bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacokinetics (PK) and Pharmacodynamics (PD) of Epoetin Alfa in Anemic Cancer Patients Receiving Cycling Chemotherapy.
Authors: Cheung Wing K. , Danneman Brian , Wacholtz Mary , Lau Henry , Miller Denis
Published: April 2020

Epoetin alfa is a recombinant human erythropoietin (EPO). The objective of this study was to compare the PK/PD of epoetin alfa after a fixed dose (40,000 IU) once weekly (QW) regimen to those after the weight based (150 IU/kg) 3 times per week (TIW) regimen for 4 weeks in healthy subjects and for 6 weeks in anemic cancer patients. In this randomized, open-label, parallel, multi-center study, PK profiles were determined during Week 1 and Week 3 in healthy subjects. For cancer patients, PK profiles were determined during Week 1 when patients were receiving chemotherapy and an optional one during Week 3 when patients were not receiving chemotherapy. Of the 33 enrolled cancer patients who received as least one dose of Epoetin alfa (safety population), 29 were evaluable for PK and 27 were evaluable for PD. Of the safety population (82% had solid tumors), ECOG scores were 0 (48%), 1 (42%), and 2 (9%). None of the patients had bone or kidney metastasis. Their ages and body weights ranged from 31–77 yrs (mean 59.2 yrs) and 50.9 –94.1 kg (mean 70.8 kg), respectively. Mean serum EPO concentrations in the cancer patients during Week 1 were higher than during Week 3. In general, the concentration-time profiles and PK parameters of anemic cancer subjects were different from those in healthy subjects during Week 1 but similar to healthy subjects during Week 3. Mean (SD) PK parameters are listed in following table. Epoetin alfa was safe and well tolerated after the two regimens in this study. The 40,000 IU QW regimen had a higher Cmax, higher exposure to epoetin alfa/erythropoietin in serum (in terms of AUC0-7days), and a lower clearance (CL/F) than the 150 IU/kg TIW regimen. Despite these PK differences, time-profiles of changes in PD parameters and their AUC (% reticulocytes, hemoglobin, and total RBCs) were similar. Mean (SD) PK parameters

150 IU/kg TIW 150 IU/kg TIW 40,000 IU QW 40,000 IU QW 1st Week 3rd Week 1st Week 3rd Week a n=3 for Week 1 and n=2 for Week 3; b n=11 for Week 1 and n=7 for Week 3. Cancer Patients (n=14) (n=4) (n=18) (n=7) Cmax, IU/mL 414 (312) 178 (58) 1077 (510) 897 (322) AUC0-7days, mIU.h/mL 43014(37270) 21082(6909) 84205(46999) 48254(17658) CL/F, mL/h/kg 20.2(15.9) 23.6(9.5) 9.16(4.69) 13.9(7.6) T1/2, h 43.7(3.9) 41.9(14.8)a 41.9(14.8)a 38.8(11.0)b Healthy Subjects (n=6) (n=6) (n=6) (n=6) Cmax, mIU/mL 163 (54) 125 (32) 1036 (238) 909 (398) AUC0-7days, mIU.h/mL 15708(4327) 12913(3249) 47469(13301) 32969(7945) CL/F, mL/h/kg 31.2 (11.5) 36.9 (10.0) 12.6 (3.1) 17.8 (3.7) T1/2, h 25.0 (7.1) 26.0 (8.9) 28.8 (8.1) 23.7 (8.5)
Need To Register Consanguinity To Avoid Transfusions in Random Fashion among Close Relatives.
Authors: Fernandez Jose , Fridman Sergio , Acevedo Mirta , Benzadon Ricardo , Dupont Juan , Garay Guy , Riveros Dardo , Cacchione Roberto
Published: April 2020

Transfusion associated graft versus host disease (TGVHD) is a complication of transfusion (T) among close relatives but this event is not usually registered. In a search in Medline, since 1966 to present, no references where found on the need to register consanguinity between donors and receptors. The object of this research was to determine the importance of recording consanguinity (C) transfusions in a random fashion to avoid TGVHD. Between January 2001 and June 2003 the presence of C between blood donors and patients (P) on the Donor’s Book and in the mother and satellite blood bags for platelets were registered. Donations (D) were directed (DD) or non-directed (ND). 10,827 D were registered, from which 18,406 units (U) of cellular hemocomponents (CH) apt for transfusion were obtained. Of these, 1,868 D (17.3%) were C with the P and 3,324 (18%) U of CH apt for transfusion were obtained. In order to investigate the chance of a random transfusion of CH to C we excluded from the probability analysis the following U: 1) ABO/Rh incompatible: 157 (4,7%). 2) Already transfused or discarded at the moment of the transfusion order: 304 (9,1%). 3) Not donated at the time of the transfusion order: 504 (15,2%). 4) Irradiated hemocomponents (for C, DD, inmunodeficiencies, etc.): 624 (18,8%). 5) Non-transfused because of absence of transfusion order for the C patient: 846 (25,5%). U from C donors ready for transfusion to a C patient was 889 from 3.324 (26,7%). According to the Accountant Book, at the moment of transfusion the pondered S from C donors was 1.067 U. In the same way, at the moment of transfusion for C receptors, the pondered S of all balance of CH not belonging to C donors was: 28,720 U. In this study the chance that a patient receives at least one U of CH from C donors in a random fashion was [1.067 /28.720 + 1.067] x 100 = 3,6 %. Although the incidence of TGVHD is unknown it is thought to be very low. The chance of 3.6% of receiving at least one U of CH from a C donor in a random way is high and justifies largely considering this event to be registered. This percentage could suffer significant variations for different modalities of Blood Banks with regards to donation, storage and supplying of CH.
Influence of Homemade and Commercial Fibrin Sealant on Alveolar Bone Healing - an Experimental Study.
Authors: Correa Maria Elvira P. , Almeida Oslei P. , Peres Danyel , Alves Marcelo C. , Costa Fernando F. , Annechinno-Bizzachi Joyce M. , Barjas-Castro Maria Lourdes
Published: April 2020

