bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of Hemotransfusion on the Arterial Compliance in the Patients with Chronic Anemia.
Authors: Barshai Josef , Nemets Anatoly , Oren Shmuel , Isakoy Irena , Lugassy Gilles
Published: April 2020

Arterial compliance disturbances are frequently observed among patients with chronic anemia. Few non-invasive studies were performed to determine hemodynamic changes in anemic patient. In this work we studied parameters of large arteries (C1) and small arteries (C2) compliance before and after blood transfusion (one packed cells unit(400cc)) by CR 2000 Hypertension Diagnostics Inc/Pulse Wave (Minneapolis MN, USA). We included 20 patients with chronic transfusion dependent anemia. The median age was 74±6 years. Twelve patients had MDS, 1 CML, 2 myelofibrosis, 2 NHL, one CLL, one thalassemia, and one patient with anemia of chronic disease. Hemoglobin range before transfusion was 7.5–9.0 g/dl. 14 patients also suffered from arterial hypertension, 5 from diabetes mellitus and received oral treatment; eight patients were smokers. Large arterial compliance before hemotransfusion was 9.8±3.8 ml/mmHgx10, and 9.2±2.9 after hemotransfusion (p<0.05). Small arterial compliance before and after blood transfusion were without significant changes (2.8±1.2 ml/mmHgx10 and 3.04±1.2 ml/mmHgx10 respectively). The decrease in C1 is explained by increased blood pressure. Systolic blood pressure rose from 135±17mmHg to 152±26mmHg (p<0.05), and diastolic pressure rose from 65±8mmHg to 72±10mmHg(p<0.05). In conclusion, hemotransfusion of one packed cells for patients with chronic anemia led to significant decrease in large artery compliance most probably due to increase in blood pressure and blood volume. Hemotransfusion increases the small artery compliance but not significantly.
Evaluation of a Non-Invasive Hematocrit Determination Device.
Authors: Berrebi Alain , Amir Orna , Weinstein Aharon , Herzenstein Olga , Dvir David , Monashkin Emanuel , Gabis Efi , Holland Paul
Published: April 2020

Background: Non-Invasive Hematocrit (Hct) measurement devices have many potential advantages in primary and secondary medical situations. Primarily, these devices prevent the pain, the potential transmission of infectious diseases, and the need for trained technicians. However, truly non-invasive techniques have not been proven to be accurate enough to eliminate the need for blood sampling. A new, non-invasive clinical device, the NBM-100, was tested for accuracy.

Aims: To examine the accuracy of Hct measurements obtained by a non-invasive clinical device as compared to values determined using venous samples.

Methods: Clinical trials using the NBM–100 device were conducted in a blood donation center (USA) and a hematology clinic (Israel). The device utilizes a finger sensor with Occlusion Red/Near-InfraRed spectroscopy (O-RNIRS) technology to non-invasively measure hemoglobin and Hct levels. Studies were carried out on a group of 304 subjects (155 Female, 149 Male), ages 18–96 (average age 54.9). All study volunteers were tested non-invasively with the NBM-100 device, and invasively, using a venous sample evaluated on a Cell-Dyn (Abbott) blood analyzer. At the blood bank center, the NBM-100 measurements were performed before donation and the venous blood sample was taken after donation. To neutralize the decrease in Hct occurring during blood donation, as described in published clinical trials, a value of 2.2% was added to the reference venous values obtained from the blood bank center.

Results: The venous Hct measurements ranged from 15 to 51%. The mean NBM-100 Hct (36%) was the same as the mean venous result. The standard deviation of the difference between the invasive and noninvasive Hct readings was found to be 3.34% and the bias was 0.40%. The mean absolute error (MAE) of their difference was 2.66%. The relative absolute error (RAE) was 7.9%. The operating staff at all centers found the NBM-100 easy to use and appreciated by subjects.

Conclusions: This study indicates the potential use of the NBM-100 device as a useful and accurate non-invasive method for measuring a wide range of Hct in both a hospital (e.g. surgery and blood transfusions) and ambulatory setting (e.g. blood banks). Good agreement was found between the non-invasive NBM-100 and the invasive Cell-Dyn Hct measurements. The non-invasive system was found to be environmentally- and user-friendly by operating staff.
Infections and ABO Blood Groups in Burn Patients.
Authors: Kowal-Vern Areta , Boral Leonard I. , Yogore Mariano G. , Patel Harita , Brown Susan
Published: April 2020

Background: Lack of isohemmaglutinins may predispose individuals to infection. The purpose of this study was to determine if there was a relationship between infections and burn patient blood groups.

Methods: This was a retrospective review of burn patient data on infections and ABO blood groups between March 1999-May 2004. Chi-square and Mann-Whitney U with one-way ANOVA statistical analyses were performed.

Results: There were 1615 burn admissions; 794 patients were typed for blood groups; 179 patients received blood transfusions. Of the study population, African-Americans constituted 59%, Hispanics 21%, Caucasians 16%, and others 4%. Although age (mean±se: 37±2 years), % TBSA (11±2%), and inhalation injury (15%) were similar in all groups, patients with B blood group had longer BICU stays compared to the O blood group, p<0.01. There was no statistically significant difference among the blood groups in the number of nosocomial wound infections, episodes of sepsis, bacteremia, urinary tract infections (UTIs) or pneumonia. The bacterial etiology was similar in all blood groups without any species preponderance. Transfused patients had a higher frequency of infections compared to those who were not transfused and significance was only reached in the comparison for UTIs, p=0.02.

