bluuebunny/crossref_metadata_2025 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

A single acquired point mutation of JAK2 1849G>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>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>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>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>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

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

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<0.001). The extent of adhesion was correlated to Lu/B-CAM expression on RBC (p<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

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

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->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

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

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

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

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.
Gene Expression Profiling of Hematopoietic Stem and Progenitor Cells in Zebrafish.
Authors: Weber Gerhard J. , Chiang Ken , Mayhall Elizabeth A. , Zhou Yi , Zon Leonard I.
Published: April 2020

The differentiation of hematopoietic stem cells (HSCs) into distinct blood lineages is orchestrated by a tight regulation of critical genes during development. To describe the genetic program during the early steps of blood and blood vessel formation in greater detail, we analyzed the expression profiles of zebrafish cloche mutants (clo), in which blood and endothelial cells are completely abrogated, and of isolated hematopoietic and vascular progenitor cell populations from FAC-sorted transgenic lmo2-GFP zebrafish embryos. Consistent with a defect in hemangioblast differentiation, clo mutants at the 5 somite stage were characterized by a prominent downregulation of transcription factors, such as scl and lmo2. As a novel finding, clo mutants at this stage strongly upregulated muscle-related genes, such as myosin, actin, or creatine kinase. FAC-sorted lmo2-GFP cells from 14 somite stage embryos expressed similar hematopoietic transcription factors including scl, lmo2, and gata1. In addition, socs genes, tgfb, fos and jun related genes, and heat shock proteins were also strongly expressed. Comparison with the expression profile of human and mouse fetal liver HSCs revealed a similar regulation between fish and mammals: Expression of 102/168 genes from lmo2-GFP positive cells were also present in mammalian fetal HSCs, indicating an evolutionary highly conserved genetic program of blood formation. In sum, gene expression profiling of zebrafish mutants and of sorted progenitor cells from transgenic zebrafish represent a powerful and versatile tool to define the genetic regulation of vertebrate hematopoietic development.
A Stem-Loop in the 3′-UTR Mediates Iron-Dependent Regulation of Alpha Hemoglobin Stabilizing Protein mRNA Stability.
Authors: dos Santos Camila O. , Duarte Adriana , Dore Louis C. , Saad Sara , Weiss Mitchell J. , Costa Fernando F.
Published: April 2020

Alpha-hemoglobin stabilizing protein (AHSP) acts as a molecular chaperone that binds and stabilizes alpha globin, minimizing the formation of cytotoxic alpha-globin precipitates in red blood cells. It has been proposed that variations in AHSP protein levels could influence disease severity in patients with beta-thalassemia. A number of genomic sequences that could be involved with AHSP transcriptional regulation have been studied. Multiple consensus GATA-1 binding motifs and a single CACCC-binding site have been identified and characterized. In vivo studies demonstrated that AHSP gene transcription is dependent on both EKLF and GATA-1 transcription factors. Here, we identify an IRE (iron response-like element) in the AHSP 3′ untranslated region that is predicted to regulate mRNA stability. IREs are regulatory elements that encode a phylogenetically defined sequence-structure motif that provide a specific recognization site for the IRE-binding proteins (IRE-BP1 or IRE-BP2). Iron starvation of cells induces high affinity binding of cytoplasmic IRE-BP to an IRE, which has at least two distinct known biological consequences. IRE in the 5′-UTR of an mRNA provides for iron-dependent regulation of translation, whereas an IRE in the 3′-UTR confers iron-dependent regulation of mRNA stability. Computer modeling identified a potential IRE stem-loop structure near the 3′-end of murine and human AHSP mRNAs. In vitro binding studies indicate that this structure recognizes an IRE-BP. Moreover, the affinity of this interaction was reduced by mutations that disrupt base pairing within the stem-loop. Iron supplementation, which is predicted to decrease IRE-BP affinity for the IRE, destabilizes AHSP in human and mouse erythroleukemia cells. These results indicate that AHSP expression is regulated by RNA stability in an iron-dependent fashion. Our findings have implications for the control of AHSP expression in beta-thalassemia.
Differential Modulation of the β-Like Globin Genes by KLFs Isolated with a γ-Globin CACCC Bait.
Authors: Moi Paolo , Porcu Loredana , Marini Maria G. , Asunis Isadora , Loi Maria G. , Ikuta Tohru , Cao Antonio
Published: April 2020

