bluuebunny/crossref_metadata_2025_split huggingface dataset:
Serial Analysis of Hematopoietic Progenitors in Mpl−/− Mice.
Authors: Kaffanke Janet , Germeshausen Manuela , Welte Karl , Ballmaier Matthias 
Published: April 2020

Abstract
Thrombopoietin (TPO) is the pivotal regulator of megakaryocytopoiesis but also an important factor for early multipotent hematopoietic progenitors. Loss-of-function mutations in the human MPL gene coding for the TPO receptor are the cause of congenital amegakaryocytic thrombocytopenia (CAMT), a rare inherited bone marrow failure syndrome presenting as isolated thrombocytopenia at birth and progressing to aplastic pancytopenia during the first years of life. In contrast, Mpl−/− mice although exhibiting thrombocytopenia and severely reduced numbers of early hematopoietic progenitors do not develop any signs of pancytopenia or generalized bone marrow failure during lifetime. To identify parallels and differences in the impact of TPO on hematopoietic development between mice and men we serially analyzed subpopulations of hematopoietic cells from early progenitors to mature peripheral blood cells from Mpl−/− mice (kindly provided by Warren Alexander, WEHI, Melbourne, Australia) over a period of 24 months. We determined frequency, clonogenicity and proliferative potential of hematopoietic progenitors from different hematopoietic tissues. Compared to wild type mice we found a strong reduction of multipotent stem cells (HSC), of common myeloid progenitors (CMP), of megakaryocyte/erythrocyte lineage restricted progenitors (MEP) but also of common lymphoid progenitors (CLP) in Mpl−/− mice. We did not find any significant changes in the frequency of these cells over a period of 24 months. For the CMP and MEP subpopulations of myeloid progenitors we found not only a decreased frequency but also a reduced clonogenicity and proliferative capacity. Interestingly, these functional restrictions significantly increased significantly during aging, a finding that was similar to our results obtained with myeloid progenitors from CAMT patients. Impaired proliferation of myeloid progenitors rather than a reduced number could be the reason for development of pancytopenia in CAMT patients.
Rab38 Expression in Platelet Dense Granule Storage Pool Disease.
Authors: Ninkovic Ivana , White James G. , Stephens Kenyatta W. , Rangel-Fihlo Artur , Wu Francsisca C. , Datta Yvonne H. 
Published: April 2020

Abstract
Platelet dense granule storage pool disease (SPD) is a bleeding disorder characterized by a lack of normal platelet dense granule function, as evidenced by decreased platelet aggregation in response to ADP, epinephrine and collagen. Platelet SPD has been studied most extensively in humans and rodents with Hermansky-Pudlak syndrome (HPS), whose phenotype is a result of defects in granule trafficking, leading to oculocutanous albinism, lysosomal storage diseases, and platelet dysfunction. We have been characterizing the fawn-hooded hypertensive (FHH) rat, which has been previously shown to have a bleeding disorder consistent with a platelet SPD and some of the features of HPS. While the platelets in the FHH rat have normal alpha granules and lysosomes, they lack dense granules as assessed by transmission electron microscopy. Platelet flow cytometric analysis of GPIb and GPIIb indicated that the FHH platelets have normal surface expression of these adhesion proteins. The FHH rat has a mutation in the Rab38 gene at the ATG start site, which is associated with the bleeding disorder. Rab38 is part of a large family of GTPases, which are involved in granule formation and secretion. Western blotting of FHH tissues revealed that there is no expression of Rab38 protein. We have used confocal immunomicroscopy to assess Rab38 in platelet formation and function. In normal rat and human platelets, there was punctate expression of Rab38. There was no Rab38 staining detected in FHH platelets. In human megakaryocytic cell lines, Dami and HEL cells, there was punctate staining of Rab38 that was mainly in the periphery of the cells, with a variable amount of perinuclear staining. There was partial colocalization of Rab38 with serotonin and VWF, and with Lamp-3, a marker of lysosomes. The degree of colocalization varied between cells. There was no clear association of Rab38 with actin and tubulin in megakaryocytes. We also examined a cohort of patients with SPD, but not HPS, for mutations in Rab38. The entire coding region and intron-exon boundaries of the Rab38 gene were sequenced in 18 patient samples collected at Emory University for the CDC Women with Bleeding Disorders and Menorrhagia Study. Ten of the patients had platelet function defects documented by standard platelet aggregation studies, and eight had no identifiable platelet function defect. No mutations in Rab38 were detected. Whereas numerous known polymorphisms were identified and confirmed, there was no association of any of them with platelet function abnormalities. In conclusion, Rab38 is expressed in platelets and megakaryocytes and may interact with other granule proteins during megakaryocyte development. Failure to express Rab38 is associated with platelet dysfunction. Further studies are needed to determine its function in megakaryocytes and platelets, and to determine whether defects in Rab38 are a cause of platelet SPD in humans.
