bluuebunny/crossref_metadata_2025 huggingface dataset:
Automated Quantitative Assessment of Bone Marrow in Normal Samples: Correlation with the Reference Method.
Authors: Zini Gina , Garzia Mariagrazia , Di Mario Antonella , Rossi Elena , Farina Giuliana , d’Onofrio Giuseppe
Published: April 2020

Bone marrow (BM) analysis is conventionally performed by microscope examination on films of about 0.3ml of aspirated bone marrow fluid, stained with Romanowsky dyes. Until 1996 the simple automated screening of marrow composition was made very difficult by a number of factors, mainly the lack of the erythroblasts quantitation and the fat interference. From 1996 last generation automated hematology analyzers provide accurate and precise erythroblasts counts; moreover same systems have improved their software reducing the problem of fat interference.

We have analyzed data from 100 normal BM samples from patients submitted for diagnostic and/or follow up purposes in our Hematology Day Hospital. BM fluid was harvested from the superior posterior iliac crest. The first 0,3–0.5 ml were used for smears, while the next 1–2 ml of BM, collected into K3-EDTA, were analysed with Coulter LH 750, a fully automated hematology analyzer which provides Complete Blood Count, White Blood Count Differential included Nucleated Red Blood Cells (NRBC) and Reticulocytes count. We used the microscope examination conventionally performed on films stained with Romanowsky dyes as reference method. Quantitative detection BM cellularity was obtained by semi quantitative evaluation based on the evaluation of hematopoietic cells in several marrow particles: physiological differences age related were also taken in account. If hematopoietic cells occupy less than 25% or more than 85% the sample is defined respectively hypocellular or hypercellular (none of our sample was as). Differential cell count was usually performed on two different slides counting 500 cells (1000 when hypercellular, but none of our sample was as).

We found a strict correlation between microscope semi-quantitative cellularity evaluation and the instrumental cell count as sum of WBC plus NRBC, the Total Nucleted Cell Count (TNCC).

The mean value of the TNCC in normal PM samples was 29,48 x109/L with a range 25,9–54,9 x109/L. These results are in good agreement with normal BM cell count reported in the literature using a cytofluorimetric method, which is 34,5 x109/L (SD28.0).

The instrumental mean percentage of BM granulocytes corrected for TNNC was 62% (range: 23,5–93,7) versus a mean microscope percentage of 58,42% (range: 40–72).

The automated NRBC BM count corrected for TNCC was 11,38% (range: 2,7 – 39,17) versus a microscopic mean value of 28% (range: 9–45). These results, including the slight NRBC underestimation probably due to partial mature cell lysis, are in line with the data of the literature.

This study confirms the feasibility of routine automated cell count using a hematology in normal BM fluid samples. Automated methods will support morphologists quickly providing accurate and precise quantitative information such as TNCC and myeloid/erythroid ratio.
Development of New Small Molecule Bone Marrow Radioprotectors.
Authors: Niu Yunyun , Kanai Anthony J. , Wipf Peter , Zhang Xichen , Greenberger Emily E. , Franicola Darcy , Epperly Michael W. , Kagan Valerian E. , Fink Mitchell P. , Greenberger Joel S.
Published: April 2020

New ways to protect the bone marrow from irradiation damage are required. We are currently developing new small molecules that can be given orally to protect the marrow from irradiation damage. One small molecule is EUK-134, a manganese superoxide dismutase (MnSOD) mimetic. A nitric oxide synthase inhibitor, and a nitroxide homologue which have been both conjugated to a mitochondrial localization peptide signal, delivers the NOS inhibitor (another radioprotector) and nitroxide to the mitochondria. In addition, we tested ethyl pyruvate (EP), which has been demonstrated to protect tissues from ischemia-reperfusion injury at the level of the mitochondria. Incubation of 32D cl 3 hematopoietic cells 1 hour before irradiation in the presence of 20 μM EUK-134 or 100 μM of NOS inhibitor demonstrated an increased shoulder on the irradiation survival curve in one case (n = 5.55 ± 0.17 for EUK-134 compared to 1.60 ± 0.38 for control irradiated, respectively, p = 0.0044) and increase in the D0 for the mitochondrial targeted NOS inhibitor compared to irradiated control 32D cl 3 cells (D0 = 3.04 ± 0.27 compared to 1.29 ± 0.01 Gy, p = 0.0236). In experiments with 32D cl 3 cells in EP before irradiation or following irradiation, there was an increase in the D0 to 2.2 ± 0.25 and 2.21 ± 0.15, respectively, compared to irradiated controls 1.42 ± 0.09 Gy for the 32D cl 3 cells (p = 0.0447 and 0.0119, respectively). Furthermore, incubation of 32D cl 3 cells in EP both before and after irradiation significantly increased the shoulder on the survival curve compared to irradiated control 32D cl 3 cells (n = 4.14 ± 1.59 compared to 1.70 ± 0.6 for the control irradiated cells, respectively, p=0.0485). To determine whether EP protected the bone marrow from irradiation in vivo, C57BL/6J mice were injected with EP intraperitoneally (70 mg/kg) 30 minutes before irradiation, daily for 5 days after irradiation, or both before and following irradiation to the LD 50/30 TBI dose of 900 cGy. Mice injected with EP either after irradiation or both before and after irradiation had prolonged survival compared to irradiated control mice or those injected with EP only before irradiation. (Percent survival at 50 days 100% and 100%, respectively, compared to 60%, p<0.05). These results demonstrate the potential effectiveness of new small molecule bone marrow radioprotectors.
Age-Related Transcription Levels of KU70 and BIK in CD34+ Hematopoietic Stem and Progenitor Cells.
Authors: Prall Wolf C. , Czibere Akos , Grall Franck , Zerbini Luiz F. , Jaeger Markus , Spentzos Dimitrios , Libermann Towia A. , Gattermann Norbert , Aivado Manuel , Haas Rainer
Published: April 2020

