bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vitro Expansion of Polycythemia Vera Progenitors Favors Expansion of Erythroid Precursors without JAK2 V617F Mutation.
Authors: Prchal Josef T. , Chang Ko-Tung , Jelinek Jaroslav , Guan Yongli , Gaikwad Amos , Issa Jean-Pierre , Liu Enli 
Published: April 2020

Abstract
A single acquired point mutation of JAK2 1849G&gt;T (V617F), a tyrosine kinase with a key role in signal transduction from growth factor receptors, is found in 70%–97% of patients with polycythemia vera (PV). In the studies of tyrosine kinase inhibitors on JAK2 1849G&gt;T (see Gaikwad et all abstract at this meeting) we decided to study the possible therapeutic effect of these agents using native in vitro expanded cells from peripheral blood. To our surprise, the in vitro expansion of PV progenitors preferentially augmented cells without JAK2 1849G&gt;T mutation.
We used a 3 step procedure to amplify erythroid precursors in different stages of differentiation from the peripheral blood of 5 PV patients previously found to be homozygous or heterozygous for the JAK2 1849G&gt;T mutation. In the first step (days 1–7), 106/ml MNCs were cultured in the presence of Flt-3 (50 ng/ml), Tpo (100 ng/ml), and SCF (100 ng/ml). In the second step (days 8–14), the cells obtained on day 7 were re-suspended at 106/ml in the same medium with SCF (50 ng/ml), IGF-1 (50 ng/ml), and 3 units/ml Epo. In the third step, the cells collected on day 14 were re-suspended at 106/ml and cultured for two more days in the presence of the same cytokine mixture as in the step 2 but without SCF. The cultures were incubated at 37oC in 5% CO2/95% air atmosphere and the medium renewed every three days to ensure good cell proliferation. The expanded cells were stained with phycoerythrin-conjugated anti-CD235A (glycophorin) and fluorescein isothiocyanate-conjugated anti-human-CD71 (transferrin receptor) monoclonal antibodies and analyzed by flow cytometry. The cells were divided by their differential expression of these antigens into 5 subgroups ranging from primitive erythroid progenitors (BFU-Es and CFU-Es) to polychromatophilic and orthochromatophilic erythroblasts; over 70% of harvested cells were early and late basophilic erythroblasts. The proportion of JAK2 1849G&gt;T mutation in clonal PV granulocytes (GNC) before in vitro expansion and in expanded erythroid precursors was quantitated by pyrosequencing (Jelinek, Blood in press) and is depicted in the Table.
These data indicate that in vitro expansion of PV progenitors favors expansion of erythroid precursors without JAK2 V617F mutation. Since three PV samples were from females with clonal granulocytes, erythrocytes, and platelets, experiments were underway to determine if the in vitro expanded erythroid cells were clonal PV cells without JAK2 V617F mutation, or derived from polyclonal rare circulating normal hematopoietic progenitors.
The Proportion of JAK2 T Allele Patients GNC T Allele (%) Expanded Cells T Allele (%) PV1 (Female) 81 10 PV2 (Male) 77 28 PV3 (Male) 44 42 PV4 (Female) 78 19 PV5 (Female) 78 28
Generation of Specific Human CD8+ T Cell Responses to the Myeloproliferative Disorder Associated V617F Mutated JAK2 Kinase by Use of Analog Peptide Vaccine Candidates.
Authors: May Rena J. , Korontsvit Tanya , Zakhaleva Victoriya , Gomez Marta , Dao Tao , Scheinberg David A. 
Published: April 2020