Classifying Leukemias Based on Epigenetic Alterations.
Authors: Jelinek Jaroslav , Estecio Marcos R.H. , Kondo Kimie , He Rong , Zavadil Jiri , Issa Jean-Pierre J. 
Published: April 2020

Abstract
Acute leukemia is caused by alterations of blood-forming stem cells leading to uncontrolled growth and diminished capacity to differentiate into mature functional blood elements. Beside genetic changes, epigenetic alterations are increasingly recognized as important events in the pathogenesis of leukemia. Cytosine methylation in CpG islands at gene transcription start regions can cause heritable gene silencing and have the same functional effects as inactivating mutations. Hundreds of genes may become epigenetically silenced in leukemia. While many of the methylated genes are not expressed in blood cells, silencing of genes critically important for control of stem cell self-renewal, proliferation, differentiation, and/or survival can contribute to the malignant phenotype. We used a genome-wide method to identify methylated genes by hybridizing a CpG island microarray with amplicons obtained by the methylated CpG island amplification technique (MCAM). We analyzed 10 leukemia cell lines with different cellular origin (myeloid cell lines KG1, KG1a, HEL, K562, and TF1; T lymphoid cell lines CEM and JTAg; and B lymphoid cell lines ALL1, BJAB, and Raji). On average, 266 genomic loci were found to be hypermethylated in these cell lines, ranging from 56 (KG1) to 483 loci (Raji), reinforcing the idea of extensive epigenome alteration in leukemia. Unsupervised hierarchical clustering showed distinct methylation pattern in the cell lines of lymphoid origin versus myeloid leukemia cell lines and a GM-CSF-dependent erythroleukemia cell line TF-1, justifying the use of methylation markers for uncovering of tumor-specific pathways of gene inactivation. There was a striking difference in the number of hypermethylated genes between two closely related myeloid leukemia cell lines: KG1 (56 methylated loci) and its undifferentiated variant KG1a (225 methylated loci). cDNA microarray analysis showed that deoxy-azacitidine treatment induced expression of genes differentially methylated in KG1a (DKKL1, GBX, HIVEP3, KCNAB1, KIAA1102, NAV2, NEIL1, and RAX) but not in KG1 cells where these genes were unmethylated. Finally, we used bisulfite PCR followed by pyrosequencing analysis to quantitatively measure DNA methylation of several genes detected by MCAM. Ongoing analyses of bone marrow samples from leukemia patients showed hypermethylation of the following genes: GDNF (in 4/22 [18%] AML and 7/20 [35%] ALL patients), HAND2 (in 5/22 [23%] AML and 7/20 [35%] ALL patients), HIVEP3 (in 9/22 [41%] AML and 6/20 [30%] ALL patients), MPDZ (in 2/6 [33%] AML and 15/20 [75%] ALL patients), and NEIL1 (in 2/20 [10%] AML and 1/12 [8%] ALL patients). Mapping of DNA methylation abnormalities may detect epigenetic markers important for leukemia classification and prognosis. Identification of pathways frequently silenced by DNA methylation may also suggest new targets for specific therapy.
