Erythropoiesis in Polycythemia Vera Is Hyper-Proliferative and Has Accelerated Differentiation during In Vitro Erythroid Expansion and Is Associated with Higher Expressions of cMYB and EPOR at Early Erythroid Progenitors

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1549-1549
Author(s):  
Hana Bruchova ◽  
Donghoon Yoon ◽  
Archana Agarwal ◽  
Eva Otahalova ◽  
Hyojin Kim ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Mononuclear Cells ◽  
Early Stage ◽  
Severe Disease ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
Differentiation Stage

Abstract Erythroid differentiation is a dynamic process leading to the production of mature red blood cells. Even small variations in this process may result in severe disease phenotype. To study this process, we used a three-phase erythroid expansion system to expand homogeneous erythroid progenitors (EPs) from peripheral blood mononuclear cells (PB-MNCs) (Bruchova H. et al, 2007, Exp. Hematology, in press). We then characterized the expanded EPs from polycythemia vera (PV) patients and healthy donors at various points of maturation comparing cell proliferation and differentiation stage. EPs from PV patients outgrew controls up to day 14 (∼12 fold for PV and ∼4 fold for control compared to day 1). Differentiation was analyzed using both FACS analysis (with CD71/CD235a staining) and morphological evaluation (Wright-Giemsa staining), and demonstrated a more rapid differentiation of PV EPs when compared to controls up to day 14. We then evaluated apoptosis/cell cycle analysis by propidium iodide staining. Although PV EPs contained larger S phase population (45%) than controls (34%) at day 11, the apoptosis proportion of PV EPs was increased ∼2 fold to control from day 14. To understand the molecular mechanism of these differences between PV and controls, we analyzed the gene expression of several known regulators in erythropoiesis - BCL2, EPOR, cMYB, p27. Two transcripts (EPOR and cMYB) showed unique profiles on PV EPs. The EPOR transcript increased earlier in PV; i.e. from day 7 until day 21 and reached a plateau at day 11, compared to day 9 until day 19 and plateau at day 14 in controls. In addition, PV EPs contained higher levels of EPOR transcripts than control on most of timepoints. Interestingly, cMYB, which is known to augment early progenitor proliferation, was highly expressed from day 7 in PV, through day 11. Control EPs also expressed cMYB from day 9 through day 11; however, cMYB levels from any stages of control EPs were markedly lower than PV EPs at day 7. In this study, we demonstrate that PV erythropoiesis has unique features of hyperproliferation and an accelerated differentiation. These features are associated with earlier and higher expressions of cMYB and EPOR at the early stage of erythropoiesis.

DYRK3 Encodes an Inhibitor of Erythroid Differentiation and Is Expressed at High Levels in Patients with Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3661-3661
Author(s):  
David P. Steensma ◽  
Julie C. Porcher ◽  
Antony Chadderton ◽  
Kevin Laubscher ◽  
Deb Jaworski ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Kinase Inhibitor ◽  
Erythroid Differentiation ◽  
Murine Models ◽  
Erythroid Progenitors ◽  
Peripheral Blood Mononuclear ◽  
Anemia Of Chronic Disease ◽  
Orally Bioavailable

