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  • 1
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    Contact and intrinsic coagulation pathways are activated and associated with adverse clinical outcomes in COVID-19
    American Society of Hematology ; 2022
    In:  Blood Advances Vol. 6, No. 11 ( 2022-06-14), p. 3367-3377
    Details
    In: Blood Advances, American Society of Hematology, Vol. 6, No. 11 ( 2022-06-14), p. 3367-3377
    Abstract: Coagulation activation is a prominent feature of severe acute respiratory syndrome coronavirus 2 (COVID-19) infection. Activation of the contact system and intrinsic pathway has increasingly been implicated in the prothrombotic state observed in both sterile and infectious inflammatory conditions. We therefore sought to assess activation of the contact system and intrinsic pathway in individuals with COVID-19 infection. Baseline plasma levels of protease:serpin complexes indicative of activation of the contact and intrinsic pathways were measured in samples from inpatients with COVID-19 and healthy individuals. Cleaved kininogen, a surrogate for bradykinin release, was measured by enzyme-linked immunosorbent assay, and extrinsic pathway activation was assessed by microvesicle tissue factor–mediated factor Xa (FXa; MVTF) generation. Samples were collected within 24 hours of COVID-19 diagnosis. Thirty patients with COVID-19 and 30 age- and sex-matched controls were enrolled. Contact system and intrinsic pathway activation in COVID-19 was demonstrated by increased plasma levels of FXIIa:C1 esterase inhibitor (C1), kallikrein:C1, FXIa:C1, FXIa:α1-antitrypsin, and FIXa:antithrombin (AT). MVTF levels were also increased in patients with COVID-19. Because FIXa:AT levels were associated with both contact/intrinsic pathway complexes and MVTF, activation of FIX likely occurs through both contact/intrinsic and extrinsic pathways. Among the protease:serpin complexes measured, FIXa:AT complexes were uniquely associated with clinical indices of disease severity, specifically total length of hospitalization, length of intensive care unit stay, and extent of lung computed tomography changes. We conclude that the contact/intrinsic pathway may contribute to the pathogenesis of the prothrombotic state in COVID-19. Larger prospective studies are required to confirm whether FIXa:AT complexes are a clinically useful biomarker of adverse clinical outcomes.
    Type of Medium: Online Resource
    ISSN: 2473-9529 , 2473-9537
    URL: Article
    DOI: 10.1182/bloodadvances.2021006620
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2022
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  • 2
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    A Prospective Study Comparing 99mtc-Sestamibi SPECT/CT with 18f-FDG PET/CT in Multiple Myeloma Staging: A Potential Resource-Constraint Image Alternative for MM Bone Evaluation
    American Society of Hematology ; 2018
    In:  Blood Vol. 132, No. Supplement 1 ( 2018-11-29), p. 3543-3543
    Details
    In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 3543-3543
    Abstract: Introduction: Multiple myeloma (MM) is a plasma cell neoplasm, characterized by plasma cell infiltration inside the bone marrow, secretion of monoclonal immunoglobulin (paraprotein), and end organ damage including lytic lesions in the bones. About 80-90% of myeloma patients suffer from osteolytic lesions during the course of the disease. 18F-FDG PET/CT is an imaging technique capable to detect active disease in patients in multiple myeloma (MM) and can be helpful in staging and prognosis. However, its routine use is still hampered by several factors, including high cost, reimbursement issues, lack of cost-effectiveness studies and limited availability. 99mTc-sestamibi (MIBI) has also been proposed as a potential tracer in MM evaluation and is more accessible with lower costs. The aim of this study was to compare these two imaging modalities at staging disease and their relation with clinical data. Materials and Methods: Sixty-four patients with newly diagnostic MM (30 male; 34 female) were submitted to 18F-FDG PET/CT and 99mTc-Sestamibi SPECT/CT before treatment. Whole body PET/CT images were acquired 60 minutes after FDG administration and anterior and posterior whole-body scans (WBS) plus SPECT/CT of chest and abdomen were obtained 10 minutes after MIBI injection. Number of focal lesions, bone marrow involvement, contiguous soft tissue impairment and extra osseous lesions were recorded. Number of focal lesions was classified in 3 groups: 0 (no lesions); 1 (1-3 lesions); 2 (4-10); 3 (more than 10). A visual degree of uptake was defined for bone marrow involvement: comparison to liver on PET/CT and to myocardium on MIBI. Standardized uptake value (SUVmax) of the hottest lesion of each patient was registered. Potentials factors contributing to progression-free survival (PFS) were assessed with Cox regression model combining baseline clinical data (including renal function, anemia, hypercalcemia, LDH, bone marrow plasma cell percentage and ISS (I, II or III)) along with PET/CT and MIBI scan status. Results: PET/CT was positive in 61 patients (95%) and MIBI in 59 subjects (92%; P = 0.15). WBS was positive in 56 patients while WBS plus SPECT/CT was positive in 59 (p= 0.08). PET/CT detected extra osseous lesions in 4 patients and sestamibi in 1 subject. Contiguous soft tissue