The globin CACCC boxes are absolutely required for the appropriate regulation of the β-like globin genes. While the β-globin CACCC box binds EKLF/KLF1, a likely adult switching factor, analogous factors, interacting with the γ-globin gene and predicted to regulate the fetal stage of hemoglobin switching, have so far been elusive. By using yeast one hybrid assay, we have isolated four KLFs, KLF1, 2, 4, and 6, that bound the γ-CACCC bait. To establish their role in globin regulation and in the switching of hemoglobins, these factors were compared to four other KLFs already established or putative globin regulators, KLF3, 11, 13 and 16, mainly evaluating their ability to bind and transactivate the ε-, γ- and β-globin gene. γ-CACCC binding at variable intensities was confirmed in band shift assay for all four isolated KLFs, for KLF3 and, faintly, for KLF13. The ε- and β-CACCC were bound by the same factors with similar affinities with the exception of KLF3 and KLF13 that bound stronger to the β- and ε- than to the γ-CACCC box. On the other hand, KLF11 and 16 did not produce any specific complex in band shift assays with anyone of the globin CACCC boxes. More relevant differences were observed among the factors in the transactivation of single and dual luciferase reporters in both K562 and MEL cells. In these assays, most factors presented peculiar modulatory properties and specific promoter tropism. Several factors presented bidirectional activity displaying in the same time the capacity to stimulate and repress different globin promoters. KLF1 and 4 were the strongest stimulators of the β-globin promoter in both cell lines, whereas KLF2 activated the β-promoter only in K562 cells. KLF1 and especially KLF4 consistently repressed ε-globin expression especially in MEL cells. KLF3 behaved always as a general globin repressor in MEL cells, but acted as a weak stimulator of the γ- and ε-promoter in K562 cells. KLF4 was the strongest inhibitor of the ε-globin gene. KLF13 significantly stimulated the γ-promoter in both cell lines, whereas KLF3, 4 and 6 showed statistically significant stimulation only in MEL cells. By RT-PCR analysis we found that KLFs were highly variable in their tissue expression and that KLF1, 3 and 13 had the highest expression in erythroid tissues. Thus the level of tissue expression should ultimately determine which factors are really active in physiological conditions. Taken together our binding and expression studies suggest that several KLFs have the potential to modulate the activity of the globin genes and that the resulting globin expression will depend on the vectorial sum of the relative activities of the factors expressed at any given time of development. Furthermore, as some KLFs, like KLF1 and 4, exert opposite effects on fetal and adult globin genes, their role in hemoglobin switching may be direct and not only dependent on their ability to mediate promoter competition for the LCR.
AHSP Is a Quantitative Trait Gene That Modifies the Phenotype of β Thalassemia.
Authors: Lai Mei I. , Jiang Jie , Silver Nicholas , Best Steve , Menzel Stephan , Garner Chad , Weiss Mitchell J. , Thein Swee Lay
Published: April 2020

Alpha hemoglobin stabilizing protein (AHSP) was identified in a screen for genes that are activated by the erythroid transcription factor, GATA-1. Studies have shown that:

AHSP binds specifically to the α chain of hemoglobin (Hb) but not to β Hb or Hb A; AHSP knock-out mice demonstrated pathological features similar to β thalassemia; and loss of AHSP exacerbates severity of disease in β thalassemia mice.

The evidence suggests that AHSP acts as a chaperone for free α Hb and that altered AHSP expression levels could modify the severity of β thalassemia in humans.

