bluuebunny/crossref_metadata_2025 huggingface dataset:
Trombophylia Prevalence in 444 Patients with Clinical Suspicion of Thrombosis.
Authors: Guerra Joao Carlos C. , Aranda Valdir F. , Mendes Claudio A. , Hamerschlak Nelson , Bacal Nydia S. , Colombini Marjorie P. , Pasternak Jacyr , Sitnik Roberta , Graca Claudia H. , Pinho Joao Renato R.
Published: April 2020

Objetives: to evaluate the prevalence of genetic polymorphism of coagulation factors: Leyden factor V (FVL), prothrombin mutation, deficiency of coagulation inhibitors, protein C, protein S and antithrombin (AT), test for the lupus anticoagulant/anticardiolipin and homocistein levels, during the laboratory investigation of patients with clinical suspicion of thrombosis.

Patients and methods: This was done as a retrospective chart review study on 444 patients admitted at our hospital between November 2003 and December 2004 ( males 188, females 256). Medium age was 46 years. Prothrombin mutation and Factor V Leyden were searched by multiplex PCR, Protein C, S and lupus anticoagulant were studied by coagulometric methods, AT by chromogenic method, anticardiolipin by immunoenzymatic assay and homocistein by immunometric assay. Statistical analysis was performed using exact Fischer test or × Pearson test.

Results and conclusions: Prevalence of FVL was 32 cases, 7.2 %, 2 homozygotes and 30 heterozygotes. Prothrombyn mutation was found in 25 cases, 5.6 %, 1 homozygote and 24 heterozygotes. On 57 cases with genetic alteration 46 (80%) had thrombosis, 39 venous and 7 arterial. On 183 cases that done anticardiolipin test 27 cases were positive, 14.7 % (18 IgG, 6 IgM and 3 IgA). On those cases 22, 81.4 % had thrombosis, 15 venous and 7 arterial. All 3 cases with lupus anticoagulant had thrombosis. Hyperhomocistein was found in 7 cases, 3.2 %, with 4 cases associated with venous thrombosis and 1 with arterial thrombosis. Deficiencies of Protein C, Protein S and AT were found in 117 cases, l2.8 %, associated with 63 cases of thrombosis, 52 venous and 11 arterial, 56.3 %. Pathogenesis of thrombosis is complex and clearly associated with genetic and environmental factors - risk factors. On populations at risk for thrombosis laboratory investigation is essential.
IL6 -174 G/C and Fibrinogen-β-455 G/A Polymorphisms in Ischemic Cerebrovascular Disease (ICD).
Authors: Moreno B Vanessa , Garcia-Raso Arantxa , Garcia-Bueno Maria Jose , Sanchez Carmen , Subira Dolores , Mata Raquel , Llamas Pilar , Martinez Jose
Published: April 2020

Ischemic cerebrovascular disease (ICD) is a multi-factorial entity whose anatomopathologic substrate is atherosclerosis. The inflammatory cells migrate to vascular endothelium and release chemokines such as interleukin 6 (IL6), resulting in the atheroma plaque development. The IL6 production induces acute phase proteins synthesis such as fibrinogen.

Objective. The purpose of this case-control study is to analyze the ICD risk associated to the polymorphisms IL6-174 G/C and beta chain of fibrinogen-455 G/A (fibri-β-455). The association of these polymorphisms, classic vascular risks factors and ICD subtypes according to TOAST classification has also been explored.

Methods and patients. A total of 159 ICD diagnosed patients (82 female and 77 male; mean age 67.7±15) were analysed. They were matched for age (±5 years) and sex with a control group (65.6±14) without thrombotic history. AHT, DM, dyslipidaemia and smoking habit data were collected for each group. Polymorphisms were analysed by PCR-RFLP. Serum IL6 was measured by ELISA and fibrinogen by Clauss method. Statistic analysis was made with the SAS v9 software.

Results. Genotype analysis showed a significant higher prevalence of the genotypes G/G and G/C IL6 -174 in the patients (OR: 1.49, IC 95%: 0.95–2.34, p: 0.049). By contrast, the fibri-β-455 genotypes were not different in patients and controls (p= 0.230). When the stroke subtypes were analysed, we found that IL6-174 G/C genotype was more frequent in cardioembolic (60.0%) and atherothrombotic ICD subtypes (58.3%). In relation to fibri-β-455 G/A, the A allele showed a increased prevalence in atherothrombotic stroke (43.2%) (p= 0.049).

We observed an association between the dyslipidaemia and IL6-174 G/C polymorphism (p= 0.034) and smoking habit with fibri-β-455G/A (p= 0.072). No synergic relation were found between IL6-174 G/C and fibri-β-455 G/A. The G allele IL6-174 G/C carriers group presented higher serum IL6 levels than C allele carriers, 5.85 pg/ml (2.9–34.5) vs. 4.94pg/ml (2.5–11.4). Also, allele A fibri-β-455 G/A was associated with a higher fibrinogen levels than G allele, 329 mg/dl (210–686) versus 280 mg/dl (107–783).