The beneficial aspects of fibrin sealants for soft tissues are well documented, but studies of their direct influence on bone healing and their effectiveness in augmenting bone graft healing have produced conflicting results. The aim of this study was to evaluate the influence of fibrin sealants (FS) in the alveolar bone healing process during a period of up to 28 days. Seventy-five Wistar rats were submitted to a superior incisor extraction after intramuscular anesthesia (kentamine chloridrate 10% – 10mg/kg and tiazina chloridrate-5mg/kg). The rats were divided into tree different groups and the sealants were introduced into the alveolar bone. The first group (25) received human homemade fibrin glue, the second group (25) FS associated to factor XIII and aprotinin (Beriplast; Aventis-Beringher) and the third group (25) was the control. Animals were sacrificed by prolonged diethyl inhalation on days 7, 14 and 28 after surgery. Animal craniums were dissected and submitted to a decalcification, and preparated for H&E light microscopy. The morphometric study was performed by means of an interactive computerized image analysis system KS400 (Zeiss, Jena). New bone formation was carefully delimited in four different alveolar regions (apical, two middle areas and cervical) of each specimen. The data were statistically analyzed using multiple regression, ANOVA and Tukey test. Results showed that the amount of alveolar bone formation (μm) in the control group and commercial sealant was statistically similar. However, alveolus receiving homemade sealant presented less amount of new bone formation comparing to commercial sealant and control group (p=0.0034) (figure 1). The present study demonstrated that homemade fibrin sealant delays osteogenic formation. Commercial sealant did not improve alveolar repair however, the amount of new bone formation was slightly higher comparing to the control, probably due to the factor XIII present in this sealant.

Figure Figure
Analyzing Variations in Blood Utilization Improves Transfusion Practice.
Authors: Arthur Kathleen R. , Menitove Jay E.
Published: April 2020

During the past five years, randomized, controlled trials and clinical guidelines provided data for making evidence-based decisions about blood transfusion. In light of this new information, we monitored transfusion practices/variations among hospitals in our region. We sought to determine whether hospitals actively involved in addressing transfusion utilization and best practices in 1999 (hospitals 1–3) experienced changes when re-surveyed in 2003. As a “control” group, we determined transfusion patterns at 9 hospitals in 2003 (hospitals 4–12), that did not participate in the 1999 survey. We compared autologous transfusion practices involving orthopedic surgery patients at 3 hospitals participating in both surveys and at 8 hospitals contrasting 2003 survey data and 1999 hospital data (no data was available for comparison from one hospital). Data comparisons involved transfusions between Oct.–Dec.1999 vs. Oct–Dec. 2003. In addition we compared transfusion practices involving cardiovascular surgery patients (DRG’s 104–107, 109–111) at 2 hospitals participating in the 1999 and 2003 surveys and 5 hospitals joining the survey in 2003. Among the hospitals participating in 1999, the number of patients donating autologous units for orthopedic surgery declined 68%(88 to 28 patients), the number of patients transfused decreased by 58% (55 to 23patients), and the number of units collected was 68% lower (151 to 49 units). In contrast, at hospitals participating in the survey for the first time in 2003, there was only a 7% decline in the number of autologous units collected (274 to 255); the number of patients donating was unchanged (156 and 157). Of note, hospitals 10 and 12 were affiliated with hospital 3 and followed the same transfusion protocols. The average number of units transfused per patient in 1999 and 2003 in both groups (~1.5 and 1.8) was essentially unchanged. The relatively low number of units infused further questions the need for autologous donations in these patients.

At 2 hospitals participating in both the 1999 and 2003 surveys, the percentage of cardiovascular surgery patients transfused with red blood cells decreased (40% to 29% and 60% to 44%). This compares to a higher percentage of patients receiving transfusions (range 46–80%) at hospitals participating only in 2003. A similar difference appears in the average number of units infused per transfused patient (2.2–3.6 vs. 2.9–4.7). No differences in patient outcomes were attributed to changes in transfusion practices. Blood utilization patterns improved at hospitals and their affiliates committed to transfusion practice benchmarking compared to hospitals not utilizing this monitoring modality. We conclude that utilization variation analysis remains an effective methods for influencing transfusion practice.