To assess variation of AHSP expression, mRNA levels were measured in peripheral blood reticulocytes of 103 healthy individuals by quantitative real-time RT-PCR. The sample was approximately 90% (91/103) female with an average age of 52 years (SD=0.14, min=18, max=76). AHSP expression relative to GAPDH, varied up to 3-fold, and did not correlate with age or sex. A systematic survey of the genomic region encompassing the AHSP locus revealed 8 sequence variants of which six were common - five single nucleotide polymorphisms (SNPs), and one homopolymer (Tn) at position 160 bp upstream of exon 1. Four variants (c.-69–237A, c.-69–160 T18, c.-4–27G, and c. 337T) showed strong association with AHSP expression and had nearly equal frequencies. The four variants are in near complete linkage disequilibrium with the minor alleles in coupling. The haplotype consisting of the four minor alleles termed clade B, was associated with relatively lower expression of AHSP. Relative expression of AHSP and AHSP/α globin ratio were both significantly higher (p<0.001) in homozygotes for clade A haplotypes compared to heterozygotes. A potential functional role of one of the variable sites, a T-homopolymer (the T18 variant being part of clade A) in the promoter was investigated in-vitro using luciferase reporter assays in K562 cells. Luciferase activity was 1.30±0.08 times higher in the T18 promoter compared to T14, consistent with genetic studies. We investigated if a shorter homopolymer could have an adverse effect on β thalassemia. Nine patients with thalassemia intermedia and a genotypic combination of heterozygous β thalassemia and 5 α globin genes (aaa/aa) were studied. Based on the allele frequencies in the healthy population, 6–7 of the 9 patients are expected to be homozygous for T18 but only two were observed. In contrast, while 2–3 patients are expected to be heterozygous for the shorter homopolymer, 3 patients were homozygous and 4 heterozygous.

In conclusion, AHSP gene expression is variable, with cis control accounting for some of its variance. The subtle altered expression might precipitate a phenotype of thalassemia intermedia in β thalassemia heterozygotes with α globin overload.
Dysregulated Arginine Metabolism and Elevated Arginase Activity in Thalassemia.
Authors: Morris Claudia R. , Kuypers Frans A. , Kato Greg , Gardner Jennifer , Larkin Sandy , Singer Titi , Vichinsky Elliott
Published: April 2020

Pulmonary hypertension (PHT) is associated with high mortality rate and is a leading factor in heart failure and death in thalassemia (Thal) intermedia. However, data on PHT and Thal major is limited. While there are several risk factors for PHT in Thal major, ongoing chronic hemolysis, despite transfusion, supports the development of hemolysis-associated PHT. Recently, we reported in sickle cell disease a novel paradigm whereby hemolysis liberates vasoactive hemoglobin and erythrocyte arginase, resulting in impaired NO bioavailability, endothelial dysfunction, PHT and death. Since arginase activity is also elevated in Thal red cells, we hypothesize that elevated arginase contributes to dysregulated arginine (Arg) metabolism in Thal. Pilot data was analyzed from transfused Thal patients (8 Thal-major, 4 E-beta Thal, 2 Hb H alpha Thal). Echocardiogram were performed in steady state in 10 of the patients; 70% (7/10) demonstrated PHT (tricuspid regurgitant jet velocity ≥ 2.5 m/s). Plasma Arg trended lower in patients with Thal (19.5 to 122mM, median 50 mM) compared to controls. Ornithine (Orn) levels were higher, and Arg/Orn ratio was lower in Thal vs. contrls. Plasma arginase activity was significantly elevated (median 0.83 mmol/cc/hr, range 0.06 – 1.17). Proline, a downstream metabolite of arginase causing pulmonary vascular remodeling was also elevated. Exhaled nitric oxide (NO) levels are significantly elevated in Thal vs. controls (49±41 parts per billion vs.18±8 ppb, p=0.02), suggesting upregulation of NO synthase in the lungs in addition to higher plasma arginase activity. Recent findings indicate diverse origins of PHT share similar pathophysiologic abnormalities, particularly decreased Arg bioavailability and a shift of metabolism towards ornithine-dependent pathways. Hemolysis is most likely a trigger for these pathologic pathyways in hemoglobinopathies. Therapies that maximize Arg and NO bioavailability may benefit patients with thalassemia.

