bluuebunny/crossref_metadata_2025_split huggingface dataset:
P27 Deficiency Results in Enhancing Hematopoiesis By Increasing IL22 Production from Bone Marrow Mesenchymal Stem Cells
Authors: Lu Rui-Nan , Wang Qian , Li Jianyong , Miao Dengshun 
Published: October 2019

Abstract
Hematopoietic microenvironment plays an important role in hematopoiesis, in which the bone marrow mesenchymal stem cells (BM-MSCs) can directly or indirectly affect the hematopoietic cells and regulate hematopoietic function. It has been reported that bone marrow hematopoietic progenitor cells were increased in p27 deficient mice, however, it is unclear whether BM-MSCs are mediated enhancing hematopoiesis resulting from p27 deficiency.
To answer this question, the conditioned medium (CM) were harvested from BM-MSC cultures derived from WT and p27 knockout (KO) mice, respectively and added to the cultures of bone marrow cells derived from WT mice. After 4 days, the sca-1+ckit+Lin- cells (hematopoietic stem cells, HSC) and sca-1+ckit+Lin+ cells (hematopoietic progenitor cells, HPC) in the resulting cells were analyzed by flow cytometry, and the assay for colony forming cells (CFCs) was performed and the colony numbers of the CFC were counted. Results showed that the HPC fraction and the number of CFCs were increased significantly, but the HSC fraction was not increased in the bone marrow cell cultures with the CM from p27 KO mice compared with those with the CM from WT mice.
To identify potential factors from the CM which enhance hematopoiesis caused by p27 deficiency, differences of protein expression profiles in the CM from the WT and p27 KO mice were examined by protein chip analysis. Five proteins were identified which were significantly increased in the CM from p27 KO mice, including interleukin-22 (IL-22), type I receptor of transforming growth factor-β, tumor necrosis factor- related apoptosis-inducing ligand, VE-cadherin and vascular endothelial growth factor B.
To determine whether IL22, as a paracrine factor, plays a role in stimulating hematopoiesis by activating JAK-STAT signal pathway mediated by IL22 receptor A1 (IL22RA1), we examined the effect of IL22 on HPCs and the expression of signal molecules of JAK-STAT pathway. Results showed that the expression levels of IL22 at both mRNA and protein levels were up-regulated significantly in BM-MSCs derived from p27 deficient mice compared with those from WT mice. The HPC fraction and the number of CFCs were increased significantly in bone marrow cultures in the present of IL22, and were decreased significantly in the cultures with the CM from p27 knockout mice containing IL22 antibody. The percentages of IL22RA1, STAT3 and p-STAT3-S727 positive HPCs were increased significantly in the bone marrow from p27 KO mice compared with that from WT mice.
Results in this study indicate that IL22, as a paracrine factor, might play an important role in stimulating hematopoiesis by activating JAK-STAT signal pathway mediated by IL22RA1.

Disclosures
No relevant conflicts of interest to declare.
Threshold Ferritin Values to Predict Control of Liver Iron Burden in Thalassemia
Authors: Lal Ashutosh , Weyhmiller Marcela G , Vichinsky Elliott 
Published: October 2019