Figure Figure
Preparing Citrated Non-Activated Gamma Globulin for Rapid Infusion.
Authors: Shanbrom Edward , Owens William J.
Published: April 2020

Human plasma is our most valuable source of therapeutic proteins, and intravenous gamma globulin (IVIg) is the most widely used plasma derivative. Traditional yields of the product from alcohol fractionation are not optimal; added to this, viral contamination and other well known side effects, including fever and aseptic meningitis, continue to be of concern. A method of precipitating very high yields of cryoprecipitable proteins with sodium citrate (“Supercryoprecipitation”) has been previously described. A by-product of “Supercryoprecipitation” is a very clean cryoprecipitate-free plasma supernatant, with a citrate concentration of 5% to 6% (w/v). When up to 10% (w/v) additional citrate is introduced into the supernatant, there is a preferential precipitation of nearly 100% of the remaining gamma globulin. Immunoglobulin G (IgG) yield was measured by radial immunodiffusion (86.3% ± 12.5%). IgG, being the largest fraction of gamma globulin, is most often used as a measure of product quality. Protein electrophoresis of the citrate precipitation product showed very little contamination by other plasma fractions (albumin: 9.8% ± 3.3%, alpha globulin: 1.6% ± 0.6%, beta globulin: 3.9% ± 1.2%, gamma globulin 84.3% ± 13.8%). Unlike present protein precipitation methods, citrate precipitation does not rely on denaturation, thereby better preserving the native structure of immunoglobulin. This may lead to improved tolerance during infusion, and may allow for more rapid infusion rates, or improved subcutaneous infusion. Stability and safety studies are currently underway. Considering the current unsettled climate in the world, the potential for improved gamma globulin therapies and supply should not be overlooked.
Further Studies of Galectin-1 (Gal-1) on CD34+ Umbilical Cord Blood Cells.
Authors: Etchegoyen Orlando J. , Morales Victor H. , Milone Jorge H. , Fink Nilda E.
Published: April 2020

The galectins are a family of carbohydrate-binding proteins that are distributed widely in vertebrate organisms. Each galectin exhibits a specific pattern of expression in various cells and tissues. Although these proteins are found mainly in the cytoplasm, some of them are secreted from cells and interact with other glycosylated molecules at the cell surface or with the extracellular matrix. There is no information about the interaction of Gal-1 and hematopoietic progenitor cells (HPC). We studied the effect of rat recombinant galectin-1 (rGal-1) on the clonogenic capability and the morphologic differentiation of the human umbilical cord blood HPC in methylcellulose cultures. The quantification of total anti-Gal-1 binding capability (total receptors) was also determined by flow citometry. First, the cells of 10 human cord blood units were collected and CD34+ selected using an indirect immunomagnetic method (MidiMACS, Miltenyi, Biotec). Ten thousand events were acquired in a flow cytometer (FACScan, Becton Dickinson), employing 105 CD34+ cells, rabbit anti Gal-1 antiserum and anti-rabbit IgG FITC antibody. Finally the CD34+ cells were cultured in a semisolid methylcellulose medium (Methocult GF+ H4435, StemCell) by triplicate at 37°C, 5% CO2 and 100% humidity during 14 days, employing 24 wells culture plates (Nunc) in presence or not of different concentrations of rGal-1 (2, 4, 6 and 20 μg/ml). We analyzed the number of GEMM-CFU (mix colonies), GM-CFU (granulocyte- macrophage colonies), BFU-E (erythroid colonies) and the presence of signals of abnormal morphologic differentiation. Control of inhibition with thiodigalactoside (TDG) and lactose (Lac) 100 mM was done in every case to determine the assay specificity. Histograms show a total receptor mean expression (mean+/−SEM) in CD34+ cells of 18.2+/−2.44% which was significantly inhibited (p<0.05) with TDG (8.8+/−0.98%) and Lac (13.5+/−3.70%). A statistically significant increase in the number of GEMM-CFU, GM-CFU and BFU-E was seen. These increases were directly proportional to the rGal-1 concentration added (p<0.05) and it was stronger for GM-CFU. This effect was partially inhibited by TDG and Lac. We observed an abnormal increase in the mesenchymal, fibroblastic-like cell growth in methylcellulose cultures. These results showed the expression of Gal-1 in CD34+ human umbilical blood cells membranes. The addition of rGal-1 to cultures would increase the proliferation of HPC and the differentiation to stromal-like cells would partially occur. The mechanism and functional meaning of this activation and differentiation must still been elucidated.
Gene-Chip Analysis of Cord Blood Derived CD34+ Cells Expanded on Bioartificial Materials.
Authors: Oswald Joachim , Steudel Christine , Salchert Katrin , Thiede Christian , Ehninger Gerhard , Werner Carsten , Schuler Ulrich , Bornhaeuser Martin
Published: April 2020

Expansion of hematopoietic stem cells from neonatal cord blood is an important issue for clinical uses since the number of CD34+ cells in individual cord blood samples is limited and often not sufficient for a successful engraftment in adult individuals. In vivo, hematopoietic stem cells reside in the bone marrow in close vicinity to stromal cells and extracellular matrix molecules. We have established a culture system for the ex vivo expansion of CD34+ cord blood cells utilizing fibrillar collagen 1 as a bioartificial matrix to enable cellular adhesion during cell culture. CD34+ hematopoietic stem cells were isolated via immunomagnetic separation from umbilical cord blood after informed consent and cultivated in presence of recombinant cytokines and reconstituted collagen 1 fibrils as matrix. After seven days of cultivation, expansion of cells, expression of surface molecules cells and expansion of colony forming units were assessed. Additionally gene expression profiling was performed with Affymetrix HG U133A chips interrogating 22,253 probe sets. As control, CD34+ cells were expanded in liquid culture without fibrillar collagen.

The overall expansion of CD34+ cells was 4.2 fold + 1.7 compared to 11.1 fold + 2.9 for the control sample. The number of colony forming units (CFU) was increased in the collagen 1 containing samples was elevated (65.1 + 10.3 compared to 26.1 + 7.6 in the control). Gene expression analysis with chip technology showed up regulation of several cytokines (e.g. interleukin 8, interleukin 1a) and also of transcription factors with antiproliferative features like BTG2. The chip data have been verified with quantitative PCR using the Taqman technology.