involvement was found in 29 and 24 patients on PET/CT and MIBI, respectively (p=0.05). PET/CT detected much more focal lesions than MIBI: 13, 11, 16 and 24 patients were in group 0, 1, 2 and 3 on PET/CT and 30, 18, 6 and 10 were on the same groups respectively on MIBI (p: 0.0001). In the figure below, a comparison between 99mTc-Sestamibi WBS (A) and 18F-FDG PET/CT (B) at staging in a 67 years-old male. SUVmax were statistically different in subjects who presented elevated LDH (p= 0.02). Seventy-five percent and 100% of patients with elevated LDH had contiguous soft tissue involvement on MIBI and PET/CT respectively. More focal lesions on PET/CT were found in patients with hypercalcemia (p=0.02), however this correlation was not observed on MIBI (p=0.45). Renal insufficiency was a negative prognostic factor for PFS (HR: 2.25). The same was observed with advanced ISS staging (HR: 4.29). However, only advanced ISS staging (III) and extramedullary disease detected by MIBI were independent predictors of worse PFS. Conclusion: There was no difference in the detection of active disease when comparing FDG PET/CT and MIBI SPECT/CT in MM staging, although the first one detected more number of lesions. Including SPECT/CT to planar images on MIBI did also not improve the number of positive scans. Elevated LDH and hypercalcemia were the only clinical parameters related to higher number of bone lesions while ISS staging (III) and extramedullary disease detected by MIBI were independent predictors of worse PFS. Our study demonstrated that sestamibi WBI detects less volume of disease compared to PET/CT, however it may substitute PET/CT in centers where it is not available or there is no reimbursement for MM staging. Figure. Figure. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2018-99-118228
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
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  • 3
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    Familial Mastocytosis: Identification Of KIT K509I Mutation and Its In Vitro Sensitivity To Imatinib, Dasatinib and PKC412
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 5267-5267
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 5267-5267
    Abstract: Mast cell diseases are myeloproliferative neoplasms characterized by an abnormal proliferation and accumulation of mast cells in different tissues. The clinical presentation of mastocytosis is heterogeneous, ranging from skin-limited disease to more aggressive variants that may be associated with multiorgan dysfunction/failure and shortened survival. In a relatively high proportion of cases, the clonal nature of the disease can be established on the basis of the demonstration of gain-of-function mutations involving the tyrosine kinase (TK) domain of KIT in skin lesions and BM cells and by the factor-independent proliferation and transforming abilities of these mutations. The tyrosine kinase inhibitor Imatinib is a treatment available for mastocytosis patients; however, some KIT mutations, specially KIT D816V, confer resistance to this drug. Aims To characterize the clinical phenotype and molecular mutations of 2 relatives with diagnosis of systemic mastocytosis (WHO 2008). We also aimed to test the in vitro sensitivity of primary bone marrow (BM) cells from both patients to tyrosine kinase inhibitors. Patients and methods Four individuals were included in the study; two patients (case 1 [mother], and case 2 [daughter] ), and the parents of case 1. DNA samples were obtained from total BM cells, CD3+ BM cells and oral mucosa of patients, and from peripheral blood of all individuals. KIT (exons 1 to 21) was submitted for Sanger sequencing analysis. Primary bone marrow cells (5X104) from the 2 patients were cultured and treated with Imatinib (5uM), Dasatinib (80nM) and PKC 412 (100nM) or with vehicle only (control cells) and submitted for proliferation (MTT) and apoptosis assays (Annexin-V/PI) at days 4, 8 and 12 of culture. Results Case 1 was a 33 year-old woman with a chronic history of pruritic skin rash who was referred to our outpatient service for evaluation of massive splenomegaly (25 centimeters in length) and pancytopenia. She had neither comorbidities nor any familial history of hematological malignancies. The patient had no siblings and had only one daughter (case 2). At biopsy, she showed extensive skin and bone marrow infiltration by mast cells. During follow up, the patient presented with spontaneous splenic rupture and had to undergo splenectomy, which led to the resolution of pancytopenia. She was diagnosed with Aggressive Systemic Mastocytosis. Her daughter (case 2), a 17 year-old woman, was also evaluated for an insidious history of diffuse skin rash. Skin and bone marrow biopsies showed massive infiltration by atypical mast cells and a diagnosis of Indolent Systemic Mastocytosis was made. The rare KIT K509I mutation was found in all DNA samples obtained from both patients, but not from the parents of case 1. This suggests that the KIT K509I was a germ line mutation acquired de novo by patient 1 that was subsequently transmitted to her daughter (patient 2). In vitro treatment of primary bone marrow cells harboring the KIT K509I mutation from patients 1 and 2 resulted in variable clinical response rates according to the drug used and the treatment duration. Imatinib treatment resulted in a significant reduction in