FLT3 Mediated MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1532-1532
Author(s):  
Christophe Desterke ◽  
Hans Hasselbalch ◽  
Dominique Bordessoule ◽  
Heinz Gisslinger ◽  
Alessandro Vannucchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cultures ◽  
Mononuclear Cells ◽  
Mapk Pathway ◽  
Primary Myelofibrosis ◽  
Mapk Activation ◽  
Specific Chemical ◽  
Mutational Status ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Myeloproliferation, myelofibrosis, osteosclerosis and neo-angiogenesis are the major intrinsic pathophysiological features of Primary Myelofibrosis (PMF). The myeloproliferation is characterized by an increased number of circulating CD34+ cells with the prominent amplification of “dystrophic” megakaryocytes (MK) through to be responsible for myelofibrosis thought fibrogenic factor release. Comparison of CD34+ and MK cell gene expression profiling between PMF patients and healthy donors revealed a global deregulation of the MAPK pathway genes. This alteration is associated with a modulation of the FLT3 tyrosine kinase gene expression in CD34+ and MK cells from patients, independently of the JAK2V617F mutation presence. Quantification of the FLT3 transcript in mononuclear cells from patients with Polycythemia Vera and Essential Thrombocythemia showed that this over expression is mainly observed in JAK2WT PMF patients. This is associated with a higher proportion of FLT3+CD34+CD41+ cells in the blood of patients. Analysis of FLT3 membrane expression in MK-derived CD34+ cultures revealed that its expression was maintained all along MK differentiation in patients in contrast to healthy donors. Such a higher expression of FLT3 is associated with an increased concentration of its ligand in the platelet rich plasma from patients, independently of their JAK2 mutational status. The role of FLT3 in the regulation of hematopoiesis incited us to analyse whether its alteration could take part in the myeloproliferation and dysmegakaryopoiesis that characterizes PMF. A flow cytometry analysis of FLT3-downstream MAPK activation in PMF CD34+ cells showed a hyperphosphorylation of p38 and JNK as compared to CD34+ cells from normal blood. This phosphorylation was maintained in PMF MK-derived CD34+ cells at day 10. Addition of PD98059, a MAPK inhibitor, induced a dose dependent restoration of the in vitro megakaryopoiesis in PMF as shown by an increase in MK ploidy with apparition of 32N cells associated with a mature cytological aspect and an increase in CD41, CD42a and CD9 MK differentiation marker expression. PD98059 also increased the MK clonogenicity of CD34+ cells from all patients tested (5/5) as compared to healthy donors. Preliminary results using a specific chemical inhibitor of FLT3 in MK-derived CD34+ cell cultures reinforced the involvement of FLT3 in PMF MK differentiation. In presence of FLT3 ligand, the FLT3 mediated MAPK hyperphosphorylation in PMF MK cultures (D6) is reversed by either PD98059 or UO126, another ERK inhibitor and is accompanied by a slight increase in proliferative MK. This effect is not observed in MK cultures from normal CD34+ cells. Surprisingly, ligation of FLT3 by a monoclonal anti-FLT3 antibody in CD34+ cell cultures resulted in an increase MK proliferation. In conclusion, this work shows a deregulation of FLT3 and MAPK pathway in the PMF CD34+ cells and suggests that the persistence of the FLT3 mediated MAPK activation participates in the dysmegakaryopoiesis of PMF patients.