There is a higher incidence of hematological clonal stem cell disorders in elderly persons. Age-related alterations of hematopoietic stem and progenitor cells (HSC) may represent one factor underlying this observation. However, the molecular changes related to stem cell aging are largely unknown. Therefore, we scrutinized gene expression patterns of HSC from umbilical cord blood (CB) as well as bone marrow from young (BM-Y, mean age 32,8, SD 12,4) and old (BM-O, mean age 88,8, SD 4,4) healthy donors. CD34+ HSC were isolated via immuno-magnetic separation and CD34+ purity was evaluated by FACS analysis. Thereafter we performed cDNA array analyses on a first set of samples (n=18). We found that the BCL2-interacting killer gene (BIK) and the gene encoding KU Antigen 70kD (KU70) show age-related mRNA expression levels. BIK is a proapoptotic gene and its expression was positively correlated with donor’s age, i.e. lowest in CB, 1.8-fold higher in BM-Y and 4.2-fold higher in BM-O. KU70 is a DNA repair gene and part of the DNA dependent protein kinase (DNA-PK). Its expression was negatively correlated with donor’s age showing highest expression levels in CB, 2.2-fold lower levels in BM-Y and 5.3-fold lower levels in BM-O. These findings were confirmed with a second set of samples (n=16) by means of quantitative RT-PCR. Elucidation of age-dependent molecular alteration in healthy HSC might facilitate a better understanding of hematological malignancies in elderly persons.
Implantation of Autologous Bone Marrow Mononuclear Cells in Patients after Acute Myocardial Infarction Via Coronary Artery.
Authors: Klabusay Martin , Navratil Milan , Koristek Zdenek , Groch Ladislav , Meluzin Jaroslav , Mayer Jiri
Published: April 2020

Background: Several populations of adult stem cells have been identified in bone marrow: hematopoietic stem cells, which are able to regenerate hematopoiesis in all of its lineages, mesenchymal stem cells, which can give rise to connective tissues (bone, cartilage and fat), and endothelial progenitor cells, which can initiate angiogenesis. Adult stem cells are found within the mononuclear cells compartment of bone marrow. Recent reports describe the effect of mononuclear bone marrow cells in reparation of ischemic tissue damage.

Methods: The authors designed the experimental protocol of cellular therapy for patients after acute myocardial infarction. Inclusion criteria were: first myocardial infarction treated with primary angioplasty and stent implantation, confirmed non-viability of myocardium by USG, PET and SPECT, elevated CK-MB, and age below 70 years. Patients with intervention on other coronary artery, in unstable condition at day 4 through 6, and with serious non-cardiac disease were excluded. Patients undergoing coronary angioplasty, who signed informed consent, were randomized into three arms: A - high dose of 1•108 mononuclear cells, B - low dose of 1•107 cells, C - no cells. The autologous bone marrow was collected within day 7 after infarction. The mononuclear cells were separated, cultured for 24 hours in serum-free medium, and implanted through catheter via coronary artery into the damaged heart muscle in 7 subsequent injections. Mononuclear cells were analyzed with multicolor flow cytometry and culture assays of CFU-GM and CFU-Meg. The cardiac perfusion, metabolism and function were evaluated with SPECT, PET and echocardiography at 3 months after cell implantation.

Results: 31 patients enrolled into the study underwent the protocol (9 in group A, 11 in groups B and C, respectively), and their cardiac functions were evaluated afterwards. There were no serious adverse effects of cell therapy procedure observed in each group. The analysis at 3 months interval showed an improvement in metabolism in the cellular therapy arms detected by PET. Left ventricle ejection fraction improved from 38 to 44%, although this improvement in global heart function was not statistically significant. However, regional heart function at the infarction site (peak systolic velocity of the infarcted wall) improved from 4.1 to 5.0 cm/s in the arm A (p<0.01), while no improvement was observed in arms B and C. A very significant improvement in metabolism and regional function of infarcted area of left ventricle was observed in three patients, all within the treatment arm A.

Conclusion: Mononuclear bone marrow cells as a potential source of adult stem cells can be enriched, cultured ex vivo, and safely used in the cellular therapy protocols for ischemic heart disease. The functional benefit of dose of 1•108 mononuclear cells can be detected in a group of patients after acute myocardial infarction.
Development of a Model for the Study of PU.1 Function in Primary Human Hematopoietic Cells.
Authors: Durual Stephane , Rideau Alexandra , Wiznerowics Maciej , Ruault Sylvie , Beris Photis , Piguet Vincent , Matthes Thomas
Published: April 2020

PU.1 is one of the best-studied transcription factors governing hematopoiesis and has been shown to regulate positively differentiation of B-lymphocytes and granulocytes. PU.1 is also expressed in early erythroid progenitors and its interaction with GATA-1 was described to directly inhibit erythroid differentiation, since GATA-1 is the key regulator of erythropoiesis. In addition, the binding of GATA-1 to PU.1 was found to repress PU.1 dependent myeloid gene expression. In order to study more in detail the effect of PU.1 in primary human hematopoietic cell differentiation, we designed lentiviral vectors which allow PU.1 overexpression and PU.1 inhibition.

For PU.1 overexpression, we cloned the PU.1 cDNA into the pWPIR-ires-GFP bicistronic plasmid and verified by transient transfection in 293T cells the production of PU.1 mRNA and of right sized protein. We analyzed PU.1 function by co-transfection assays into 293T cells using the pWPIR-PU.1 vector and CAT reporter genes governed by PU.1 responsive elements. By CAT ELISA assay we observed a dramatic increase in OD. The production of PU.1 mRNA and protein in Hela cells was verified by stable transduction with complete lentivectors.

For PU.1 inhibition, we constructed a lentiviral vector encoding a siRNA specific for PU.1. After transduction of the K562 erythroleukemic cell line, both PU.1 mRNA and protein became undetectable, as verified by RT-PCR and Western blot, respectively, whereas GATA-1 mRNA and protein expression remained unchanged.