Abstract
The recent identification of the JAK2 V617F mutation in myeloproliferative diseases including polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis has the potential to serve as a new therapeutic target for these disorders. We hypothesized that the mutated JAK2 kinase sequence could generate new immunogenic peptide-epitopes that could be recognized by the human immune system. Therefore, such sequences could be potential vaccine candidates. The myeloproliferative diseases, especially PV and ET, are ideal diseases for a vaccine approach as they are indolent disorders and also not suitable for use of possibly toxic chemotherapies. Using computer based predictive algorithms, potentially immunogenic nonamer peptides spanning the V617F region of JAK2 were identified. Since JAK2 is a normally expressed self-protein, the goal is to break immune tolerance to the mutated JAK2 sequence without allowing significant recognition of the native sequence. Therefore, synthetic analogs of the V617F mutated peptides with stronger predictive binding to MHC were designed by introducing single amino-acid substitutions into the HLA-A0201 and A0301 binding positions. Corresponding non-mutated native sequences showed little predicted binding. The binding of the mutated and synthetic analog peptides was confirmed in vitro using an MHC class I stabilization assay on TAP deficient cells. Native sequences bound poorly as expected. JAK2 peptides derived from V617F sequences as well as their analogs were used to elicit MHC-restricted, peptide-specific CTL responses in an in vitro model using T-cells from either HLA-A0201 or A0301 healthy donors after stimulation with autologous monocyte-derived dendritic cells. The analog peptides were shown to be immunogenic in vitro, and activated CD8+ T cells that recognized the synthetic analog peptide pulsed targets as determined by an IFN-gamma ELISPOT assay. In addition, and most importantly, these T cells cross-reacted with cellular targets that were pulsed with the mutated V617F peptide associated with the myeloproliferative diseases, but not with the native JAK2 peptide. These analog peptides thus satisfied our initial goals for a vaccine candidate. Further studies with these peptides and others eliciting CD4+ responses will determine their potential for successful use as vaccine candidates
Increased Adhesion of Red Blood Cells from Patients with Polycythemia Vera Is Mediated by Lu/B-CAM and Laminin alpha 5.
Authors: Wautier Jean-Luc , Gane Pierre , El Nemer Wassim , Rain Jean-Dider , Le Van Kim Caroline , Wautier Marie-Paule 
Published: April 2020

Abstract
Polycythemia vera (PV) is associated to a high risk of vascular thrombosis which is related to red blood cell (RBC) volume and platelet activation. We have investigated whether, beside high hematocrit, RBCs exhibit qualitative abnormalities. Along this line we have measured RBC adhesion to human umbilical vein endothelial cell (HUVEC) under static and flow conditions. A group of 38 PV patients and one of 36 normal subjects were explored. Adhesion molecule expression using specific antibodies (anti-CD36, CD49d, ICAM-4, Lu/B-CAM, CD147, CD47) and flow cytometry were determined on RBCs. In addition antibodies directed against adhesion molecules or recombinant Lu/B-CAM or VCAM molecules were tested in the adhesion assays. PV-RBC adhesion was found to be increased 3.33 folds (2–4.5) compared to normal RBCs (p&lt;0.001). The extent of adhesion was correlated to Lu/B-CAM expression on RBC (p&lt;0.001) and was inhibited by polyclonal or monoclonal anti-Lu/B-CAM antibodies and soluble Lu/B-CAM recombinant molecules but not VCAM molecules. Adhesion of PV-RBCs activated HUVEC and stimulated VCAM expression, but stimulation of HUVEC by TNF did not increase PV-RBC adhesion. The increase of adhesion was apparently limited to a population of RBCs which are not reticulocytes and which cannot be detached from HUVEC even by a shear stress above 2 Pa. On HUVEC side the adhesion can be blocked by anti-laminin alpha 5 chain antibodies but not anti-Lu/B-CAM. These results indicate that Lu/B-CAM on PV-RBC is responsible for the increased adhesiveness to laminin expressed on HUVEC. This may be one of the factors involved in thrombotic events.
Gab2 Is Constitutively Phosphorylated in Cells Expressing the Polycythemia Vera-Associated Jak2 V614F Mutant and Links Jak2 to Erk1/2 Activation.
Authors: Robinson Lisa J. 
Published: April 2020

