bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ferroportin1-Deficient Zebrafish Treated with Iron Dextran Develop Anemia Despite Increased Macrophage Iron Stores.
Authors: Fraenkel Paula G. , Traver David , Donovan Adriana , Zon Leonard I. 
Published: October 2019

Abstract
Patients with type 4 hemochromatosis exhibit iron accumulation in the reticuloendothelial system and a tendency to develop anemia following venesection. These patients are heterozygous for mutations in the intestinal and macrophage iron exporter, ferroportin1 (fpn1), however it is not known whether these mutations cause a gain or loss of function. Of current animal models, only the Tp85c allele of the zebrafish anemia mutant weissherbst (weh) encodes a missense mutation (L167F) in the conserved region of fpn1, between transmembrane helix 3 and 4, where several missense mutations have been identified in individuals with type 4 hemochromatosis. Tp85c homozygote zebrafish die within 10–14 days post-fertilization, however administering iron dextran injections allows them to survive. To explore the effects of fpn1 deficiency on blood development and iron homeostasis in adult zebrafish, we raised Tp85c homozygotes along with control cohorts of injected and uninjected heterozygotes and wild types. Iron dextran injections were discontinued at 4 months of age in all cohorts. Although iron accumulation was observed in all iron-injected fish, only homozygotes exhibited iron accumulation in the intestinal epithelium on Perl’s staining, consistent with a block in enterocyte export. At 6 months of age, none of the cohorts were anemic, however at 12 months of age, only the homozygotes developed hypochromic anemia, characterized by reduced levels of hemoglobin per erythrocyte and impaired late erythroid maturation. Histologic evaluation of the anemic homozygotes revealed increased numbers of iron-loaded macrophages in the liver and in the kidney, the zebrafish bone marrow equivalent. Quantitative realtime rt-PCR, performed on liver and intestinal tissues at 1 year of age, revealed that homozygotes had increased intestinal expression of fpn1 compared to the other cohorts. Hepatic transcript levels of the secreted iron-regulator hepcidin were profoundly decreased in homozygotes, consistent with response to anemia. In contrast, hepatic transcript levels for transferrin, fpn1, and DMT1 were not significantly different among the cohorts. In summary, we provide evidence that Tp85c is a loss of function mutation affecting enterocytes and macrophages. After iron dextran injections were discontinued, Tp85c homozygotes developed anemia, despite increased macrophage iron stores. These studies support the hypothesis that fpn1-deficiency impairs iron cycling and suggest this as a possible mechanism in type 4 hemochromatosis.
Management of Severe Intractable Sickle Cell Pain with Intrathecal Opioid Analgesia.
Authors: Ballas Samir K. , Kucler Marcia , Tabbaa Kutaiba 
Published: October 2019

Abstract
Management of severe Sickle Cell Pain (SCP) often entails the utilization of opioids via the intravenous route. In this abstract, we describe the utilization of Intrathecal Morphine Analgesia (IMA) infusion in the management of severe SCP. Two African American women were the subjects of this study: a 50-year-old patient with sickle cell anemia and a 51-year-old patient with sickle-b+-thalassemia. Complications of their disease included frequent acute painful episodes, S/P cholecystectomy, iron overload, and severe avascular necrosis (AVN) of the left hip in the first patient and frequent acute painful episodes, S/P cholecystectomy, retinopathy, and severe AVN of the right hip in the second patient. Both patients were admitted frequently to the hospital due to severe acute painful episodes involving mostly the hips and lower extremities. In both patients, a Medtronic Indura Catheter was inserted intrathecally under fluoroscopy guidance. The entry level was at the L3–L4 lumbar space and the tip of the catheter reached the T8 level. The catheter was attached to an Isomed pump that was filled with a mixture of preservative free morphine and clonidine and placed in a subcutaneous pouch in the abdominal wall. The first patient required 0.5mg of morphine and 25mg of clonidine daily, and the second patient required 1.5mg of morphine and 150mg of clonidine daily. The pumps were refilled every 2–3 months. No side effects were noted and the vital signs of both patients remained stable. Both patients continue to use IMA for 2–3 years to date with no complications. The utilization of IMA resulted in significant decrease in the frequency of acute painful episodes treated in the emergency room or hospital, as well as the number of hospital days and the amount of oral opioid consumption expressed in morphine sulfate (MOSO4) equivalent as shown in the table. Moreover, the quality of life of both patients improved: the first patient resumed social and family activities and the second patient could be gainfully employed. The data show that utilization of IMA in selected patients with severe intractable pain below the diaphragm has a significant salutary effect on the frequency and duration of hospital admissions and the total amount of opioids consumed.
Effect of IMA on Annual Utilization of Medical Facilities and Opioid Consumption
Hospital Amissions Hospital Days ER Visits PO MOSO4 Equivalent Patient #1 Before IMA 15 222 14 17585 mg After IMA 7 88 7 6885 mg Patient #2 Before IMA 9 19 21 8415 mg After IMA 5 18 9 3310 mg
Neutropenia in Patients with Thalassemia Major.
Authors: Galanello Renzo , Origa Raffaella 
Published: October 2019