proliferation (days 4, 8 and 12 of culture) and an increase in apoptosis (days 8 and 12) in cases 1 and 2 (all p≤0.03). Although Dasatinib resulted in decreased proliferation in both patients at day 12 (all p≤0.008), a significantly higher apoptosis ratio was observed only for patient 1 at day 12 of culture (p=0.03). PKC412 had a negative effect over cell growth in patient 1 (days 4 and 8) and in patient 2 (day 4) (all p≤0.03); however, no effect in apoptosis ratio was seen. Conclusions We herein provide a report of a KIT K509I mutation in familial mastocytosis. This mutation has been previously described in the literature in one case of familial mastocytosis. Although rare, the screening for KIT K509I mutation should be considered in all cases of familial mastocytosis. Based on in vitro studies, mastocytosis patients harboring the KIT K509I mutation could benefit from treatment with Imatinib, Dasatinib and PKC 412. However, Imatinib may be more effective in inducing neoplastic mast cells apoptosis. Both patients described were started on Imatinib in June 2013. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.5267.5267
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 4
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    IRS2 Associates With JAK2 and May Be Involved In Cell Proliferation Pathways In Chronic Myeloproliferative Neoplasms
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 1598-1598
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 1598-1598
    Abstract: Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are BCR-ABL1 negative Chronic Myeloproliferative Neoplasms (MPN) characterized by increased myeloid proliferation, with predominant erythroid, megakaryocytic and megakaryocytic/granulocytic expansion, respectively. The finding of a recurrent mutation in the gene of the tyrosine-kinase Janus kinase 2 (JAK2 V617F) in these diseases has raised the hypothesis that this could be the main cause of their development. However, the evidence that MPN patients have a very similar response to JAK2 inhibitors regardless of JAK2 mutation status, and the knowledge that many receptors and substrates may lead to the activation of JAK/STAT, Ras/Raf/MAP kinases and PI3K/Akt/mTOR pathways, indicate the need to investigate other crucial proteins involved in the physiopathology of these diseases. Insulin receptor substrate 2 (IRS2) mediates mitogenic and antiapoptotic signaling from IR, IGF-IR, EPO-R and TPO-R. Previous studies performed on non-hematological cell lines have shown the association of IRS2 with JAK/STAT, PI3K/Akt/mTOR and Ras/Raf/MAP kinases pathways, giving rise to the hypothesis that IRS2 could participate in the activation of crucial signaling pathways in MPN through direct interaction with JAK2 or through alternative mechanisms. Aims To identify the JAK2/IRS2 protein interaction and to study the effects of pharmacological JAK1/2 inhibition (Ruxolitinib) over IRS2 phosphorylation in leukemia cell lines harboring or not the JAK2 V617F mutation; to characterize IRS2 expression in CD34+ cells from patients with MPN and its correlation with clinical data including JAK2 mutation status. Methods Leukemia cell lines carrying JAK2 V617F mutation (HEL) or not (HL60) were used for immunoprecipitation and immunobloting with IRS2 and JAK2 antibodies. Cells treated or not with JAK1/2 inhibitor Ruxolitinib were also submitted to immunoprecipitation and immunobloting with IRS2 and anti-phosphotyrosine antibodies. Peripheral blood mononuclear cells from 28 healthy donors and 97 patients with MPN (PV=28, ET=38, PMF=31) were included, and CD34+ cells were submitted to quantitative PCR (q-PCR). Relative expression of IRS2 was correlated with clinical data and with JAK2 V617F mutation status. Results Immunoprecipitation analysis showed that IRS2 associates with JAK2 in leukemia cell lines harboring (HEL) or not (HL60) the JAK2 V617F mutation. Furthermore, treatment of HEL cell line with the JAK1/2 selective inhibitor Ruxolitinib resulted in decreased IRS2 tyrosine phosphorylation. IRS2 mRNA expression in CD34+ cells were significantly higher in patients with ET when compared to healthy donors (1.70 [0.42-10.60] versus 0.87 [0.01-11.22] , p=0.03). There was no difference in IRS2 mRNA expression in PV or PMF patients when compared to healthy donors. Furthermore, significantly higher levels of IRS2 mRNA expression were observed in patients harboring JAK2 V617F mutation when compared to the wild type JAK2 for ET (2.37 [0.96-10.60], n=14 versus 1.54 [0.42-1.54] , n=22; p=0.01); and for PMF (2.27 [0.003-10.59], n=20 versus 0.60 [0.02-2.42] , n=11; p=0.02). Although there was also a significant difference in IRS2 mRNA expression in mutated versus non mutated JAK2 in PV (p=0.02), the number of non mutated samples was low (n=2). Conclusions Our data indicate that IRS2 is a binding partner of JAK2 in myeloproliferative neoplasms and suggest that this protein association may be involved in cell proliferation in these diseases. The higher IRS2 expression in mutated samples (JAK2 V617F) might be associated with the constitutive activation of JAK2 in these samples. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.1598.1598
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 5
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    Proteomic Analysis of Waldenstrom Macroglobulinemia (WM) Using Nanoscale Protein Micorarray Techniques.
