Unless there were contraindications, patients received: (1) IV iron (200 mg, Venofer, Vifor, Saint Gallen, Switzerland), three doses starting on admission; (2) Vitamin B 12 (1 mg) intramuscularly on admission; and (3) Folic acid (5 mg/day) for the entire duration of hospitalization. Patients presenting with preoperative haemoglobin levels < 13 g/dL also received a single dose of recombinant human erythropoietin (40,0 0 0 IU, sc; Eprex, Janssen-Cilag, Madrid, Spain) 24 h after admission [25] (link) .
Eprex
Manufactured by Johnson & Johnson
Sourced in Switzerland, France, Denmark, United Kingdom
Eprex is a pharmaceutical product manufactured by Johnson & Johnson. It is a recombinant human erythropoietin (EPO) used for the treatment of anemia.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Isotonic saline, by B. Braun (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Human stem cell factor, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
Cd34 microbead kit, by Miltenyi Biotec (1 mentions)
Interleukin 3 (il 3), by Cell Signaling Technology (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Heat inactivated fetal calf serum, by Merck Group (1 mentions)
Recombinant mouse cytokines, by Thermo Fisher Scientific (1 mentions)
Iscove modified dulbecco media (imdm), by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
Gallios analyzer, by Beckman Coulter (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
Cd235a pe cy7, by BD (1 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
22 protocols using eprex
1
Preoperative Anemia Management Protocol
2
Intravenous Iron and rhEPO for Anemia
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According to hemoglobin level at the start of the therapy, the women were treated either with intravenous iron and rhEPO or with intravenous iron only twice weekly as described elsewhere [21 (link), 26 (link)] (Figure 1 ).
Patients with an Hb level between 9.0 and 9.9 g/dl (33 patients) received 200 mg iron sucrose (VENOFER®, Vifor Int., St. Gallen, Switzerland) intravenously twice weekly (Figure 1 ). If response to therapy was poor (i.e., Hb increase <0.7 g/dl) after 2 weeks (13 patients), patients additionally received rhEPO (10,000 U EPREX®, Janssen-Cilag, Baar, Switzerland). On the basis of our previous experience we choose this cut-off for adequate primary response [21 (link), 26 (link), 27 (link)]. Patients with an Hb between 8.0 and 8.9 g/dl (17 patients) received iron sucrose (VENOFER) and rhEPO (EPREX) twice weekly from the start of therapy.
Sufficient overall response to therapy (the difference of baseline hemoglobin and that after therapy) was defined as Hb increase >1.0 g/dl. The maximum total iron dose was 1,600 mg; therefore therapy was stopped if the maximal iron sucrose dose was administered, or target Hb > 10.5 g/dl was achieved.
Side-effects (hypotensive and hypertensive response, allergic reaction, and thromboembolic complications) were registered.
Patients with an Hb level between 9.0 and 9.9 g/dl (33 patients) received 200 mg iron sucrose (VENOFER®, Vifor Int., St. Gallen, Switzerland) intravenously twice weekly (
Sufficient overall response to therapy (the difference of baseline hemoglobin and that after therapy) was defined as Hb increase >1.0 g/dl. The maximum total iron dose was 1,600 mg; therefore therapy was stopped if the maximal iron sucrose dose was administered, or target Hb > 10.5 g/dl was achieved.
Side-effects (hypotensive and hypertensive response, allergic reaction, and thromboembolic complications) were registered.