Abstract
Monitoring of the neutrophil count has become an important issue in the management of thalassemia patients after the introduction of the new oral chelator deferiprone, as this chelator has been associated with agranulocytosis and milder neutropenias. Although a clear relation between deferiprone and agranulocytosis has been demonstrated, it is unclear whether milder neutropenias are due to deferiprone use or to other factors such as hypersplenism, infections, autoantibodies, or other drugs. In this study we evaluated 7,708 consecutively blood counts and leukocyte differential obtained between August 2003 to June 2004 from 344 patients with thalassemia major, aged 2 to 40 years, treated with deferiprone (N=39), desferrioxamine (N=179), ICL670 (N=45), or a combination of deferiprone and desferrioxamine (N=81). Complete blood count and differential were performed with Coulter LH 750 automated haematology analyzer (Beckman Coulter, Miami, FL), which can detect nucleated red blood cells (NRBC) and correct the white blood cell (WBC) count in the presence of NRBC, platelets clumps, unlysed or fragmented RBC. The capability to correct the WBC count is particularly relevant in the monitoring of the neutrophil counts of patients with excessive NRBC, which could lead to potential errors in the differrential count. Neutropenia was defined as a neutrophils count below 1500 cells/μL, whereas agranulocytosis was defined as a neutrophil count below 500 cells/μL. No episodes of agranulocytosis were observed. Episodes of neutropenia (range 850 – 1490 neutrophils/μL) were observed in 29 (8,4%) patients. Repeated episodes of neutropenia were observed in 7 of the 29 patients. At the time of neutropenia, 23 (79%) of the patients were being chelated with desferrioxamine, 2 (7%) with deferiprone, 3 (10%) with a combination of both chelators, and 1 (3%) with ICL670. Considering the type of chelation, neutropenia was observed in 12,8% of patients being treated with desferrioxamine, 5% in patients treated with deferiprone, 3,7% in patients treated with combination therapy, and 2.2% in patients treated with ICL670. The severity of neutropenia, assessed as absolute neutrophil count, was not different on the basis of the type of chelation (p=0,26). Neutropenia was more frequent in children than in adults (31% vs 7.8%, respectively, p=0.0001). No statistically significant difference in the incidence of neutropenia was found between males and females (10.2% vs 6.5% respectively, p=0.2), anti-HCV positive and negative (7.7% vs 10.3% respectively p=0.43), or splenectomized and non-splenectomized patients (11.1% vs 8.1% respectively, p=0.5). Neutropenia appears to be a common event in patients with thalassemia major, irrespectively of the chelation therapy used. The higher incidence in children may be secondary to a greater frequency of viral infections at this age, which may produce neutropenia with several mechanisms including redistribution, sequestration and destruction of neutrophils.
Molecular Analysis of Alpha Hemoglobin Stabilizing Protein (AHSP) in Caucasian Patients with different Beta-Thalassemia Phenotypes.
Authors: Cappellini M. Domenica , Refaldi Chiara , Bignamini Daniela , Zanaboni Laura , Fiorelli Gemino 
Published: October 2019