Abstract Background: Many patients (pts) with anemia due to impaired erythropoiesis fail to respond to currently available erythropoiesis-stimulating agents or do not wish to receive these agents due to safety concerns. For these pts, novel approaches are needed. DYRK3, an evolutionarily conserved member of an emerging family of serine-threonine kinases, is expressed at high levels in erythropoietic progenitors; murine models suggest that DYRK3 selectively inhibits red cell production during stress erythropoiesis. We sought to determine DYRK3 expression in pts with anemia, and whether in vitro inhibition of DYRK3 augments erythropoiesis. Methods: We performed quantitative RT-PCR for DYRK3 and 4 control genes using whole peripheral blood (WPB) from 14 healthy persons, 5 pts with anemia due to multiple myeloma (MM), and 3 pts with anemia of chronic disease (ACD); in addition, peripheral blood mononuclear cells (PBMCs) were assayed in the 3 ACD and 5 MM pts and bone marrow mononuclear cells (BMMCs) in the MM pts. Erythrocyte subpopulations (CD36+, CD71+, and dual CD36+/CD71+) were quantified by flow cytometry. CFU-E growth was measured from PBMCs and BMMCs in the presence of varying concentrations of GSK626616, an orally bioavailable first-generation DYRK3 kinase inhibitor. Results: Normalized expression of DYRK3 in WPB was 7.2 fold higher in MM pts (p=0.0001) and 3.4 fold higher in pts with ACD (p=0.026) compared to healthy controls. DYRK3 expression was proportional to the degree of anemia, and WPB and PBMC expression of DYRK3 in MM pts correlated well with BMMC expression. The level of DYRK3 expression was proportional to the population of marrow CD36+/CD71+ erythroid progenitors, and inversely proportional to the size of the more mature CD36−/CD71+ population, suggesting that high DYRK3 expression is associated with maturation arrest in humans at a stage of erythroid differentiation roughly corresponding to pre-Ter119pos/CD71high inhibition observed in murine models. Although incubation of pt-derived BMMC or PBMC with GSK626616 at concentrations up to 30 μM, either in the presence or absence of physiological concentrations of erythropoietin, did not augment in vitro CFU-E formation, CFU-E growth overall was poor in the pt samples studied. Conclusion: DYRK3 is expressed at high levels in pts with anemia due to neoplasia or inflammation, and elevated DYRK3 expression is associated with decreased numbers of CD36−/CD71+ red cells. Further studies of the effects of DYRK3 antagonists on human erythropoiesis in vitro are necessary, and clinical trials in anemic patients will be required to determine if DYRK3 antagonists can reverse DYRK3-associated inhibition.

In Vitro Erythroid Cell Expansion Analysis in Polycythemia Vera.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4916-4916 ◽  
Author(s):  
Donghoon Yoon ◽  
Hana Bruchova ◽  
Archana M. Agarwal ◽  
Josef T. Prchal ◽  
Jaroslav F. Prchal
Keyword(s):  
Polycythemia Vera ◽  
Molecular Mechanism ◽  
Mononuclear Cells ◽  
Biological Behavior ◽  
Erythroid Cell ◽  
Erythroid Progenitors ◽  
Peripheral Blood Mononuclear ◽  
Expansion Protocol ◽  
Differentiation And Proliferation

Abstract Polycythemia vera (PV) is an acquired myeloproliferative clonal stem cell disorder characterized by cytokine hypersensitivity. The erythroid colony forming assay has been useful in PV diagnosis. However, studies aiming to elucidate molecular mechanism in PV pathogenesis often require large numbers of cells from a specific erythroid stage. For this purpose, we had adapted the mouse expansion assay described by Karur et al (Blood, 2006 in E-Pub) and differential display method described by Zhang et al (Blood, 2003, 102; 3938) to develop a human erythroid expansion protocol. In this study, we used peripheral blood mononuclear cells (PB-MNCs) from PV patients and healthy donors, and expanded them along erythroid lineage in 21 day culture. Through the culture, we took samples at 8 timepoints (days 1, 7, 9, 11, 14, 16, 19, 21) to evaluate differentiation patterns. We had generated 5 different regions using CD71 (transferrin receptor) and CD235a (glycophorin A) and characterized these regions by standard morphology analysis. This protocol allowed differentiation of PB-MNCs to all erythroid stages ending with late normoblast, reticulocyte, and mature erythrocytes (which do not survive well in this culture environment). The erythroid differentiation and proliferation patterns were different between PV and normal. In the first week of culture, there was no significant proliferation difference observed between PV and normal. In contrast, the differentiation progressed more rapidly in PV, likely reflecting the differentiation of Epo independent PV population (since the first week culture contains no Epo). In the second week of culture (Epo present), there was a markedly increased expansion of PV erythroid cells. However, the differentiation pattern of PV resembled that of normal. In conclusion, we demonstrated that our in vitro expansion method allows expansion of PB-MNCs along erythroid lineage with 60~80% stage homogeneity as to the stage of differentiation in both PV and normal. The differences of differentiation and proliferation pattern between PV and normal reflected the expected biological behavior of erythroid progenitors. Thus, the expansion protocol is useful to study molecular mechanism of PV pathogenesis, which requires large number of erythroid progenitors of various stages.