    American Society of Hematology ; 2005
    In:  Blood Vol. 106, No. 11 ( 2005-11-16), p. 504-504
    Details
    In: Blood, American Society of Hematology, Vol. 106, No. 11 ( 2005-11-16), p. 504-504
    Abstract: The objective of this study was to determine the molecular alterations that occur at the protein level in patients with WM in order to identify novel targets of therapy, determine new markers of prognosis, and begin to delineate the pathogenesis of WM. Five bone marrow samples were obtained after informed consent from patients with symptomatic WM. Two bone marrow samples were obtained from age-matched controls and were pooled. All samples were purified with anti-CD19+ beads with over 90% purity. Protein quantification was performed and 1ug of protein was obtained for each sample and control. The nanoscale protein micorarray technique (BD Clontech, CA) was used to measure changes in the patterns of protein expression between WM samples and control lymphocytes. This is a new technique that detects differences in protein abundance between the tumor and control samples by hybridizing fluorescently labeled (Cy3 and Cy5) protein mixtures onto slides spotted with 512 monoclonal antibodies against human polypeptides. It requires minimal amount of protein. Two microarray slides were used for each experiment and a control experiment of control versus control was performed for normalization of the data. The slides were scanned using the Axon GenePix 4000B scanner. Two ratios were generated from the spot images for each protein target. The mean of the ratios of Cy5/Cy3 of both slides were analyzed using Clontech software and used to calculate an Internally Normalized Ratio (INR = ÷Ratio1/Ratio2, ratios 1 and 2 correspond to slides 1 and 2) for each spot on the array. The INR values were input into GeneSpring 6.0 software (Silicon Genetics, Redwood City CA). The data was normalized to the mean INR of the control samples. Proteins whose expression fold change relative to control was greater than 1.3 fold were determined. All samples were of symptomatic WM. There were 3 females and 2 males. The median age was 61 years (range, 47–83). Four patients were newly diagnosed and one had received prior rituximab, CHOP ad thalidomide therapy. Clustering analysis did not demonstrate a difference between newly diagnosed and treated samples. There were 72 proteins up or downregulated by 1.3 fold in all WM samples as compared to control. These included proteins in the PI3K pathway such as VHR, PTP1B, PI3K (p110alpha) and Rb2. Protein kinases such as PKCi, PKCbeta, PKC gamma, PKC delta, PLCgamma were all upregulated in WM samples. Other proteins included the B-cell specific activator protein PAX-5, the ubiquitin protein UBCH6, the STAT kinase STAT4, the GTPase Rho A-binding kinase ROK alpha, and the apoptosis protein SMAC/DIABLO. We demonstrate that several isoforms of the PKC family of proteins are upregulated in WM. PKC proteins regulate apoptosis, proliferation and migration in many cancer cells. These proteins may be useful targets of therapy in future clinical trials in WM. Other inhibitors that may be useful in WM include ubiqutin/proteosome inhibitors such as bortezomib and PI3K pathway inhibitors such AKT or mTOR inhibitors. Our results also confirm the presence of PAX-5 in WM consistent with previous cytogenetic studies. Supported in part by an ASH scholar award and Research Fund for Waldenstrom.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V106.11.504.504
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2005
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  • 6
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    Abnormal Expression of TRAF Adapter Proteins in Waldenstrom’s Macroglobulinemia.