Variable NL Control (n=36) Thalassemia (n=14) p* Arginine (μM) 67±18 57±26 (50) 0.15 Ornithine (μM) 62±22 85±68 0.05 Arg/Orn ratio 1.2±0.5 0.79±0.4 <0.01 Proline (μM) 161±48 258±116 <0.001 Citrulline (μM) 25±11 42±17 <0.001 Arginase (μmol/cc/hr) 0.33±0.2 (n=45) 0.71±0.3 <0.001
A Randomized, Placebo Controlled, Double Blind Trial of the Effect of Combined Therapy with Deferoxamine and Deferiprone on Myocardial Iron in Thalassaemia Major Using Cardiovascular Magnetic Resonance.
Authors: Tanner Mark A. , Galanello Renzo , Dessi Carlo , Westwood Mark A. , Smith Gillian C. , Khan Mohammed , Nair Sunil V. , Walker J. M. , Pennell Dudley J.
Published: April 2020

Introduction: Beta thalassaemia major (TM) is a hereditary anaemia affecting 60 000 births worldwide each year. Survival is dependent upon lifelong blood transfusions with cardiac failure secondary to myocardial iron loading being the commonest cause of death. Conventional treatment with the parenteral iron chelator deferoxamine improves mortality but prognosis remains poor and it has been reported that approximately two-thirds of patients maintained on this treatment have myocardial iron loading. More recently, the oral iron chelator, deferiprone has been demonstrated to remove myocardial iron and it has been proposed that in combination with deferoxamine it may have an additive or synergistic effect. Myocardial iron can now be rapidly and reproducibly quantified using the validated cardiac magnetic resonance (CMR) T2* technique. CMR is therefore well suited to assess the efficacy of new therapies for the removal of myocardial iron in TM.

Purpose: To report the primary outcome measure (heart T2*) from a randomized placebo controlled trial testing the hypothesis that the combined therapy of deferiprone and deferoxamine is superior in the removal of myocardial iron than the standard therapy of deferoxamine alone.

Methods: A mobile CMR scanner (1.5T Siemens Sonata) was transported to Cagliari, Italy on 3 occasions. The myocardial T2* was assessed in 167 patients with TM. 65 patients (male 27, female 38, age 30+/−5.2years) with mild-moderate myocardial iron loading (T2* 8–20ms) were randomized to receive either deferoxamine and placebo, or deferoxamine and deferiprone. The primary outcome measure of myocardial T2* was assessed at baseline, 6 and 12 months. Normal myocardial T2* is greater than 20ms.

Results: In the combined treatment group myocardial T2* improved (reducing myocardial iron) from a baseline of 11.7ms to (at 6 months) 14.6ms (+25%; CV 11.9%; p=0.007) and at 12 months 16.8ms (+40%; CV 14.2%; P<0.001). In the placebo controlled group myocardial T2* improved from a baseline of 12.4ms to (at 6 months) 14.2ms (+14%; CV 14%; p=0.15) and at 12 months 15.2ms (+21%; CV 16%; p=0.02). Analysis of covariance showed a significant difference between groups (p=0.017) with the combined group showing greater effects in improving myocardial T2* (figure 1).

Conclusion: In mild-moderate cardiac iron loading the combined therapy of deferiprone and deferoxamine is superior to deferoxamine alone in the removal of myocardial iron in TM.

Figure 1 Figure 1.
Reduced Intensity Conditioning Regimen for Unrelated Cord Blood Transplantation in Adults. A Multicentric Phase I-II Trial.
Authors: Rio Bernard , Belhocine Ramdane , Renaud Marc , François Sylvie , Gluckman Eliane , Blaise Didier , Rocha Vanderson
Published: April 2020