Abstract
Beta-thalassemia is a inherited hemoglobin disorder characterized by absent or reduced synthesis of the b globin chains. The pathophysiology and the severity of b-thalassemias reflect the degree of globin chain imbalance and the excess of free a globin chains that precipitate and cause oxidative damage in red cell precursors inducing their premature destruction in the bone marrow (ineffective erythropoiesis). Although the phenotype of b thalassemias can be modified by inherited factors such as different number of a globin genes or increased fetal hemoglobin production, other mechanisms appear to be involved. Recently, a protein, named alpha hemoglobin stabilizing protein (AHSP), that acts as a molecular chaperone specifically for free a globin chains, preventing their precipitation in red cell precursors, has been identified.
To establish whether AHSP might have a role in modifying the clinical outcome of b thalassemias, we have analyzed the AHSP gene in 70 Caucasian b thalassaemic subjects: 26 patients with b°/b° genotype (Thalassaemia Major),24 patients with Thalassemia Intermedia (b°/b+ or b+/b+) and 20 patients with a Thalassaemia Intermedia phenotype but with only one mutation in the b globin gene, a normal a globin genotype and no other causes of anemia. In all the subjects, we have performed Denaturing High-Performance Liquid Chromatography (DHPLC) of the three exons and the direct genomic sequencing of coding and noncoding regions (~ 1.5 kb) of AHSP gene. No mutations able to modify the structure or function of AHSP have been found, however we identified eight single nucleotide polymorphisms (SNPs) spanned along the whole gene that segregate in four different aplotypes. To evaluate a possible relationship between a particular aplotype and b thalassemia severity, the allele frequency of each single aplotype in the tree groups has been established and compared to that of 33 Caucasian normal controls: no statistically significant association has been proved. Even though the loss of AHSP aggravates the b thalassaemia phenotype in mice, in Thalassemic Caucasian population the AHSP apparently doesn’t make changes in the clinical severity of b thalassemia confirming the results recently found in Thai population.
Massive and Selective Ex Vivo Generation of Matured and Functional Human Red Blood Cells (RBC) from Hematopoietic Stem Cells of Diverse Origins: Towards the New Concept of “Cultured RBC”.
Authors: Douay Luc , Kobari Ladan , Lapillonne Helene , Chalmers David , Kiger Laurent , Cynober Therese , Marden Michael C. , Wajcman Henri , Giarratana Marie-Catherine 
Published: October 2019

Abstract
We report a technological approach permitting, for the first time, the massive (up to 2x106-fold cell expansion) and selective (100%) ex vivo production of mature RBCs (cRBCs) starting from CD 34+ cells from peripheral blood (PB), bone marrow (BM) or cord blood (CB) into mature red cells in three steps: firstly, cell proliferation and erythroid differentiation were induced in serum free media supplemented with SCF, IL-3 and Epo for 8 days. Secondly, cells were co-cultured with additional Epo alone on either the murine MS-5 stromal cell line or human mesenchymal cells for 3 days. In the third step, all exogenous factors were withdrawn and cells were incubated on a simple stroma for 4 to 10 days.
These cultured erythroid cells (reticulocytes and mature RBCs) displayed characteristics identical to those of native cells, in terms of MCV, MCH, MCHC, enzyme content (G6PD and PK) and deformability. The nature of the Hb produced depended on both the origin of the CD34+ cells and the culture conditions. cRBCs derived from PB or adult BM contained adult Hb (95±1%) whereas cRBCs derived from CB contained essentially HbF (64±13%). As for native RBCs, Hb was able to fix and release oxygen. CFSE-labelled-reticulocytes ex vivo generated from leukapheresis were injected into NOD-SCID mice. The transfused reticulocytes were found in the circulation to the same extent as native RBCs and fully matured into RBCs. This methodology is applicable for fundamental analysis of the mechanisms of terminal erythropoiesis and hemoglobin synthesis. Moreover, large scale cRBCs production could be possible with such a protocol. It can therefore be extrapolated to a wide range of clinical applications in the field of gene therapy, infectious diseases and particularly transfusion medicine with a pointed interest for the generation of a cell population homogeneous in age, thus achieving the new concept of cultured RBCs transfusion.
Identifying Mechanisms of Enhanced NK Cell Cytotoxicity Using Permanent NK Cell Lines.
Authors: Suck Garnet , Branch Donald R. , Vergidis Joanna , Fahim Soad , Keating Armand 
Published: October 2019