Conclusions: Our data shows that burn patients with B and AB blood groups did not have an increased frequency of infections compared to the patients with A and O blood groups. Burn patients were immunosuppressed as part of their injury. Those who received transfusions probably had a further perturbation of their immune system, since blood products cause immunosuppression. Other factors such as inhalation injury and %TBSA also predisposed burn patients to infection. Transfusions rather than blood groups were associated with an increased number of nosocomial UTIs in burn patients.

Demographic Characteristics of the Burn Population by ABO Groups Blood Groups AB A B O mean ± s.e. Study Population # (%) 27 (3) 258 (32) 134 (17) 375 (47) Study African American (%) 4 29 21 46 Study Hispanic (%) 1 36 7 56 Study Caucasian (%) 3 37 14 46 BICU stay (days) 15±6 13±2 23±4 11±1 PRBC Transfusions (units) 7±2 9±2 13±3 8±1 UTI/Transfused # (%) 5 (100) 12 (92) 8 (89) 20 (60) UTI/Not Transfused # (%) 0 1 (8) 1 (11) 13 (40)
Umbilical Cord Blood Stem Cells Induced into Myeloid, Erythroid, or Megakaryocytic Pathways Are Susceptible to Infection by Porcine Endogenous Retroviruses (PERV).
Authors: Morgan Doris A. , Argaw Takele , Wilson Carolyn A.
Published: April 2020

Susceptibility of human hematopoietic stem cells to infection with porcine endogenous retroviruses (PERV) is of prime importance in determining the feasibility and safety of transplanting pig organs into humans. Under the influence of lineage specific cytokines, umbilical cord blood stem cells were evaluated for their permissiveness to PERV prior to linage commitment as well as after induction into the myeloid, erythroid or megakaryocytic pathways. CD34+ cells were plated in serum-containing nutrient medium supplemented with stem cell factor (SCF) to induce proliferation - a requirement for successful retroviral infection. Various microenvironment conditions included addition of lineage specific secondary cytokines for myeloid (G-CSF), erythroid (EPO), and megakaryocytic (TPO) commitment. After seven days, one-half of each culture was exposed to retroviral pseudotypes composed of replication competent wildtype PERV-NIH virions that also carry an MLV-based retroviral vector genome encoding β-galactosidase (β-gal) (Wilson et al, J. Vir,2000); control cultures were not exposed to the virus. Analyses for PERV infection were implemented after 72 hours’ viral exposure with the following observations: (1) exposure to PERV had no detected effect on proliferation nor on terminal differentiation of all three hematopoietic lineages; (2)PERV infected (β-gal +) cells (Fig.1) were detected in all cultures exposed to PERV regardless of microenvironment variations; and (3) PERV reverse transcription (DNA) and gene expression (RNA) as analyzed by PCR were positive only in virus exposed cultures (Fig. 2). Control cultures were negative in all analyses for PERV entry and expression. Preliminary observations indicate the erythroid lineage (SCF+E) may be more efficient in supporting PERV infection than the uninduced CD34+ cells (SCF) as well as those committed to the myeloid (SCF+G) and megakaryocytic (SCF+T) lineages. These results extend the initial observation that PERV could infect established hematopoietic cells lines to now show that the in vitro tropism of PERV also includes primary hematopoietic cells representing three lineages.

Fig. 1 Nuclear staining of PERV+ cells. Upper cell is in mitosis Fig. 1. Nuclear staining of PERV+ cells. Upper cell is in mitosis Fig. 2 qPCR Data of PERV-Exposed Cells Fig. 2. qPCR Data of PERV-Exposed Cells
Absence of nNOS Increases Longevity of Long Term Bone Marrow Cultures and Radiation Resistance.
Authors: Cao Shaonan , Greenberger Emily E. , Epperly Michael W. , Kanai Anthony J. , Greenberger Joel S.
Published: April 2020

Neuronal nitric oxide synthase (nNOS) has been shown to be localized to the mitochondrial membrane. The mitochondria play an important role in irradiation-induced apoptosis. Following irradiation, there is increased production of superoxide as well as an induction of nitric oxide in mitochondria. The combination of superoxide and nitric oxide results in production of peroxynitrite, a very strong oxidant that produces lipid peroxidation. Previous data have demonstrated that lack of nNOS protects the bladder from ionizing irradiation damage. To determine the role of nNOS in hematopoiesis, we established long term bone marrow cultures (LTBMCs) from nNOS deletion recombinant negative (knockout) mice as well as nNOS+/+ littermate mice. LTBMCs from nNOS knockout mice compared to +/+ control demonstrated increased total cumulative cell production (32.2 x 106 and 15.9 x 106 non-adherent cells, respectively), cobblestone island formation (5073 and 1106, respectively), and increased cumulative generation of day 14 CFU-GEMM colony forming cells per 105 plated (325 ± 30.4 and 9 ± 2.5 colonies, respectively) over 20 weeks in culture. IL-3 dependent non-adherent cell lines derived from the nNOS-/- and the nNOS+/+ cultures were tested for radiosensitivity. Cells from the nNOS−/− cell line demonstrated decreased radiation apoptosis 24 hours following irradiation, 5.89 ± 0.71% apoptotic cells compared to 10.42 ± 1.19% for the control littermates (p=0.041). Cell cycle analysis of littermate cells at 24 hours after 10 Gy demonstrated a G0/G1 and a G2/M block while there was no change in the cell cycle distribution of the nNOS−/− cells. The data demonstrate that absence of nNOS increases the longevity of hematopoiesis in LTBMCs and increases the radiation resistance of hematopoietic cell lines derived from such cultures.
BMI-1 Overexpression in 32Dcl3 Cells Suppresses Granulocytic Differentiation and Ameliorates Their Growth under IL-3 Diminished Conditions.
Authors: Hishida Asahi , Yamamoto Kazuhito , Sawa Masashi , Naoe Tomoki
Published: April 2020

The BMI-1 gene is one of the polycomb group (PcG) genes that have been shown to play essential roles in the self-renewal of both normal and leukemic stem cells. BMI-1 is reported to be highly expressed in primitive cells and its expression level decreases as bone marrow cells differentiate. We hypothesized that overexpression of BMI-1 might modulate the growth, differentiation and survival of 32D cells, and examined this hypothesis by generating BMI-1 overexpressing 32D cells. 32D cells were infected with MIG-BMI-1-IRES-EGFP vector or MIG vector, and the GFP positive rates of BMI-1 overexpressing 32D cells gradually increased under IL-3 diminished conditions, and the consecutive MTT assays using cells sorted by GFP positivity confirmed this finding, suggesting that BMI-1 could confer growth advantages on 32D cells under IL-3 diminished conditions.