We tested both viral constructs in an in vitro culture system, in which CD34+ hematopoietic precursors obtained from bone marrow aspirates, differentiate into mature red cells under the influence of SCF, IL-3 and Epo or into mature granulocytes by stimulation with thrombopoietin, SCF and Flt-3L. Results for cultures with PU.1 transduced cells showed inhibition of erythroid cell differentiation by 40% ± 10% (mean of three experiments) and increased myeloid proliferation, whereas cultures with siPU.1 transduced cells showed no influence on erythroid cells and strong decrease of myeloid cell proliferation (50 – 60x) and differentiation (90 % decrease of CD13+ cells).

In conclusion, our model gives us the opportunity to test the function of PU.1 overexpression and/or inhibition in primary hematopoietic cells, to test the effect on target genes in various stages of differentiating precursors and the interaction with other transcription factors like GATA-1, and to analyze pathologic conditions like some forms of acute myeloid leukemia, where PU.1 was described to be mutated or downregulated.
Upregulation of HOXB4 Message in Human CD34+ Cells Transduced with TAT-NF-YA Fusion Protein.
Authors: Domashenko Alevtina D. , Heyward Christa , Hexner Elizabeth , Zhu Jiang , Emerson Stephen G.
Published: April 2020

We have recently found that the trimeric transcription factor NF-Y is a direct stimulator of hematopoietic stem cell (HSC) renewal, activating the transcription of several genes involved in HSC self-renewal and differentiation including the Hox4 paralogs HoxB4, HoxC4, HoxD4 as well as LEF1, Notch1, p27 and telomerase (Zhu J. et al. PNAS, 102:11728–11733; 2005). HIV-TAT peptide transduction offers a distinct therapeutic advantage over viral vectors due to the temporary and regulatable nature of the induction. We have previously modeled the delivery of NF-YA protein fused with the protein transduction domain of the HIV-1 TAT protein to K562 cells, demonstrating upregulated HOXB4 to levels similar to that achieved by retroviral overexpression (Domashenko A. et al. Blood, 104:964a; 2004). We have now asked whether TAT-NF-YA protein induction can be modified for direct application to normal human CD34+ cells. Recombinant protein was designed by fusion of the human NF-YA sequence with the TAT protein transduction domain and a GST-tag at the N-terminus, and purified under native conditions. Binding activity of fusion protein to HOXB4 promoter probe was confirmed by electrophoretic mobility shift assay (EMSA). Human peripheral blood CD34+ cells were cultured with GST-TAT-NF-YA for 1.5h and displayed bright fluorescence in the cytoplasm and nucleus after staining with anti-GST-Alexa Fluor 488 Ab. To evaluate the effect of TAT-NF-YA fusion protein on HOXB4 promoter activity in primary hematopoietic cells, the level of HOXB4 mRNA was measured by real-time PCR with normalization to GAPDH. It was found that delivered NF-YA was able to increase endogenous HOXB4 message up to 3.5-fold in 2h after incubation with the protein. To minimize the recombinant protein toxicity and maximize the efficiency of delivery we investigated the possibility of having the same functional effect at low protein concentration using lysosomotropic agents (chloroquine and sucrose), to facilitate fusion protein nuclear uptake. The effect of varying concentrations of each agent on protein delivery and cell survival was analyzed by determination of endogenous HOXB4 message using quantitative PCR and trypan blue staining. Both chloroquine and sucrose increased the effect of upregulation of the HOXB4 message by GST-TAT-NF-YA 1.5 – 2.5 fold. Chloroquine exhibited substantial toxicity to the cells whereas sucrose was improving cell survival; therefore we have used sucrose in subsequent experiments. A sustained several fold activation of HOXB4 expression in CD34+ cells exposed to GST-TAT-NF-YA (40 nM) and sucrose (250 mM) has been observed for at least 24 h following exposure to the protein. In conclusion, our results showed the ability to transduce functionally active NF-YA protein into human primary hematopoietic cells. These findings support the further employment of protein transduction techniques for development of new therapeutic strategies.
Gene Regulation during the Erythrocytic Differentiation of Embryonic Stem Cells.
Authors: Carrier Ewa , Kausal Shermila , Srivastava Anand S.
Published: April 2020

We have studied the in vitro differentiation of murine embryonic stem cells (ES cells) towards erythropoiesis and expression of genes during this process. It has been reported that dexamethasone directs ES cells towards erythrocytic differentiation but the mechanism of gene regulation induced by dexamethasone is not well understood. We hypothesized that dexamethasone induces upregulation of erythropoietic genes such as GATA-1, FLK-1, EPO-R and directs ES cells towards erythropoietic differentiation. Murine ES cells (129 CCE) obtained from Dr. Nagy laboratory, Canada (Nagy et al., Histochem Cell Biol., 2001; 115:49–58) were subjected to the in vitro primary hematopoietic differentiation media containing methylcellulose, IMDM, IL -3, IL-6 and SCF (stem cell factor) without LIF (leukemia inhibitory factor) to promote embryoid body (EB) formation. Total RNA was collected on day 3, 5 and 9 EBs for gene expression studies using RT-PCR. On day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combination 1) SCF, EPO, dexamethasone, IGF, 2) SCF, IL-3, IL-6, TPO, 3) SCF IL-3, IL-6, TPO, EPO. Total RNA from day12 of secondary differentiated ES cells was collected to study cytokines and growth factors dependent erythrocytic differentiation and gene regulation, using RT-PCR. Our results demonstrate upregulation of Gata-1, Flk-1, HoxB-4, Epo-R and globin genes (α-globin, BH-1 globin, β-major globin, e -globin and z-globin) in the 9 days old EBs, whereas, RNA collected from 5 days old EBs showed expression of HoxB-4, e-globin, γ-globin, BH1-globin and FLK-1. Three days old EBs showed only HoxB-4 and FLK-1 gene expression and lack of expression of globin genes, indicating that erythtropoiesis-specific genes activate later. Gene expression studies of RNA collected from secondary differentiated ES cells and media containing dexamethasone showed downregulation of GATA-3 and upregulation of GATA-1, Flk-1 and Epo-R in comparison to the two other cytokines and growth factors media combination. These results confirm our hypothesis that dexamethasome induces erythropoiesis by down regulating GATA -3 and upregulating erythropoietic-related genes such as GATA-1, Flk-1 and Epo-R. The morphological characteristics of cells after secondary differentiation showed enhanced production of erythrocytic precursors in dexamethasone containing media, which corresponded with molecular studies. Further studies will address the role of wnt/β-catenin and E-cadherin in this process.
The Combination of Stem Cell Factor and Oncostatin M Maintains Cord Blood-Derived NOD/SCID-Repopulating Cells.
Authors: Gilfillan Siv , Kraner Simone , Goetze Katharina S. , Schiemann Matthias , Marz Stefanie , Peschel Christian , Oostendorp Robert A.J.
Published: April 2020