Conclusion. There is an association between-174 IL6 G/C polymorphism and ischemic stroke. By contrast, we did not find any association between fibri-β-455 G/A, and ICD. Besides, the analysis performed by ICD subtypes showed an association of this polymorphism and atherothrombotic subtype. These results suggest a relation between smoking habit and fibri-β-455 G/A, and dislypidaemia and-174 IL6 G/C polymorphism.
Argatroban Anticoagulation for Heparin-Induced Thrombocytopenia in Elderly Patients.
Authors: Bartholomew John R. , Pietrangeli Carolynn E. , Hursting Marcie J.
Published: April 2020

Argatroban, a direct thrombin inhibitor, is used for prophylaxis or treatment of thrombosis in heparin-induced thrombocytopenia (HIT). Although its clearance is reduced in elderly, vs younger, healthy volunteers (4.3 vs 5.0 mL/min/kg), argatroban therapy in elderly patients with HIT has not been specifically described. In this retrospective analysis of a 118-patient, multicenter registry database, we evaluated argatroban dosing, anticoagulant responses, and clinical outcomes in adults aged ≥65 years with clinically diagnosed HIT or history of HIT administered argatroban therapy. From the registry, we identified 62 argatroban-treated patients with HIT (n=54) or history of HIT (n=8). Baseline characteristics, argatroban doses and responses, and outcomes were summarized for patients overall and stratified by age (65–74 years, n=31; 75–84 years, n=26; ≥85 years, n=5). Indications for anticoagulation were similar among groups, typically cardiac-related. Argatroban was infused and adjusted to achieve activated partial thromboplastin times (aPTTs) 1.5–3 times baseline. In each group, the initial median argatroban dose as determined by the treating physician was 1.0 mg/kg/min (a similar initial dose was used for all registry patients, irrespective of age) and maintained at or near that dose during therapy (median, 5–7 days). Overall, therapeutic aPTTs occurred within 11.5 hours; the median aPTT during therapy was 54.7 seconds. By regression analysis, mean argatroban dose decreased ~0.1 mg/kg/min with each 0.2-mg/dL increase in serum creatinine but was unaffected by patient age, total serum bilirubin, calculated creatinine clearance, or blood urea nitrogen. Platelet counts in HIT patients increased a median 41% from baseline during therapy. Within 37 days of argatroban initiation, 13 (21%) patients died (1 while on argatroban), 5 (8%) had new thrombosis (2 while on argatroban), and none experienced amputation or major bleeding. The oldest group had no event. By univariate regression analysis, the risk of developing new thrombosis decreased with increasing mean argatroban dose (hazard ratio=0.020; 95% CI, 0.001–0.757; p=0.035), yet was not significantly affected by age, gender, baseline platelet count, or mean aPTT. We conclude that argatroban at a median initial dose of 1.0 mg/kg/min, adjusted to achieve aPTTs 1.5–3 times baseline, provides safe and effective anticoagulation in elderly patients with HIT or history of HIT. For patients ≥65 years old, age is not a significant factor determining therapeutic argatroban doses or thrombotic risk.
Thrombotic Complications in Young Adults at High Altitude Areas: An Indian Experience.
Authors: Nair Velu , Sharma Ajay , Das Satya R. , Mishra D.K. , Kotwal J. , Agarwal Mohan B.
Published: April 2020

High altitudes(HA) have been a source of great adventures to sea land natives during short visits. But they also result in life threatening situations. There is no definite protocol to predict these likely complications. Certain thrombotic tendencies have been identified as possible risks for these complications but there have been some times such complications can occur in the individuals otherwise not known to have any known abnormalities of thrombosis. Also the duration of exposure to high altitude required to trigger this complication is uncertain. We present 24 cases of high altitude associated thrombotic complications. All patients were incidentally males in an age group of 22–38 years (median age 28 years). They were all otherwise healthy individuals having undergone a basic medical examination before moving to HA (defined as an altitude above 9000 ft.). They all moved to HA by air and underwent standard acclimatization program before they were allowed to move out there. The duration of stay ranged between 2 weeks to 4 months. The complications encountered included deep vein thrombosis (DVT) of lower limbs (n- 18) pulmonary embolism(PE; n- 9), cortical venous thrombosis (CVT) in 5 cases and splenic infarctions in 5 cases. 25 of these also developed features of pulmonary hypertension as confirmed by echocardiography in these patients and pulmonary angiography in 3 cases who had developed pulmonary embolism. They all were treated with heparin followed by oral anticoagulation. These all patients were told to move out of HA at earliest possible opportunity. They were all investigated for possible thrombotic abnormalities. These tests included assays for Protein C,S,AT, APC resistance and mutation analysis for MTHFR and Factor V leiden. Two of the Five patients, who had developed splenic infarction were detected to have sickle cell trait. These patients underwent splenectomy besides anticoagulation. The anticoagulation was stopped 6 months later. They all later moved out of HA and were advised to avoid re-exposure to HA. These individuals arte on regular follow up and are free of any subsequent recurrence of thrombotic complications. We conclude that HA itself may be a risk for various thrombotic complications especially in individuals exposed to HA for a longer duration.
Evaluation of the Hemochron Junior Signature Analyser for Oral Anticoagulant Point of Care Testing.
Authors: Bogo Peter H. , Maddox Jamie , McGregor Ewan , McKay Jayne , Bancroft Anne , Pippard Martin J. , Kerr Ron
Published: April 2020

The community anticoagulant service in Tayside, Scotland provides point of care testing for around 5000 patients at 25 sites using 43 analysers. Since December 2004 this service has used the Hemochron Junior Signature (HJS) analyser (International Technidyne Corporation) for INR measurement.

Following satisfactory initial laboratory evaluation, we have compared results of capillary samples analysed using the HJS to venous samples collected simultaneously and analysed in Ninewells Hospital using a Sysmex CA1500 analyser. Reagents used were Citrate PT test cuvettes (International Technidyne Corporation) which uses rabbit brain thromboplastin for the HJS and Dade Innovin (Dade Behring) for the CA1500. Samples for comparison were collected randomly from approximately every tenth patient tested in the anticoagulant clinics for a 12 month period. Results of the comparison are given for 3 separate periods within the 12 months of study as ongoing analysis of data during the study period led to the introduction of a correction factor for results obtained using the HJS analyser. The first correction factor was introduced after the first 4 weeks of study and there was a subsequent revision of this correction factor after a further 5 months.