    American Society of Hematology ; 2006
    In:  Blood Vol. 108, No. 11 ( 2006-11-16), p. 4640-4640
    Details
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 4640-4640
    Abstract: Background: Waldenström’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma with as yet unknown genetic basis for its pathogenesis. Several TNF family members (CD40L, APRIL and BAFF/BLYS) are known to regulate WM growth and survival. TRAFs are a novel family of adapter proteins that facilitate pro-apoptotic (TACI) or pro-survival/differentiation (CD40, BAFFR, BCMA) receptor signaling mediated by TNF family ligands. Therefore, understanding the TRAF system in WM may yield important clues about WM growth and survival. Methods: WM cell lines (BCWM.1 and WSU-WM), IgM secreting low-grade lymphoma cell lines (MEK1, RL, Namalwa), and primary bone marrow CD19+ selected lymphoplasmacytic cells (LPC) from 20 WM patients and 6 healthy donors were evaluated for TRAF (TRAF 2, 3, 5, 6) expression using semi quantitative RT-PCR and/or western blot analysis. Results: The TNF familiy receptors CD40, BAFFR, BCMA, and TACI were expressed in all cell lines tested as well as in CD19+ selected LPC from WM patients and healthy donors. Moreover, TRAF 2, 3, 5, 6 were expressed in all cell lines by both RT-PCR and western blot analysis. In contrast, we observed loss or abnormally low expression of both TRAF 2 and 5 in 6/20 (30%) patients, whilst TRAF 3 was absent or abnormally low in 3/30 (15%) patients. TRAF 6 was expressed in all patients. Among healthy donors, we observed expression of all TRAF adapter proteins. Conclusion: Up to one third of WM patients demonstrate loss of TRAF 2 and 5 adapter proteins which facilitate signaling through the pro-apoptotic receptor TACI. Ongoing studies including gene sequencing and siRNA knockdown models are delineating a role for TRAF loss in the pathogenesis of WM.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V108.11.4640.4640
    RVK:
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    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
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  • 7
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    Quiescence/Mobilization of Hematopoietic Immature Cells Induced By Polyphenols through Modulation of APC/EPCR/PAR-1 Axis
    American Society of Hematology ; 2019
    In:  Blood Vol. 134, No. Supplement_1 ( 2019-11-13), p. 4996-4996
    Details
    In: Blood, American Society of Hematology, Vol. 134, No. Supplement_1 ( 2019-11-13), p. 4996-4996
    Abstract: The mechanism underlying quiescence and/or mobilization of hematopoietic stem cells and their bone marrow progenitors (HSPC) into circulation are tightly regulated for the continuous supply of peripheral blood cells; however, non-physiological or stress conditions, such as infections, can accelerate these mechanisms. Our results have shown that polyphenols modulate quiescence/mobilization of HSPC, but do not affect mature populations. Thrombin has been reported to induce the rapid HSPC mobilization through coagulation thrombin/PAR-1 axis, and quiescence is maintained across the APC/EPCR/PAR-1 axis (Nat. Med. 2015, 21:1307-17). Our objective was to investigate the effect of polyphenols on thrombin/PAR-1 and APC/EPCR/PAR-1 axis. C57BL/6J mice (6-8 weeks old) were treated with polyphenols from green tea extract (250 mg/kg body weight) orally (gavage) once every seven days and injected (i.p.) at day 7 with lipopolysaccharide (LPS) (100μg;Sigma) (n=6). The control group received vehicle and was injected with LPS (n=6). After 24h of LPS injection, mice were anesthetized for blood collection, and then sacrificed for bone marrow collection. PAR-1 and EPCR expression, nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) phosphorylation were evaluated in HSPC by flow cytometry. The functional ability of HSC was assessed by competitive repopulation assay. Vascular permeability was studied using Evans blue. After LPS injection, mice showed reduced expression of EPCR in bone marrow LSK parallel to an increase of PAR-1 expression in circulating immature and mature cells. Treatment of these mice with polyphenols partially prevented the reduced expression of EPCR in bone marrow LSK (13±3 vs 54±12; p 〈 0.05), but did not affect the increased PAR-1 expression in circulating immature and mature cells. Evans blue assay revealed a reduction in the vascular permeability of the bone marrow of LPS-injected mice treated with polyphenols (3.9±0.5 vs 2.1±0.1; p 〈 0.05). To assess whether