Successful Treatment of HU-Refractory Polycythemia Vera with Atorvastatin and Low Dose Hydroxyurea. Results from a Pilot Study in Bolivia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5621-5621
Author(s):  
Ricardo Amaru ◽  
Ariel Amaru ◽  
Hortensia Miguez ◽  
Gina Torres ◽  
Josue Mamani ◽  
...  
Keyword(s):  
Pilot Study ◽  
High Risk ◽  
Polycythemia Vera ◽  
Mononuclear Cells ◽  
Jak2v617f Mutation ◽  
World Health ◽  
La Paz ◽  
Healthy Donors

Abstract Background Polycythemia Vera (PV) is a clonal myeloproliferative neoplasm, characterized by the JAK2V617F mutation. The main goal of current therapies for PV is to prevent thrombotic events and delay transformation to Myelofibrosis (MF) or Acute Myeloid Leukemia (AML).Treatment for PV to keep an hematocrit (Hct) level <45 %, has been associated with a reduction in cardiovascular deaths and thrombotic events (Marchioli, R et al. NEJM 2013). Currently, low-risk PV patients (<60 years and no previous thrombotic events) are treated with aspirin and phlebotomy while high-risk patients require additional cytoreductive therapy, usually with Hydroxyurea (HU). Resistance to HU is associated with an increased risk of transformation and reduced survival. This is why for HU-refractory patients, second line treatments with interferon alpha, anagrelide or even ruxolitinib are recommended. In Latin America, because of high cost and drugs availability, this last group reflects difficulties to be treated. Because statins have been reported to modulate the erythroid clonogenic activity of normal BM erythroid colonies we performed a pilot study to investigate in vitro and in vivo the biologic and clinical activity of atorvastatin in PV patients Patients and Methods Ten high risk PV patients with a median age of 64.3 years (range 58-73) entered into this study. The diagnosis of PV was done according to the 2008 World Health Organization diagnostic criteria and patients were stratified according to an algorithm proposal provided by Griesshammer et al. (Ann Hematol, 2015). The definition of HU resistance (Barosi, G et al.: BJH 2009) was applicable to five patients (median age 63.9 years) failing to achieve a satisfactory hematologic response upon treatment with more than 2 g of HU, 100 mg of Aspirin and phlebotomies. The assessment of the JAK2V617F mutation was performed as previously described (Guerini et al.: Leukemia 2009). Colony assay, proliferation and apoptosis tests were performed with or without Simvastatin (3.5 uM), as previously described (Amaru, A, Experimental Hematology 2012), on cell lines (UKE1 and K562) and bone marrow mononuclear cells obtained from PV patients and healthy donors. Patients with HU refractory PV (n=5) and high risk PV with hypercholesterolemia (n=5) were eligible to receive Atorvastatin (20 mg/day) added on the top of the ongoing treatment with phlebotomies, Aspirin (100 mg/day) and cytoreductive HU therapy (500 mg/day). All treated patients were high altitude residents (> 3.600 m.a.s.l.) of La Paz (Bolivia) where the normal Hct level of healthy subjects is 48-57% for men and 44-54% for women. This pilot study was approved by the Review Board of the Hospital and the University of San Andres, La Paz. Results In a preliminary set of in vitro proliferation cell assays, simvastatin (3.5 uM), added for 5 days, induced a 33% inhibition of cell proliferation of UKE-1 (JAK2V617F mutated) as compared to 5 % of K562 (BCR/ABL positive). A comparable result was obtained in a 7-day clonogenic cell assay where the colony inhibition was 50 % for UKE-1 and 10 % for K562. On the basis of these results similar experiments were also performed using BM mononuclear cells derived from PV patients and healthy donors. In these experiments performed with the addition of simvastatin, it induced a 41% of inhibition in BFU-E colonies of PV patients and a 25% of inhibition in healthy donors. Furthermore, BFU-E colonies inhibited by simvastatin presented a decrease in hemoglobinization and the size of colonies. HU refractory PV patients and High-risk PV patients with hypercholesterolemia treated with the addition of Atorvastatin, Aspirin, cytoreductive HU and phlebotomies; after a follow-up of 2.6 years (1-7 years), induced a decrease of WBC from 16.500 to 9.270/ul, Hct 61.1 to 52.3% and PLT 457.900,000 to 324.7000/ul. The number of required phlebotomies is reduced in comparison to the required at starting treatment. None of the patients presented thrombotic or cardiopulmonary event. One patient died within two years of starting treatment, due to complications of diabetes mellitus. Conclusions In vitro and in vivo, statins showed some evidence of inhibitory activity of the hematopoiesis of PV patients. These preliminary results might indicate the opportunity to further investigate the potential clinical value of these molecules in the treatment of PV. Disclosures Off Label Use: Atorvastatin was used for its antiproliferative activity on myeloid progenitor cells shown by in vitro experiments.