Abstract
Although NK cells are promising candidates for adoptive immunotherapy and at least one permanent cell line is in clinical trials, further studies evaluating efficacy and mechanisms of action are warranted. As a first step towards identifying the most potent effector cells, we investigated the molecular mechanisms of cytotoxicity of the three natural killer lines, KHYG-1, NK-92 and YT, and the NK-T cell line, SNT-8, under standardized culture conditions with human serum as the only serum source. We confirmed the previously established differential killing potential of the 4 cell lines against target K562 cells using a new method based on detecting Annexin V (+) target cells by flow cytometry. By labeling the NK cells with specific antibodies, the assay is designed to screen any target cell for NK cytotoxicity. In contrast to previous reports, we found KHYG-1 the most cytotoxic, followed by NK-92, SNT-8 and YT. Genotypic and transcriptional phenotypic analysis of the cell lines for killer cell Ig-like receptors (KIRs) by SSP-PCR showed that inhibitory KIRs outnumbered activating KIRs in all cases but did not explain the differential cytotoxicity. A correlation with cytotoxicity was found with expression of the activating type II C lectin-like receptor, NKG2D: KHYG-1, 99%+; NK-92, 91%+; SNT-8, 6%+ and YT, 2%+. Moreover, the ITAM-bearing adaptor molecule DAP12, involved in the alternative activation signaling pathway via NKG2C-CD94 and activating KIRs, was detected only for KHYG-1 by immunoblotting, These data suggest that the superior cytotoxicity of KHYG-1 may be due, in part, to the additional activation of this alternative pathway that is not triggered in the other lines. The downstream signaling molecules involved in NK cytotoxicity, including the tyrosine phosphatases SHP-1, SHP-2 and SHIP-1 (inhibitory), as well as SHIP-2, the tyrosine kinases ZAP-70, Syk, PI3K and the MAP kinase phospho-ERK-2 (activating) were compared among the lines by immunoblotting followed by densitometry normalized to b-actin or ERK-2 for phospho-ERK-2. We found that the activating kinase Syk was expressed only in NK-92 and KHYG-1 at even higher levels. Also, phospho-ERK-2, was hyperphosphorylated only in KHYG-1. Perforin, granzyme A and granzyme B, present in cytotoxic granules, were compared by RT-PCR and intracellular flow cytometry and/or immunoblotting. Perforin was found to be almost exclusively fully processed to the active 60 kD form only in KHYG-1, in contrast to the other lines, which displayed approximately half the levels of the active form. These data provide a further explanation for the superior cytotoxicity of KHYG-1 and demonstrate the value of comparing cell lines with diverse cytotoxic potential as a means of elucidating cell killing mechanisms. It is conceivable that targeted modifications to the signaling pathways for cytotoxicity in this model will lead to the generation of activated NK cells with even greater efficacy.
Continuous Versus Subsampling Analysis of Thrombin Generation.
Authors: Lau Alice , Berry Leslie , Chan Anthony K.C. 
Published: October 2019

Abstract
Thrombin generation measurement is becoming a useful technique to assess the hemostatic system. Subsampling methods though labour intensive, measure activities directly. The continuous method has become an alternative option. Studies by Hemker et.al. (1) showed that both methods gave comparable results but had some differences. We decided to further assess the variation between the methods. For the continuous method, 125μl of plasma, 15μl of Pefabloc (50mg/ml) and 12.5μl of Pefachrome TG5134 (0.5mM) were added to a 96 well ELISA plate and prewarmed to 37°C. The reaction was started by adding 97.5μl of 1/12.5 Thromborel S in 25.64mM CaCl2/Tris buffer. The optical density at 405nm was recorded at an interval of 15sec for 15 min. For the subsampling method, 125μl of 1/16 Thromborel S in 20mM CaCl2/Tris buffer was added to 125μl of defibrinated (Arvin) plasma. Subsamples were taken into 5mM EDTA solution at 15sec intervals. Chromogenix S2238 was used for measuring thrombin in the subsampling method. The continuous method gave a peak height of ~410nM and area under the curve of 860–900 nM.min whereas the subsampling method gave a peak height of ~360nM and an area under the curve of 550–600nM.min. This difference disappeared when PefachromeTG5134 was added into the reaction mixture prior to running subsampling assays. Thus, substrate likely slowed inhibition of generated thrombin in the continuous assay, which confirms previous findings by Hemker et.al. (1). Further examination revealed that the concentration of α2M-IIa complex was very different between the two methods. α2M-IIa complex calculated from the continuous method (80nM) was much greater than α2M-IIa complex obtained experimentally by the subsampling method (38nM) even in the presence of TG5134. However, addition of a large excess of antithrombin and heparin at the final equilibrium time point of the continuous assay reduced the α2M-IIa complex to the same level (38nM) as that of the subsampling method. We have shown that the slow-acting substrate Pefachrome TG5134 gave an increased estimate of both free and α2M-IIa complex. Thus, a significant amount of free thrombin remains at the end of thrombin generation that is not accounted for in the continuous method. This estimate of final α2M-IIa complex concentration may be important in determining the relative protection against long-term plasma thrombin activity in pediatric patients with elevated α2M levels.
SB 497115-GR, a Low Molecular Weight TPOR Agonist, Does Not Induce Platelet Activation or Enhance Agonist-Induced Platelet Aggregation in Vitro.
Authors: Erhardt Joseph , Erickson-Miller Connie L. , Tapley Peter 
Published: October 2019