We here investigated whether receptor tyrosine kinase signaling (ckit, flt3) was sufficient to maintain hematopoietic stem cells (HSC) in culture, or whether, like in embryonic stem cells, signaling through gp130 is also required. Sorted CD34+ AC133+ (CD33/ CD38/ CD71)- cells from human umbilical cord blood (CB) were cultured in the presence of combinations of ckit-ligand stem cell factor (SCF), flt3-ligand (FL) and different stimulators of gp130 (IL-6, IL-11, LIF, OSM, CT1 and CNTF). Of these cytokines, SCF stimulated cell division by itself. Gp130-stimulating cytokines did not give any additional cell division activity. When SCF was included in the cytokine cocktail, progenitor levels (CFC, CAFCw2, CAFCw6) was very similar for all combinations of cytokines tested after this initital 6-day culture. To study more immature progenitors, cells were harvested after 6 days of serum-free culture and cultured for another 6 weeks on FBMD-1 stromal cells (LTC-CAFC). These experiments showed that in initial 6 day cultures with FL or gp130-stimulators alone, very little hematopoietic activity remained. SCF maintained CFC, and LTC-CAFCw2 and LTC-CAFCw6 at about half the original level. However, combinations of SCF with OSM consistently increased the number of LTC-CAFCw2 and w6 about two-fold. This finding suggests that very immature hematopoietic progenitors were at least maintained in serum-free cultures. To study the most immatore cells, we performed in transplantations into sublethally irradiated NOD/SCID mice. When CB cells were cultured with SCF alone, NOD/SCID-repopulating cell (CRU) frequency (1 in 900) dropped to about 20% in 6 days (1 in 5500). Similarly, cultures without growth factors or with FL or OSM showed a decrease to less than 10% of the original CRU number. In contrast, the combination of SCF and OSM maintained CRU at about half the original level (1 in 1900). This study shows that signals through ckit and gp130 synergize to maintain CRU level. However, to achieve expansion of human NOD/SCID-repopulating cell numbers, additional signaling pathways need to be stimulated.
Chemokines and Cytokines Required for Migration of Mesechymal Stem Cells into Marrow or Injured Tissues.
Authors: Tondreau Tatiana , Nathalie Meuleman , Alain Delforge , Marielle Dejeneffe , Martine Massy , Christine Mortier , Dominique Bron , Laurence Lagneaux
Published: April 2020

Background: Intravenous infusion of mesenchymal stem cells (MSC) after chemotherapy are currently used to accelerate the hematopoietic recovery but their potential of migration and homing to bone marrow is poorly understood.

Methods: Bone marrow MSC (BM-MSC) were isolated with the classical plastic adhesion method. Cultured MSC were identified through their expression of SH2, SH3, CD44, CD105 but lacked typical hematopoietic antigens: CD14, CD31, CD34, CD45 and CD62-e. The MSC migration was evaluated using matrigel invasion chamber assay. MSC were used at 5.105 cells/mL in the upper chamber. In the bottom chamber, different cytokines were tested for their chemoattractive properties: SDF-1 (100ng/mL), PDGF-bb (100ng/mL), IGF (0,5 μg/mL) and IL-6 (100ng/mL) in a serum free medium. BM-MSC conditionned medium (CM) at 10% was also used as chemoattractant. After 24 hours, cells that had not migrated were removed from the top of the insert by scrubbing with a cotton swab and cells that migrated through the membrane were counted after staining with 0,5% crystal violet.

Results: We observed that MSC migration was greater in the presence of PDGF-bb, IL-6 or with 10% CM with respectively an increase of 6,6; 5,6 and 5 fold compared to spontaneous migration. Only a small number of MSC migrated in response to SDF-1, and this observation correlates with the very low expression of CXCR4 receptor (0,67±0,08%). Indeed, we demonstrated that CXCR4, the SDF-1 receptor was mainly intracytoplasmic expressed (74,9±1,3%).

Conclusions: IL-6 and PDGF-bb are important cytokines produced by the bone marrow microenvironment or by injured tissues. In our study, we thus demonstrated that these chmokines could be involved in the migration of MSC to bone marrow as well as to damaged tissues.

Mesenchymal stem cells migration Ctrl SDF-1 IGF-1 10%CM IL-6 PDGF-bb *number of cells evaluated/3 fields of view. Migrated MSC * 13,7±2,7 24,9±5 42,2±12,6 69±17,9 77,1±14,1 90,5±5,3
Identification of Erythroid Cell Stimulating Factor as a Unique Protein, Quiescin Q6: Role in Erythropoiesis.
Authors: Udupa Kodetthoor B. , Bose Chhanda
Published: April 2020