Curative potential of allogeneic transplantation from related or unrelated donors is limited by tranplant related mortality (TRM) and donor compatibility. After reduced intensity conditioning regimens (RIC) transplantation can be extended to older patients, those with poor performance status, organ dysfunction or intensive prior therapy. In absence of HLA-matched donor, unrelated cord blood transplant (UCBT) is a possible source of hematopoietic stem cells. In order to confirm previous report of RIC after UCBT in adults we conducted a phase I–II trial. Primary endpoint was neutrophil recovery and chimerism. Secondary endpoints were early TRM, acute GVHD incidence, early relapse and survival. Between October 2003 and March 2005, 18 patients (pts) were enrolled: median age: 46y (19–64), weight: 62.5 kg(45–90), gender: 13 females and 5 males, CMV serpositivity in 8 pts, ABO major incompatibility in 8 pts. The median of follow-up was 4.5 mo (0.5–16). Disease status were 2 ALL (CR1: 1; CR2: 1), 10 AML (CR1: 3, CR2: 6, CR3: 1), 1 AP CML, 1 CLL, 1 Waldenström disease, 3 follicular lymphoma relapsed after autologous transplantation. Infused nucleated and CD34+ cells number were respectively: 2.3x107/kg (1.7–3.7) and 0.7x105/kg (0.4–1.2). Only a single cord blood was used. HLA disparity was 0/6 in 1, 1/6 in 6 and 2/6 in 11. RIC regimen consisted of cyclophosphamide 50 mg/kg D-6, fludarabine 200 mg/m2 and 2Gy TBI at D-1. CsA and MMF from D-3 were used in GVHD prophylaxis. Pts received GCSF until 5 G ANC/l.

Results: Median day for 0.5 G/l ANC recovery was 14 days (0–28) and for 20 G/l platelets was 28 days (0–48). Mixt chimerism was present until D+56 and full chimerism at D+80/100 in all pts except 2 who relapsed. Mucositis was not observed, CMV reactivation in 2 pts, bacteremia in 3, SRV pneumonitis in 1 pt, bone aspergillosis in 1 pt. Regressive limbic encephalitis in 2 pts. Two pts recovered normal ovarian function 5 mo and 12 mo after UCBT. Grade 2 acute GVHD was observed in 2 pts and limited chronic GVHD in 3 out of 8 pts at risk. TRM was 0%. Two pts relapsed and died at D+100 and D+171. EFS was 80+/−13%. In conclusion, in spite of the short follow-up and few patients enrolled in this trial, these results confirm those reported by Barker J et al. Importantly there was a rapid neutrophil recovery with acquired late full chimerism and absence of early TRM. The encouraging results justify the use of cord blood cells in RIC and prospective trials in order to compare phenoidentical peripheral blood stem cell transplantation and UCBT with the same RIC regimen.
Efficacy of Performing a Second Non-Myeloablative Allogenic Stem Cell Transplant.
Authors: Byrne Brian J. , Antin Joseph H. , Alyea Edwin P. , Choa Nelson J. , Rizzieri David A.
Published: April 2020

Introduction: Non-myeloablative stem cell transplants are being performed for a variety of benign and malignant hematologic conditions. The reported graft failure rates have been as high as 20%. Little is reported on the safety and efficacy of a second attempt at a nonmyeloablative allogeneic transplant.

Methods: Patients who underwent at least 2 nonmyeloablative allogenic transplants at either Duke University or Dana Farber Cancer Centers were included.

Results: 9 patients were identified who underwent at least 2 preparatory regimens with stem cell infusion. 7 patients underwent related donor stem cell infusions and 2 patients had an umbilical cord donor. 4 patients, including the 2 with an umbilical cord donor, had primary graft failure. The remaining had secondary graft failure within 1–9 months from viral disease/therapy or disease progression. 5 of 9 received a 5 day regimen of fludarabine 125mg/m2, campath100mg and cyclophosphomide 2 gm/m2 total for a secondary preparatory regimen (FCC). Two patients received a one-day prep with fludarabine 30mg/m2, 200cGyTBI, cyclophosphamide 2gm/m2 and campath 20mg (FCC/TBI). The two umbilical cord patients received a one day prep that included TBI, cyclophosphamide 500mg/m2, fludarabine 30 mg/m2, and campath 30 mg (FCC/One). One patient with refractory CML failed to engraft, though 8/9 did (1 required a 3rd attempt), including 4 patients who had the same donor as their first transplant. Long-term follow-up revealed the graft was intact in 5 patients at 6, 7, 8, 21, 39, and 63 months. Three patients died.

Conclusion: Patients who have graft failure can be transplanted with a second nonmyeloablative allotransplant. Response and long-term engraftment is possible, even using the same donor.