polyphenols altered NO production, we measured NO levels and eNOS phosphorylation in immature LSK EPCRhigh (or LT-HSC) cells. NO production is activated by eNOS phosphorylation at Ser1177 and negatively regulated by eNOS phosphorylation at Thr495. LPS injection rapidly increased NO levels and eNOS phosphorylation at Ser1177 in bone marrow LSK of mice. Treatment of these mice with polyphenols reduced the percentage of bone marrow LSK EPCRhigh cells with higher intracellular NO (52±2.8 vs 28±5.6; p 〈 0.01) and increased eNOS phosphorylation at Thr495 in immature LSK. In order to evaluate the action of polyphenols on the functional ability of HSC, a competitive bone marrow repopulation assay was performed. Donor mice (C57BL/6J) received or not polyphenols followed by LPS injection (treated group: Polyphenols+LPS; control group: LPS), and bone marrow cells were transplanted (1:1) together with bone marrow cells of competitors (B6.SJL-PtprcaPepcb/BoyJ) in lethally irradiated recipients (B6.SJL-PtprcaPepcb/BoyJ). Mice were followed for 16 weeks and hematological analysis revealed no difference in circulating leukocytes, platelets or hemoglobin levels. Transplanted mice (recipients) presented a higher percentage of CD45+ cells from Polyphenols+LPS donors (33.7±13 vs 78.6±0.9; p 〈 0.05) in the peripheral blood, as well as increased number of T lymphocytes (6.7±4.5 vs 37.2±2.9; p 〈 0.05) and myeloid cells (68.5±1.7 vs 82.5±3.5; p 〈 0.05) from Polyphenols+LPS group. After 16 weeks, mice were euthanized and a higher percentage of LSK (or HSC) and LSK EPCRhigh (or LT-HSC) cells from Polyphenols+LPS donors were detected in the bone marrow, although only the percentage of LSK EPCRhigh was statistically different (0.0014±0.0001 vs 0.0032±0.001; p 〈 0.05). Taken together, our results indicate that polyphenols increased the functional ability of HSC in LPS-injected mice showing increased percentage of bone marrow LSK EPCRhigh cells, which are the most quiescent stem cells with strong self-renewal ability. Polyphenols reduced EPCR expression and NO production in immature cells of LPS-injected mice, exhibiting an anti-inflammatory effect that leads to the maintenance of barrier integrity and quiescence of cells, which was corroborated by reducing vascular permeability in the bone marrow. Thus, polyphenols appear to modulate quiescence/mobilization of HSPC through APC/EPCR/PAR-1 axis. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2019-127354
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2019
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  • 8
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    NRF2 Is Targeted By the Polyphenol Quercetin and Induces Apoptosis, in Part, through up Regulation of Pro Apoptotic Mirs
    American Society of Hematology ; 2019
    In:  Blood Vol. 134, No. Supplement_1 ( 2019-11-13), p. 2529-2529
    Details
    In: Blood, American Society of Hematology, Vol. 134, No. Supplement_1 ( 2019-11-13), p. 2529-2529
    Abstract: Nuclear factor-erythroid 2 related factor (NRF2) is involved in cell defense and survival against endogenous and exogenous stress. Constitutively active Nrf2 in malignant cells increases the expression of cytoprotective genes and consequently, enhances proliferation via metabolic reprograming and inhibition of apoptosis (Leinonem, Advances in cancer Research, 2014). NRF2 is persistently activated in many human tumors, including acute myelogenous leukemia thus, inhibition of NRF2 activity may be a promising strategy for leukemia therapy. Flavonoids, present in vegetables, fruit and propolis, might exert antitumoral effects through induction of apoptosis and chromatin remodeling (Link, Biochem Pharmacol., 2010). A previous study from our group showed that Quercetin (Qu), a natural polyphenolic flavonoid compound, induced apoptosis, partly due to its DNA demethylating activity, through HDAC inhibition and by the enrichment of H3ac and H4ac in the promoter regions of genes involved in the apoptosis pathway, leading to their transcription activation (Alvarez, Clinical epigenetics 2018). In the present study, we evaluated the effect of Qu as a modulator of NRF2. This study was performed in vivo in human xenograft acute myeloid leukemia (AML) models, and in vitro using leukemia cell lines. Qu treatment (50 µM Qu) for 48h downregulated HDAC4, NRF2 and p-NRF2 at protein levels (p 〈 0.05; p 〈 0.005; p 〈 0.005 respectively). Imaging Flow Cytometry (AMNIS, ImageStream ISX mkIITM) and Confocal Microscopy evidenced a decrease in NRF2 nuclear localization. Furthermore, combined treatment with the proteasome inhibitor MG132 prevented degradation of NRF2, indicating that treatment increased proteasomal degradation. Loss of NRF2 decreases HDAC4, a redox sensitive histone deacetylase, resulting in an increased expression of miR-1 and miR-206 (Singh, J Clin Invest. 