Our data support the idea that direct contact of CD34+ cells with fibrillar collagen 1 results in a delay in cell cycle progression which prevents a subsequent differentiation into more committed progenitors. Therefore fibrillar collagen 1 may serve as supportive matrix for the ex vivo expansion of cord blood derived CD34+ cells.
Comparative Analysis of Methods for Assessment of Circulating Endothelial Progenitor Cells Indicated Heterogeneity of Subpopulations at Various Stages of Differentiation.
Authors: Deutsch Varda R. , Shmilovich Haim , Abashidze Anastasia , Miller Hylton , Keren Gad , Naparstek Elizabeth , George Jacob
Published: April 2020

The cells of the vasculature are derived from pluripotent stem cells, known as hemangioblasts, which also give rise to blood. Angiogenesis, the formation of new blood vessels is considered to result from the proliferation and migration of mature endothelial cells from existing blood vessels or from the recently described, but not yet well characterized endothelial progenitor cells (EPCs). The number and properties of EPC in disease states is of considerable interest due to the promising therapeutic potential of these cells. EPCs have been shown to be mobilized from the bone marrow and contribute to angiogenesis following vascular injury, organ ischaemia and tumor progression. However, mechanisms that drive the EPC response to injury remain elusive and the lack of definitions of EPC subpopulations and the many methods used by different groups to identify these cells make correlation of results difficult. To begin to understand the potential of these cells, we performed a comparative analysis of several methods used for circulating EPC assessment in 40 healthy individuals (mean age of 33±9 years) and correlated them with humoral factors known to influence their numbers. Peripheral blood mononuclear cells were obtained and evaluated by flow cytometeric analysis with antibodies to CD34, CD45, CD133 and KDR, and the remaining cells grown under endothelial cell conditions for assessment of colony forming unit (CFU) numbers and adhesive properties. Levels of circulating vascular endothelial growth factor (VEGF), erythropoietin and C-reactive protein were determined and correlated with each of the EPC markers. CFU numbers did not correlate with the number of CD34/KDR (VEGFR2) or CD34/CD133/KDR positive cells and negatively correlated with CD34/CD133 (which includes hematopoietic progenitors). CD34/KDR number correlated with CD34/CD133/KDR but not with CD34/CD133. Only the VEGFR2 positive cells populations (CD34/KDR and CD34/CD133/KDR) correlated with VEGF serum levels. The number of EPC adhering to fibronectin and endothelial-cells correlated with CFU numbers but not with EPC membrane markers. Our results indicate for the first time that EPC, like hematopoietic precursors may be a heterogeneous cell population comprised of progenitors at various stages of differentiation, having varied proliferative capacity. This could explain the lack of correlation in results obtained using different methods for quantitatively assessing the numbers of circulating EPC. These data also suggest that co-expression of CD34, CD133and KDR-VEGFR2 is appropriate for defining a population of circulating EPC whereas CFU are more likely to reflect the proliferative capacity of the progenitors.
Acquisition of CD34 Correlates with Increased Hematopoietic and Self Renewal Activity of CD34−CD133+ Cord Blood Cells.
Authors: Götze Katharina S. , Schiemann Matthias , Marz Stefanie , Peschel Christian , Oostendorp Robert A.J.
Published: April 2020

CD34 is a sialomucin expressed on hematopoietic cells, endothelial cells and muscle satellite cells. Within the hematopoietic system, CD34 expression has been associated with very immature progenitor cells as well as hematopoietic stem cells (HSC), and it is widely used to assess stem cell activity in clinical protocols. In the past, HSC activity was thought to be retained exclusively in the subset of cells expressing CD34. This view has been challenged by recent observations in mice in which HSC activity was also found in the CD34-negative fraction. These findings have since been reproduced using human marrow and cord blood cells. However, the exact relationship between CD34+ and CD34− stem cells remains unclear. We investigated the regulation of CD34 expression as dependent on cell division history. To follow cell division, human cord blood cells were labeled with the fluorescent dye CFSE. Lin-CD34−CD133+CFSE+ (CD34−) and CD34+ populations were almost indistinguishable in their ability to produce CAFCweek6 content. After three days of serum-free culture with stem cell factor, Flt3 ligand and thrombopoietin, almost all initially CD34− cells had acquired expression of CD34, including all undivided cells. We found that, in cultures initiated from CD34− cells, virtually all CAFCweek6 were produced from the divided, now CD34+ cells, indicating these cells had self-renewed. In contrast, similar cultures from initially CD34+ cells demonstrated that hematopoietic activity associated with the undivided cell fraction. We did not find any hematopoietic activity in the cell fraction that remained CD34− or the fraction that lost CD34 after division. Analysis of mRNA expression showed that CD34− and CD34+ cells expressed almost equal levels of CD34, AC133, Flt1, Flk1 and Flt4, while CD34− cells expressed significantly lower levels of Tie1 and Tie2 than CD34+ cells. The expression of CD34 message in CD34− cells was explained by our observation that these cells contained intracellular CD34, indicating that they are “primed” to express the antigen on their cell surface. In conclusion, Lin−CD34−CD133+ cells acquire expression of CD34, even in the absence of cell divisions. These CD34− cells self-renew more rapidly in vitro than cells initially expressing CD34, and self-renewal is preceded by acquisition of CD34 antigen.
Identification of Quantitative Trait Loci Regulating Steady State Hematopoiesis and Progenitor Mobilization in AKRB6F2 Mice.
Authors: van Os Ronald , Ausema Albertina , Vellenga Edo , de Haan Gerald
Published: April 2020