Abstract
Although chronic myeloid leukemia was one of the first hematologic disorders for which a molecular pathogenesis could be defined, the molecular abnormalities responsible for other myeloproliferative disorders have remained largely unknown. Several recent studies, however, have identified an activating mutation in Janus kinase 2 (Jak2 V614F) as a frequent abnormality in polycythemia vera. Substitution of phenylalanine for valine 614 in Jak2 was found to result in constitutive kinase activity. In erythroid precursors expressing this mutant, erythropoietin hypersensitivity was seen, and, in an animal model, the mutation was sufficient to produce an erythrocytosis resembling PV. Jak2 is a key intermediate in erythropoietin signal transduction and its phosphorylation and activation of Stat proteins is well-characterized. Its involvement with other downstream pathways, however, is less well-defined but potentially important to the pathogenesis of polycythemia vera. We therefore investigated signaling events downstream from the activated Jak2 V614F mutant in the human erythroleukemia (HEL) cell line. We discovered that the scaffolding protein Gab2 was constitutively phosphorylated in cells expressing the Jak2 mutant; moreover, its phosphorylation was blocked when cells were treated with the Jak2-specific inhibitor AG490. This suggested that Gab2 might represent an additional Jak2 target and one that could link Jak2 to multiple downstream signaling pathways including PI3kinase-Akt and Shp2-Erk1/2. We investigated whether Jak2 could phosphorylated Gab2 in vitro using a Gab2-GST fusion protein and found that purified, activated Jak2 could phosphorylate site(s) in the C-terminus of Gab2. This region contains two tyrosine residues that lie within a consensus sequence for Jak2 phosphorylation (pY-X-X-I/V/L): Gab2 tyrosines 614 and 643. Mutagenesis of tyrosine 643 resulted in a marked inhibition of Jak2-dependent Gab2 phosphorylation in vitro and in vivo. Mutation of tyrosine 643 also inhibited Shp2 association with Gab2 and activation of Erk1/2. These results suggest that Gab2 may provide a link between the activated Jak2 and the Shp2-Erk2 pathway and thereby play a role in the neoplastic effects of mutant Jak2. Interestingly, Gab2 is also implicated in the pathogenic effects of Bcr-Abl, suggesting that the protein interactions of this molecular scaffold could represent a target for therapeutic intervention that could be of benefit in multiple myeloproliferative disorders.
The First Homozygous RPS19 Mutation Identified in a Diamond-Blackfan Anemia Patient Is Not Deleterious.
Authors: Cretien Aurore , Rince Patricia , Leblanc Thierry , Ducrocq Rolande , Proust Alexis , Marie Isabelle , Delaunay Jean , Narla Mohandas , Tchernia Gil , Da Costa Lydie 
Published: April 2020

Abstract
Mutations in ribosomal protein S19 (RPS19) gene have been found in 25% of patients affected with Diamond-Blackfan anemia (DBA). Two of the RPS19 mutations identified in DBA patients are located in the sequence upstream of the translational start site: a missense mutation g-&gt;t at −460 and a 4 bp insertion, gcca, 3 nt upstream of the 5′ end of the RPS19 promoter. All of the RPS19 mutations identified to date are expressed in the heterozygous state. In the present study, in a DBA patient from Ivory Coast we identified the gcca insertional mutation on both alleles. Diagnosis of DBA was made at the age of 3 months. No malformations were noted. The patient responded neither to steroid nor to IL-3 therapies. The patient was regularly transfused but without an effective iron chelation therapy. As a consequence DBA was complicated by a severe hemochromatosis with thyroid, parathyroid, and liver damage. The patient was enrolled into a metoclopramide protocol in our hospital when he was 12 year old. He had a partial response, with increased time intervals between transfusions. While analysis of the RPS19 gene did not identify a mutation in the coding sequence, the 4 bp insertion, gcca at nt − 631 was noted on both alleles. Mother was heterozygous for this mutation and strikingly the father was homozygous. Both parents are apparently healthy. In screening of 200 Caucasian control chromosomes and 100 chromosomes from Ivory Coast we did not identify the 4bp insertion, ruling out the possibility that it is a common polymorphism. A parental disomy was eliminated by a genescan microsatellite analysis using the microsatellites: D19S200, D19S197, and LIPE. To evaluate the effect of this mutation on erythroid differentiation, we isolated 30,000 CD34+ cells from peripheral blood of the patient and normal individuals. CD34+ cells were cultured for 7 days in methylcellulose with EPO, SCF, and IL-3. At day 7, erythroid colonies were isolated and cultured for additional 3 and 5 days in liquid medium. At D7, D10 and D12, aliquots of cells were collected and cloning efficiency and apoptosis was quantitated. RPS19 mRNA was assayed by quantitative RT-PCR and level of protein expression determined by Western blot analysis. The cloning efficiency of DBA erythroid progenitors was decreased by 2.6 fold compared to normal between D7 and D10 and by 3.5 fold between D10 and D12. No difference in apoptosis was noted between DBA and normal erythroid progenitors. Strikingly, during terminal erythroid differentiation, RPS19 mRNA and protein expression levels was similar in the DBA and normal erythroid cells. The presence of the homozygous mutation in the healthy father in conjunction with normal expression of RPS19 in the DBA erythroid cells imply that the gcca insertion at nt −631 is not deleterious and cannot by itself account for the DBA phenotype of our patient.
CD28-Mediated Regulation of Multiple Myeloma Cell Proliferation and Survival.
Authors: Lee Kelvin P. , Bahlis Nizar J. , King Anne M. , Kolonias Despina , Carlson Louise M. , Hussein Mohamad , Terebolo Howard , Byrne Gerald E. , Levine Bruce L. , Boise Lawrence H. 
Published: April 2020