2013). Herein, expression profile of 84 miRNAs (Apoptosis miRNA PCR array) were performed in samples from human xenograft model. Treatment up-regulated the expression profile of 5% (n=4) of the 84 miRNAs evaluated, corresponding exclusively to miRNAs that target anti-apoptotic genes and to miRNAs that have been demonstrated to have pro-apoptotic functions. Furthermore, expression levels of miR-1, miR-133a/b, which target anti-apoptotic genes and miR-206, a pro apoptotic miR, were validated in xenograft model samples, resulting in a significant up-regulation of the expression levels in treated animals compared to controls (p 〈 0.05). In addition, lentiviral sh down regulation of NRF2, led to an increased apoptosis, decreased cell survival and an up regulation of miRNA 206 expression in Qu treated cells. In summary, Qu might induce programmed cell death in part, by decreasing NRF2 nuclear localization, by inducing NRF2 proteasomal degradation and down regulation of HDAC4 which led to up-regulation of pro apoptotic miRNAs. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2019-130982
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2019
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  • 9
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    Serum Free Light Chain in Waldenstrom Macroglobulinemia.
    American Society of Hematology ; 2006
    In:  Blood Vol. 108, No. 11 ( 2006-11-01), p. 2420-2420
    Details
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-01), p. 2420-2420
    Abstract: Background: Waldenstrom macroglobulinemia (WM) is a low-grade lymphoplasmacytic lymphoma characterized by the involvement of the bone marrow with lymphoplasmacytic cells and the production of IgM monoclonal protein. The serum IgM level is an important marker of diagnosis and response; however, it does not correlate with prognosis. Because of it’s long half-life, it is not a sensitive test for response, indicating the need for more sensitive serum markers that predict tumor burden. The level of serum beta 2 microglobulin (B2M), cytopenias, and age are poor prognostic markers in WM. In this study, we sought to determine the value of serum free light chain (sFLC) in WM. Method: We analyzed 159 patients, 99 patients with WM and 70 patients with IgM-MGUS. sFLC levels were performed using Freelite™ reagents on a Dade-Behring Nephelometer. The Freelite assay (The Binding Site, UK) uses antibodies directed against the free light chain (FLC) epitopes. The clonal free light chain was considered the involved immunoglobulin free light chain (FLC), whether kappa or lambda. Results: The median age at diagnosis was 63 years (range, 37–90). The male/female ratio was 1.5. Forty-one% of the patients with WM required therapy at the time of this study. In the patients diagnosed with WM, the median serum B2M was 2.3 mg/L, median hemoglobin 10 gm/dL, median serum viscosity 1.9 cp, and median platelet count 240 ×109/L. Kappa light chain was present in 74% of the total cohort, with no difference between WM and IgM-MGUS. The mean sFLC was significantly higher in WM as compared to IgM-MGUS, with131.2 mg/L (95% CI 72–189) in WM and 39.2 mg/L (95% CI 21–56) in IgM-MGUS, p=0.003. In addition, sFLC correlated with the serum IgM level (r=0.27; p=0.008). To determine whether sFLC predicted for poor prognosis, we analyzed the relation of sFLC with prognostic markers. Elevated sFLC strongly correlated with high serum B2M (r=0.33; p=0.001), anemia (hemoglobin 〈 10 gm/dL) (r=0.39; p 〈 0.001), and thrombocytopenia (platelet count 〈 120×109/L) (r=0.22; p=0.034). There was no correlation with age at diagnosis. We next defined the threshold of sFLC 50 mg/L, based on its ability to correctly classify MGUS and WM with an 88% specificity to identify MGUS in the group with low sFLC. This cut off was able to correctly classify patients with low B2M ( 〈 = 3 mg/L) and high B2M ( 〉 3 mg/L), (p 〈 0.001). Prognostic models were then developed based on B2M, anemia (hemoglobin 〈 10gm/dL) and thrombocytopenia (platelet count 〈 120×109/L). A point was given for elevated B2M of 〉 3mg/L, anemia, or thrombocytopenia, with score of 3 indicating elevated B2M in combination with anemia and thrombocytopenia. Elevated sFLC ( 〉 =50 mg/L) was able to correctly classify patients with a score of 2 points or more versus 1 point in this model (p 〈 0.005). Conclusion: sFLC clearly differentiated patients with WM and IgM MGUS. In patients with WM, sFLC significantly correlated with poor prognostic markers. Future studies are needed to validate the role of sFLC as a prognostic marker of survival in WM. ASM and XL are co-first authors.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V108.11.2420.2420