FLT3-Mediated MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 963-963 ◽  
Author(s):  
Christophe Desterke ◽  
Chrystele Bilhou-Nabera ◽  
Bernadette Guerton ◽  
Carole Tonetti ◽  
Denis Clay ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Mapk Pathway ◽  
Primary Myelofibrosis ◽  
Qrt Pcr ◽  
Mutational Status ◽  
Pathway Gene ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Abstract 963 Introduction: Flt3, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis. Primary myelofibrosis (PMF) is a Ph-negative (Ph−) myeloproliferative neoplasm (MPN) characterized by a myeloproliferation with increased hematopoietic progenitors (HPs) and a prominent proliferation of “dystrophic” megakaryocytes (MK). The JAK2V617F mutation is present in about 50% of PMF patients. Previous results from our group have revealed a MAPK pathway gene deregulation associated with an Flt3 transcript modulation in PMF patients. Since activation of Flt3 receptor is known to activate MAPK pathway, which plays a role in megakaryopoiesis, we studied the functional impact of MAPK and Flt3 abnormalities on PMF dysmegakaryopoiesis. Patients and Methods: The study included a group of 106 PMF patients. Transcriptome and QRT-PCR studies were performed on MACS selected CD34+ HPs, megakaryocytes (MK)-derived from CD34+ cell cultures and peripheral blood mononuclear cells (PBMNC) from PMF patients and healthy donors. Cell phenotype associated with Flt3 expression as well as phosphorylation levels of Flt3 and MAPK effectors were analyzed by flow cytometry. Functional studies (FL-induced stimulation and migration) were performed on MK-precursors at day 6 of CD34+ culture. Effect of i) MAPK inhibitors: PD98059, targeting ERK1/2; SB202190, SB203580, PD169316, targeting p38 and, SP600129, targeting JNK, ii) Flt3 inhibitors and iii) Flt3 monoclonal antibody was tested on PMF MK cell cultures. MAPK-induced transcripts were quantified by QRT-PCR in MK-precursors during an 18-hour FL-stimulation kinetic. FL mRNA level was evaluated by using QRT-PCR in bone marrow stromal cells and FL protein was quantified in plasma by using ELISA. Results: Comparative transcriptome analysis of CD34+ HPs and MK cells from PMF patients (with or without JAK2 mutation) and healthy donors showed that the MAPK pathway gene deregulation was independent of the presence of the JAK2V617F mutation. This alteration was associated with a modulation of mRNA Flt3 level in both types of cells. PMF patients also had a higher proportion of circulating Flt3+CD34+CD41+ cells as compared to healthy donors. This population demonstrated an increased phosphorylation of Flt3 on tyr591 and of MAPK (p38, p42/p44, JNK). MAPK effector phosphorylation was also increased in PMF CD34+ cells and MK-derived from CD34+ cell cultures, independently of JAK2 mutational status. In contrast to healthy donors, Flt3 membrane expression was maintained at all stages of in vitro megakaryocyte differentiation in PMF patients. The FL level was increased in the plasma of patients and was mainly expressed by bone marrow stromal cells. In contrast to healthy donors, in MK-derived from PMF CD34+ cell cultures, activation of Flt3/FL axis by addition of exogenous FL induced a MAPK hyper-phosphorylation, especially of p38 and p42/p44 as well as an up-regulation of downstream p38 transcripts (ATF-2, NFATC4, p53, AP-1, IL-8). Addition of chemical inhibitors targeting either MAPK or Flt3 and of an antibody directed against Flt3 reduced the phosphorylation of p38 and of its pathway effectors (MKK3/MKK6, MSK1, ATF2, HSP27 and MAPKAPK2) and normalized the PMF altered megakaryopoiesis. Lastly, in contrast to healthy donors, MK-derived from PMF CD34+ cells showed a FL-induced migration that was reversed by addition of p38αβ inhibitors. Conclusion: Our results demonstrated an increase in the FL circulating level in PMF patients that was mainly secreted by stromal cells. This was associated with an aberrant expression of Flt3 in CD34+ and MK cells and an alteration of the MAPK pathway activation in patients, independent of their JAK2 mutational status. The persistence of Flt3-mediated MAPK activation that participates in the PMF dysmegakaryopoiesis, suggests that drugs targeting “FL/Flt3-MAPK” axis could be promising agents for rescuing the altered megakaryopoiesis observed in patients. Our demonstration that FL, a cytokine mainly produced by stromal cells, participates in the altered megakaryopoiesis in PMF patients strengthens the hypothesis highlighting the crucial role of stroma cells in the hematopoietic deregulation that characterizes the disease. Disclosures: No relevant conflicts of interest to declare.

Aberrant Megakaryocyte Gene Expression Contributes to Primary Myelofibrosis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2867-2867
Author(s):  
Laure Gilles ◽  
Christy Finke ◽  
Terra L Lasho ◽  
Animesh Pardanani ◽  
Ayalew Tefferi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Trial ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Primary Myelofibrosis ◽  
Gene Set Enrichment Analysis ◽  
Gene Expression Program ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Expression Program