Abstract

Transfusion-dependent (TD) and non-transfusion dependent (NTD) forms of thalassemia are associated with progressive iron overload reflected by increase in serum ferritin and liver iron concentration (LIC). Despite the adoption of non-invasive methods, measurement of LIC is available to only a minority of patients or can only be performed annually. Maintenance of low systemic iron usually depends on frequent determination of ferritin, a less reliable marker whose level can vary among individuals with similar LIC. We evaluated the relationship between LIC and ferritin in TD and NTD (including un-transfused (NT) or intermittently transfused (TI)) individuals to determine the validity of average annual ferritin to identify inadequate control of liver iron (defined as LIC >7 mg/g dry-liver-weight).
After excluding individuals with active hepatitis C virus infection, the LIC (measured by Ferritometer) and serum ferritin values were available from 115 TD (729 observations), 36 TI (139 observations) and 14 NT (28 observations) individual patients.
The median (range) ferritin and LIC were 1,684 (91-14,155) ng/mL and 10.2 (0.14- 59.4) mg/g for TD, 469 (50-4,536) ng/mL and 8.8 (2.4-33.1) mg/g for TI, and 407 (30-1,061) ng/mL and 5.7 (1.3-22.8) mg/g for NT groups, respectively. The slope of the regression line between ferritin and LIC was significantly different between the 3 groups (p=0.019), being lowest in TD, intermediate in TI, and highest in NT. The predicted LIC for ferritin level of 1000 ng/mL was 8.1 mg/g for TD, 12.1 mg/g for TI, and 13.5 mg/g for NT groups. Receiver operating characteristics (ROC) analysis showed that in NTD group (NT+TI), a ferritin threshold of >200, >300 and >500 ng/mL failed to correctly identify 7%, 21% and 38% of individuals with LIC >7 mg/g, respectively. In contrast, in the TM group, these ferritin values were associated with false negative rate of 0%, 0% and 2%, respectively. In a subgroup analysis restricted to TM patients with ferritin <1,000 ng/mL, LIC varied from 0.14 mg/g to 21.1 mg/g. ROC analysis in this subgroup showed no ability of ferritin to discriminate between individuals with LIC <3 mg/g or ≥3 mg/g (AUC 0.54, P=n.s.).
In conclusion, transfusions significantly alter the relationship between serum ferritin and LIC in thalassemia. In TM individuals, ferritin levels do not identify those with very low LIC. NTD patients with seemingly safe serum ferritin levels may have clinically significant hemosiderosis and be at risk for organ injury. These relationships should be factored into making decisions about screening or treatment of iron overload in untransfused or intermittently transfused individuals with thalassemia.

Disclosures
No relevant conflicts of interest to declare.
Nrf2 Activation and Fetal Hemoglobin Induction in Sickle Cell Disease
Authors: Krishnamoorthy Sriram , Gupta Dipti , Hobbs William E. , Loh Christine , Light David R. , Peters Robert , Sturtevant Sarah , Pace Betty , Nakamura Yukio , Lucas Brian , Vieira Benjamin F. 
Published: October 2019

Abstract
Sickle Cell Disease (SCD) is caused by a point mutation in the beta- chain of hemoglobin, triggering a complex pathophysiology resulting in recurrent, painful vaso-occlusive events (VOCs) and chronic hemolytic anemia. Induction of fetal hemoglobin (HbF), is a well-established approach towards the treatment and potentially cure for SCD. This is exemplified by the condition known as Hereditary Persistence of Fetal Hemoglobin (HPFH), which when present in SCD patients results in asymptomatic disease. Reactivation of fetal hemoglobin can be achieved by different methodologies both genetic manipulation and pharmacological agents. The mechanism of action of hydroxyurea, the only FDA approved drug for the treatment of SCD, is by activating HbF. We have investigated the role of Nrf2 in activating fetal hemoglobin and it potential to ameliorate the symptoms of SCD. Nrf2 is a basic leucine zipper transcription factor that is bound to a cytosolic protein Keap1, a Kelch domain containing protein, which targets the factor to the proteasome. Previous studies have shown that chemical activation of Nrf2 leads to induction of HbF. Here we show that siRNA based knockdown of Keap1 led to ~70 % reduction in Keap1 mRNA levels in Human Umbilical cord Derived Eryrthroid Progenitor cells (HUDEP), and concomitantly mediated a 2-fold induction of g-globin mRNA levels. Peripheral blood derived mononuclear cells differentiated into erythroid progenitors treated with bardoxolone-Me, a potent Nrf2 activator, resulted in a significant 4-fold induction in γ-globin mRNA, 2.5 fold induction in F-cells and up to 2.5-fold up-regulation in both Aγ and Gγ globin protein. Bardoxolone-Me treated erythroid progenitors also demonstrated a significant reduction in hypoxia-induced shape changes of sickle cells - about 42 % decrease at 100 nM and 55 % decrease at 250 nM. Bardoxolone-Me was also evaluated in a Townes SCD mouse model carrying an inducible γ-globin gene. A single oral dose of bardoxolone-Me at 30mg/kg, showed 9-fold induction in γ-globin mRNA levels at 6 h compared to untreated mice. Based on these findings, Nrf2 activation, can potentially be utilized for HbF induction in the treatment of SCD.