Exposure of the GFP sorted 32D cells to G-CSF revealed that BMI-1 overexpression suppressed the granulocytic differentiation of 32D cells by G-CSF. The expressions of CD11b and Gr-1 were both suppressed by the overexpression of BMI-1, and real-time PCR analyses indicated that the expression levels of MPO was remarkably suppressed, while those of C/EBPα were not significantly changed.

Our experiments revealed that BMI-1 delayed granulocytic differentiation and ameliorated the growth of 32D cells under IL-3 diminished conditions.
Comparison of Stem Cells Harvested from Adipose Tissue and Bone Marrow.
Authors: Ogawa Rei , Fujimura Juri , Hanawa Hideki , Hirai Yukihiko , Kurai Toshiyuki , Mizuno Hiroshi , Hyakusoku Hiko , Shimada Takashi
Published: April 2020

Backgrounds: We have studied tissue engineering using both adipose-derived stem cells (ASCs) and bone marrow-derived stem cells (BSCs), and previously demonstrated that these stem cells differentiate into a variety of cell lineages both in vitro and in vivo.

Purposes: In this study, we investigated the biological differences between ASCs and BSCs harvested from mice. We studied the differences from the viewpoint of 1. morphology, 2. cell surface antigens, 3. proliferation potency, 4. pluripotency, and 5. gene transfer efficiency.

Methods: ASCs and BSCs were isolated from 5-week-old C57BL/6 mice. After subculture in a control medium, the cell surface markers expressed on the ASCs and BSCs were studied by flow cytometry. The cells were also incubated in osteogenic, chondrogenic and adipogenic media and differentiation was assessed by electron microscopy, special staining and RT-PCR. Gene transfer efficiency was studied using adenoviral, lentiviral, retroviral, adeno-associated viral (AAV) type 1, type 2, and type 5 vectors.

Results: BSCs were generally much more anaplastic than ASCs. However, ASCs and BSCs had generally similar properties as targets of viral vector mediated gene transfer, except that ASCs were particularly sensitive to lentiviral and oncoretroviral mediated gene transfer. The proliferation potency of ASCs was better than that of BSCs, but pluripotency and cell surface antigens were similar.

Conclusions: ASCs can be easily harvested and proliferated; ASCs may be better than BSCs for further experiments on stem cell biology and tissue engineering.
Differentiation of Human ES Cell into the Hemangioblast.
Authors: Carrier Ewa , Kausal Shermila , Srivastava Anand S.
Published: April 2020

We hypothesize that during early human embryo development a common endothelial/hematopoietic precursor is formed. This precursor, named the hemangioblast, is an early endothelial/hematopoietic precursor responsible for angiogenesis and both primitive and definite hematopoiesis. The aims of this work are 1) to establish the ES/EB model system for genotypic studies of early vasculo/hematopoiesis. 2) to phenotype human hemangioblast and establish standard culture/storage condition that will stabilize the phenotype of these cells for a defined period and permit consistent, predictable outcomes of experiments. Human ES (H-1 line) cells (NIH code WA01) has been maintained on g -irradiated MEF cells supplemented with DMEM/F12 media containing 4ng/ml b-FGF and 15% knockout serum by changing media everyday and passaging cells every 3–4 days. EBs were generated either in liquid or 1% methylcellulose cultures (1 x 104 ES cells per 35-mm Petri dish) in medium containing Iscove modified Dulbecco medium (IMDM), 15% FCS, 2 mM glutamine, 450 μM MTG, 50 μg/mL ascorbic acid, and 20% BIT (1% bovine serum albumin (BSA), 10 μg/mL insulin, and 200 μg/mL transferrin. In order to induce EB formation two days old hES cells were plated in 1% methyl cellulose in IMDM media supplemented with 50mg/ml ascorbic acid, 10mg/ml insulin, 200 mg/ml transferrin, 20% Bits, 15% FCS, 450mM MTG, 2mM-L-glutamine for 3days. To generate hemangioblasts, 1 x 104/mL EB cells were replated in 1% methylcellulose in the presence of IMDM, 10% PDS (bovine platelet-poor plasma-derived serum; Biomedical Technologies, Stoughton, MA), 2 mM glutamine, 450 μM MTG, 25 μg/mL ascorbic acid, 20% BIT, 5 ng/mL human vascular endothelial growth factor (hVEGF), 50 ng/mL SCF, 10 ng/mL human fibroblast growth factor 2 (hFGF-2), and 2 U/mL hEPO. RNA from day 0, 2, 3 and 4 days old EBs was collected for gene regulation studies. We have chosen to study the key regulatory genes (FLK-1, Runx-1, SCL, HEX, GATA-1) during this process. RNA collected from undifferentiated hES cells has been used as control. We observed expression of stemness genes: Sox-2 and Oct-4, in the undifferentiated ES cells while there was an upregulation in expression of Brachury on day 2 and FLK-1 and Hex genes in day 3 of differentiation. In hemangioblast forming cells (BL-CFC), we observed upregulation of FLK-1, SCL, Runx and downregulation of Brachury and Hex. This sptio-temporal expression profile of genes suggest that during hemangioblast formation and at the initial stages of differentiation hemangioblast fate is decided by Hex, while after day 2 the control is taken by FLk-1, SCl and Runx genes. Ongoing studies will define signaling pathways involved in this process. Understanding of molecular processes involved in hemangioblast formation will allow directed generation of this progenitor from human ES cells in the future.
HOVON 50/GMMG-HD3-Trial: Phase III Study on the Effect of Thalidomide Combined with High Dose Melphalan in Myeloma Patients up to 65 Years.
Authors: Goldschmidt Hartmut , Sonneveld Pieter , Breitkreuz Iris , van der Holt Bronno , Benner Axel , Barge Renee M.Y. , Salwender Hans , Bos Gerard M.J. , Glasmacher Axel , Raymakers Reinier , Scheid Christof , Huijgens Peter C. , Naumann Ralph , van Oers Marinus H.J. , Hänel Annette , Vellenga Edo , Martin Hans , Wijermans Pierre W. , Ho Anthony D. , Westveer Petra H.M. , Verhoef Gregor E.G. , Mazitschek Uta , Lokhorst Henk M.
Published: April 2020