3
Differentiation of β-Thalassemia Patient CD34+ Cells
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This study was performed in accordance with the Helsinki declaration and was approved by the Mahidol University Institutional Review Board (MU-IRB), approval number 2013/022.1103. Written informed consent was obtained from all individual participants included in the study. Human CD34 + cells were isolated from peripheral blood obtained from six βIVS2-654-thalassaemia/HbE patients. Briefly, peripheral blood mononuclear cells were isolated by centrifugation on Lymphoprep (Axis-Shield, Oslo, Norway) and subsequently enriched for CD34 + cells by immunomagnetic separation using the CD34 MicroBead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) as per the manufacturer’s instructions. The CD34 + cells were cultured for 14 days in two-phase liquid culture. The first phase, cells were cultured for 4 days in Iscove’s Modified Dulbecco Medium (IMDM; Gibco) supplemented with 20% FBS (Sigma-Aldrich), 100 U/mL penicillin/streptomycin (Gibco), 300 μg/mL holo-human transferrin (PromoCell, Heidelberg, Germany), 50 ng/mL human stem cell factor (Miltenyi Biotec), 10 ng/mL human interleukin-3 (Miltenyi Biotec), 2 U/mL erythropoietin (Epo; EPREX; Janssen-Cilag, Schaffhausen, Switzerland). The second phase, cells were subsequently stimulate towards erythrocytes differentiation for 10 days in IMDM containing 20% FBS, 100 U/mL penicillin/streptomycin, 300 μg/mL holo-human transferrin and 5 U/mL Epo.
4
Erythropoietin Induced MSC Differentiation
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The culture conditions of MSC are described in detail in the Online Supplementary Materials and Methods. Cells were treated with erythropoietin alfa (10–100 IU/mL, EPREX, Janssen-Cilag). In most experiments, differentiation medium was applied for 10 days. The dosage of erythropoietin was chosen according to previous publications,3 as well as personal experience showing that 50 IU/mL erythropoietin are required to induce anabolic effects in MSC. In certain experiments, cells were treated with parathyroid hormone intermittently for 8 h three times a week (PTH, 100 ng/mL, Preotact, Nycomed) and/or lithium chloride continuously (25 mM).
5
Erythropoietin Therapy for Trauma Care
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EPO (recombinant human Erythropoietin, Eprex, 4000 IU/ml, Janssen-Cilag, France) was administered intraperitoneally 30 min after trauma, and thereafter once daily in a dose of 5000 IU/kg throughout the study. Animals in the control group received an identical volume of saline (isotonic NaCl 0.9%, Fresenius Kabi, Sweden) intraperitoneally.
6
Altitude Exposure and rHuEPO Effects
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The present study was performed during the altitude trial arm of a larger study which aimed to determine biological markers for detection of blood doping at altitude [6] . Pre data was acquired after three days upon arrival to moderate altitude (2,320 m), before the rst rHuEPO injection. Participants were randomly assigned either to blinded treatment with rHuEPO, consisting of intravenous injections of 20 IU × kg bw -1 epoetin alpha (Eprex, Janssen, Birkerød, Denmark), or to placebo consisting of intravenous injections of ~0.3 mL isotonic saline (B. Braun, Melsungen, Germany). Participants wore a blindfold during injections to maintain blinding. Treatment started on the third day following arrival to altitude and was given every second day for three weeks, resulting in a total of eleven injections. Post data was collected a day after the last injection. In this period, subjects trained daily including both resistance and endurance training. Details of the training program are reported elsewhere [23] .
7
Preparation and Characterization of Carbamylated Recombinant Erythropoietin
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RhC-EPO was prepared from rhEPO (Eprex, Janssen Cilag, France) as previously described (Leist et al., 2004) . Briefly, one volume of rhEPO (1 mg/ml) was mixed with one volume of 1 M Na-borate (pH 8.8) and recrystallized KOCN was added to a final concentration of 1 M. The mixture was incubated at 37 • C for 24 h. Samples were immediately dialyzed against Mill-Q water and subsequently against 20 mM Sodium citrate in 0.1 M NaCl, pH 6.0. After dialysis, the samples were concentrated using Centricon with pore size of 3 kDa (Amicon). The protein content was determined with a Bradford dosage. Efficient carbamylation of rhEPO was confirmed by testing the resistance of native and carbamylated rhEPO to proteolysis by Lys-C proteinase (Leist et al., 2004) . Briefly, 10 g of rhEPO or rhC-EPO were incubated with 0.4 g of Lys-C proteinase at 37 • C for 20 h. Laemmli buffer was then added and samples were boiled for 5 min. The digestion products were finally separated by SDS-PAGE electrophoresis on 15% acrylamide gel and proteins were visualized by silver staining.