Abstract
Although interactions with bone marrow stromal cells are essential for multiple myeloma (MM) cell survival, the specific molecular and cellular elements involved are largely unknown due to the complexity of the bone marrow microenvironment. The CD28 receptor, which costimulates survival signals in T cells, is also expressed on normal plasma cells and myeloma cells. In MM, CD28 expression correlates significantly with disease progression, also suggesting a pro-survival function. In contrast to T cells however, activation and function of CD28 in myeloma and plasma cells is almost entirely undefined. We found that direct activation of myeloma cell CD28 by anti-CD28 mAb alone induced activation of NFkappaB, suppressed MM cell proliferation and protected against serum starvation and dexamethasone-induced cell death. We hypothesized that the specific CD80/CD86 expressing stromal cell partner of this interaction is a professional antigen presenting cells, in particular dendritic cells. Histological studies demonstrated DC were extensively interdigitated throughout the myeloma infiltrates in patient bone marrow biopsies. In vitro coculture with DC also elicited CD28-mediated effects on MM survival and proliferation, and could be blocked by CD28Ig. Our findings suggest a previously undescribed myeloma:DC cell-cell interaction involving CD28 that may play an important role in myeloma cell survival within the bone marrow stroma. These data also suggest that CD28 may represent a therapeutic target in the treatment of multiple myeloma.
Frequency of Anti-Erythropoietin Antibodies in Patients with End Stage Renal Disease Treated with Recombinant Erythropoietin.
Authors: Hoppensteadt D. , Tobu M. , Fareed D. , Iqbal O. , Baltasar F. , Patel C. , Bansal V. , Demir M. , Fareed J. 
Published: April 2020

Abstract
End Stage Renal Disease (ESRD) manifests hematologic, cardiovascular, inflammatory, and immunologic dysfunctions. Erythropoietin deficiency is present in ESRD leading to anemic states. ESRD patients receive regular/repeated administration of commercially available recombinant erythropoietin (r-E) (Epogen® Procrit® Patients treated with r-E have been known to develop thrombotic complications during r-E therapy. Antibodies to erythropoietin are generated in some patients and lead to erythropoietic aplasia. The mechanisms of the anti-erythropoietin antibody induced aplasia and reported hypercoagulability state in patients treated with r-E are not fully understood. Anticoagulant drugs such as heparin are used to reduce the incidence of thrombotic complications during r-E therapy. To understand pathogenesis leading to hypercoagulable state and its potential link with r-E treatment, blood samples taken from a group of 60 patients with ESRD were drawn prior to initiation of antithrombotic therapy. These patients on periodic hemodialysis were administered r-E 6–8 weeks prior to blood drawn. Anti-erythropoietin antibody titer, anti-phospholipid antibody (APA) titer, CRP levels, fibrinopeptide-A (FPA), nitric oxide (NO) levels, asymmetric 1,3-dimethylarginine (ADMA) levels, and thrombin activatable fibrinolytic inhibitor (TAFI) levels were measured. Five of 60 patients (8.3%) showed higher titers of anti-erythropoietin antibodies. Three of 60 patients (5%) exhibited positive titers of anti-phospholipid antibodies. Marked elevation of the CRP, FPA, NO, ADMA, and TAFI levels were noted in comparison to match controls. Patients with positive levels of anti-erythropoietin antibody titer (3 to 5 folds) exhibited a simultaneous elevation of FPA, CRP and TAFI suggesting the potential role of anti-erythropoietin antibody in mediating these responses. These observations indicate that ESRD patients treated with r-E are at a higher risk of developing thrombotic complications. The upregulation of NO and ADMA in these patients is suggestive of ongoing inflammatory processes. Anticoagulants such as low molecular weight heparins may be useful in the management of these patients.
Molecular Heterogeneity in Recombinant Erythropoietin Using Surface Enhanced Laser Induced Desorption Ionization (SELDI). Clinical Implications.
Authors: James E. , Maddineni J. , Fareed J. , Florian-Kujawski M. , Baltasar F. , Jeske W. , Hoppensteadt D. , Tobu M. , Demir M. , Iqbal O. , Bansal V. 
Published: April 2020