The hematopoietic system is a complex organized tissue with a hierarchical structure. Identification of organizational pathways within the hematopoietic system would be relevant for a better understanding of hematopoiesis in health and disease. We have analyzed numerous hematopoietic parameters in AKRB6F2 mice, a cross between AKR and C57Bl/6 mice that differ in various stem cell traits. Specifically, we have measured stem cell numbers, progenitor cell cycling, G-CSF induced stem cell mobilization, as well as neutrophil numbers in blood and bone marrow. We used these data to map quantitative trait loci for these parameters in order to assess whether distinct traits are associated with each other and by which genetic loci these parameters are regulated. When the different hematopoietic parameters were compared, surprisingly few significant correlations were found and only two quantitative trait loci (QTL) could be identified for all hematopoietic parameters. We confirm that a region on chromosome 11 is involved in progenitor cell cycling and we found an additional QTL for stem cell numbers, measured as CAFC day 35 per femur, on chromosome 2. A suggestive QTL was found for the number of progenitors in the bone marrow and the number of neutrophils in the blood. In the first of two series of offspring mice we found a strong association between stem cell mobilization and B6 alleles at the chromosome 3 locus D3Mit88 or AKR alleles on chromosome 15 (D15Mit241). However, this could not be confirmed in the second series. In addition, breeding of an F3 generation from the best mobilizers (6479 ± 1544 CFU-GM/ml blood vs. 3481 ± 3076 overall) of each series did not result in high mobilization capacity of the offspring (1333 ± 844 CFU-GM/ml). Taken together, our data illustrates that different hematopoietic parameters are independently regulated. This suggests that hematopoiesis is highly structured and regulation at all levels of stem cell development can lead to distinct mechanisms of stem cell maintenance resulting in normal levels of mature blood cells. Additionally, variation based on epigenetic determinants that do not transmit to the next generation may play an important role in the regulation of steady state hematopoiesis and stem cell mobilization.
Recurrent Abnormalities in TGIF RNA Sequence in Blasts from Patients with Acute Myeloid Leukemia.
Authors: Hamid Rizwan , Vnencak-Jones Cindy L. , Martincic Danko , Means-Powell Julie A. , Cogan Joy D. , Brandt Stephen J.
Published: April 2020

To identify genes with prognostic potential in acute myeloid leukemia (AML), we applied microarray analysis and real-time PCR to RNA from cryopreserved bone marrow or venous blood samples from 61 patients with newly diagnosed or relapsed AML, and the expression of one gene, TG-interacting factor (TGIF) was found to be highly predictive of relapse and survival. TGIF is a transcriptional repressor belonging to the TALE (three amino acid loop extension) class of homeobox proteins, and deletion or mutation of a single allele of TGIF is associated with the craniofacial genetic disorder holoprosencephaly (HPE). TGIF transcripts in blasts from patients with AML have now been analyzed for mutations using reverse transcriptase-based PCR followed by dideoxy fingerprinting and automated sequencing. Using this approach, we identified the identical, aberrantly spliced TGIF transcript in independent RNA samples from a subset of patients (10/48) with low TGIF expression and poor long-term survival. Sequencing of this novel product showed that it lacked part of exon 1, all of exon 2, and most of exon 3 and eliminated all of the known interacting domains of TGIF (DNA binding, SMAD2, and mSin3A interacting). In contrast, Southern blot analysis of DNA from these patients did not identify any gross abnormalities in TGIF gene structure. This in combination with the RNA results suggests the likelihood of aberrant splicing. In sum, these results identify a novel, recurrent genetic abnormality in AML. The presence of aberrant splicing in AML contrasts with HPE, which is associated with mono-allelic mutations or deletions. As the protein product of this transcript would not be expected to have any of the known functions of TGIF in the cell, reduced expression of functional protein product may have a role in relapse or progression of AML.
Dissecting the Mechanism of Endogenous GATA-2 Transcription: GATA Factor Interplay at Multiple Upstream Regulatory Regions Establishes Long-Range Transcriptional Control.
Authors: Bresnick Emery H. , Martowicz Melissa M. , Grass Jeffrey A. , Boyer Meghan E.
Published: April 2020