Abstract Abstract 2867 Primary myelofibrosis (PMF) is a clonal hematologic malignancy, which results from the transformation of a pluripotent hematopoietic progenitor cell. A major consequence of this transformation is increased hematopoiesis and an overproduction of abnormal blood cells. PMF is associated with bone marrow fibrosis, extramedullary hematopoiesis, increased numbers of circulating CD34+ cells, splenomegaly, and a propensity to evolve to AML. Patients also display anemia and thrombocytopenia and harbor abnormal, immature megakaryocytes (Mks) in their bone marrow and spleen. PMF patients can present well known mutations including JAK2V617F (65%), MPL (10%), TET2 (17%), CBL (6%), IDH (4%,), which are not specific to the disease and are also present in polycythemia vera, essential thrombocythemia and AML. We hypothesize that the genetic events associated with PMF, including MPL and JAK2 mutations, contribute to defects in Mk maturation, but that additional changes are needed to explain the striking abnormalities seen in PMF relative to the other myeloproliferative diseases. Although there have been studies to examine the aberrant gene expression program of CD34+ cells of PMF patients, we chose to examine the changes that occur in gene expression specifically in Mks as a way to better understand their abnormal differentiation and to determine their contribution to the disease. Primary CD34+ cells from PMF patients and healthy donors were cultivated in serum free media supplemented with recombinant TPO, BSA, liposomes, insulin and transferrin to support the growth of Mks. After 10 days of differentiation, we evaluated the cultures for proliferation, apoptosis and differentiation by flow cytometry. We found that PMF specimens gave rise to a lower percentage of mature (CD41+CD42+) cells as compared to healthy donors, but showed, a lower ploidy level, a greater proliferation and increased survival. These observations are consistent with the clinical observations that PMF bone marrow is characterized by an increased number of immature, dysplastic Mks. We used flow cytometry to collect two populations of cells for analysis: immature CD41+CD42− Mks, and CD41+CD42+ mature MKs. After sorting, we extracted RNA and performed whole genome microarray analysis with Illumina Human HT12-v4 arrays on cohorts of PMF and control specimens. Gene expression data were analyzed by GeneSpring and Gene Set Enrichment Analysis (GSEA). We found that the CD41+CD42− MKs derived from PMF progenitors showed reduced expression of GATA1 as compared to control cells, as expected based on previous study by Dr. Alessandro Vannuchi. GeneSpring analysis revealed that myeloid transcription factors, including CEBPa, GFI1, and SPI1 (PU.1), which are not expressed in normal MKs, are strikingly and significantly overexpressed in PMF samples. Moreover, c-myb, which regulates the erythroid/Mk cell fate decision, FOG-1 and AML1, are also overexpressed in PMF Mks. This aberrant myeloid gene expression program in PMF Mks is reminiscent of a similar defect we observed in Mks with reduced expression of GATA-1 and GATA-2. We predict that reduced levels of GATA-1 protein in PMF Mks, as reported by Dr. Alessandro Vannucchi and colleagues, is in part responsible for the aberrant growth and differentiation of the PMF Mks. Our data support the model that PMF Mks are defective in their ability to properly regulate expression of hematopoietic regulators. Further analysis by GSEA revealed that hematopoietic and cytokine pathways are among those that are highly enriched in PMF Mks. We recently reported that the molecules dimethylfasudil (diMF) and MLN9237 are able to selectively increase ploidy, Mk surface marker expression, and apoptosis of malignant Mks. We treated Mks derived from PMF progenitor cells with diMF and observed a high increase in polyploidization accompanied with a reduction of Mks proliferation. Thus, diMF is able to partially restore Mk differentiation of PMF cells, supporting the testing of polyploidy inducers in myelofibrosis patients. Disclosures: Pardanani: Sanofi-Aventis: Clinical trial support Other; YM BioSciences: Clinical trial support, Clinical trial support Other; Bristol-Myers Squibb: Clinical trial support, Clinical trial support Other.

scholarly journals Colony formation of clone-sorted human hematopoietic progenitors

Blood ◽  
1990 ◽  
Vol 75 (10) ◽  
pp. 1941-1946 ◽  
Author(s):  
H Ema ◽  
T Suda ◽  
Y Miura ◽  
H Nakauchi
Keyword(s):  
Single Cell ◽  
Cell Cultures ◽  
Mononuclear Cells ◽  
Direct Action ◽  
Single Cells ◽  
Accurate Estimation ◽  
Hematopoietic Progenitors ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Sorting System

Abstract To characterize human hematopoietic progenitors, we performed methylcellulose cultures of single cells isolated from a population of CD34+ cells by fluorescence-activated cell-sorting (FACS) clone-sorting system. CD34+ cells were detected in bone marrow (BM) and peripheral blood (PB) cells at incidences of 1.0% and 0.01% of total mononuclear cells, respectively. Single cell cultures revealed that approximately 37% of BM CD34+ cells formed colonies in the presence of phytohemagglutinin-leukocyte conditioned medium and erythropoietin. Erythroid bursts-, granulocyte-macrophage (GM) colony-, and pure macrophage (Mac) colony-forming cells were 10% each in CD34+ cells. Approximately 15% of PB CD34+ cells formed colonies in which erythroid bursts were predominant. CD34+ cells were heterogeneous and fractionated by several antibodies in FACS multicolor analysis. In these fractionated cells, CD34+, CD33+ cells formed GM and Mac colonies 7 to 10 times as often as CD34+, CD33- cells. Most of the erythroid bursts and colonies were observed in the fraction of CD34+, CD13- cells or CD34+, CD33- cells. The expression of HLA-DR on CD34+ cells was not related to the incidence, size, or type of colonies. There was no difference in the phenotypical heterogeneity of CD34+ cells between BM and PB. About 10% of CD34+ cells were able to form G colonies in response to granulocyte colony-stimulating factor (G-CSF) and to form Mac colonies in GM-CSF or interleukin-3 (IL-3). Progenitors capable of generating colonies by stimulation of G-CSF were more enriched in CD34+, CD33+ fraction than in CD34+, CD33- fraction. Thus, single cell cultures using the FACS clone-sorting system provide an accurate estimation of hematopoietic progenitors and an assay system for direct action of colony-stimulating factors.