Defining the Leukemia Epigenome: Distinct Genome Wide Histone H3 Modification Patterns Exist in AML, ALL and Healthy Hematopoietic Progenitor Cells.
Authors: Muller-Tidow Carsten , Homme Claudia , Klein Hans-Ulrich , Hascher Antje , Koschmieder Steffen , Becker Anke , Wang Yipeng , McClelland Michael , Stadt Udo zur , Battke Florian , Nieselt Kay , Thiede Christian , Ehninger Gerhard , Berdel Wolfgang E. , Dugas Martin , Serve Hubert 
Published: April 2020

Abstract
Aberrant transcriptional regulation plays a crucial role in the pathogenesis of acute leukemias. Chromosomal translocations and other mutations frequently affect transcription and chromatin regulation-associated genes in these diseases. Based on these findings we hypothesized that distinct global chromatin modification patterns exist that can distinguish between progenitor cells (CD34+ HSC) and acute leukemia as well as its subtypes. We used high density oligonucleotide ChIP-Chip assays querying more than 31,000 genomic loci to analyze global Histone H3 acetylation (H3Ac) and Lysine 9 (H3K9me3) trimethylation patterns each in a large number of AML (n=115), ALL (n=30), CD34+ HSC (n= 21) and peripheral blood cell (N=18) specimens. Class comparisons and predictions as well as unsupervised analyses were performed. Bioinformatic analyses led to histone modification maps across the genome with Histone H3 acetylation levels peaking around the predicted transcriptional start sites. More than 1000 loci differed in H3 acetylation and H3K9me3 between AML and CD34+ specimens (5% FDR). Among the regulatory classes over-represented among the altered genes were those involved in oncogenesis and cellular proliferation/differentiation. Histone H3 acetylation and H3K9me3 patterns also differed at hundreds of loci between ALL and AML samples (5% FDR). Genes involved in transcriptional regulation were significantly altered between the two leukemia subtypes. Specific differences in global chromatin modifications allowed support vector machine-based classification of CD34+ progenitor cells, AML, and ALL, with about 90% sensitivity and specificity based on Histone H3 acetylation patterns. A similar classification power was observed for H3K9me3. Within the AML patients, several groups of patients were identified that clustered together in unsupervised hierarchical cluster analysis due to similar histone modification patterns. These patterns did not primarily depend on patients′ karyotypes,, indicating that it may be possible to define some, as yet unknown, different types of AML based on chromatin modification. On a global scale, acute leukemias are associated with specific histone modification patterns that distinguish AML from ALL and CD34+ hematopoietic progenitors. Taken together, these first data on the leukemia epigenome provide the basis for improved understanding of genes involved in leukemia pathogenesis.
A New Plate Based Plasma von Willebrand Factor (VWF): Ristocetin Cofactor Activity Assay.
Authors: Chen Dong , Pruthi Rijiv K. , Nichols William L. , Heit John A. , Owen Whyte G. 
Published: April 2020

Abstract
Introduction: Assays of plasma von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) are essential for the laboratory diagnosis of von Willebrand disease (VWD). However, the current manual platelet aggregation/aggregometry method has relatively poor operating characteristics. Our goal was to develop a simple, accurate and sensitive platelet based assay.
Methods: VWF protein from normal or patient plasma was first captured on the polyclonal anti-VWF antibody coated plastic plate surface. VWF:RCo activity was assayed by the binding of mitotracker (a red fluorochrome)-labeled, formalin-fixed normal platelets in the presence of ristocetin (1mg/ml) and under agitation (200 rpm by vortex). The fluorescence from the bound platelets, measured by a fluorescence microtiter plate reader (Fluor4000, BioRad), was related to the calibrator plasma signal (6∼150% or IU/dL), and reported as % or IU/dL. We tested plasma samples from normal donors (n=16) and known VWD patients (type 1, n=6; type 2, n=9) based on clinical history and levels of plasma VWF antigen (VWF:Ag), VWF:RCo activity (manual platelet aggregation/aggregometry method), factor VIII activity and VWF multimer analysis.