We demonstrated earlier the presence of an erythroid cell stimulating factor (ESF) in mouse serum that enhanced the erythropoietin-dependent erythroid cell proliferation in vitro. Employing a highly specific monoclonal antibody generated in our laboratory, we showed further that ESF had an essential role in normal erythropoiesis in vivo [Blood98, 296a, 2001, Exp Hematol.31, Supp. 1, 165, 2003]. In order to clone and express this protein, ESF was purified from mouse serum and on Western blotting using the monoclonal anti-ESF antibody the purified protein was stained as bands of ~62/64 kDa. These bands were analyzed by Mass Spectrometry. The data were processed using Protein Lynx 2.1 (Waters) and the resulting peak list was submitted for database searching on Mascot. One of the peptide sequences had a highest similarity with Quiescin Q6 (Q6). This protein was then BLAST searched for mouse DNA sequences in the NCBI database for nucleotides corresponding to the mouse Q6. We designed the primers to reclone Q6 ORF (Open reading frame) having restriction sites NCO1 and ECOR1. Amplified product was sequenced to confirm DNA fragment. Fragment was cloned in pGEMT vector and subsequently re-cloned in expression vector pET30a(+). After each cloning step, the orientation of the insert was analyzed using restriction enzyme digestion. His-tagged Q6 protein was over-expressed in E.coli BL21 Codon Plus (DE3)-RP cells. Western blot analysis using anti-ESF antibody identified 62/64 kDa-bands of Q6 in the soluble fraction of the bacterial extract. Further characteristics of this recombinant protein and its CFU-E enhancing properties are being investigated and will be reported. Thus we have identified ESF to be a unique protein, quiescin Q6 and successfully cloned and expressed it. Expressed protein is similar to ESF as regards its antigenic property.
Influence of Valproic Acid on Cytokine Expression of Spleen Cells and Acute Graft-Versus-Host-Disease in a Murine Model.
Authors: Jang Pil-Sang , Lee Dae-Hyung , Park Young-Shil , Chung Nak-Gyun , Cho Bin , Jeong Dae-Chul , Kim Hack-Ki
Published: April 2020

Inhibition of histone deacetylase, one of the mechanisms for the immunologic changes by antiepileptic drugs, induces gene expression which, in turn, alters cytokine secretion. Valproic acid (VPA), the most commonly used antiepileptic drug, inhibits TNF-α and IL-6 secretion induced by PHA in THP-1 cells, a human monocytic leukemia cell line. We examined the changes in cytokine secretion induced by differing concentrations of VPA and whether VPA could prevent lethal GVHD in murine allogeneic bone marrow transplantation (BMT).

For the evaluation of inhibitory effect of VPA, C3H/He (H-2k) splenocytes, irradiated BALB/c (H-2d) splenocytes, and PHA were cocultured with 0.1 mM, 1 mM, and 10 mM VPA. Concentrations of 0.1 mM and 1 mM VPA did not inhibit cell proliferation but 95% was inhibited at 10 mM. Apoptosis was induced more than 99% cells with 10 mM VPA but less than 5% of cells with 0.1 mM, and 1 mM. RT-PCR was performed to evaluate the changes and concentrations of each cytokine. TNF-α and IL-1β secretion, determined by the RNA quantity, was inhibited significantly more at 1 mM VPA than 0.1 mM (P<0.05). Decrease in IFN-γ concentration and increase in TGF-β concentration were significantly more at 1 mM VPA than 0.1 mM (P<0.05). However, differences in IFN-γ and TGF-β secretion, determined by the RNA quantity, were not statistically significant. Lethally irradiated BALB/c mice were transplanted with C3H/He bone marrow cells and splenocytes. Recipients were given 30 mg/kg and 100 mg/kg VPA intraperitoneally once daily from day −3 to day +14, respectively, whereas controls received phosphate buffered saline. There were no significant differences in the GVHD severity and mortality.

These results show that VPA inhibits the secretion of inflammatory cytokines and stimulates the secretion of an antiinflammtory cytokine without inhibition of cell proliferation. Although lethal GVHD was not prevented or controlled in a major and minor histocompatibility complex-mismatched murine allogeneic BMT model, further investigations concerning the effects of VPA on GVHD prevention using a congeneic murine model should be performed.
ATM and ATR Are Not Required for DNA Damage-Dependent Accumulation of Chk1 at the Centrosome.
Authors: Loeffler Harald , Bochtler Tilmann , Ho Anthony D. , Jeggo Penny A. , Bartek Jiri , Kraemer Alwin
Published: April 2020

ATR-Seckel syndrome and ataxia-teleangiectasia are autosomal recessive disorders associated with hematologic malignancies. They share the feature of genetic instability and are caused by defects in the DNA damage response: underlying causes are a hypomorphic ATR mutation and inactivating ATM mutations, respectively. A common substrate of ATM and ATR in the DNA damage response is the nuclear checkpoint kinase Chk1, which we have recently shown to localize to interphase centrosomes and thereby negatively regulate entry into mitosis by preventing premature activation of cyclin B-Cdk1 (Nat Cell Biol6: 884–891, 2004). Here, we demonstrate that DNA damage by ultraviolet radiation or hydroxyurea treatment enhances the centrosomal localization of Chk1. In fibroblasts derived from ATR-Seckel or ataxia-teleangiectasia patients, this DNA damage-dependent centrosomal accumulation of Chk1 was rather more pronounced in comparison with control fibroblasts. Accordingly, this cellular reaction is not dependent on ATR or ATM. These results indicate that centrosomal accumulation of Chk1 might contribute a novel regulatory mechanism to the DNA damage checkpoint. Our study might help to further dissect the contributions of ATR and ATM to the DNA damage response, and to better understand their role in the development of hematologic malignancies.
Both G0/G1 Cell Cycle Arrest and Continuous Activation of MAPK Induce Autophagic Cell Death in bcr-abl-Positive Leukemic Cells.
Authors: Mishima Yuko , Terui Yasuhito , Mishima Yuji , Takizawa Toshihiro , Kimura Shinya , Maekawa Taira , Hatake Kiyohiko
Published: April 2020

Back ground;

Accumulating evidence suggests that programmed cell death is not defined as apoptosis but cells use different pathways for active self-destruction as reflected by different morphology. Autophagic cell death (APCD), which also be designated type II programmed cell death and appears to be a phylogenetically old phenomenon, is observed in physiological and disease state. Indeed, APCD is observed in several neurodegenerative diseases and cancer cells, however, the APCD in leukemic cells has not been reported yet. Here, we found APCD was occurred in bcr-abl-expressing CML cell lines.