Disclosures
Krishnamoorthy: Biogen: Employment, Equity Ownership, Other: share holder. Gupta:Biogen: Employment, Equity Ownership, Other: share holder. Hobbs:Biogen: Employment, Equity Ownership, Other: shareholder. Loh:Biogen: Employment, Equity Ownership, Other: share holder. Light:Biogen: Employment, Equity Ownership, Other: share holder. Peters:Biogen: Employment, Equity Ownership, Other: share holder. Sturtevant:Arietis: Employment. Pace:Augusta University: Employment; Biogen: Research Funding. Nakamura:Riken Institute: Employment. Lucas:Biogen: Employment, Equity Ownership, Other: share holder. Vieira:Biogen: Employment, Equity Ownership, Other: share holder.
Hydroxyurea Is Associated with Early Onset Malaria Episode in Treated Sickle Cell Disease Patients
Authors: Guindo Aldiouma , Sarro Yeya dit Sadio , Toure Boubacari Ali , Fane Baba , Coulibaly Mody , Diallo Oumahane Ouane , Dorie Alain , Diallo Dapa Aly 
Published: October 2019

Abstract
Hydroxyurea (HU) is the only drug which has demonstrated efficacy in terms of reduced incidence of VOC and ACS, reduced need for hospitalization and reduced number of blood transfusion in sickle cell disease. African countries are among those with highest rate of both malaria and sickle cell disease. Despite of that the heterozygous form (HbAS) is known to protect against malaria, there is no documented studies on the clinical effect of HU on malaria. 36 patients aged 4 to 60 years old and treated by HU at the sickle cell disease research and control center of Bamako for at least 2 years. The treated group was compared to a matched untreated sickle cell patients group. During the follow-up, malaria incidence and onset to clinical episode of malaria were compared between treated SCD patients and untreated group. Results of the comparison show that there is no increased incidence of malaria in the treated group after 2 years follow-up (P=0.9). However, a reduction on the time to first malaria episode within the sickle cell population treated with HU was observed (P=0002).Based on this observed reduced time to first malaria episode in HU treated SCD patients, the use of HU in malaria endemic areas needs to be revaluated. It is howether clear that a more comprehensive approach would hopefully reduce the morbidity due to HU use for SCD affected children.

Disclosures
No relevant conflicts of interest to declare.
Thromboelastography and the Functional Confirmation of Heparin-Induced Thrombocytopenia in Donor Whole Blood
Authors: Ero Mike , Yamini Bynagari , Lewis Bradley , Powers Kathleen , Cota Jacob , Switala Lauren 
Published: October 2019

Abstract
Thromboelastography (TEG) presents a novel testing environment to detect functional antibodies associated with heparin-induced thrombocytopenia (HIT). TEG is considered a qualitative and qualitative method for assess global hemostasis in a test environment that includes shear stresses. In this study we explore the sensitivity of TEG to detect a HIT-associated reaction when whole blood or platelet rich plasma (PRP) is mixed with heat inactivated positive HIT sera in the presence of low dose (0.2U/mL) or high dose (100U/mL) sodium heparin. TEG is designed to record the kinetic changes in whole blood or plasma-based samples during clot initiation, propagation and fibrinolysis. Here we show that HIT positive sera can cause a reaction in both whole blood and PRP when exposed to reactive normal donor whole blood or PRP. Curiously, whole blood was shown to be more sensitive to the presence of HIT positive sera than PRP. The ration of heat-inactivated sera to donor whole blood or PRP was 1 to 2 parts. HIT positive sera was obtained from a patient that was clinical diagnosed with HIT type II and had a clinically significant positive ELISA with an optical density of 1.735 ± 0.032. HIT positive sera with ELISA optical densities above 1.000 or associated with an enhanced likelihood of developing thrombosis. Additionally, the HIT positive sera was functionally positive with 13 qualified, reactive HIT normal platelet donors with a washed-platelet heparin-induced platelet activation (wp-HIPA) average result of 66 ± 11%. The wp-HIPA is an established reference method or GOLD STANDARD assay for the diagnosis of HIT. The positive TEG detection of this functional HIT antibody may reveal another, more rapid test approach for the hospital and reference laboratory diagnosis of HIT. Additional studies are warranted to further characterize the comparative sensitivities of TEG and wp-HIPA and other reference methods for the diagnosis of HIT.