Results for these 3 periods are as follows (all results given are INR units).

Period 1:

Mean INR 2.8 CA 1500 vs. 3.8 HJS, mean difference −1.0, 95% upper limit of agreement 0.5, 95% lower limit of agreement −2.5 (n=145).

Period 2:

Mean INR 2.6 CA 1500 vs. 2.7 HJS, mean difference −0.1, 95% upper limit of agreement 1.1, 95% lower limit of agreement −1.2 (n=345).

Period 3:

Mean INR 2.7 CA 1500 vs. 2.8 HJS, mean difference 0.0, 95% upper limit of agreement 1.3, 95% lower limit of agreement −1.3 (n=662).

Additionally, local external quality assurance was performed on 13 occasions (683 total tests) during the period of study. Cumulative results demonstrate that 86% of results were within 15% of the median.

We have demonstrated an approach to quality assurance for oral anticoagulant point of care testing and conclude that the HJS is a suitable analyser for this purpose.
In Vitro Evaluation of Apixaban, a Novel, Potent, Selective and Orally Bioavailable Factor Xa Inhibitor.
Authors: Luettgen Joseph M. , Bozarth Tracy A. , Bozarth Jeffrey M. , Barbera Frank A. , Lam Patrick Y. , Quan Mimi L. , Pinto Donald J. , Wexler Ruth R. , Rendina Alan R. , Knabb Robert M.
Published: April 2020

Apixaban, previously known as BMS-562247, is a high affinity, highly selective, orally-active, reversible inhibitor of coagulation factor Xa (fXa), in clinical studies as a therapeutic agent for prevention and treatment of thromboembolic diseases. The in vitro characteristics of apixaban were evaluated in purified systems and in human blood from healthy volunteers. Detailed kinetic analysis of apixaban inhibition of human fXa showed that it is a readily reversible, potent and competitive inhibitor versus a synthetic tripeptide substrate with a Ki of 0.08 nM, an association rate of 2 × 107 M−1s−1and a dissociation half life of 3.4 min. Weak affinity (Ki ~3 μM) is observed for thrombin, plasma kallikrein, and chymotrypsin. Affinity for trypsin and all other serine proteases tested is negligible with Ki > 15 μM. Apixaban is an effective inhibitor of free fXa and of prothrombinase, in buffer, platelet poor plasma, and whole blood. The anticoagulant activity of apixaban was determined in platelet-poor human plasma. Apixaban causes concentration dependent prolongation of the fXa mediated clotting assays. The human plasma concentration required to produce a doubling of the clotting time is 3.6 μM for prothrombin time, 7.4 μM for activated partial thromboplastin time and 0.4 μM for HepTest. To support preclinical efficacy and safety studies purified fXa from rabbit, dog and rat plasma was also found to be inhibited by apixaban (0.17, 2.6, and 1.3 nM, respectively). In summary the in vitro properties of apixaban show that it is a highly selective and potentially potent antithrombotic agent for venous and arterial thrombotic diseases.
Expression of Tumor-Associated Antigens (TAAs) in Acute Myeloid Leukemia (AML) Correlated with Specific T Cell Responses and Survival.
Authors: Greiner Jochen , Schmitt Michael , Li Li , Giannopoulos Krzysztof , Bosch Katrin , Dohner Konstanze , Schmitt Anita , Schlenk Richard F. , Pollack Jonathan R. , Dohner Hartmut , Bullinger Lars
Published: April 2020

Several Tumor-associated antigens (TAAs) are expressed in acute myeloid leukemia (AML) and elicit specific immune responses of CD8 positive T cells. These specific T cell responses against leukemic blasts expressing TAAs might play a critical role in the control of minimal residual disease (MRD) in AML. Therefore, we investigated whether TAAs inducing specific immune responses in AML patients were associated with the clinical outcome. A DNA-microarray analysis of 116 AML samples was performed to correlate expression of TAAs to the clinical outcome. In these AML patients specific T cell responses to TAAs were assessed by ELISPOT analysis, tetramer staining and chromium release assays. Quantitative RT-PCR based validation of our results demonstrated the power of DNA microarray technology. We found a significant correlation of high mRNA expression of the TAA G250/CA9 with a longer overall survival (P=0.022), a trend for better outcome in patients with high expression levels of PRAME (P=0.103), and a hint for RHAMM/HMMR. In contrast, for other TAAs like WT1, TERT, PRTN3, BCL2, and LAMR1 we found no correlation with clinical outcome of AML patients. Moreover, co-expression of RHAMM/HMMR, PRAME and G250/CA9 provided a favorable prognostic effect (P=0.005). We found specific T cell responses at high frequency for these three antigens in AML patients. Positive immune reactions were detected in 8/17 (47%) AML patients for RHAMM/HMMR-R3-derived, in 7/10 (70%) for PRAME-P3-derived, and in 6/10 (60%) for newly characterized G250/CA9-G2-derived peptides. We detected a significant increased immune response of AML patients in complete remission compared to AML patients with refractory disease (P<0.001). Furthermore, we could demonstrate specific lysis of T2 cells and AML blasts presenting these epitope peptides RHAMM/HMMR-R3, PRAME-P3 and G250/CA9-G2. In conclusion, the expression of the TAAs RHAMM/HMMR, PRAME and G250/CA9 can induce strong anti-leukemic immune responses of CD8 positive T cells possibly enabling the control of MRD in AML patients. Thus, the antigens RHAMM/HMMR, PRAME and G250/CA9 represent interesting target structures for polyvalent immunotherapeutic approaches in AML.
Hematopoietic Stem and Progenitor Cell Biology in the Zebrafish.
Authors: Traver David , Bertrand Julien , Kim Albert , Cisson Jennifer , Violette Emily
Published: April 2020