The joint phase-III HOVON50/GMMG-HD3 trial was designed to assess the effect of thalidomide in induction treatment and as maintenance after high-dose therapy (HDT) and autologous stem cell transplantation (SCT) for multiple myeloma (MM). The standard treatment arm comprised 3 cycles of VAD, mobilisation with CAD+G-CSF (cyclophosphamide 1000 mg/m2, day 1; adriamycin 15 mg/m2, days 1–4; dexamethasone 40 mg, days 1–4; G-CSF until end of harvest), HDT with 1 or 2 cycles of melphalan 200 mg/m2, followed by autologous peripheral blood SCT (PBSCT), and maintenance with interferon-alpha (9 mio. U per week). In the experimental arm, TAD (thalidomide, 200 mg for HOVON / 400 mg for GMMG; adriamycin 9 mg/m2, days 1–4; dexamethasone 40 mg, days 1–4, 9–12, 17–21) was used for induction treatment. Mobilisation and HDT were identical to the standard arm. Experimental maintenance was thalidomide (50 mg per day). A first group of 406 patients (of 1050 included) are evaluable for the comparison of VAD vs. TAD and response after 1st HDT. A trend for a higher toxicity was observed in the TAD- compared with the VAD-arm (drop out: 15% vs. 8%, p= 0.10). Low molecular weight heparin was effective in the prevention of deep venous thrombosis during TAD-treatment (DVT-incidence 8% vs. 4% p= 0.15). The median number of stem cell collections to harvest at least one autograft was 1 in both arms (p= n.s.). Treatment-results are presented in table 1. In a subgroup of 90 GMMG-patients, 78% and 74% compared with 62% and 54% still received thalidomide compared with interferon-alpha at 12 and 24 months after start of maintenance. In summary, thalidomide+AD induce a significantly higher response rate, but this effect is completely offset by HDM. Therefore, results on EFS/PFS are necessary before thalidomide containing regimens can be defined as a standard for induction treatment before HDT. The maintenance treatment with thalidomide is better tolerated compared with interferon-alpha.

Treatment results (n=406) after VAD/TAD and after first HDT After VAD After TAD p-value PR 60% 73% <0.001 CR 3% 7% 0.11 PR/CR 63% 80% 0.001 After VAD/HDT After TAD/HDT p-value PR 75% 72% 0.8 CR 13% 19% 0.3 PR/CR 88% 91% 0.4
Regulation of Globin Gene Expression and Erythroid Differentiation by Sp/KLF Factors.
Authors: Hong Jie , Stamatoyannopoulos George , Song Chao-Zhong
Published: April 2020

Sp/Krüppel-like factor (KLF) family of proteins are characterized by the presence of three highly homologous Cys2His2 type zinc-fingers near the C-terminus that bind GC/CACCC boxes, which are one of the most common regulatory elements found in promoters of many cellular and viral genes. Currently, more than 20 members have been identified in the family. This family of factors plays important roles in cell growth, differentiation, development and homeostasis by regulating the expression of their target genes. The GC and GT/CACCC boxes in the globin gene promoters and the beta globin locus control region play an important role in the tissue- and developmental stage- specific expression of globin genes. We have carried out extensive studies to identify the KLF factors that regulate gamma globin expression and erythroid differentiation. Gene expression analysis revealed that most of the Sp/KLF factors are expressed, albeit at variable levels, in human fetal liver and adult blood cells. To determine the role of the Sp/KLF factors in gamma globin expression and erythroid differentiation, functional studies using systematic RNAi to knockdown selected Sp/KLF factors were performed. We used a lentiviral mediated siRNA expression system for specific silencing selected Sp/KLF factors. Effective knockdown of Sp/KLF factors was achieved as judged by a 70–90% decrease in their mRNA levels in the cells. Analyses of globin gene expression showed that the knockdown of some Sp/KLF factors resulted in changes in globin gene expression in K562 cells. We also observed that knockdown of specific Sp/KLF factors resulted in erythroid differentiation of K562 cells. These results suggest that specific Sp/KLF factors may play a role in regulation of globin gene expression and erythroid differentiation.
The Structure of the Gene for miR-223 in Humans, a MicroRNA Highly Expressed in Hematopoietic Tissues.
Authors: Silva Wilson A. , Kodama Kelson R. , Molfetta Greice A. , Zanette Dalila L. , Valente Valeria , Araujo Amelia G. , Rego Eduardo M. , Zago Marco A.
Published: April 2020