Disclosures
No relevant conflicts of interest to declare.
Beyond Prothrombin Time and Activated Partial Thromboplastin Time: Clot Formation Rate and Clot Structure Index As Novel Parameters for Coagulation Test Running Head: Novel Parameters of Coagulation Test
Authors: Song Jaewoo , Jihoon Yoon , Minyong Lee , Tae Hyun Yoon 
Published: October 2019

Abstract
BACKGROUND: Coagulation analyzers based on turbidimetry provide clot waveform data. Clotting times are defined only a point on this curve and have limitations to represent the whole characteristics of curve. We investigated to represent the whole curve shape by additional parameters and explored its clinical utility.
METHODS: We applied the Gompertz growth model to the waveform and the two-dimensional curve shape can be expressed with novel parameters. The model was evaluated in terms of fit, applicability, and reproducibility. To understand how the properties of parameters affected by various conditions, we controlled fibrinogen concentration, clot structure, and factor activity. For clinical application of these parameters, a reference interval was established, and the distribution was studied in different populations (normal, lupus anticoagulant-positive, and hemophilia). Finally, they were examined for use as indicators of disease severity in hemophilia and direct oral anticoagulant monitoring.
RESULTS: The model was near-perfectly fitted to the waveform, mostly applicable, and reproducible. Two parameters of the model, κ and α, determined the shape of waveform; κ was linked to the fibrinogen content and clot structure, and α reflected clotting activity and influenced on κ inversely. The clot formation rate (CFR) and clot structure index (CSI), defined using κ and α, provided additive clinical information for hemostatic function evaluation, disease severity estimation, and anticoagulant monitoring beyond the clotting times.
CONCLUSIONS: The clotting curve model enabled to utilize the overall shape of clot waveform. CFR and CSI which derived from the model can be used as additional informative parameters of coagulation test.

Disclosures
No relevant conflicts of interest to declare.
Adherence Rates Between Once and Twice Daily Direct Oral Anticoagulants - Results of a Single University Pharmacy Cohort Study
Authors: Shatzel Joseph , Daughety Molly , Tao Derrick , Kim Edward , Olson Sven , Olyaei Ali , Deloughery Thomas G. 
Published: October 2019

Abstract
INTRODUCTION
Direct oral anticoagulants (DOACs) have been shown to have equal or superior efficacy and safety when compared to Vitamin K antagonists, and have become first line therapy for many disease processes. While both the once daily drug rivaroxaban and the twice daily drug apixaban have been shown to be highly effective anticoagulants, it is unknown if differences in their dosing frequency affects patient compliance and, consequently, the likelihood of anticoagulant failure. In this study, we retrospectively evaluated the medication adherence rates of all individuals prescribed therapeutic rivaroxaban or apixaban over a one year period in our pharmacy.
METHODS
In this retrospective cohort study, we obtained a list of all patients prescribed treatment dose rivaroxaban or apixaban from July 2015 to July 2016 (including the initial loading and maintenance dose) at our university-affiliated pharmacy. We excluded patients who received less than three separate refills or patients on prophylactic or decreased doses. Medication possession ratio for each individual was calculated based on refill dates and means were compared using an unpaired t-test.
RESULTS
During the study period, there were 756 therapeutically-dosed rivaroxaban or apixaban prescriptions. After excluding individuals who received fewer than three separate refills at our pharmacy (49.3%), 105 rivaroxaban refills and 278 apixaban refills were included (mean prescription size of 32.2 tabs for rivaroxaban and 65.7 tabs for apixaban). In the rivaroxaban group, the mean medication possession ratio across the cohort was 0.9321 (SD=0.1057). In the apixaban group, the mean medication possession ratio across the cohort was 0.9051 (SD=0.1467). The mean difference of the possession ratios was 0.0269 (95% confidence interval -0.0433 to 0.0971, p = 0.4472), thus there was no statistically significant difference in possession ratios between the study drugs.
CONCLUSIONS
Analyzing our internal pharmacy data, we found that among individuals who filled at least three times, medication adherence was high and not dissimilar between the two groups. Thus, the likelihood of compliance does not appear to be dependent on dosing frequency. However, it should be noted that nearly half of patients filled fewer than three times at our pharmacy and were thus excluded. It is unclear whether these patients completed their prescribed course, discontinued the drug all together or elected to refill at an alternative location, but it is likely that this may have caused an overestimation of drug adherence. While larger studies are needed to further assess adherence between the two drugs, these findings are reassuring that medication compliance between rivaroxaban and apixaban are similar in those who continue the drug.