Results: For both normal donors and VWD patients, VWF:RCo activities by the plate assay vs. manual platelet aggregation/aggregometry method correlated closely (R2=0.84), and VWF:RCo/VWF:Ag ratios did not differ significantly. Platelet binding to VWF-coated plastic wells is both VWF specific and ristocetin dependent.
Conclusions: Although this new platelet based VWF:RCo plate assay is still being optimized and validated, the preliminary data suggest that it is simple, accurate and sensitive.
Establishment of an International Registry of Patients with Inherited FXIII Deficiency.
Authors: Ivaskevicius Vytautas , Seitz Rainer , Kohler Hans P. , Muszbek Laszlo , Ariens Robert A.S. , Seifried Erhard , Ichinose Akitada , Nugent Diane J. , Oldenburg Johannes 
Published: April 2020

Abstract
Inherited factor XIII (FXIII) deficiency is a rare autosomal recessive disorder affecting approximately one out of one to three million people. FXIII deficiency is characterized by a lifelong bleeding tendency, impaired wound healing and spontaneous abortions in females. In 1993, the European Thrombosis Research Organization (ETRO) Working Party on FXIII initiated a Europe-wide questionnaire on inherited FXIII deficiency. Since 2005, the registry has been endorsed by the Factor XIII Subcommittee of the Scientific and Standardization committee (SSC) of the ISTH. The analysis of 104 European patients demonstrated that the most common bleeding symptoms were subcutaneous bleeding (57%) followed by delayed umbilical cord bleeding (56%), muscle hematoma (49%), hemorrhage after surgery (40%), hemarthrosis (36%), and intracerebral bleeding (34%). Prophylactic treatment was initiated in about 70% of all patients. FXIII-B subunit-deficient patients had a milder phenotype than patients with FXIII-A subunit deficiency.The most frequent mutation affecting the F13A gene was a splice site mutation in intron 5 (IVS5-1G&gt;A).This mutation was found in eight (17%) of 46 analyzed families.The haplotype analysis of patients carrying the IVS5-1A allele was consistent with a founder effect. Recently, we created a new FXIII database website (http://www.f13-database.de) with information about FXIII proteins, genes, mutations and polymorphisms. This website also includes a new questionnaire. Information provided by this questionnaire will allow better understanding of the differences of diagnostic and treatment possibilities in various parts of the world, and it will help to understand the impact of reduced FXIII activity in heterozygous relatives and finally, it will generally increase our knowledge on this rare disease. We hope that our initiative to establish a new international FXIII registry will be actively supported by the community involved in caring for FXIII deficient patients.
FDG-PET in the Serial Assessment of Patients with Lymphoma in Complete Remission.
Authors: Zinzani Pier Luigi , Stefoni Vittorio , Ambrosini Valentina , Derenzini Enrico , Musuraca Gerardo , Marchi Enrica , Alinari Lapo , Fina Mariapaola , Tani Monica , Pellegrini Cinzia , Fanti Stefano , Baccarani Michele 
Published: April 2020

Abstract
FDG-PET role in the assessment of lymphoma patients is well established but only few papers evaluated the usefulness of FDG-PET during follow up.
