is used for up to 7 days. Further evaluation of tinzaparin in patients with severe renal insufficiency is required.
Figure Figure
Active Site-Inhibited rFVIIa (ASIS) Blocks Platelet Adhesion/Aggregation on Intact Endothelium Activated with TNFα and Shiga Toxin-1.
Authors: Grabowski Eric F. , Nestoridi Eirini , Bussel James B. 
Published: April 2020

Abstract
Human blood flowing over monolayers of endothelial cells (ECs) in vitro under controlled flow conditions constitutes a promising model of blood-EC interactions, especially in a system permitting real-time imaging of single platelets (1 μm resolution or better) and platelet aggregates. We have developed such a model and have used it to study platelet adhesion/aggregation on monolayers of human renal microvascular ECs grown on optically clear vinyl slides and activated with TNFα (20 ng/ml; hr 0 to 22) and Shiga toxin-1 (Stx; 10 pM; hr 18 to 22), in simulation of probable events in the childhood hemolytic uremic syndrome (HUS). Normal donor blood, collected into 4U/ml low MW heparin (dalteparin) and 10 μM mepacrine (platelet, EC, and granulocyte label), was drawn at shear rates of 270–650 sec−1 through a parallel-plate flow chamber for which one wall was one of the above slides. Such shear rates give rise to shear stresses which approximate the 20–25 dynes/cm2 estimated to exist in glomerular arterioles. Platelets, ECs, and granulocytes were imaged in real time using epifluorescence digital videomicroscopy. With activated, but not with control ECs, clusters of platelets deposit on the monolayers in strings (Fig.1, upper right, with flow from top to bottom) superposed upon ECs, for which the cytoplasm is fluorescent. The strings are tethered at the upstream end, as has been observed by others using platelet-rich plasma. Granulocytes, identifiable by size (Fig. 1, upper middle), also adhere to activated ECs. In five paired experiments, monolayer preincubation × 30 min with 50 nM active-site inactivated recombinant factor VIIa (ASIS; courtesy of Dr. Ulla Hedner, Novo Nordisk Pharmaceuticals) largely eliminated the platelet strings, while reducing the number of adherent granulocytes to zero. In control experiments, preincubation of monolayers with 100 nM of a monoclonal antibody directed against human tissue factor similarly largely eliminated both platelet strings and adherent granulocytes. In other control experiments, the use of the above concentration of low MW heparin did not by itself block fibrin formation (by immunostaining) in the boundary region near the slide-blood interface. These findings may be explained on the basis that ASIS both interrupts the tissue factor pathway of coagulation and blocks tissue factor-associated upregulation of endothelial cell E-selectin. ASIS therefore may have promise as a therapy for the prothrombotic and proinflammatory effects of childhood HUS. Studies with the TAB monoclonal antibody and ALEXA 555 are in progress to permit platelet-specific labeling and quantitation of platelet adhesion/aggregation with and without ASIS.
Figure Figure
Hematopoietic Stem Cells Regulate Niche Development.
Authors: Taichman Russell S. , Jung Younghun , Williams Benjamin , Song Junhui , Krebsbach Paul H. 
Published: April 2020

Abstract
Events localized to endosteal surfaces are critical for the maintenance of hematopoetic stem cells (HSCs). Here we explored whether HSCs themselves regulate their microenvironment directly by regulating cytokine expression by osteoblasts (OBs) in response to physiologic demands, or influence the developmental pattern of mesenchymal lineages and thereby indirectly modulate cytokine expression. To test the these possibilities, marrow was isolated from mice 48h after stressing the animals with a single acute bleed (removing 30% of the calculated blood volume by jugular vein venipucture) and in a second control group of non-stressed (puncture only) animals. The Sca-1(+) hematopoietic cells were co-cultured with confluent murine bone marrow stromal (BMSC) and calvarial digested OBs. The presence of HSCs stimulated the basal production of IL-6, SDF-1 and osteoclacin by OBs and BMSCs as determined by ELISA. Co-cultures of HSCs derived from the stressed group produced more IL-6, SDF-1 and osteocalcin relative to the non-stressed group. Newborn dermal fibroblasts did not respond in a similar fashion. To determine if HSCs influence the developmental pattern of the marrow, HSCs derived from stressed and non-stressed animals were separated from either murine OBs or BMSCs using TranswellR membranes and the ability of target cells to differentiate along the osteoblastic lineage was evaluated. A significant proportion of the colonies established from calvarial-derived OB cultures were able to mineralize their extracellular matrix relative whole BMSC population (CFU-OB). In the presence of HSCs, the proportion of non-mineralized (CFU-F) and mineralized colonies from the OB populations significantly increased. Further enhancement of both colony types were induced by the HSCs derived from stressed and non-stressed animals. Adherent cells derived from mixed BMSCs also responded to the presence of HSCs by increasing the generation of CFU-F and OBs relative to the No HSC groups. HSCs derived from stressed vs. the non-stressed groups of animals were better able to induce CFU-OB differentiation. Microarray of HSCs derived from stressed vs. the non-stressed groups, and QRT-PCR of highly purified HSCs (CD150(+), CD48(−), CD41(−), Sca-1(+), cKit(+)) suggested that BMP-2 and BMP-6 were responsible for the activities. Antibody neutralization studies confirmed these observations that BMP-2 and BMP-6 derived from the HSCs themselves alters the developmental pattern of the marrow microenvironment. In conclusion, cross-talk between HSCs-OBs is essential for the development of both cellular populations. These studies demonstrate that at least 2 mechanisms whereby HSCs might set up a paracrine loop with OBs to establish the HSC niche; (i) HSCs directly regulate cytokine expression by OBs in response to physiologic demands or, (ii) HSCs may influence the developmental pattern of mesenchymal lineages and thereby indirectly modulate cytokine expression in the marrow.