scholarly journals Role of protein kinases JNK and p38 in regulation of mononuclear leucocytes apoptosis in oxidative stress

2008 ◽  
Vol 7 (3) ◽  
pp. 38-43 ◽  
Author(s):  
N. Yu. Chasovskikh
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Transmembrane Potential ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Stress Induction ◽  
Healthy Donors ◽  
Mononuclear Leucocytes

Programmed cell death of peripheral blood mononuclear leucocytes taken from healthy donors and cultivated with various concentration of Н2О2, selective inhibitors of JNK (SP600125), 38 (ML3403) and in case of pneumonia was investigated. Intensification of intracellular production of reactive oxy р МАРК - gen species was accompanied by the increase in number of apoptotic and TNFR1-presented cells and mononuclears with reduced value of mitochondrial transmembrane potential in a case of oxidative stress induction with 1 mM hydrogen peroxide and in blood taken from patients with pneumonia. Action of inhibitors SP600125 and ML3403 in vitro in oxidative stress conditions prevents the increase in number of annexin- positive mononuclear cells, that confirms the participation of JNK and 38 -kinases in mechanisms of oxidative stress-mediated apoptosis dysregulation.

Effect of a TNFα Blocker and Peginfa on Polycythemia Vera Clonal Hematopoiesis and Suppressed Normal Dormant Hematopoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1820-1820
Author(s):  
Sabina Swierczek ◽  
Soo Jin Kim ◽  
Mohamed E Salama ◽  
William L. Heaton ◽  
Michael W. Deininger ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
X Chromosome ◽  
Mononuclear Cells ◽  
Myeloproliferative Neoplasms ◽  
Intracellular Cytokine Staining ◽  
Advisory Board ◽  
Erythroid Progenitors ◽  
The Impact ◽  
Allelic Burden

Abstract Polycythemia vera (PV) is a clonal disorder arising from a single stem cell while normal stem cells are present in the marrow but are suppressed by the PV clone by an unknown mechanism. Pegylated interferon alfa-2a (PegInfa), a better-tolerated form of Infa induces clinical remission, reduces JAK2V617F allelic burden, and may convert clonal to polyclonal hematopoiesis (EL Liu, Blood 2003). Tumor necrosis factor-α (TNFa) levels are increased in patients with myeloproliferative neoplasms, including PV (Fleischman, Blood 2011). We previously reported that transcripts of TNFα mRNA are higher in CD34+ cells compared to more differentiated cells. TNFa is markedly reduced in those PegInfa-treated patients with decreased JAK2V617F allelic burden and/or return of polyclonal hematopoiesis (detected in females by X-chromosome allelic usage ratio), while no such decrease was seen in PV with hydroxyurea-induced normalization of elevated hematocrit/platelets/leukocytes (Swierczek, ASH 2012). To directly interrogate the role of TNFα in inducing suppression of normal hematopoiesis, we used a TNFα blocking antibody (adalimumab) and examined its role on PV erythropoiesis using a 3-week liquid culture system characterized by synchronized differentiation of expanding erythroid progenitors (Bruchova, Exp Hemat, 2007). In vitro expanded PV erythroid progenitors were grown with or without adalimumab or PegInfa. We evaluated apoptosis, proliferation and differentiation at different stages of erythroid maturation and correlated these parameters with TNFα transcripts, JAK2V617F allelic burden and, in females, clonality. Although the initial mononuclear cells represented a heterogeneous population, the expansion process favors erythroid progenitors and results in their synchronized differentiation. The JAK2V617F allelic burden increased concomitant with erythroid expansion and reached its peak at day 11 (when the majority of cells are proerythroblasts and basophilic erythroblasts), indicating a preferential expansion of erythroid progenitors, and then declined progressively. The addition of adalimumab markedly reduced TNFα mRNA and JAK2V617F allelic burden compared to controls. We previously reported that in this in vitro liquid expansion system, PV erythroid progenitors exhibit accelerated differentiation at days 7-14 and increased proliferation at days 9-14, with a larger S-phase population (40%) than controls (20%) at day 11 (Bruchova Exp Hemat, 2007). Compared to controls, adalimumab increased proliferation and delayed differentiation at early stages of PV erythropoiesis, with the proportion of apoptotic cells consistently decreased compared to erythroid cells expanded without adalimumab. Furthermore, X-chromosome-based clonality assays revealed preferential expansion of normal progenitors in 1 informative female patient. We also measured the impact of TNFα inhibition with adalimumab on burst-forming units-erythroid (BFU-E) colonies from PV patients cultured ex vivo. As expected, JAK2WT, JAK2WT/V617F and JAK2V617F BFU-E colonies were detected in cultures performed in the absence of adalimumab, while the addition of adalimumab preferentially abrogated JAK2V617F homozygous BFU-Es. In analogous experiments, PegInfa markedly decreased TNFα mRNA and JAK2V617F allelic burden, and decreased differentiation, but unlike adalimumab, it decreased proliferation and had no demonstrable effect on apoptosis. Ongoing studies are being performed to correlate the TNFα mRNA expression changes seen with adalimumab treatment in erythroid progenitors with differences in TNFα protein levels using intracellular cytokine staining and flow cytometry. These data suggest that blocking TNFα can suppress the JAK2V617F clone, rescue normal dormant hematopoiesis, and provide a foundation for a combined PV therapy using TNFα blockers with either PegInfa or JAK2 inhibitors. Disclosures Deininger: BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMS, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