Methods;

Bcr-abl-positive cell line, K562, were cultured with 2nM of TPA up to 48hours. Subsequently, we examined the morphological change by light microscopy and electronmicroscopy. To evaluate the APCD, TPA-treated K562 cells were labeled with autofluorescent agent Monodansylcadaverin(MDC), which specifically accumulates in autophagosomes and we measured accumulation of autophagosmes in cytoplasm by fluorescent microplate reader. TPA continuous activated MAPK of K562 cells and induced cell cycle arrest in G0/G1 phase. In order to elucidate the role of activation of MAPK in APCD, MAPK in K562 cells were inactivated by MAPK inhibitor, U0126 or dominant negative MAPK-transfection, and then, the cells were treated with TPA. Furthermore, to evaluate the involvement of cell cycle arrest in APCD, bcr-abl-transfected murine leukemic cell line, BAF3, was treated with cell cycle arrest inducer, mimosine. In addition, we investigated the expression of an autophagy-related molecule, Beclin 1 by immunohistochemical analysis.

Result;

The 48 hours’ treatment with TPA induced cell death in K562 cells, which accumulated plenty of autophagosomes in cytoplasm. MDC labeling assay revealed that accumulation of autophagosomes were increased in time- and dose-dependent manner. On the other hand, MAPK-inactivated K562 cells exhibited resistance to TPA-induced APCD. Furthermore, the bcr-abl-transfected BAF3 cells exhibited continuous activation of MAPK and underwent APCD by cell cycle arrest by mimosine. Immunohistochemical analysis revealed that Beclin 1 shuttled between nuclear and cytoplasms and co-localized with MAPK during APCD progression.

Conclusion:

APCD in bcr-abl-positive leukemia cells were closely involved in G0/G1 cell cycle arrest and continuous activation of MAPK. These findings are useful in developing the novel strategy to treatment of Ph1 positive leukemia via alternative pathways and another type of cell death.
Frequency of Mesenchymal Colony-Forming Cells (CFU-F) from Human Cord Blood and the Umbilical Vein.
Authors: Oostendorp Robert A.J. , Mohaupt Nicole , Kaltz Nikolas , Hippauf Sandra , Peschel Christian
Published: April 2020

Umbilical cord blood (UCB) is frequently collected for the purpose of hematopoietic stem cell therapy. However, the use of these cells is infrequent. To investigate other possible applications we recently showed that UCB is an excellent source of endothelial colony-forming cells which improve vessel density and heart function ofter myocardial infarction (Ott, Keller, et al., FASEB J, 2005;19:992–4). We here investigated whether UCB or the umbilical cord would be suitable as a routine source of mesenchymal colony-forming cells (CFU-F). CD34+ and AC133+ cells were selected by MACS (Miltenyi) from mononuclear UCB cells. The resulting cells were cultured at limiting dilutions on fibronectin in medium with 2% FCS (BioWhittaker: EGM-2). CFU-F were isolated from 3 of 7 (42%) UCB donors investigated. However, the CFU-F frequency is extremely low: only 1.3 in 100 ml of UCB. Similraly to the endothelial cells we previously described, these few UCB-derived stromal cells (CBSC) could easily be expanded. In Parallel, we also investigated CFU-F from the human umbilical vein: human umbilical vein stromal cells (HuvSC). Here, CFU-F were isolated from all donors when veins were prepared within 6 hours of collection. The CFU-F frequency was estimated to be 3 to 5 per 10 cm of umblical vein. Like the UCB-derived CFU-F, HuvSC were easily expandable. To find out whether these stromal cells showed characteristics of immature cells, we investigated the presence of embryonic markers by RT- and realtime PCR. We confirmed the expression of POU5F1 (Oct4), SSEA-4 and Stella-related (DPPA3). Immature HuvSC were shown to express mRNA of many genes associated with mesenchymal lineages but did not express the hematopoietic markers CD45 or CD34. HuvSC were shown to differentiate into adipogenic, osteogenic and myogenic lineages, though their differentiation is not as pronounced as bone marrow derived MSC. HuvSC did also support long-term production of immature cobblestone area-forming cells week 6. In conclusion, UCB and the umbilical vein contain CFU-F with characteristics of immature mesenchymal stem cells. However, not all UCB collections contained CFU-F and the CFU-frequency is very low. Thus, UCB can, in our view, not be used as routine MSC source. On the other hand, CFU-F grew from small stretches of umbilical vein (HuvSC), making these cells excellent source of mesenchymal progenitors for tissue engineering purposes.
Hematopoietic Differentiation of ES Cells from P230 Bcr/Abl Transgenic Mouse.
Authors: Tanosaki Sakae , Inokuchi Koiti , Sawaguchi Kazuhiro , Takeuchi Jyunko , Tamai Masato , Uchida Naoya , Watanabe Ayako , Inami Mituharu , Yamaguchi Hiroki , Dan Kazuo
Published: April 2020