Given the simplicity of the DNA sequence that mediates binding of GATA transcription factors, GATA motifs reside throughout chromosomal DNA. However, analyses of GATA-1 occupancy by chromatin immunoprecipitation (ChIP) revealed that GATA-1 discriminates exquisitely among these sites. GATA-2 selectively occupies the −2.8 kb region of the GATA-2 locus in the active state, despite there being numerous GATA motifs throughout the locus. The GATA-1-mediated displacement of GATA-2 is tightly coupled to repression of GATA-2 transcription. We have used high-resolution ChIP to show that GATA-1 and GATA-2 occupy two additional regions, −3.9 kb and −1.8 kb of the GATA-2 locus. GATA-1 and GATA-2 had distinct preferences for occupancy at these regions, with GATA-1 and GATA-2 occupancy highest at the −3.9 kb and −1.8 kb regions, respectively. Activation of an estrogen receptor fusion to GATA-1 (ER-GATA-1) induced similar kinetics of ER-GATA-1 occupancy and loss of GATA-2 occupancy at the sites. As the distinct preferences for GATA factor occupancy were not evident from electrophoretic mobility shift assay analysis in vitro, establishment of the preferences requires the native nucleoprotein structure of the GATA-2 locus. In the transcriptionally active state, the −3.9 kb and −1.8 kb regions formed strong DNaseI hypersensitive sites (HSs), and the −2.8 kb region formed a weak HS. Whereas ER-GATA-1-instigated repression abolished the −1.8 kb HS, the −3.9 kb HS persisted in the inactive state. Transient transfection analysis provided evidence that the −3.9 kb region functions distinctly from the −2.8 kb and −1.8 kb regions. Molecular studies, including capturing chromosome conformation (3C) analysis, are underway to dissect the structure/function of the upstream regulatory regions. Based on the high sequence conservation and occupancy by GATA factors in vivo, targeted deletions of the −2.8 kb and −1.8 kb regions were generated. Mutant mice are being developed, and functional analysis is being conducted via in vitro differentiation of homozygous mutant ES cells. Our results support a model in which dynamic changes in GATA factor occupancy and chromatin structure at the −3.9 kb, −2.8 kb, and −1.8 kb regions are intimately controlled by Friend of GATA-1 and additional coregulators. This GATA factor interplay is essential for GATA-2 transcriptional regulation and therefore the control of hematopoiesis.
Histone Acetylation at the β-Globin Locus Changes with Developmental Age.
Authors: Barkess Grainne , Sun Jin , Fang Xiangdong , Cao Hua , Li Qiliang , Stamatoyannopoulos George
Published: April 2020

The β-globin locus contains a number of developmentally regulated genes that undergo switching of expression. In the embryo the ε-globin gene is expressed with a subsequent switch to γ-globin in the fetal liver, followed by a final switch to β-globin in definitive cells. Recently, the importance of chromatin structure in regulating gene expression has been recognized, and it has been proposed that modifications of histones (i.e. acetylation, methylation) have a direct effect on gene regulation. This hypothesis would suggest that changes in the histone code underlie the differences in globin gene expression during development. To address this we studied the pattern of histone acetylation across the β-globin locus at different stages of human development. Two different time points of human fetal erythropoiesis (approximately 55dpc and 98dpc) were studied. At these stages the level of γ mRNA is approximately 98%, there is no detectable ε mRNA, while β mRNA is about 2% of total mRNA. The adult stage of erythropoiesis was represented by erythroblasts derived from cultured adult CD34+ cells where γ mRNA was approx. 25% of total mRNA. Nuclei from the fetal and adult erythroblasts were analyzed by Chromatin Immunoprecipitation (ChiP) using antibodies to histone H3 acetylation and H4 acetylation. Quantification of enrichment was carried out by SYBR green real-time PCR, with PCR primers spanning the globin locus. The LCR region showed high levels of enrichment for acetylation in all stages of development. The ε-globin gene showed minimal levels of enrichment in all stages of development. In the 55dpc human erythroblasts γ-globin showed the highest level of enrichment of all globin genes, with the acetylation spreading across the promoter as well as the whole γ-coding region. In the 98dpc fetal erythroblasts, the acetylation at γ-globin was also enriched.

In the cultured adult erythroblasts a reduced level of γ-globin acetylation was present, which was expected since there was less γ-globin synthesis in these samples. There was a higher level of acetylation at the δ- and β-globin genes in the adult cells, as would be expected for the adult stage of development. These results demonstrate that dynamic changes in histone acetylation take place as the human β-globin gene region undergoes its developmental switches. Whether these changes represent primary events in the course of globin gene switching remains to be determined.
Single Administration of Antiapoptotic Cytokines Soon after High Dose Irradiation Prevents Monkeys from Thrombocytopenia.
Authors: Drouet Michel , Grenier Nancy , Delaunay Christophe , Mourcin Frederic , Mayol Jean-Francois , Lataillade Jean-Jacques , Herodin Francis
Published: April 2020

Preservation of hematopoietic stem and progenitor cell survival is required for recovery from radiation-induced myelosuppression. We recently showed the capacity of a single injection of antiapoptotic cytokines, namely stem cell factor, FLT-3 ligand, megakaryocyte growth and development factor, and interleukin-3 in combination (4F, each factor given intravenously at 50 μg/kg) administered shortly after a 5 Gy gamma total body irradiation to prevent monkeys from myelosuppression. As cytokine efficacy depends on the residual stem cell pool, the aim of the present study was to assess the efficacy of 4F in case of high dose irradiation. Adult monkeys (n=7) were globally and frontally irradiated at 7 Gy gamma using a Co60 source (dose rate 20 cGy/mn). Three animals received 4F 2 hours after irradiation and four control animals were injected with the diluant at the same time. Moreover, one of the treated monkeys was grafted with allogeneic mesenchymal stem cells (MSC) to enhance hematopoietic recovery. Human MSCs were infused directly in humerus simultaneously with 4F injection, allowing cell tracking. Treated monkeys experienced a very short period of thrombocytopenia as compared with untreated animals (platelet [PLT] count < 20 x 109/L: 1 ± 1.7 day versus 27 ± 18 days). Areas under the curve (AUC) of PLTs and red blood cells (RBCs) between days 0 and days 90 were higher in treated animals than in controls. The animal treated with 4F + MSC did not differ from the two 4F monkeys. In contrast with 5 Gy irradiated animals previously studied, both treated and untreated monkeys experienced a prolonged period of neutropenia (neutrophil [ANC] count < 0.5 x 109/L: 12 ± 3 days versus 10.3 ± 7.3) and AUCs of ANC did not significantly differ. This study shows that the 4F treatment, as a single dose regimen, prevents thrombocytopenia in high dose irradiation setting and could act as an emergency treatment for nuclear accident or terrorism victims.
Pretreatment Serum Soluble Interleukin-2 Receptor Levels Predict the Response after Immunosuppressive Therapy of Aplastic Anemia.
Authors: Min Chang-Ki , Lee Jong-Wook , Min Woo Sung , Kim Chun Choo , Kim Myungshin , Lim Jihyang , Kim Yonggoo , Han Kyungja
Published: April 2020