Over the past decade, the development of forward genetic approaches in the zebrafish system has provided unprecedented power in understanding the molecular basis of vertebrate blood development. Establishment of cellular and hematological approaches to better understand the biology of resulting blood mutants, however, has lagged behind these efforts. We have recently developed the means to identify zebrafish hematopoietic stem cells (HSCs), transgenic lines to mark hematopoietic precursors and their progeny, and the assays to test these populations functionally. Like other vertebrates, zebrafish demonstrate differential waves of hematopoiesis during embryogenesis. These waves can be visualized directly by fluorescent transgenesis in living embryos. The earliest blood-forming cells in the zebrafish embryo express the scl and lmo2 genes. By directing expression of GFP to early blood precursors using the lmo2 promoter, we have isolated early hematopoietic cells by flow cytometry and tested them functionally by transplantation. Transplantation of lmo2::GFP+ cells isolated from embryos at 14 hours post-fertilization (hpf) resulted in only transient reconstitution of erythrocytes, suggesting that the earliest identifiable blood-forming cells are committed to the erythroid lineage. Later in embryogenesis, lmo2:GFP+ GATA-1:dsRED+ cells are found in the posterior blood island (PBI) from approximately 30–60 hpf. Molecular and functional characterization of these cells suggests that they possess limited myeloid and erythroid, but not lymphoid differentiation potentials. This suggests that committed progenitors with definitive hematopoietic potential arise in the embryo before HSCs can be identified. Additional studies have suggested that the first multipotent HSCs are born later in the zebrafish aorta/gonad/mesonephros (AGM) region. We have visualized putative HSCs in the AGM by their expression of the lmo2 and cd41 transgenes. Using confocal timelapse imaging in living embryos, lmo2::GFP+ cells have been observed to emigrate from the AGM region into circulation. Transplantation studies are underway to test putative HSC populations for repopulation activity. Taken together, our findings suggest that at least three independent waves of blood cell precursors are formed during zebrafish embryogenesis.
Interferon-γ Attenuates the Survival Activity of G-CSF through PI3K/Akt Signaling Pathway in Mouse Multipotent Progenitor Cells.
Authors: Liu Hong , Mihara Keichiro , Tanaka Hideo , Kimura Akiro
Published: April 2020

Aplastic anemia (AA) is characterized by cytopenias caused by loss of stem cell and immunological relevance. We found out that impaired stem cell could be caused by excess of interferon-gamma (IFN-γ) in the serum or hypersensitivity to IFN-γ in patients with AA, notably severe AA. However, it is not clear how IFN-γ works in terms of the etiology of AA, as it exhibits diverse biological effects on cells. Thus, we investigated the essential role and the mechanism for the apoptotic function of IFN-γ against hematopoietic stem and/or progenitor cells. Although G-CSF augmented the surviving, proliferative, and differentiating activity in 32D cells, mouse multipotent progenitor cells, those effects of G-CSF were abolished by IFN-γ and they were susceptible to apoptosis with IFN-γ treatment. We focused on Akt, which was associated with the control of stem cell fate. IFN-γ did not affect the expression of Akt in 32D cells treated with G-CSF. Intriguingly, although Akt was strongly phosphorylated at Ser473 with G-CSF, IFN-γ attenuated phospho-Akt expression in dose- and time-dependent manner. As Akt is known to be phosphorylated through phosphatidylinositol-3-kinase (PI3K) pathway, we analyzed whether wortmannin, a specific inhibitor of PI3K, might have any synergistic effect on Akt phosphorylation in conjunction with IFN-γ. Wortmannin enhanced the inhibitory effect on Akt phosphorylated by G-CSF in collaboration with IFN-γ, suggesting that the activity of IFN-γ might converge on PI3K pathway. Next, to explore the downstream molecule of Akt, we examined the expression of BAD and NF-kB, which work downstream of Akt. IFN-γ increased the BAD protein reduced by G-CSF. Regardless of addition of IFN-γ, there was no alteration of NF-kB expression. IFN-γ exerted apoptosis against 32D cells through the caspase pathway. Taken together, these results suggest that IFN-γ could exert the inhibitory action on stem cells and/or progenitors by the interference of the PI3K / Akt signaling pathway in AA patients. Our findings may provide a suggestion of the treatment potential for AA such as anti-IFN-γ antibody and contribute the further understanding of AA in terms of immunological aberration.
Defective Ribosomal RNA Maturation in Patients with Diamond Blackfan Anemia.
Authors: Quarello Paola , Aspesi Anna , Flygare Johan , Karlsson Stefan , Ellis Steven R.
Published: April 2020

Diamond Blackfan Anemia (DBA) is a rare congenital red cell aplasia due to a selective defect of erythroid progenitor cell maturation. DBA patients exhibit a wide array of developmental abnormalities and can be predisposed to cancer. Heterozygous mutations in the gene encoding Rps19, a ribosomal protein of the small subunit, have been identified in 25% of DBA cases. By using human cell lines (TF-1) containing small interfering RNA (siRNA) designed to silence RPS19 we have shown that Rps19 deficient cells present an altered ribosomal profile which conveys a decrease in the 40S subunit population. This decrease is due to faulty maturation of the 21S precursor to functional 18S rRNA as illustrated by pulse chase and northern blot analysis of total RNA extracts from the cell lines. Analysis of these intermediates in CD34− cells from the bone marrow of DBA patients harboring mutations in RPS19 revealed a pre-rRNA processing defect similar to that observed in TF-1 cells where RPS19 expression was reduced. This defect was observed to a lesser extent in CD34+ cells from DBA patients with mutations in RPS19. Thus, our data reveal that rRNA processing is disrupted in cells from DBA patients with mutated RPS19.
Ex Vivo Proliferation and Differentiation of CD34+ Cells into Myeloid Progenitor Cells: Cord Blood Versus Mobilized Peripheral Blood.
Authors: De Bruyn Cecile , Delforge Alain , Ngirabacu Marie-Christine , Bron Dominique
Published: April 2020