Chronic neutrophilic leukemia (CNL) is a distinct disease form with less aggressive phenotype mimicking chronic myelogenous leukemia (CML). The patients present lower white blood cell counts consisting principally of neutrophilia, more severe thrombocytosis, less severe anemia, and delayed or absent transformation to blastic crisis. CNL sometimes has the translocation (9; 22) results in the transcription of an e19/a2 type Bcr/Abl mRNA that codes for a 230-kD Bcr/Abl protein (P230). Recently, we generated the P230 Bcr/Abl-transgenic mice, whose disease phenotype is myeloproliferative disese (MPD) with thrombocytosis. To investigate the disease phenotype of p230 Bcr/Abl-expressing MPD, we derived embryonic stem (ES) cells from the P230 Bcr/Abl transgenic mouse and non-transgenic control mouse. We differentiated ES cells on OP9 stromal cells, which supportive for hematopoietic differentiation. After day 5, ES cells were differentiated into megkaryocytes with TPO, erythrocytes with EPO and myeloid cells without cytokines. At day 13, floating cells were analyzed with lineage specific markers of megakaryocyte (CD61), erythroid (TER119) and myeloid (CD11b) cells by FACS analysis. P230 Bcr/Abl ES cell had more tendency of myeloid differentiation than control ES cell. With methylcellulose colony assays, we found that P230 Bcr/Abl ES cell produced more colony forming units. Intriguingly, the number of myeloid colonies was significantly increased and that of erythroid colonies was slightly decreased with no significance. It is reported that P210 Bcr/Abl markedly suppressed erythroid colony development. These data suggested that P230 Bcr/Abl enhances myeloid development in hematopoiesis and that it suppresses erythropoiesis not as strongly as P210 Bcr/Abl.
Overexpression of MEL1 as a Novel Fusion Partner of AML1 in the Blastic Phase of Chronic Myeloid Leukemia with the Recurrent Cryptic Translocation t(1;21)(p36.3;q22).
Authors: Hazourli Sawcene , Chagnon Pierre , Fetni Raouf , Busque Lambert , Hebert Josee
Published: April 2020

Located at 1p36.3, MEL1 is a member of the MDS1/EVI1 gene family and encodes a zinc finger protein with a N-terminal PR-domain. It has been proposed that the overexpression of a modified version of MEL1 lacking the PR domain is oncogenic, whereas MEL1 retaining the PR domain is anti-tumorigenic. MEL1 is known to be overexpressed in some myeloid malignancies with reciprocal translocation t(1;3)(p36.3;q21) characterized by trilineage dysplasia and poor prognosis. It is suggested that the translocation of RPN1 gene at 3q21 in the vicinity of MEL1 gene might activate MEL1 expression through an enhancer element. Here we characterized a recurrent cryptic translocation t(1;21)(p36.3;q22) that fuses MEL1 to AML1 gene in a blastic transformation of chronic myeloid leukemia (CML). Fluorescence in situ hybridization (FISH) analysis with BAC/PAC clones revealed that the breakpoints are in intron 1 of MEL1 and between intron 1 and exon 8 of AML1. RT-PCR analysis showed that AML1-MEL1, but not the reciprocal MEL1-AML1 was expressed. Many splicing variants are present, and all fusions splice the 5′ end of AML1 that contains the RUNT domain with almost the entire MEL1. Furthermore two fusion transcripts contained open reading frames making possible the translation of two forms of AML1-MEL1 fusion proteins. To investigate if AML1-MEL1 leads to an inappropriate expression of MEL1 we performed a quantitative RT-PCR with primers outside and within the fused MEL1 allowing the detection of the normal and the rearranged allele respectively. Interestingly, our data show that while no normal MEL1 transcript was detected, there was an overexpression of the fused MEL1. These results suggest that similarly to AML1-EVI1 gene, overexpression of MEL1 could be regulated by the AML1 promoter in leukemic cells with the AML1-MEL1 fusion gene and might also play an important role in the progression of CML. Moreover, in contrast to previous studies showing an antioncogenic role for the PR domain, our findings indicate that in some leukemias, the overexpression of MEL1 is not restricted to the MEL1 PR-lacking form. This suggests that the mechanism by which the PR domain has his effect, is more complex than previously thought.
Molecular Characterization of NRG Gene, a Novel Partner, Fused to NUP98 Gene as a Result of the t(3;11)(q29q13;p15) Translocation in an Acute Myeloid/T Lymphocytic Leukemia.
Authors: Chen Sai-Juan , Zhu Yong-Jin , Pan Qin , Gu Bai-Wei , Xie Zhi-Yao , Cai Xun , Pan Jin-Lan , Wu Ya-Fang , Shi Jing-Yi , Li Guo , Xiong Shu-Min , Xue Yong-Quan , Chen Zhu , Chen Sai-Juan
Published: April 2020

The NUP98 gene has been reported to be fused with at least 17 partner genes in leukemia with 11p15 translocation. An adult patient with de novo acute myeloid/T lymphocytic leukemia harboring t(3;11)(q29q13;p15) has been investigated to characterize the genes involved in that translocation. Through molecular cytogenetic analysis, we identified a fusion transcript between NUP98 gene and a novel partner gene named as NUP98 related gene (NRG) at 3q29. Further molecular analysis showed that exon 13 of NUP98 was fused in-frame to exon 10 of NRG. Moreover, the segment from 3q13 to 3q29 translocated at 11p15 had been inverted and accompanied by the deletion of the distal portion of breakpoint at chromosome 3q29. Interestingly, the NUP98-NRG protein showed nuclear and cytoplasmic distribution, a pattern different from that of wild type NUP98 or NRG. When assayed in a GAL 4 reporter system, the fusion gene showed an aberrant trans-regulatory activity. Transfection in HL-60 cells demonstrated that NUP98-NRG could promote cell proliferation, survival and arrest differentiation. Therefore, NUP98-NRG may exert transforming effects by interfering with the cellular mechanism of transcriptional regulation. Our data provide thus new evidence that NUP98-related molecular abnormality is a recurrent genetic event in leukemogenesis.
Derivative (1) t(1;1) (p36; q21) Produces the Fusion Protein with Phosphatase 10 and MEL1 (MDS1/EVI1-Like Gene 1) in a AML (M5a) Case.
Authors: Noguchi Mitsuho , Tashiro Haruko , Shirasaki Ryosuke , Goto Moritaka , Kawasugi Kazuo , Shirafuji Naoki
Published: April 2020

Objective: A 25 years-old Japanese male was diagnosed as AML (M5a) with 46, XY, der (1) t(1; 1) (p36; q21). To determine the leukemogenesis in this case, we analyzed the fusion product of this chromosomal translocation.