Severe aplastic anemia (SAA), characterized by pancytopenia and a hypocellular marrow, has heterogenous pathophysiologic mechanisms including a stem-cell defect or a T-cell disease. Immunosuppressive (IS) therapy based on antilymphocyte globulin (ALG) and cyclosporine (CsA) improved the response rate in patients with SAA not eligible for stem cell transplantation. The goal of IS regimens in SAA is elimination of T cells effecting the immune-mediated destruction of hematopoietic progenitor and stem cells. Increased levels of soluble interleukin-2 receptor (sIL2R) have been detected in patients with high immune activity such as autoimmune diseases, allograft rejection, and severe infection. Twenty-eight patients with SAA (15 males and 13 females, median age 27 years, range 16–62) were given an ALG (Lymphoglobuline, Institut Pasteur Merieux, 15mg/kg/day intravenously from day 1 to 5) and CsA (5mg/kg/day orally from day 12 to 180). We measured the serum sIL2R levels using ELISA test kit (CELLFREE IL-2R Medex, Kyowa, Japan) in 28 patients with SAA before and after therapy with ALG+CsA. The mean serum sIL2R levels at baseline, 7 and 60 days after ALG treatment were 572.8 pg/ml (range 181–1715), 2156.3 pg/ml (range 211–7315) and 488.0 pg/ml (range 168–1411), respectively. The baseline sIL2R levels were significantly higher in our patients when compared with normal control subjects (n=26, 379±164 pg/ml, P=0.01). Overall response, defined as transfusion independence, was achieved in 12/28 (42.9%) patients on 180 days after treatment. The baseline serum sIL2R levels were elevated more than the normal control mean+1SE (541 pg/ml) in 7 of 12 patients with response but in 4 of 16 without response (Pearson Chi-Square, P=0.047). According to the baseline sIL2R levels, the positive predictive value (the number of patients with response/>control mean+1SE) was 63.6% (7/11) and the negative predictive value (the number of patients who failed to obtain the response/<control mean+1SE) was 70.6% (12/17). The sIL2R levels on both day 7 and 60 did not show any difference between the groups. Overall, these findings demonstrate that serum sIL2R levels increase in patients with aplastic anemia. Pretreatment serum levels of sIL2R might be closely related to clinical outcome following IS therapy, reflecting the immunological abnormalities in individual patients.
CERA (Continuous Erythropoietin Receptor Activator): Dose-Response Trial of Subcutaneous (SC) Administration Once Every 3 Weeks (Q3W) to Patients with Aggressive Non-Hodgkin’s Lymphoma and Anemia Receiving Chemotherapy.
Authors: Osterborg Anders , Hellmann Andrzej , Couban Stephen
Published: April 2020

CERA is an erythropoiesis-stimulating agent (ESA) acting differently at the receptor level with a prolonged half-life. In this ongoing, multicenter, randomized, open-label, Phase II dose-response study, CERA was administered subcutaneously in a Q3W schedule to 93 transfusion-independent patients with aggressive (intermediate or high grade) B-cell non-Hodgkin’s lymphoma (NHL) and anemia receiving combination chemotherapy. Eligible patients met the following inclusion criteria: age ≥18 years, hemoglobin (Hb) <11 g/dL, combination chemotherapy scheduled to be administered throughout the 12-week treatment period, life expectancy >6 months, and Eastern Cooperative Oncology Group (ECOG) performance status grade 0–2. Major exclusion criteria included transferrin saturation <20% and platelet count <50 x 109/L. No patient had received an ESA in the 8 weeks prior to the first dose of CERA. Patients were randomized to receive CERA 2.1 μg/kg (n=31), 4.2 μg/kg (n=30), or 6.3 μg/kg (n=32) administered once every 3 weeks for 12 weeks. The primary efficacy variable was time-adjusted average change in Hb from baseline during 12 weeks, until end of initial treatment (last observed value before dose change or transfusion). Enrollment has been completed; the treatment phase of the trial is nearing conclusion. Demographics show similar baseline characteristics in all three patient subgroups [mean age: 62.9 years (2.1 μg/kg); 59.1 years (4.2 μg/kg); 64.3 years (6.3 μg/kg)]. Diffuse large B-cell lymphoma was the most common lymphoma in all three subgroups (87%, 70%, and 84% of patients in the 2.1, 4.2, and 6.3 μg/kg subgroups, respectively). To date, the majority of patients receive an anthracycline-containing chemotherapy regimen, either standard CHOP or CHOP plus rituximab. Preliminary efficacy and safety results will be presented. Ongoing assessments have indicated a safety profile consistent with that seen in patients with aggressive NHL and anemia receiving chemotherapy. This Phase II trial will help further characterize the ability of CERA to safely correct anemia when administered at an extended dosing interval (Q3W) to patients with aggressive NHL receiving chemotherapy.
Inhibition of T Lymphocyte (TL) Proliferation by Mesenchymal Stem Cells (MSCs): Role of Cell Contact between MSCs and TL, Role of MSCs Dose Effect, and Role of Soluble Factors and Adhesion Molecules Expressed by MSCs.
Authors: Nasef Aisha , Chapel Alain , Benshidom Morad , Zang Yizhuo , Mazurier Christelle , Bouchet Sandrine , Lopez Manuel , Gorin Norbert Claude , Thierry Dominique , Fouillard Loic
Published: April 2020