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
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  • 10
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    Early Molecular Response Is Predictive Of Overall, Progression-Free and Event-Free Survival In Chronic Myeloid Leukemia Using Second-Generation Tyrosine Kinase Inhibitors After Imatinib Treatment
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 1326-1326
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 1326-1326
    Abstract: Second-generation tyrosine kinase inhibitors (2G-TKI), nilotinib or dasatinib used after imatinib failure can induce complete cytogenetic response (CCR) in 50% of chronic myeloid leukemia (CML) patients. BCR-ABL transcript levels reduction in the initial months of treatment has been associated improved outcome. Aims to evaluate the predictive value of early molecular responses, at 3 and 6 months after treatment with 2G-TKI in CML patients with imatinib failure or intolerance; to correlate these responses with CCR, overall survival (OS), progression-free survival (PFS) and event free survival (EFS). Methods Between September 2007 and August 2012, 71 consecutive patients with CML resistant or intolerant to imatinib were treated with dasatinib (n= 50) or nilotinib (n=21). BCR-ABL transcripts were measured in peripheral blood using real-time quantitative PCR (RQ-PCR) at 3 months intervals. Results were expressed as BCR-ABL/ABL ratio, with conversion to the international scale (IS). Major molecular response (MMR) was defined as a transcript level ≤ 0.1% (IS). Cytogenetic analysis was performed at baseline, 3, 6, 12 and 18 months after starting therapy with 2G-TKI. BCR-ABL mutation analysis by direct sequencing was investigated in resistant patients. Probabilities of OS, PFS and EFS were calculated using Kaplan-Meier method. An event was defined as the loss of CHR, CCR, progression to advanced phases, death or 2G-TKI discontinuation. The CCR probabilities according to molecular responses were calculated by c2 method and cumulative incidence, considering as competitive event death or progression. Results 71 patients were analyzed, median age of 47 years (15-81); Disease status before 2G-TKI was: 50 (71%) CP; 13 (18%) AP and 8 (11%) BC. Responses: 59/71 (83%) obtained CHR; 38/55 (69%) CCR and 37/60 (62%) MMR. At 3 months of therapy, 81.5% (44/54) had a BCR-ABL ratio ≤10% and at 6 months 66% (33/50) had ≤ 1%. At 3 months, CCR was obtained 65% (19/29) pts with ≤10% RQ-PCR and 16% (1/6) with 〉 10% RQ-PCR (p= 0.06). At 6 months, CCR was 100% (12/12) in pts with RQ-PCR ≤ 1% and 14% (1/7) in those with 〉 1% (p 〈 0.0001). The probability to achieve RQ-PCR 〈 10% at 3 month was 43% (95% CI 32-54%). During treatment 3 (4%) progressed to AP and 5 (7%) to BC. The 5-year probability of OS was 78% (95% CI: 68-88%) albeit by disease status was 86% in CP, 92% in AP and 12% in BC (p 〈 0.0001). OS was inferior in pts with RQ-PCR 〉 10% at 3 months (60 vs 84%, p= 0.03) and 〉 1% at 6 months (68 vs100%, p= 0.006). PFS was 68% in 5-year, and was lower in BC pts (p 〈 0.0001) and pts with RQ-PCR 〉 1% at 6 months (p= 0.004). EFS was 53% and lower in BC pts (p 〈 0.0001), in pts with RQ-PCR 〉 10% at 3 months (p= 0.005) and 〉 1% at 6 months (p 〈 0.0001). RQ-PCR at 3 and 6 months were also predictive of a worse survival when patients in CP were analyzed separately. 2G-TKI was discontinued in 44% (31/71) due to: resistance (n=18), intolerance (n=5), death (n= 3), HSCT (n=3) and loss of follow-up (n=2). Eleven BCR-ABL mutations were detected in 36 pts; 3 previously 2G-TKI (L387M-1, E255K-1, M351T-1) and 9 after therapy (T315I-5, M244V-2, E255V-1, Y253H-1). OS by mutation was 45% with any mutation and 88% with no mutation (p= 0.05). Conclusion BCR-ABL transcript levels at 3 and 6 months can identify patients with worse prognosis and less chance to obtain CCR with 2G-TKI after imatinib treatment. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.1326.1326
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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