Abstract
Several erythropoietin preparations (EPO) are used globally for anemia management. Repeated EPO use has been reportedly associated with thrombotic complications. Molecular profiles of these recombinant proteins vary widely, impacting their clinical profile. This may be due to cellular/receptor interactions and other modulatory properties. There are WHO and FDA regulatory guidelines regarding molecular profile; data on protein profiling is usually not provided by the manufacturers. Purpose of this investigation was to compare various clinically-used recombinant erythropoietin products Epogen (Amgen), Vintor (Emcure), Eprex (Janssen-CILAG) and Neo Recormon (Roche) utilizing protein chip array technology (Ciphergen, Fremont, CA). All products were analyzed using anionic SAX2 chips and molecular profile was obtained in the range of 0–150 KDa. Epogen and Vintor exhibited a main component at 32.4 KDa range with minor peaks at 13.5, 21.7 and 43.2 KDa. Intensity of the main component varied widely even within the 3 batches of Epogen. The Epogen preparations and Vintor brand contained albumin in different quantities. One batch of Epogen contained 4x higher amount of this material with a heterogeneous distribution. Epex and Neo Recormon did not have any albumin. One of the Epogen product also contained a component at 110.0 KDa. Glycosylation profiles of each of these preparations also differ. These data suggest that molecular heterogeneity exists within different recombinant erythropoietin preparations. Molecular heterogeneity among these preparations may be partly responsible for differential interactions within erythropoietin receptors on cells and anti-epo antibody generation. Protein chip array technology may be helpful in examining the protein components within different recombinant erythropoietins and may be helpful in establishing molecular profile guideline to minimize pharmacodynamic variance in erythropoietin preparations. Furthermore, molecular profiling of these recombinant proteins may be helpful in establishing the purity and homogeneity of these drugs.
Differential Changes in Platelet VWF Receptor Following Refrigeration for Short or Long Periods.
Authors: Josefsson Emma C. , Bergmeier Wolfgang , Wagner Denisa D. , Stossel Thomas P. , Hartwig John H. , Hoffmeister Karin M. 
Published: April 2020

Abstract
One pathway that mediates the rapid removal of transfused platelets exposed to cold for short periods (≤ 2 h) is the clustering of the von-Willebrand factor (VWF) receptor complex. Clustered VWF receptors are recognized by the αMβ2 integrin on hepatic macrophages, which, through its lectin domain, binds exposed βN-acetylglucosamine (βGlcNAc) residues on the clustered GPIbα subunits (Cell112: 87, 2003). Coverage of the exposed βGlcNAc residues by galactosylation prevents clearance of platelets from the circulation. We have now investigated detailed changes of the VWF complex on platelets during short (≤ 2 h) and prolonged refrigeration (≥ 48 h). The VWF receptor structure in chilled and rewarmed platelets was probed by flow cytometry using a panel of mAbs directed towards the GPIbα N-terminal (N–T) and GPIX (FITC-AN51 (aa36–59), PE-Cy5-HIP1 (aa59–81), FITC-SZ2 (aa268–282), and PE-GPIX mAb). Binding of the anti-GPIbα mAbs HIP1 and SZ2 as well as the anti-GPIX was diminished 40–50 % following 0.5 h of cooling when compared with room temperature (RT) platelets. The anti -GPIbα N-T mAb AN51 bound similarly to cooled and RT platelets. In contrast, the binding of all anti-VWF complex mAbs was decreased by 50–60 % after refrigeration of platelets in plasma for 48 h when compared to fresh RT platelets. Immunoblots demonstrated that loss of binding did not result from GPIbα shedding. VWF and fibrinogen binding were also increased by ~ 5 % or ~ 20 %, respectively, and P-selectin exposure increased by ~ 30 % in 48 h refrigerated platelets compared to the RT platelets stored for 48 h as determined by flow cytometry. These results suggest that the conformation of the VWF receptor is altered by long term refrigeration which leads to VWF binding and partial platelet activation. Loss of antibody binding is not simply due to GPIbα clustering as incubation of platelets with 5 μM latrunculin A for 1 h, which induces profound GPIbα clustering, increased binding by ~ 30 % compared to untreated platelets, but did not alter the diminished mAb binding to platelets induced by chilling. Refrigerated (≤ 14 days) platelets still adhere well to collagen-coated surfaces under flow (1000 seconds−1 for 2 min) demonstrating that VWF receptor function remains intact. We conclude that refrigeration of platelets in plasma for ≥ 48 h induces both conformational changes and clustering of the VWF complex, to prime it for activation by inducing VWF binding.