The purpose of our study was to evaluate the capacities of cord blood (CB) CD34+ cells to proliferate and differentiate ex vivo into myeloid lineage in response to cytokines and to compare them with mobilized peripheral blood (MPB) cells. Briefly, 2.5 × 104 CD34+ cells, isolated from CB (n=10) and MPB (n= 9), were cultured in 5 ml MacoBiotech HP01 (Macopharma) with SCF, Flt3-L, IL-3 and G-CSF. At day 9, 106 cultured cells were replated for 5 additional days. Cells were counted and evaluated for their CD34, CD13 and CD15 expression. Differentiation into myeloid compartment was assessed by CD11b and CD16 coexpression on CD15+ cells. We observed that

leucocyte expansion was significantly higher in CB than in MPB at day 9 (24.3±3.8 vs 15.2±1.9) and at day 14 (224.7±54.2 vs 72.9±20.0). A similar difference was observed for CD34+ cell expansion (8.7±1.4 vs 3.4±0.5 at day 9 and 31.3±4.6 vs 7.6±2.4 at day 14). at day 9, despite superior CB leucocyte expansion, CD13+ and CD15+ cell number produced per CD34+ cell seeded at day 0 were similar in CB and in MPB (18.5±2.4 vs 14.4±2.5 for CD13+ and 7.1±1.3 vs 6.0±1.2 for CD15+). Increasing the culture period led to higher numbers of CD13+ and CD15+ cells in CB than in MPB. This increase was due to a total leucocyte expansion rather than to high CD13+ and CD15+ cell percentage. The distribution of CD11b−CD16−, CD11b+CD16− and CD11b+CD16+ subpopulations in CD15+ cells was comparable in CB and in MPB after 9 days of culture, with a majority of relatively immature CD11b−CD16− myeloid progenitor cells.

However, after 5 additionnal days of culture, MPB CD15+ cells expressed a more mature phenotype than CB CD15+ cells, with a dramatic increase of CD11b+CD16− cells (promyelocytes and myelocytes). In conclusion, our study suggests that, despite the high CB cell capacity of expansion in our culture conditions, CB CD34+ cell differentiation process into myeloid lineage appears to be slower. This difficulty of CB cells to reach maturation in vitro is likely to be related with the longer delay of neutrophil recovery after CB transplantation.
Productive Infection of Human Cord Blood Stem Cells and Lineage Progenitors by Porcine Endogenous Retrovirus (PERV).
Authors: Morgan Doris A. , Fitzsimmons John , Argaw Takele , Wilson Carolyn
Published: April 2020

Endogenous retroviruses were once considered “junk DNA” and merely residual evolutionary genetic material. In the human system, recent reports have suggested a link between activation of human endogenous retroviruses (HERV) and certain diseases. Activation may be restricted to an up-regulation of viral specific transcripts or may involve gene translation with the assembly of virus-like particles (Morgan, Exp.Hem., 2004). Against this background of a possible link between activated endogenous retroviruses and disease, the potential risks of porcine to human transmission in the context of xenotransplantation becomes more plausible. The principal focus of this report was to determine the susceptibility of human primary cells to porcine endogenous retroviruses (PERV). Expression of a receptor for PERV has been described to be widespread in human tissues (Ericsson,PNAS,2003) and was expressed in hematopoietic stem cells and progenitors of our study. We exposed human umbilical cord blood (UCB) stem cells and cytokine-induced erythroid, myeloid and megakaryocytic progenitors 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, J. Vir., 2000; Harrison, J. Vir., 2004). Three days after exposure to PERV, cells were washed in order to remove unbound virus and assayed for viral RNA and DNA using quantitative PCR and RT-PCR (Argaw, J. Gen. Vir. 2002). Cell pellets from stem cells as well as lineage-induced cells were consistently positive for viral DNA and RNA and persisted during a kinetic study 3,5,6,and 7 days post-exposure to PERV. Control cells that were not exposed to PERV were negative in all parameters. There were no detectable adverse effects of PERV infection on cell proliferation or on terminal maturation of the progenitors of all three lineages. PERV infection does not require lineage commitment and provides a mechanism by which self-renewing stem cells may serve as an in vivo reservoir of virus in human bone marrow. Of note, the natural course of disease (anemia) of a related retrovirus, feline leukemia virus, also relies upon infection of hematopoietic progenitors in the bone marrow.
A Gene Dosage Requirement for Transcription Factor Gfi1 in the Regulation of Myelopoiesis and Myeloproliferative Disorders.
Authors: Grimes H. Leighton , Horman Shane R.
Published: April 2020