Aim: to prospectively investigate the value of serial FDG-PET scans in the follow up of lymphoma patients in complete remission. All lymphoma patients who achieved a complete remission were prospectively enrolled in the study and scheduled for serial FDG-PET scans at 6, 12, 18 and 24 months; further scans were then carried out on annual basis (overall 421 pts, 160 pts with Hodgkin’s Disease (HD) and 261 pts with non-Hodgkin Lymphoma (NHL) were studied). All patients had a final assessment using other imaging procedures and/or biopsy and/or clinical evolution. FDG-PET findings were reported as positive, indeterminate or negative for relapse; after comparison with all available data, PET results were categorized as true positive (TP), true negative (TN), false positive (FP), indeterminate turned out to be relapse (I+) and indeterminate turned out to be complete remission (I-).
Results: PET documented relapse in 42 cases at 6 mo (14 HD (8.8%) and 28 NHL (10.7%); in 31 cases at 12 mo (14 HD (9.5%) and 17 NHL (7.3%); in 27 cases at 18 mo (6 HD (4.5%) and 21 NHL (3.2%); in 9 cases at 24 mo (3 HD (2.4%) and 6 NHL (3.2%); and in 5 cases at &gt; 36 mo (2 HD (2.8%) and 3 NHL (6.5%). Out of 125 scans reported as positive for relapse, 109 turned out to be TP (PPV of 87%); no false negative scan was recorded, and in the great majority of cases PET detected the presence of relapse before clinical evidence. Our results confirm that FDG-PET is a valid tool for lymphoma patients follow-up. The higher incidence of relapse occurred in both HD and NHL quite early after complete remission (at 6 and 12 months for HD and at 6, 12 and 18 months for NHD), thus confirming the usefulness of performing FDG-PET scans at these times in order to identify recurrence. The role of serial PET at later times (after 18 months for HD and 24 months for NHL) was found less relevant.
NK Cells Induce Alloreactive Donor T Cell Apoptosis and Decrease Proliferation in GVHD Induction.
Authors: Olson Janelle A. , Leveson-Gower Dennis B. , Beilhack Andreas , Negrin Robert S. 
Published: April 2020

Abstract
Natural Killer (NK) cells have the ability to suppress graft-versus-host disease (GVHD) while inducing a graft-versus-tumor response (GVT) during allogeneic bone marrow transplantation (BMT). Previous studies in allogeneic BMT models have shown NK cell trafficking to and proliferation in lymphoid organs and GVHD target organs, which are also sites of donor T cell trafficking. This study aims to investigate the impact of NK cells on alloreactive, GVHD-inducing donor T cells. Interleukin-2 activated allogeneic NK cells isolated from C57Bl6 (H–2b) or FVB (H–2q) animals were transplanted along with T cell-depleted bone marrow into lethally irradiated BALB/c (H–2d) mice, followed 2 days later by luciferase-expressing CD4+ and CD8+ conventional T cells from the same donor strain (NK+Tcon group). Control mice received lethal irradiation and T cell-depleted bone marrow on day 0, and luciferase-expressing T cells on day 2 after transplant (Tcon group). Bioluminescence imaging of NK+Tcon mice revealed a significantly lower T cell bioluminescent signal (p=0.03 for FVB into BALB/c on day 6) than from Tcon mice. CFSE proliferation analysis of alloreactive T cells on day 3 after transplant showed no significant change in the percent of donor T cells that have divided in the spleen, and only a slight decrease in the percent of T cells that have divided in the lymph nodes when NK cells are present. However, at this timepoint 82% of the proliferating cells have divided past the third generation, in contrast to 64% in the NK+Tcon mice. Donor T cells in both groups become equally activated in vivo, expressing similar levels of the early-activation marker CD69. T cells re-isolated from NK+Tcon animals at day 5 stained 2 to 10-fold higher for the TUNEL apoptosis marker than those from Tcon mice in the mesenteric and peripheral lymph nodes, respectively (p&lt;0.0001). Additionally, decreased numbers of T cells were re-isolated from the peripheral lymph nodes in the NK+Tcon group as compared to the Tcon group. This increase in TUNEL staining was not seen when the transplanted NK cells were isolated from a perforin-deficient donor. This indicates that NK cells in lymph nodes use a perforin-dependent mechanism to increase apoptosis in proliferating, alloreactive donor T-cells, which are syngeneic to the transplanted NK cells. Donor T cells re-isolated from the lymph nodes of transplanted mice up-regulate the NKG2D ligand Rae1γ as compared to naïve T cells, as shown by FACS. This suggests that NK cells may cause direct lysis of alloreactive donor T cells in vivo during GVHD induction, mediated by the NK cell activating receptor NKG2D. This study provides crucial mechanistic information regarding the function of NK cells in suppressing GVHD.