Abstract
SB-497115-GR is a low molecular weight, orally active molecule that requires Tpo receptor (TpoR) expession for activity and activates JAK/STAT signalling in platelets. The ability of recombinant Tpo to enhance agonist-induced platelet aggregation is well-documented, thus this study was undertaken to compare the ability of rhTpo (150 ng/ml) and SB-497115 (0.01–10 uM) to affect platelet activation. Platelets were obtained from healthy human volunteers, following written informed consent, by standard venipuncture technique. Citrated whole blood was used directly in platelet activation and P-selectin assays within 15 minutes. Platelet aggregation was assessed by light transmittance aggregometry. Aggregation was induced by a submaximal concentration of adenosine diphosphate (ADP) (1.0 or 1.5 uM) in experiments examining synergy with rhTpo (150ng/mL) or SB-497115 (0.01–10 uM). For examination of potential inhibitory actions of SB-497115 on platelet function, aggregation was induced by ADP (3 uM), collagen (2 ug/mL), or the thrombin receptor activating peptide (TRAP; 20 uM). Pre-treatment of platelet samples with rhTpo, but not SB-497115, potentiated the effects of 1.0 or 1.5 uM ADP. No inhibitory effect on normal ADP, Collagen or TRAP-induced aggregation was seen with either SB-497115-GR or rhTpo. In experiments investigating the expression of platelet P-selectin, an early marker of platelet activation, platelets labeled with anti-CD62P antibody were analyzed by flow cytometry. Data was expressed as percent of platelets positive for CD62P within the defined platelet gate. Treatment with SB-497115 (0.1 – 10 uM) demonstrated no induction of platelet CD62P expression above background levels. In contrast, rhTpo produced a modest but significant and reproducible increase in the percent of platelets positive for CD62P. This study demonstrates that, in contrast to rhTpo, the orally active small molecular weight agonist of the Tpo receptor, SB-497115-GR, does not mimic the ability of rhTpo to enhance ADP-induced aggregation of platelets. SB-497115-GR also did not directly induce P-selection expression on human platelets. Finally, SB-497115 has no antagonistic effect on ADP-, TRAP-, or collagen-induced platelet aggregation. Thus, SB-497115 is differentiated from rhTpo with respect to platelet activation.
ATRA Resolves the Differentiation Block in t(15;17) Myeloid Leukemia by Restoring PU.1 Expression.
Authors: Mueller Beatrice U. , Pabst Thomas , Osato Motomi , Tenen Daniel G. 
Published: October 2019

Abstract
The transcription factor PU.1 is crucial for hematopoiesis. pu.1 knockout mice lack mature neutrophils, macrophages, B- and T-cells. PU.1 regulates important taget genes, like the M-CSF-, the G-CSF-, and the GM-CSF receptor promoter. Heterozygous mutations of the PU.1 gene have been found in some patients with AML, but not in AML with the t(15;17) encoding the PML-RARA fusion protein. These facts let us to hypothesize that decreased expression of PU.1 might contribute to the block in myeloid differentiation in t(15;17) AML. We therefore screened 141 AML patients for PU.1 mRNA expression using real-time PCR technology. We detected that t(15;17) AML patients (n=18) had significantly less PU.1 mRNA than AML-M2 patients (n=35), and 16fold less PU.1 mRNA than mature neutrophils. Moreover, the amount of PML-RARA transcripts in APL patients was inversely correlated to PU.1 mRNA levels. Conditional expression of PML-RARA in U937 cells suppressed PU.1 mRNA and PU.1 protein. HT93A cells derived from a t(15;17) AML-M3 patient nearly lack PU.1 mRNA and protein expression, and binding activity to the PU.1 site in the M-CSF-Receptor promoter is suppressed. Treatment of HT93A cells with all-trans retinoic acid (ATRA) powerfully restores PU.1 mRNA and protein and induces neutrophilic differentiation. We identified a region of extending −122 bp upstream of the translation start site in the PU.1 promoter mediates ATRA-induced activation of the PU.1 promoter in HT93A cells. Finally, conditional expression of PU.1 in HT93A cells is sufficient to trigger neutrophilic differentiation. This is the first report demonstrating that PU.1 is a critical target of the PML-RARA protein and an early ATRA-responsive gene.