MicroRNAs (miRNA) are highly conserved noncoding regulatory RNA found abundantly in multicellular organisms. They derive from long primary transcripts (pri-miRNA) and act in a variety of regulatory pathways, including cell proliferation, apoptosis, fat metabolism and organogenesis. Over 200 human miRNA were identified by computational analysis and little is known about the gene hosts. Thus far, only 4 genes specific for microRNA have been characterized. Here we describe the structure of the gene for miR-223, a microRNA highly abundant in hematopoietic tissues. One cluster among 21,134 ESTs that we obtained from human bone marrow matched GenBank CD109334. Sequencing of cDNA from normal and leukemic cells did not correspond to known or potential protein structure, and revealed two alternative forms, with two or three “exons”. Phylogenetic analysis showed that our 234-nucleotide (nt) sequence matched DNA sequences of pri-miRNA of 6 non-human primates, with absolute identity in a 38-nt region that encompasses the 21-nt of the mature form. Secondary structure analysis of the transcribed RNA using M-Fold showed the stem-loop structure characteristic of a pri-miRNA. Northern blot with a probe designed to span the three exons of the genome sequence confirmed the expression of multiple bands in normal bone marrow, spleen, and acute and chronic leukemias, whereas other tissues were negative. Real time PCR with specific probes demonstrated that the expression of both alternate forms was restricted to the same cells. Northern blot revealed that mature miR-223 was observed only in hematopoietic cells. Thus, we describe for the first time the structure of a human non protein-coding gene specific for miRNA, spanning 4,450 nt of the genome in chromosome Xq12, with two transcript isoforms with 1,096 and 971 nt respectively, comprising three exons with 27, 125 and 944 nt, and show that the mature mRNA derives from the 3rd exon. Finally, there is an inverse relationship between the abundance of primary and mature species of miR-223 in normal and leukemic hematopoietic tissues, probably owing to a balance between transcription and processing of primary miRNA.
TNF Family Protein Regulation in Megakaryocytes and Platelets.
Authors: Crist Scott A. , Sprague Daniel L. , Lentz Steven R. , Ratliff Timothy L.
Published: April 2020

Activated platelets express and release several TNF family proteins with diverse immunological and inflammatory functions. While the biological effects of these potent immunological modifiers are an area of intense study, there are no studies to date that have investigated the mechanism of TNF protein regulation in megakaryocytes. Understanding the mechanisms of TNF protein regulation in megakaryocytes may allow for the design of new therapeutic strategies for the treatment of chronic inflammatory and autoimmune diseases. Our data show that CD40 Ligand, Fas Ligand and TNF-Related Apoptosis Inducing Ligand (TRAIL) expression are induced during megakaryocyte-differentiation of hematopoietic precursor cells. Promoter analysis and expression studies reveal multiple mechanisms of TNF family protein regulation. Megakaryocyte-specific regulation of TRAIL and Fas Ligand are primarily dependent on Sp1 and Sp3 transcription factor binding to proximal Sp1 binding elements within the respective promoter regions. Our preliminary data demonstrates differentiation-induced expression of CD40 Ligand on the other hand, is mediated via an Ets-1 site within 50 nucleotides of the transcription start site. In addition, endogenous CD40 Ligand but not TRAIL or Fas Ligand is constitutively expressed with ectopic expression of Fli-1, in the absence of differentiation-stimuli. Fli-1 is an Ets-1 family transcription factor that modulates several megakaryocyte-restricted genes. Further regulation studies reveal megakaryocyte expression of CD40 Ligand is diminished by the peroxisome proliferator-activated receptor (PPAR) agonist prostaglandin J2 and enhanced by 9-cis retinoic acid. Conversely, TRAIL and Fas Ligand expression are enhanced by prostaglandin J2 and diminished by retinoic acid. Taken from these data is there are differential mechanisms of differentiation-driven and induced regulation of the TNF family proteins. Continuing studies include the determination of the mechanisms of PPAR agonist, retinoic acid and differentiation-modulated TNF protein expression. Our studies demonstrate novel TRAIL and Fas Ligand regulatory pathways in the context of megakaryocyte differentiation and represent the first investigation into megakaryocyte-associated regulatory mechanisms for the platelet-derived CD40 Ligand, Fas Ligand, and TRAIL.
A Stromal Cell Derived Factor 1 (SDF-1) Agonist Stimulates Neutrophil Mobilization in Normal Humans after Subcutaneous Injection.
Authors: Wong Donald , Korz Walter , Merzouk Ahmed , Salari Hassan , William Bensinger I.
Published: April 2020