Table 1: Patients who have undergone greater than one mini allogeneic transplant. Disease Prep #1 Donor Engraftment Prep #2 Donor Engraftment Response Key: FCC: Fludarabine, campath, cyclophosphamide; ATG FM: Fludarabine, melphalan, ATG FCC/TBI: TBI, fludarabine, campath, cyclophosphamide, FCC/One: TBI, fludarabine, cyclophosphamide, campath Thalaseemia FCC 6/6 Yes FCC 6/6 RFLP 75% CR Aplastic anemia FCC 4/6 No FCC/TBI same RFLP 99% CR Myelofibrosis FCC/TBI 6/6 RFLP 63% FCC same RFLP 98% CR CML FCC 4/6 RFLP 61% FCC 4/6 No PD Renal Cell FCC 3/6 RFLP 30% FCC same RFLP 94% PD AML/MDS FCC 4/6 RFLP 5% FCC/TBI same RFLP 89% PR AML FCC/TBI 3/6 RFLP 44% FCC 3/6 RFLP 98% CR AA/MDS ATG,FM UCB 4/6 No FCC/One UCB 4/6 RFLP 100% CR AA/MDS ATG,FM UCB 4/7 No FCC/One UCB 4/7 RFLP 100% CR
Kinetics of Early Donor Chimerism after Nonmyeloablative Haematopoietic Stem Cell Transplantation Monitored with High-Resolution Real Time Quantitative PCR.
Authors: Masmas Tania N. , Petersen Soren L. , Madsen Hans O. , Ryder Lars , Dickmeiss Ebbe , Svejgaard Arne , Vindelov Lars
Published: April 2020

Purpose: From May 2003 to March 2005, 37 patients with haematological malignancies were transplanted with peripheral blood stem cells after nonmyeloablative conditioning with fludarabine 30 mg/m2 i.v. once daily on day −4 to −2 and 200 cGY of total body irradiation on day 0. Post-transplant immunosuppression consisted of oral cyclosporin 12.5 mg/kg/day and mycophenolate mofetil 30 mg/kg/day. Three patients were not eligible for follow-up due to lack of informed consent or informative markers for chimerism measurement. The purpose of this study was to evaluate kinetics of early chimerism of CD4+, CD8+, and, CD15+ cells immediately post transplant and furthermore to identify factors associated with rejection of graft.

Methods: Blood samples were collected post transplant on day +1–3, +5–7, and then weekly. Samples were separated with immunomagnetic beads and DNA was salt extracted. Chimerism analysis was performed by automated high-resolution real-time quantitative PCR based on short insertion or deletion polymorphisms or single nucleotide polymorphisms.

Results: The kinetics of CD15+ chimerism differed from that of CD4+ and CD8+ chimerism (Figure). CD15+ donor chimerism was low with a median of 0.9% donor cells on day +1–3, and remained low until around day +14. Subsequently the donor chimerism increased rapidly towards 100%. The CD4+ and CD8+ donor chimerism in contrast started higher with a median of 16.3% and 15.1% respectively on day +1–3, increased slowly, and took months to reach 100%. CD4+, CD8+ or CD15+ donor chimerism on day +1–3 did not predict later development of acute graft versus host disease. Two patients had primary rejection of the donor stem cells, while four patients had secondary rejection. The patients with primary rejection had no measurable donor chimerism in any cell lineages from day +1–3. No patient with CD15+ donor chimerism above the median (0.9%) on day +1–3 rejected, while 40% (6/15) of the patients with CD15+ donor chimerism below the median rejected (LogRank test, p=0.01). No differences were found in total number of nucleated cells, CD34+, CD3+, CD4+, or CD8+ cells in the donor harvest between patient who rejected and those who did not.

Conclusions: The kinetics of early CD15+ donor chimerism differed from the kinetics of early CD4+ and CD8+ donor chimerism. Early CD15+ donor chimerism was markedly lower than CD4+ and CD8+ donor chimerism. CD15+ donor chimerism increased toward 100% donor chimerism more rapidly than the CD4+ and CD8+ chimerism. Primary rejection of donor stem cells can be identified early, as these patients have no measurable donor CD4+ or CD8+ chimerism on day +1–3. Furthermore, patients with CD15+ donor chimerism above the median on day +1–3 have a low risk for rejection.

Figure Figure