Mesenchymal stem cells (MSCs) can inhibit T lymphocyte (TL) proliferation in mixed lymphocyte reaction (MLR) and are candidate to induce tolerance of allogeneic haematopoietic stem cell graft. Results of previous studies evaluating the role of cell contact between MSCs and TL are contradictory. Some soluble factors produced by MSCs have been shown to be involved in TL inhibition such as Transforming Growth Factor-b (TGF-b), Hepatocyte Growth Factor (HGF), and Indoleamine 2,3 dioxygenase (IDO).

In this study inhibition of TL proliferation in MLR was evaluated in cultures with MSCs-TL contact and in cultures without MSCs-TL contact (transwell), at different concentration of MSCs (ratio MSCs/TL: 0.3, 0.1, 0.03, 0.01). Expression of mRNA encoding cytokines and adhesion molecules produced by MSCs were analysed by semi-quantitative RT-PCR. Inhibitory cytokines studied were IDO, HLA-G, LIF, IL-10, TGF-b and HGF; adhesion molecules studied were VCAM and LFA3.

Results of MSCs dose effect. In MLR with MSCs-TL contact, inhibition of proliferative index was related to the dose of MSCs. The percentage of inhibition was 74%, 60%, 48%, and 28% at ratio 0.3, 0.1, 0.03 and 0.01 respectively (p<0.05). In MLR without MSCs-TL contact, no dose effect was observed: the percentage of inhibition was 48%, 46% and 46% at ratio 0.3, 0.1 and 0.03 respectively; no inhibition was observed at ratio 0.01. Results of comparison between inhibition with cell contact and without cell contact. At ratio 0.3 the percentage of inhibition was 74% with cell contact and 48% without cell contact (p<0.05), at ration 0.1 the percentage of inhibition was 60% and 46% respectively (p<0.05). These results confirmed that TL inhibition is mediated by soluble factors but is increased when MSCs and TL are in contact. Results of mRNA RT-PCR in MSCs showed an overexpression of IDO, HLA-G and LIF in cultures with cell contact and without cell contact. In cultures with cell contact an overexpression of IL10 and TGF-b was observed, but not in cultures without cell contact. Adhesion molecules VCAM and LFA3 were overexpressed in both types of cultures with and without cell contact. In conclusion although cell contact is not mandatory to inhibit T cell proliferation, inhibition is higher when TL and MSCs are in contact, concomitantly an overexpression of additional inhibitory molecules is observed.
Stimulation of the Supportive Function of Marrow Stromal Cells by Anticancer Agents: Critical Role of Marrow Macrophages.
Authors: Ohta Kensuke , Nakamae Hirohisa , Koh Ki-Ryang , Nakaie Kiyotaka , Li Hong-Zhang , Yamane Takahisa , Takubo Takayuki , Hino Masayuki
Published: April 2020

Anticancer agents used for the treatment of hematological malignancies are known to cause long-term toxicities to the structure and function of bone marrow microenvironment. These obbservations are based predominantly on the in vivo experiments with anticancer drug-treated mice and also on the clinical features of the patients who survived after inhtensive chemotherapies. In the present study, in order to evaluate immediate gene response of human marrow stromal cells to anticancer drugs, we tested with real-time PCR for the expression of various cytokine mRNAs from the stromal cells treated with anticancer drugs. Contrary to our expectation, we found that treatment of stromal cells with cytarabine (Ara-C; 0.1–100 μmol) for 3 to 14 days significantly and dose-dependently enhances the expression of mRNAs of stem cell factor (SCF) and leukemia inhibitory factor (LIF) while downregulates interleukin-6 (IL-6) mRNA. On the other hand, carboplatin (0.1–100 μmol) up-regulated only SCF mRNA and adriamycin (0.001–1 μmol) did not affect these gene expressions (n=6). In order to determine the responsive cell elements of stromal cells for these novel responses to Ara-C, mRNAs were independently quantified after separating CD14 positive macrophages and CD45 negative mesenchymal cells from Ara-C-treated stromal cells (n=6). In this additional experiments, we found that the above-mentioned changes in gene expression by Ara-C were provoked predominantly in the stromal macrophage fraction. Based on these observations, we treated the stromal cells established from 6 patients with AML and 11 with non-leukemic subjects with Ara-C for 2 weeks, followed by washing 3-times to eliminate Ara-C. Allogenic CD34 positive cells were then recharged and supportive functions of the stromal cells were evaluated with standard 2-stage long-term cultures. Indeed, the results showed that transient treatment with Ara-C significantly and dose-dependently upregulates supporting function of premature hematopoietic cells in non-leukemic and, although to the less extent, in leukemic stromal cells. These novel short-term stimulatory effects of Ara-C to marrow microenvironment may provide new explanations for some clinical events such as mechanism of rapid recovery of normal hematopoiesis and stem cell mobilization observed several days after completion of intensive chemotherapy to acute leukemia. In addfition, the pattern of gene response of stromal cells induced by Ara-C is of biologically great interest since it is quite different from the already known gene response of human stromal cells provoked by IL-1.