Gab1 Is Involved in Erythropoietin Receptor-Mediated Survival Signal through Activation of the Erk Pathway.
Authors: Fukumoto Tetsuya , Kubota Yoshitsugu , Kitanaka Akira , Waki Fusako , Imataki Osamu , Ohnishi Hiroaki , Ishida Toshihiko , Tanaka Terukazu 
Published: April 2020

Abstract
Erythropoietin (EPO) is required for the survival, proliferation and differentiation of erythroid progenitor cells. The scaffolding adaptor protein Grb2-associated binder-1 (Gab1) is tyrosine phosphorylated upon stimulation of EPO in several cell lines and erythroid progenitor cells, and interacts with signaling molecules such as SHP2 phosphatase and the p85 subunit of phosphatidylinositol 3-kinase (PI3K). However, biological functions of Gab1 in EPO receptor (EPOR)-mediated signaling has not yet been established. In this study, to explore the biological functions of Gab1 in vivo, Gab1-deficient F-36P human erythroleukemia cells were generated by means of transfection of the expression vector of siRNA against Gab1. WST-1 assay revealed that growth of Gab1-deficient F-36P cells was reduced to 61% and 77%, respectively, 5 days after incubation with lower concentrations of EPO (0.001 and 0.01 ng/ml), compared with that of mock-transfected F-36P (F-36P-mock) cells. In contrast, growth of Gab1-deficient F-36P cells at sufficient concentration of EPO (10 ng/ml) was similar to that of F-36P-mock cells. Analysis of apoptosis by flow cytometry using FITC-labeled annexin-V showed that the percentage of annexin-V-positive apoptotic cells in Gab1-deficient F-36P and F-36P-mock cells was increased to 19% and 34%, and 8% and 17%, respectively, 72 h after incubation with 0.01 and 0.001 ng/ml of EPO. These results indicate that Gab1 plays a crucial role in transducing EPOR-mediated survival signals. Next, we examined the molecular mechanism of EPOR-mediated signaling involved in survival of erythroid cells through Gab1. Western blot analysis showed that EPO-induced phosphorylation of threonine 202/ tyrosine 204 on Erk-1 and Erk-2 in Gab1-deficient F-36P but not in F-36P-mock cells was significantly suppressed. Interestingly, phosphorylation of serine 473 on Akt in Gab1-deficient F-36P cells in response to EPO was slightly suppressed in comparison with that in F-36P-mock cells. Therefore, Gab1-mediated survival signals appear to be mainly transmitted to downstream through activation of the Erk pathway, although the PI3K/Akt pathway may be involved in EPO-initiated survival signal transduction mediated by Gab1. Furthermore, EPO induced the association of SHP2 with EPOR in Gab1-deficient F-36P cells. Gab1 was associated with SHP2 in EPO-treated F-36P cells. In addition, Gab1 was constitutively associated with Grb2 in F-36P cells. Taken together, EPO induces the recruitment of Gab1 to EPOR through binding of Gab1 to SHP2, which is associated with EPOR. Because the guanine nucleotide exchange factor SOS1 is known to bind to the SH3 domain of Grb2, SOS1-Grb2 complex is recruited to vicinity of Ras at the plasma membrane to activate this GTP-binding protein through the interaction of Grb2 with Gab1, leading to activation of Erk.