Stromal cell-derived factor-1 (SDF-1) is a well known key signaling molecule in the proliferation, homing, engraftment and expansion of hematopoietic stem cells and leukocytes. CTCE-0214, a peptide produced by rational drug design, is an analog of SDF-1 and agonist of the SDF-1 receptor, CXCR4. In vitro and in vivo models have shown that CTCE-0214 mobilized blood and progenitor cells and enhanced the survival and expansion of cord blood cells (Letters in Drug Design and Discovery.1:126, 2004; Exp Hematol.32:470, 2004; Exp Hematol.32:300, 2004; Stem Cells. In press). In this first phase I clinical trial of CTCE-0214, the safety and tolerability of single doses of CTCE-0214 given subcutaneously to healthy subjects as well as the pharmacokinetic profile and the pharmacodynamic effects were studied. The randomized, double-blinded, placebo-controlled dose-escalation trial enrolled 24 subjects in six dose-escalation groups. Four healthy human subjects in each cohort received either CTCE-0214 or Placebo, randomized in a 3:1 ratio. The starting dose for CTCE-0214 was 0.2 mg/kg. Successive dose cohorts received 0.5 mg/kg, 0.8 mg/kg, 1.5 mg/kg, 2.0 mg/kg, and 3.0 mg/kg. CTCE-0214 was shown to be safe with no serious adverse events reported in any of the dose levels studied. The most common drug related adverse events were injection site pain and erythema which were transient and resolved without intervention. The severity of injection site pain appeared to have an association with the overall quantity of study drug administered. Moderate or severe pain was reported only in subjects who received at least 80 mg of CTCE-0214 per syringe. Dilution across more than one syringe appeared to be effective in reducing injection site pain. Pharmacokinetic analysis of CTCE-0214 plasma concentrations showed that Tmax was reached at 0.25 hours post-administration for all CTCE-0214 treated cohorts. The apparent terminal elimination half-life (t1/2) values were estimated to be 0.41 to 0.32 hours. CTCE-0214 administration was associated with significant dose-dependent increases in total white blood cell and neutrophil counts in treated subjects, peaking at around 6 hours post-injection. In the 3.0 mg/kg arm, the mean difference in neutrophil count from baseline was more than three times the corresponding figure in the Placebo arm. The same trend was apparent in the 2.0 and 1.5 mg/kg arms with a greater than two times and two times increases, respectively. These results suggest that SDF-1 agonists may potentially be used in patients with low neutrophil count receiving chemotherapy with or without the use of G-CSF. The potential for CTCE-0214 to mobilize neutrophils and other blood cells merits serious consideration.
CD34+ Cells Mobilized with the New CXCR4 Antagonist AMD3100 + G-CSF Show Increased Anti-Apoptotic, Cell Cycle, DNA Repair and Cell Motility-Associated Gene Expression When Compared to G-CSF Mobilization Alone.
Authors: Seeger Timon , Maier Patrick , Li Li , Laufs Stephanie , Eckstein Volker , Bridger Gary , Calandra Gary , Wenz Frederik , Goldschmidt Hartmut , Ho Anthony D. , Fruehauf Stefan
Published: April 2020

AMD3100 is a new CXCR4 antagonist which induces a rapid release of hematopoietic progenitors from the bone marrow to the peripheral blood. We conducted a clinical study where patients with multiple myeloma and Non-Hodgkin’s lymphoma were treated with AMD3100 (A) to increase the number of peripheral blood progenitor cells (PBPC) when given an mobilization regimen of granulocyte colony-stimulating factor (G-CSF, G). Since experimental data suggest that A+G-mobilized PBPC are functionally different from G-mobilized PBPC we were interested in an intraindividual comparison of the gene expression profile of CD34+ cells in the two different settings.

To this end peripheral blood CD34+ cells of 3 patients (3 G, 3 A+G samples) were isolated by immunomagnetic followed by flow cytometric sorting to a purity of >99%.

Total RNA was purified. Differentially expressed genes were analysed by using the GeneChip Human Genome U133 Plus2.0 (Affymetrix). The expression analysis was performed with the SAS-MAS 1.0.3 software (SAS Institute Inc.). We found a pattern of unanimously higher (81 genes, fold-change > 2e0.5, p<10e−4) or lower (29 genes, fold-change > 2e−0.4, p<10e−4) expressed genes in the G+A-mobilized vs. G-mobilized CD34+ PBPC. The significant changes noted in the microarray analysis were validated by representative quantitative real-time PCR of four genes in these three patients and in further four independent patients. Genes were grouped according to gene function. Only increased expression was found in the categories anti-apoptosis (e.g. MPO, HSPA1B), cell cycle (e.g. MS4A3, RRM2), replication/DNA repair (e.g. DNTT RRM2), cell motility (e.g. TNFSF4, HMMR) and oxygen transport. Only decreased expression occurred in the pro-apoptosis gene group (e.g. MDA5 BCL10). A mixed pattern of decreased and increased gene expression was found for the categories transcription, cytoskeleton, immune response, metabolism and signalling.

CXCR4 receptor gene expression itself was significantly increased 1.5-fold in the A+G vs. G group. Interestingly also regulating molecules upstream of CXCR4 such as Id1, MYC, CREBP1, CEBP were increased in their expression while molecules downstream of CXCR4, e.g. in the MAP kinase pathway were significantly decreased (EGR1, EGR3) or increased (DUSP1, DUSP6), suggesting activity of feedback loops. We conclude that A+G mobilizes CD34+ PBPC with increased gene expression in categories which potentially promote superior engraftment after myeloablative therapy than G-mobilized CD34+ PBPC.
Quantitative Analysis of Growth Factor Production in the Mechanism of Fibrosis in Agnogenic Myeloid Metaplasia.
Authors: Wang Jen-Chin , Chang Tsong S. , Goldberg Amit , Novetsky Allan D. , Lipton Jeffrey
Published: April 2020

There are previous reports that growth factors including TGF-β1, PDGF and FGF produced by megakaryocytes are responsible for the etiology of fibrosis in agnogenic myeloid metaplasia (AMM). Due to the technical difficulty in isolating enough megakaryocytes from the inaspirable bone marrow from patients with AMM, a quantitative analysis of these growth factors produced by the megakaryocytes and correlated to the degree of myelofibrosis has not been able to be performed. The present study employed the cell culture technique to grow megakaryocytes from blood CD 34+ cells and then perform the quantitative analysis of these growth factors, compared with other sources such as monocyte-macrophage lineages and correlated to the degree of bone marrow fibrosis. We found TGF-β1, PDGF and FGF produced by the megakaryocytes were significantly elevated in AMM compared with normal controls (p<0.05) and TGF-β1 was more abundantly produced than PDGF or FGF. While these growth factors are several fold elevated in AMM compared with other MPD including essential thrombocythemia (ET), polycythemia vera (PV), it was not statistically significant. A quantitative analysis of these growth factors produced by the CD 14+ cells in the blood and bone marrow showed that these growth factors were not significantly elevated in AMM compared with other MPD or controls and were significantly elevated only in some patients (defined as elevation of more than 2 SD of the controls). The correlation of these growth factors produced by the megakaryocytes or monocyte-macrophage lineages with degree of myelofibrosis in 12 patients with AMM were r=0.73 and 0.23 respectively (Non parametric (Spearman) correlation with two-tailed analysis was used to calculate the correlation). We concluded:

In AMM, these fibrosing growth factors are mainly produced by the megakaryocytes and in some patients, monocyte-macrophage lineages may contribute to the production of these growth factor production. TGF-β1 is more abundantly produced from the megakaryocytes than PDGF or FGF confirming TGF-β1 is the most important fibrosing growth factor in the pathogenesis of myelofibrosis in AMM. A statistically significant correlation of the growth factor and degree of myelofibrosis in AMM suggests that these fibroing growth factors produced by the megakaryocytes are main etiology of bone marrow fibrosis in AMM.
Danazol Stimulates Proplatelet Formation of Megakaryocytes Via Rac Activation Pathway.
Authors: Murai Kazunori , Kowata Shugo , Ishiguro Mamiko , Ishida Yoji
Published: April 2020

Danazol (C 22 H 27NO2; 17a-2,4-pregnadien-20-yno[2,3-d]isoxazol-17-ol) have been widely used for patients with endometriosis. Recently, many reports showed that danazol was effective especially in thrombopoietic action in patients with refractory chronic idiopathic thrombocytopenic purpura, apalstic anemia or myelodysplastic syndrome-refractory anemia (MDS/RA). The thrombopoietic mechanism of danazol has been unknown. In this study, we investigated the thrombopoietic effects of danazol on megakaryocytes, especially proplatelet formation (PPF).

Murine megakaryocytes were isolated from bone marrow cells by Percoll®‚ density centrifugation and BSA gradient method. Isolated megakaryocytes (300/well) were cultured for 24 hrs in serum free condition with danazol and several kinase specific inhibitors including PPI (Src family kinase inhibitor), SU6695 (Src family kinase inhibitor), or LY294002 (PI3K inhibitor). PPF stimulation index (%) by danazol was calculated as (PPF with danazol)/(PPF without danazol). It was stimulated by danazol in a dose dependent manner(0μM: 100%, 0.2μM: 118±14.29%, 2μM: 136±10.3% (p<0.05), 12μM:176.7±17.4%(p<0.001), mean±SD, n=5).

The inhibitiory index(%) by kinase specific inhibitor was calculated as (PPF with danazole and each kinase specific inhibitor)/(PPF without them). Src family kinase inhibitor including PPI or SU6656 did not affect the PPF of murine megakaryocytes at 0, 0.2, 2.4, and 12 μM concentrations of danazol. While, LY294002 inhibited PPF of murine megakaryocytes at 0μM of danazol (LY294002; 0μM: 100%, 10μM: 75.33±4.5% (p<0.01), 100μM: 61.3±9.4% (p<0.01) (n=4)), as shown in Figure 1. PPF with danazole 0.2μM/ LY294002 100μM was 86% of that with danazol 0.2μM/ LY294002 0μM, that with danazol 2.4μM/ LY294002 100μM was 78%(p<0.05) of PPF with danazol 2.4μM/ LY294002 0μM, and that with danazol 12μM/ LY294002 100μM was 60%(p<0.01) of PPF with danazol 12μM/ LY294002 0μM (Figure 1). As a result, the similar inhibitory effects of LY294002 on PPF of murine megakaryocytes at 0, 0.2, 2.4 and 12.0μM of danazol were also observed.

These results strongly suggest that danazol is a powerful stimulating agent for PPF and that PI3K activation, followed by Rac activation might be the signal transduction pathway in PPF of megakaryocytes, based on that PI3K and Src family kinase stimulate Rac activation.

Figure 1. %PPF by danazol and/ or LY294002(P13K inhibitor) Figure 1. %PPF by danazol and/ or LY294002(P13K inhibitor)
The G Protein-Coupled Receptor cysLT1 Signals Via ERK/MAP-Kinase and Pyk2, but Not NF-kappa B, in CD34+ Progenitor Cells, Explaining Its Dual Effect on Both, Cell Migration and Proliferation.
Authors: Boehmler Andreas M. , Seitz Gabriele , Kanz Lothar , Möhle Robert
Published: April 2020

The G protein-coupled receptor (GPCR) cysLT1 is expressed in CD34+ cell lines and immature hematopoietic progenitor cells (HPC), while its ligand leukotriene D4 (LTD4) is produced by bone marrow endothelium and stromal cells when deprived of hematopoietic cells. In the present study, we analyzed the signal transduction pathways of cysLT1 in progenitor cell lines and primary CD34+ HPC isolated from the mobilized peripheral blood. Of note, the effects of LTD4 on intracellular calcium fluxes, a typical event related to signaling of Gi-coupled GPCR, were substantially stronger than those observed after stimulation of the HPC-associated chemokine receptor CXCR4 with optimal doses of the corresponding ligand stromal cell-derived factor-1 (SDF-1, CXCL12). In cytokine-supplemented liquid cultures in vitro (containing interleukin-3, stem cell factor, and FLT3-ligand), addition of LTD4 resulted in a significantly greater expansion of both, committed and immature CD34+ progenitors. The effect of LTD4 in these cultures was comparable to that induced by the early acting hematopoietic growth factor FLT3-ligand, whereas the CXCR4 ligand SDF-1 (CXCL12) was unable to expand the number of CD34+ HPC when added to similar cytokine-supplemented liquid cultures. In both, CD34+ cell lines and primary CD34+ progenitor cells, LTD4 induced a rapid phosphorylation of ERK/MAP-kinase, a signaling step related to cell proliferation, and Pyk2, which is connected to downstream signaling pathways resulting in cell migration. Signaling through NF-kappa B, representing an alternative pathway of GPCR signaling associated with cell proliferation, was unaffected. In conclusion, the GPCR cysLT1 activates multiple signal transduction pathways in CD34+ HPC leading to cell migration and proliferation. In contrast to the CXCR4 ligand SDF-1 (CXCL12), the effect of the cysLT1 ligand LTD4 on cell proliferation was more pronounced and even comparable to that induced by early acting hematopoietic growth factors.
Comparison of Human IL-11 and PTH: Effect on Blood and Bone Parameters in Adult Ovariectomized Mice.
Authors: Liu Ling , Zeng Qing Qiang , Zeng Wei , Cain Ricky , Ma Yan Fei
Published: April 2020