Method: After obtaining informed consent, RNA was extracted from his bone marrow cells (blastic cells were occupied in almost 100%) with GTC method, and poly-A rich RNA was separated with oligo-dT latex particles. 1st strand cDNA was synthesized with oligo-dT primer. Oligo-dA stretch was added with TdT to make 5′ RACE. 1st PCR was performed with the synthetic antisense-1 primers from several candidates including p73, and MEL1, and with QT primer (5′-TGAGCCAGAGTGACGAGGACTCGAGCTCAAGCT17-3′) as a sense primer. 2nd PCR was performed with internal antisense-2 primers from several candidates, and Q0 primer (5′-CCAGTGAGCAGAGTGACG-3′) as the sense primer. The reaction of DNA sequencing was performed with BigDye Terminator Cycle Sequencing kit (Applied Biosystems), and analyzed with ABI Prism 3700 DNA analyzer.

Result and Discussion: The fusion cDNA product was obtained, which was consisted of MEL1 and Phosphatase 10. The breakpoint of MEL1 was exon 4, and PR domain of MEL1 was disrupted. This domain is reported to be the key function of MEL1, and when MEL1 without PR domain is expressed, myeloid differentiation is blocked. In this case the fusion of Phosphatase 10 and MEL1 is seemed to be the main cause of leukemogenesis.
Activation of the Unfolded Protein Response in Acute Leukemia and Myeloma.
Authors: Yujiri Toshiaki , Tanimura Atsuko , Tagami Kozo , Nakamura Yukinori , Tanizawa Yukio
Published: April 2020

Accumulation of unfolded or misfolded proteins within the endoplasmic reticulum triggers the unfolded protein response (UPR). Evidence from several studies suggests that the UPR is activated in various tumors and might play a crucial role in tumor growth. For example, in vitro activation of the UPR alters the sensitivity of tumor cells to chemotherapeutic agents. The role of the UPR in hematological malignancies remains unclear, however. We therefore used real-time RT-PCR to quantitatively assess expression of UPR-related genes in clinical samples [peripheral blood (PB), bone marrow (BM) and lymph node (LN)] from 9 newly diagnosed patients with acute myeloid leukemia (AML), 5 with acute lymphoid leukemia (ALL), 4 with multiple myeloma (MM), 12 with Non-Hodgkin’s lymphoma, 1 with Hodgkin’s lymphoma and 6 with infectious lymphadenopathy. Thirteen healthy controls were also examined. Evaluated was the mRNA expression of a spliced form of X-box DNA-binding protein (XBP1), ER degradation enhancing a-mannosidase-like protein (EDEM), C/EBP-homologous protein (CHOP) and glucose regulated protein 78 (GRP78). We found the transcription levels of all four of these genes to be significantly higher in leukemic blast cells and myeloma cells than in control BM cells or PB mononuclear cells. In particular, leukemic cells from Philadelphia chromosome-positive ALL and aggressive MM cells showed higher levels of UPR-related gene expression. Thus, activation of the UPR might be associated with the malignant characteristics of leukemia and myeloma. By contrast, expression of UPR-related genes was not increased in lymphoma cells compared to LN cells from the patients with infectious lymphadenopathy.

The present study provides the first evidence that UPR-related genes are activated in acute leukemia and multiple myeloma cells. Thus, activation of the UPR might play an important role in the development and progression of some hematological malignancies. More comprehensive in vitro studies will be necessary to determine the magnitude of the role played by the UPR, to identify the most important components of the pathway, and to determine which stages of tumor development are regulated by the UPR.
Analysis of IGFBP7 Expression Level and Its Correlation with Associated Genes in AML Patients.
Authors: Hu Shao-yan , Chen Zi-xing , Cen Jian-nong , Gu Wei-ying , Zhao Ye , Gu Min
Published: April 2020

IGFBP7 (insulin-like growth factor binding protein 7) is a newly-identified protein which can bind with insulin-like growth factors(IGFs), with 45%~65% homology to other IGF binding proteins. Several studies reported that IGFBP7 may be involved in the development of meningioma, breast cancer and prostate cancer, while its correlation with human leukemia had not been reported yet. We have observed an induced expression of IGFBP7 in human leukemia K562 cells which were transfected with and overexpressed RbAp46 gene. Besides, the expression level of WT1, RbAp46 and IGFBP7 was coordinately regulated during the induced differentiation of NB4 acute promyelocytic leukemia cells (data not shown), suggesting a role of IGFBP7 gene in human leukemia.. To investigate the expression level of IGFBP7(previous known as Insulin-like growth factor binding protein related protein 1)in bone marrow (BM) of leukemia patients and its correlation with other leukemia biomarkers, Real-time quantitative reverse transcription polymerase chain reaction (QRT-PCR) method was established for detecting IGFBP7 expression levels in BM of 127 patients with acute leukemia (AL), 24 with chronic myelogenous leukemia in chronic phase (CML-CP), 9 with CML in blast crisis (CML-BC) and 27 with non-leukemias. The M-Estimators of RbAp46N in 63 patients with newly diagnosed AMLs were higher than those of 27 with ALLs and 27 non-leukemic controls and 10 with AMLs in complete remission(CR)(193.80 v.s 88.84, 81.30 and 67.82, respectively). Although IGFBP7N was 101.13 in CML-BC and 168.77 in CML-CP, difference between them was not statistically significant. Difference in IGFBP7N values among CML-BC, AML, ALL and control groups was also not significant. The M-Estimators of IGFBP7N were lowest in M4 and highest in M5 among initial acute leukemia subtypes, and there was statistical differences between M4 and M5, and between M4 and M2. IGFBP7N expression level was not correlated to the expression of fusion genes BCR/ABL and AML1/ETO, but it was positively correlated with the expression level of PML/RARαfusion gene, the WT1 and RbAp46 gene. The correlation coefficiency was 0.71, 0.31 and 0.52, respectively. Our results suggested that IGFBP7 might act in WT1 mediating pathway to participate in acute myelocytic leukemias.