8
Erythroid Differentiation of CD34+ Cells
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CD34+ cells were individually cultured in a 2-phase medium systems to drive the cellular commitment to the erythroid lineage and differentiate into mature red blood cells. Cells from each donor were cultured in Phase I medium: Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco®, Thermo Fisher Scientific, Inc., MA, USA) containing 20% FBS (Sigma-Aldrich®, Sigma-Aldrich, Inc., MO, USA), 300 μg/mL holo-Transferrin (holo-TF, PromoCell®, PromoCell GmbH, Heidenberg, Germany), 50 ng/mL human Stem Cell Factor (hSCF, CellSignaling Technology®, Cell Signaling, Inc., MA, USA), 10 ng/mL interleukine-3 (IL-3, CellSignaling Technology®), 2 U/mL erythropoietin (EPO, EPREX®, Janssen-Cilag, Auckland, NZ) in the presence of 100 U penicillin/streptomycin (Gibco®). On day 5, cells were replaced with fresh Phase II medium: IMDM containing 20% FBS, 300 μg/mL holo-TF, 5 U/mL EPO and 100 U penicillin/streptomycin at 37°C under 5% CO2 and 100% humidity. Fig 1 displays a flowchart of experimental design for cell culture, lentiviral transduction settings, and strategies for erythroid differentiation.
9
Hematopoietic Progenitor Cell Culture
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The cells were cultured for 6‐7 days in IMDM (Sigma‐Aldrich) with 20% heat‐inactivated fetal calf serum (Sigma‐Aldrich), 100 U/ml penicillin (Sigma‐Aldrich), 0.1 mg/mL streptomycin (Sigma‐Aldrich), and 50‐200 μmol/L β‐mercaptoethanol (Thermo Fisher Scientific). The medium was supplemented with 20 ng/mL IL‐3 and 100 ng/mL stem cell factor, or 80 ng/mL stem cell factor, 20 ng/mL IL‐3, 50 ng/mL IL‐9, and 2 U/mL erythropoietin. All cytokines were recombinant mouse cytokines (Peprotech, Rocky Hill, NJ) except the erythropoietin (Eprex; Janssen‐Cilag, High Wycombe, UK), which was human.
10
Erythroid Differentiation of CD34+ Cells
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A three-step protocol35 (link) was adapted to generate mature RBCs from mock- and LV-transduced CD34+ cells. From days 0 to 6, cells were grown in a basal erythroid medium supplemented with the following recombinant human cytokines: 100 ng/mL SCF (Peprotech, London, UK), 5 ng/mL IL3 (Peprotech, London, UK), and 3 IU/mL of erythropoietin (EPO) Eprex (Janssen-Cilag, Issy-Les-Moulineaux, France) and hydrocortisone (Sigma, St. Louis, MI, USA) at 10−6 M. From days 6 to 9, cells were cultured onto a layer of murine stromal MS-5 cells in basal erythroid medium supplemented only with 3 IU/mL EPO Eprex. Finally, from days 9 to 20, cells were continued to be cultured on a layer of MS-5 cells in basal erythroid medium but without cytokines. Erythroid differentiation was monitored by May Grunwald-Giemsa staining, flow cytometry analysis of the erythroid surface markers CD36 (CD36-V450, BD Horizon, Franklin Lakes, NJ, USA), CD71 (CD71-FITC, BD PharMingen, Franklin Lakes, NJ, USA) and glyophorin A (GYPA) (CD235a-PECY7, BD PharMingen, Franklin Lakes, NJ, USA). We used the nuclear dye DRAQ5 (eBioscience, San Diego, CA, USA) to evaluate the proportion of enucleated RBCs. Flow cytometry analyses were performed using the Gallios analyzer and Kaluza software (Beckman-Coulter, Brea, CA, USA).
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