The generation of mature myeloid lineage cells from hematopoietic stem cells (HSCs) requires a precise synergy between cytokine signaling and lineage-specific transcription factors. Gfi1 (Growth Factor Independence 1) is a zinc finger transcription factor that is necessary for normal myelopoiesis. Gfi1 knockout mice display an abnormal ratio of phenotypic common myeloid progenitors (CMP) and granulocyte/monocyte progenitors (GMP), and such mice completely lack mature neutrophils. In contrast, the Gfi1 heterozygote mouse presents no obvious phenotype. Here we show a gene dosage requirement for Gfi1 in the differentiation of mature myeloid cells. While bone marrow from wild type littermates generates granulocytic, monocytic and mixed methylcellulose colonies, Gfi1 knockout bone marrow cells yield mainly monocytic colonies. In comparison to wild type littermates, Gfi1+/− bone marrow cells generate lower numbers of granulocytic colonies and higher numbers of monocytic colonies. Interestingly, methylcellulose colonies from both Gfi1 knockout mice and heterozygotes display increased serial replating capacity in comparison to wild type littermates. These data suggest that Gfi1 gene dosage may control self renewal and may relate to oncogenic transformation. Activating mutations in K-Ras are common genetic abnormalities in human acute myeloid leukemia and myeloproliferative disease (MPD). However, expression of activated Ras from endogenous regulatory sequences (knock-in) results in an MPD of varying lethality. The severity of the K-Ras-induced MPD depends on unknown genetic modifiers, as lethality is increased in a Balb/c genetic background. Given the effect of Gfi1 gene dosage on serial replating, we have analyzed the effect of Gfi1 gene dosage on the activated Ras knock-in mouse model of MPD. Preliminary data indicate that lowering Gfi1 gene dosage modifies the phenotype of activated Ras MPD by dramatically increasing the number of immature circulating myeloid progenitors. These results reveal that Gfi1 is a modifier of Ras induced disease pathogenesis.
Cell Cycle Influence of Stem Cell Differentiation in Culture.
Authors: Zomorodian Toska J. , Abedi Mehrdad , Dooner Gerri , Greer Debbie , Johnson Kevin , Demers Delia , Quesenberry Peter J.
Published: April 2020

Hierarchical models of hematopoiesis suppose an ordered system in which stem cells and progenitors with specific fixed differentiation potentials exist. We show that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell cycle transit. To address whether the cell cycle plays a role in the differentiation of stem cells, we co-cultured murine bone marrow Lin- Sca-1+ cells, at different points in their cycle, with the OP9-DL1 system. OP9-DL1 stromal cell layer has been transduced to allow T-cell differentiation in culture. We first induced cell cycle synchrony by exposing the isolated cells to a cytokine cocktail of TPO, Flt-3 and Stem Cell Factor. The cells were exposed to this primary culture for 0, 6, 24, 32 and 40 hours and were subsequently cultured on an OP9-DL1 stromal cell layer grown in 6-well plates. Cells were co-cultured for 8 days and 21 days, in the presence of IL-7 and Flt-3. Cultured cells were evaluated for CD4, CD8, B220, CD19, NK1.1, and Mac-1 surface markers, using flow cytometry. On Day 8, we found a significant hotspot at 32-hours (early-S phase) for B220+ cells (34.3 %), while Mac-1 positive cells demonstrated a 24-hour hotspot (18.1 %). As expected, terminal T and B-cell differentiation (CD 4, CD8, and CD19) was undetectable at 8 days. Three separate short-term (8 day) experiments have confirmed these data. Cells in culture for 21 days similarly show variation in differentiation outcome. CD4 cells demonstrate a peak at the 40 hour time point (mid-S phase) (69.9%), while CD8 positive cells were significantly increased at the 32 hour time point (34.4%). These data indicate both B and T cells show reversible differentiation fluxes linked to cell cycle. This work supports previous evidence that marrow hematopoiesis at the stem cell level is regulated on a continuum and that stem cells have reversible, cycle-related differentiation capacity.
MiRNA Transcription Is Regulated by the PML-RARα Oncoprotein in Acute Promyelocytic Leukemia.
Authors: Nigten Jeannet , Knops Ruth , Nikoloski Gorica , de Witte Theo M. , van der Reijden Bert A. , Jansen Joop H.
Published: April 2020

The discovery of the microRNA (miRNA) molecules has led to new insights into the regulation of gene expression. They are able to bind specific mRNA sequences, and due to inhibition of translation or mRNA degradation, miRNAs cause downregulation of their target genes. To date, several hundreds of unique human miRNAs have been described. So far, for only few of them the target mRNAs have been experimentally confirmed. MiRNAs have been linked to several important biological processes as early stage development, cell growth, cell differentiation and apoptosis. In addition, impaired miRNA expression has been implicated in tumorigenesis. Leukemia is often associated with mutated transcription factors and, as a consequence, deregulated gene expression and impaired proliferation and differentiation. Acute Promyelocytic Leukemia (APL), is characterised by the expression of the mutated transcription factor PML-RARα, which may interfere with the normal function of the retinoic acid receptor α (RARα), a nuclear hormone receptor that acts as a ligand-dependent transcription factor. APL is uniquely sensitive to treatment with the RARα ligand, all-trans retinoic acid (ATRA), which results in the expression of genes that induce terminal granulocytic differentiation of the leukemic blasts. To investigate whether miRNA expression is regulated by ATRA in APL, we performed Taqman miRNA assays for 157 different mature miRNAs in the APL cell line NB4 before and after treatment with ATRA. We found that ATRA induced a more than 10 fold upregulation of 18 miRNAs and a more than 10 fold downregulation of 2 miRNAs. These expression patterns were confirmed in primary APL patient cells before and after treatment with ATRA. To study whether the miRNA expression pattern was dependent on the PML-RARα fusion protein, we used U937 cells stably transfected with a zinc-inducible PML-RARα expression cassette (U937PR9, a kind gift of Dr Pelicci). Upon ATRA treatment, we found that several miRNAs were only induced in the presence of PML-RARα, suggesting that PML-RARα is implicated in the expression of these miRNAs. To investigate whether the PML-RARα fusion protein binds to the endogenous miRNA genes chromatin immunoprecipitation assays were performed with PML-RARα transfected 293 cells. We demonstrated the presence of PML-RARα protein on the miRNA genes. This indicates that the oncoprotein PML-RARα directly influences the expression of these miRNAs. The function of the PML-RARα targeted miRNAs in APL cell differentiation is currently being studied using retroviral expression vectors.
AML1/Runx1 Negatively Regulates the Number of Quiescent Hematopoietic Stem Cells in Adult Hematopoiesis.
Authors: Ichikawa Motoshi , Asai Takashi , Nakagawa Masahiro , Kawazu Masahito , Goyama Susumu , Takeshita Masataka , Chiba Shigeru , Ogawa Seishi , Kurokawa Mineo
Published: April 2020