Interleukin-11 (IL-11) is a multi-functional cytokine with effects on hematopoiesis and bone remodeling. IL-11 is known to promote osteoclast formation by RANKL-dependent and independent processes. Recently, it was reported that IL-11 receptor signaling is required for normal bone turnover. To understand recombinant human IL-11 (rhuIL-11) effects on bone remodeling in vivo, we compared rhuIL-11 and PTH treatment in adult ovariectomized mice. IL -11 or PTH was administered s.c. daily for 6 weeks to 7 month-old mice that had been permitted to lose bone for 1 month post-ovariectomy. IL-11 at either 50 or 200 microg/kg/day increased platelets and neutrophils in the peripheral blood indicating that a therapeutic dose was achieved, but there was not a significant effect of rhuIL-11 on the plasma concentrations of osteocalcin as compared to vechicle control. In contrast, treatment with 30 micro/kg/day PTH significantly increased plasma osteocalcin. Interestingly, both IL-11 and PTH increased bone mineral density (BMD) of whole femur at the end of the 6-week study. Further studies are required to understand the effects of IL-11 on bone formation and resorption.
Enhanced Apoptosis in Response to Chemotherapeutic Agents in Leukemic Cells Is Associated with Post-Transcriptional Modification of Cyclin A1.
Authors: Ekberg Jenny , Brunhoff Cecilia , Landberg Göran , Perssson Jenny L.
Published: April 2020

A critical cell cycle regulatory protein, cyclin A1 has previously been demonstrated to initiate acute myeloid leukemia (AML) in transgenic mice. We have recently shown that the cellular level and distribution of cyclin A1 correlates with the status of the leukemic phenotype. The aim of our present study is to investigate the role of cyclin A1 in mediating the response of leukemic cells to drug-induced apoptosis, and to define the molecular pathways that cooperate with cyclin A1 to enhance drug-induced apoptosis. We have shown that apoptosis was enhanced in U-937 cells when treated with chemotherapeutic agents including staurosporine and TNF. Interestingly, the increased rate of apoptosis was associated with concomitant increase in cyclin A1 expression. Treatment with a general caspase inhibitor blocked the cleavage of caspase-3 but did not prevent the up-regulation of cyclin A1. We have found that cyclin A1 protein is normally undergoing ubiquitin-mediated degradation in myeloid cells. However, in cells undergoing apoptosis induced by TNF, cyclin A1 protein is prevented from degradation and ubiquitination, suggesting that post-transcriptional modification of cyclin A1 protein is involved. Two dimensional gel analyses further verified that cyclin A1 protein is modified following the increased rate of apoptosis induced by TNF. We have further established a link between cyclin A1 and CDC25C, which might contribute to the protein modification of cyclin A1 and thus contribute to the enhanced apoptosis in response to TNF in leukemic cells. To further define the functional role of cyclin A1 in drug-induced apoptosis, we are currently using myeloid cells from cyclin A1 deficient mice and wild-type controls.
Vascular Endothelial Growth Factor Contributes to Adverse Patient Outcome in Acute Myeloid Leukemia by Promoting Cell Growth and Survival and by Reducing the Sensitivities of Leukemic Cells to Drug-Induced Apoptosis.
Authors: Brunhoff Cecilia , Ekblad Jenny , Persson Jenny Liao
Published: April 2020

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that contributes to the regulation of hematopoietic stem cell development, extracellular matrix modelling and inflammatory cytokine generation. To study the potential role of VEGF in proliferation and apoptosis in acute myeloid leukemia, we evaluated the VEGF protein expression and its association with disease characteristics and patient outcome in 42 patients with acute myeloid leukemia (AML). In contrast to its weak expression in myeloid progenitors in normal bone marrows, high levels of VEGF expression was detected in 22 of the AML patients. Interestingly, VEGF expression significantly correlated with the expression of several major cell cycle regulatory proteins including cyclin A1, cyclin A2, cyclin E, CDK2 and p27. Log-rank test stratified by levels of VEGF expression revealed a clear trend with worse overall survival for patients with high levels of VEGF compared to those with low levels. Treatment of U-937 cells with recombinant VEGF protein resulted in an increased rate of proliferation. This suggested that VEGF may promote cell growth by mediating the cell cycle regulatory pathways. We further demonstrated that induced expression of VEGF promoted survival of leukemic cells and protected the cells from ATRA-induced apoptosis. Taken together, our studies demonstrated that increased expression of VEGF in AML patients was associated with worse patient outcome. Elevated levels of VEGF may contribute to the adverse outcome by promoting cell growth and survival of leukemic cells and reducing the sensitivity of leukemic cells to drug-induced apoptosis in AML patients.