Disclosures
No relevant conflicts of interest to declare.
Band 3 Phosphorylation Induces Irreversible Alteration of Stored Red Blood Cells
Authors: Azouzi Slim , Colin Aronovicz Yves , Pereira Catia , Romana Marc , Peyrard Thierry , Amireault Pascal , Le Van Kim Caroline 
Published: October 2019

Abstract
Introduction: Storage of red blood cells (RBCs) for transfusion purposes is accompanied by a number of morphological and biochemical changes (storage lesions) that reduce post-transfusion survival/efficacy and increase risk for adverse reactions in the recipients. The clearance of altered and older RBCs from circulation is triggered by the clustering of Band 3, an aggregate state that is recognized by a low-affinity naturally occurring IgG antibody (Nab). Considering the key role of Band 3 in the maintenance of RBC structure and survival, elucidation of functional and structural modifications of Band 3 during storage should lead to new approaches aiming to improve RBC storage and post-transfusion viability.
Results: Immunoblot analysis of RBC membrane proteins using an anti-phosphotyrosine antibody showed a progressive increase in the phosphorylation status of Band 3 during RBC storage (Figure 1).
In addition, using the quenching fluorescence of eosin-5-maleimide (EMA), we showed an increase of the mobile fraction of Band 3. These findings are consistent with previous demonstration that tyrosine phosphorylation of Band 3 reduces its affinity for ankyrin, leading to the release of the immobile fraction of Band 3 from the skeleton complex, and enhancement of the lateral mobility of Band 3 into the lipid bilayer. Immunoblot experiments using an antibody that specifically recognizes the clustered form of Band 3 revealed an increase of Band 3 cluster formation from the 28th day of storage. We also showed that the release of microparticles (MPs) that occurs during RBC aging increases from the 28th day of storage (Figure 2).
Finally, stopped-flow-based functional studies showed a decrease of the anion exchanger activity of Band 3 from the 28th day of storage.
Conclusion: Altogether, our results suggest that the 28th of storage represents a key moment for the molecular processes leading to irreversible lesions of RBCs and allow us to propose a new Band 3 phosphorylation/clustering-based mechanism of RBC aging.
Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures
No relevant conflicts of interest to declare.
Loss of Src-like Adaptor Protein 2 Expression Increases the Transforming Potential of Oncogenic FLT3-ITD
Authors: Moharram Sausan A. , Kazi Julhash U. , Rönnstrand Lars 
Published: October 2019

Abstract
The receptor tyrosine kinase FLT3 is found to be a mutated oncogene in hematological malignancies including acute myeloid leukemia (AML). FLT3 inhibitors in combination with chemotherapy display promising results in a clinical setting, but patients relapse after short-term treatment due to the development of resistant disease. Therefore, targeting signaling proteins downstream of FLT3 can be an alternative approach for the treatment of patients carrying mutant FLT3. Activated FLT3 is constitutively phosphorylated on several tyrosine residues. These tyrosine residues facilitate association of SH2 domain-containing signaling proteins. By using a panel of SH2 domain-containing proteins we identified SLAP2 as a potent interaction partner of FLT3. The interaction in between FLT3 and SLAP2 occurs when FLT3 is activated and an intact SH2 domain of SLAP2 is required for the interaction. SLAP2 associates with FLT3 mainly through its SRC binding sites and expression of SLAP2 inhibited oncogenic FLT3-ITD-mediated cell proliferation and colony formation in vitro, and tumor formation in vivo. By analysis of patient expression data, we found that loss of SLAP2 expression correlates with poor prognosis of AML patients carrying FLT3-ITD. SLAP2 inhibits FLT3-mediated downstream signaling such as activation of AKT, ERK, p38 and STAT5. Inhibition is partially mediated through ubiquitination-mediated degradation of FLT3. Taken together our current study demonstrates that SLAP2 is an important regulator of FLT3-mediated oncogenic signaling and thus modulation of the SLAP2 expression levels can be an alternative approach for the treatment of FLT3-ITD positive malignancies.

Disclosures
No relevant conflicts of interest to declare.