Transcription factor AML1 (also called Runx1), which was initially isolated from the t(8;21) chromosomal translocation frequently found in the acute myelogenous leukemia FAB M2 subtype, is essential for the development of multilineage hematopoiesis in mouse embryos. By analyzing conditional AML1 knockout mice, we have previously shown that AML1 negatively regulates the number of immature hematopoietic cells defined as lineage-negative, CD34− Sca-1+ c-Kit+ (34KSL) cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. The former is a significant observation because an increase in hematopoietic stem/progenitor cells due to defective AML1 function may be closely related to the development of human leukemia. In support of this is the fact that mice in which leukemic chimeric protein AML1/ETO is expressed in hematopoietic cells are subject to myeloproliferative disease and develop leukemia after additional mutation. However, it has remained yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population (SP) fraction are increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We quantitatively evaluated HSCs by bone marrow transplantation assays using limiting dilution and found a significant increase in HSC number within the AML1-deficient bone marrow. These results indicate that the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia.
The Effects of Glycosaminoglycans on Thrombopoietin-Induced Megakaryocytopoiesis.
Authors: Takahashi Kenji , Kashiwakura Ikuo , Toki Tsutomu , Takagaki Keiichi
Published: April 2020

The extracellular matrix (ECM) plays an essential role in normal hematopoiesis. Proteoglycans and glycosaminoglycans (GAGs) are major components of the ECM. In this study, the effects of various GAGs on the proliferation and differentiation of CD34+ megakaryocytic progenitor cells (CFU-Meg) were evaluated in vitro. CD34+ cells were highly purified from steady-state human peripheral blood. The GAGs tested were hyaluronic acid (from human [HA-h], pig and rooster), keratan sulfate, heparan sulfate, chondroitin sulfate (from whale [CS-A], shark or squid cartilage) and dermatan sulfate (DS). When used alone, none of the GAGs supported the clonal growth of CFU-Meg; however, in cultures stimulated by recombinant human thrombopoietin (TPO), HA-h, CS-A and DS significantly enhanced such growth. In particular, the addition of DS resulted in increases of about 1.3-fold, 1.6-fold and 2.0-fold in the numbers of total cells, megakaryocytes and CFU-Meg, respectively, compared with the control culture stimulated by TPO alone after 9–12 days of serum-free liquid culture. Furthermore, DS induced the generation of hyperploid megakaryocytes and promoted pro-platelet formation. Chemical fragmentation and desulfation of DS showed that a chain of at least 12 saccharides is required for colony-promoting activity and that the sulfate groups play an essential role. DS acts on an immature population of CD34+ cells, stimulates the proliferation of CFU-Meg, and enhances the terminal maturation of megakaryocytes and thrombopoiesis. These results suggest that DS has a wide spectrum of action in promoting megakaryocytopoiesis and thrombopoiesis.
PBI-1402: A Low Molecular Weight Synthetic Hematopoietic Growth Stimulant.
Authors: Grouix Brigitte , Julien Nathalie , Lagraoui Mouna , Morin Marie-Josée , Krosl Gorazd , Roy Denis-Claude , Penney Christopher , Gagnon Lyne
Published: April 2020

PBI-1402 is a non-toxic, well-defined low molecular weight synthetic hematopoietic growth stimulant. PBI-1402 promotes the proliferation and maturation of hematopoietic progenitors (myeloid and erythroid populations) yielding a biological efficacy comparable to G-CSF, GM-CSF and EPO in in vitro human bone marrow cell proliferation and colony formation assays. An additive effect is observed when PBI-1402 is combined with G-CSF, GM-CSF and EPO. In human bone marrow colony assay, PBI-1402 enhances the differentiation of pluripotent stem cells: CFU-GEMM, CFU-GM with a predominant effect on BFU-E. Furthermore, PBI-1402 exerts its activity via a different mechanism of action than EPO and stem cell factor (SCF) and at an earlier stage on more immature hematopoietic progenitors. PBI-1402 is targeted as an adjunct to cancer chemo/radiotherapy, bone marrow transplantation and diseases involving neutropenia and anemia.
In GM-CSF Restrain Mitochondria Pathway Apoptosis NF-kB Is Not the Only Way.
Authors: Jiang Bo , Zhang Ri , Wu De Pei
Published: April 2020

After stimulated by GM-CSF, Akt and IkB kinase (IKK) can be phosphorylated by activated PIK3-kinase. Activated IKK phosphorylate IkB, making NF-kB released and translocate to nucleus. Granulocyte-macrophage colony-stimulating factor (GM-CSF) restrain TF-1 cell mitochondria pathway apoptosis through PI3K pathway, so we asked if NF-κB is the only way in this process.