scholarly journals Failure of Gamma Interferon but Not Interleukin-10 Expression in Response to Human Papillomavirus Type 11 E6 Protein in Respiratory Papillomatosis

2004 ◽  
Vol 11 (3) ◽  
pp. 538-547 ◽  
Author(s):  
James A. DeVoti ◽  
Bettie M. Steinberg ◽  
David W. Rosenthal ◽  
Lynda Hatam ◽  
Andrea Vambutas ◽  
...  
Keyword(s):  
T Cells ◽  
Human Papillomavirus ◽  
Mononuclear Cells ◽  
Severe Disease ◽  
Interleukin 2 ◽  
Cytokine Expression ◽  
Gamma Interferon ◽  
Peripheral Blood Mononuclear ◽  
Ifn Γ

ABSTRACT Recurrent respiratory papillomatosis (RRP) is a chronic, debilitating disease of the upper airway caused by human papillomavirus type 6 (HPV-6) or HPV-11. We describe responses of peripheral blood mononuclear cells (PBMC) and T cells from RRP patients and controls to the HPV-11 early proteins E6 and E7. PBMC were exposed in vitro to purified E6 or E7 proteins or transduced with fusion proteins containing the first 11 amino acids of the human immunodeficiency virus type 1 protein tat fused to E6 or E7 (tat-E6/tat-E7). TH1-like (interleukin-2 [IL-2], gamma interferon [IFN-γ], IL-12, and IL-18), and TH2-like (IL-4 and IL-10) cytokine mRNAs were identified by reverse transcription-PCR, and IFN-γ and IL-10 cytokine-producing cells were identified by enzyme-linked immunospot assay. These studies show that HPV-11 E6 skews IL-10-IFN-γ expression by patients with RRP toward greater expression of IL-10 than of IFN-γ. In addition, there is a general cytokine hyporesponsiveness to E6 that is more prominent for TH1-like cytokine expression by patients with severe disease. Patients showed persistent IL-10 cytokine expression by the nonadherent fraction of PBMC when challenged with E6 and tat-E6, and, in contrast to controls, both T cells and non-T cells from patients expressed IL-10. However, E7/tat-E7 cytokine responses in patients with RRP were similar to those of the controls. In contrast, E6 inhibited IL-2 and IL-18 mRNA expression that would further contribute to a cytokine microenvironment unfavorable to HPV-specific, T-cell responses that should control persistent HPV infection. In summary, E6 is the dominant inducer of cytokine expression in RRP, and it induces a skewed expression of IL-10 compared to the expression of IFN-γ.

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