All group were plused TF-1 cells 2.5X105/250μl, group A plused hGM-CSF (100ng/ml ) 50μl; group B plused hGM-CSF (100ng/ml) 50 μl and SN50 (50 μmol/L) 20μl; group C plused hGM-CSF 50μl and SN50 200μl. After cultured for 48 hours, all groups were dyed with Annexin and PI, analyzed in FCM. At the same time all groups were dyed by diluted MitoCapture to analyze mitochondria membrane electrical potential. Drop experimental group B 10μl on slips, so as group C, then observe it under Fluorescence microscopes FL1 channel.

Group A cells that have normal mitochondria membrane electrical potential can form poly-MitoCapture in it, and inspired red fluorescence. Group C cells that have monomer MitoCapture was inspired green fluorescence. The red fluorescence intensity of control, group A, B, C means is 368.93, 1137.59, 1033.61 and 618.37 respectively. Group A and B retained a statistically significant difference with control (P<0.01). Group C retained a statistically significant difference with control (P<0.01), group A and group B (P<0.01). This investigation proves that GM-CSF restrain TF-1 cells can be incompletely blocked by SN50, which is the specificity inhibitor of NF-kB. So we get the conclusion that NF-kB is an important but not the only pathway in the process of GM-CSF restrain TF-1 cells apoptosis. It also suggests that there maybe some other pathway in this process.
CD73-Independent Toxicity of Fludarabine on Adult Normal Bone Marrow Mesenchymal Cells (NBMMC).
Authors: Pebrel-Richard Celine , Veyrat-Masson Richard , Boiret-Dupré Nathalie , Rapatel Chantal , Berger Juliette , Tournilhac Olivier , Boisgard Stephane , Chassagne Jacques , Berger Marc G.
Published: April 2020

The purine analogue fludarabine is efficient in treating advanced, chronic B-cell lymphocytic leukemia (CLL). We and others have shown that front-line fludarabine treatment impaired the subsequent mobilization of hematopoietic CD34+ cells by G-CSF. This might imply an effect on hematopoietic and/or mesenchymal compartments. We also previously showed that native mesenchymal cells (MC), like expanded MC, express the 5′-ecto-nucleotidase (CD73) which should aid the entry of fludarabine into the cell. So we investigated the toxicity of fludarabine on NBMMC.

We incubated expanded (P1) and fresh NBMMC from femoral heads with increasing doses (0.1 to 6 mMol) of fludarabine for 3 days, and assessed its effect on cell survival (annexin V staining), CFU-F frequency, cell proliferation and immunophenotyping. We used α,β, methylene-adenosine 5 diphosphate (MADP) to inhibit CD73 in order to evaluate its role in the trans-membrane passage of fludarabine.

We observed a direct, dose-dependent toxicity of fludarabine on expanded mesenchymal cells, after 3 days incubation, characterized by apoptosis of 48% of MC. The mesenchymal progenitor cells were more sensitive; their frequency amongst the viable cells, being decreased about 5-fold from the 1mMol dose, to 13-fold at the highest dose, where there were far fewer colonies containing>50 cells. Even after fludarabine removal, viable MC showed an impaired ability to proliferate at the next passage. Fresh NBMMC, incubated under same conditions, showed a similar but lower sensitivity than did expanded (P1) mesenchymal cells, as indicated by a shift in the dose-response curve to a 5-fold decrease at the highest dose. Again, we observed a decreasing proportion of large colonies. MADP provided no significant protection against the effect of fludarabine on fresh or expanded cells. The phenotype of progeny was comparable to that of the controls.

In conclusion, we have shown for the first time that adult NBMMC are sensitive to fludarabine and could prove to be an unexpected in vivo target. The differences between un-manipulated and expanded cells suggest a biologic alteration during culture. The lack of protective effect of MADP and the absence of lower expression of CD73 on resistant cells suggest another route of trans-membrane passage for fludarabine. The consequences of low dose fludarabine incubation on MC properties are currently being studied.
Usefulness of Wharton’s Jelly, Cord Blood, and Adipose Tissue as Alternative Sources of Mesenchymal Stem Cells.
Authors: Jang Jun Ho , Lee Hyun Woo , Eom Young-Woo , Kang Seok Yun , Park Joon Seong , Choi Jin Hyuk , Kim Hugh C.
Published: April 2020

Mesenchymal stem cells (MSCs) are a highly promising source of adult stem cells for purposes of cell therapy and tissue repair in the field of regenerative medicine. Although the most studied and accessible source of MSC is the bone marrow, the clinical use of bone marrow-derived MSCs (BMSCs) has presented problems, including pain, morbidity, and low cell number upon harvest. For those reasons, we isolated, cultured, and characterized MSCs from a number of tissues; including wharton’s jelly, cord blood, and adipose tissues that were discarded routinely in the past, and evaluated the usefulness of these MSCs compared to BMSCs. Proliferation ability of Wharton’s jelly-derived MSCs (WJ-MSCs), Cord blood-derived MSCs (CB-MSCs), or adipose tissue-derived MSCs (ASCs) was lost at passage 8–10 (22–27 population doubling), passage 7–10, or passage 7–12 (45–50 population doubling), respectively. WJ-MSCs, CB-MSCs, and ASCs expressed CD73, CD90, and CD105, CD90, CD105, and CD166, and CD44, CD73, CD90, and CD166, respectively, were absent for CD14, CD31, and CD45, and differentiated into osteoblast, adipocyte, and chondrogenic lineages under appropriate culture condition. In this study, like BMSCs, WJ-MSCs, CB-MSCs, and ASCs expressed similar cell surface antigens, were able to differentiate into mesenchymal lineages, and possessed highly proliferation potential. Therefore, MSCs isolated from wharton’s jelly, cord blood, and adipose tissue may become useful alternative sources of MSCs to cell therapy and tissue repair in the field of regenerative medicine.