Expression of HMGA2 in PB Leukocytes and Purified CD34+ Cells from Normal Individuals and Patients with Myelofibrosis and Myeloid Metaplasia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2435-2435
Author(s):  
Joris R. Andrieux ◽  
Chrystèle Bilhou-Nabera ◽  
Jean Loup Demory ◽  
Olivier Pierre-Louis ◽  
Brigitte Dupriez ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Hematologic Malignancy ◽  
Consensus Conference ◽  
Myeloid Metaplasia ◽  
Clonal Proliferation ◽  
Low Concentrations ◽  
Healthy Donors ◽  
Normal Individuals ◽  
Cd34 Cells ◽  
Enhanced Expression

Abstract Background: Myelofibrosis with myeloïd metaplasia (MMM) is a rare myeloproliferative disorder which associates clonal proliferation of multipotent hematopoietic progenitors and reactive fibrosis. Deregulation and overexpression of HMGA2 has recently been demonstrated in MMM. Using quantitative RT-PCR on blood mononuclear cells from 27 patients, we found various levels of expression. It could be hypothesized that HMGA2 expression reflected the variable increase in circulating CD34+ progenitors in MMM though we failed to establish significant correlation between the level of expression and the blood enumeration of CD34+ progenitors. Material and Methods: We studied 24 patients diagnosed as primary MMM according to the set of criteria recently updated through an Italian Consensus Conference. Six individuals free from hematologic malignancy were used as controls. We also studied-purified CD34+ cells: 8 samples were isolated from the PB of MMM patients; given the very low concentrations of progenitors in normal PB, 2 samples pooling 8 healthy donors each were also prepared. In addition, 7 CD34+ cell samples were purified from the BM of consenting subjects collected during surgery for hip replacement. Results: We assayed HMGA2 expression in purified CD34+ populations from MMM patients and controls: both expressed HMGA2 but markedly more the CD34+ cells from the patients. We also evidenced transcripts in their CD15+ granulocytic cells suggesting that the enhanced expression of the gene affects the whole pathological clone. HMGA2, not expressed in normal blood cells, is involved in benign solid tumors of mesenchymal origin. Conclusion: Our data support the hypothesis that its reactivation in the clonal progenitors contribute to the pathogenesis of MMM.

scholarly journals Association of Bax Expression and Bcl2/Bax Ratio with Clinical and Molecular Prognostic Markers in Chronic Lymphocytic Leukemia

2016 ◽  
Vol 35 (2) ◽  
pp. 150-157 ◽  
Author(s):  
Ksenija Vucicevic ◽  
Vladimir Jakovljevic ◽  
Natasa Colovic ◽  
Natasa Tosic ◽  
Tatjana Kostic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Mononuclear Cells ◽  
Prognostic Markers ◽  
Statistical Significance ◽  
Genetic Alterations ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Aberrant Expression ◽  
Mutational Status

Summary Background: In chronic lymphocytic leukemia (CLL), in vivo apoptotic resistance of malignant B lymphocytes results, in part, from the intrinsic defects of their apoptotic machinery. These include genetic alterations and aberrant expression of many apoptosis regulators, among which the Bcl2 family members play a central role. Aim: The aim of this study was to investigate the association of pro-apoptotic Bax gene expression and Bcl2/Bax ratio with the clinical features of CLL patients as well as with molecular prognostic markers, namely the mutational status of rearranged immunoglobulin heavy variable (IGHV) genes and lipoprotein lipase (LPL) gene expression. Methods: We analyzed the expression of Bax mRNA and Bcl2/Bax mRNA ratio in the peripheral blood mononuclear cells of 58 unselected CLL patients and 10 healthy controls by the quantitative reverse-transcriptase polymerase chain reaction. Results: We detected significant Bax gene overexpression in CLL samples compared to non-leukemic samples (p=0.003), as well as an elevated Bcl2/Bax ratio (p=<0.001). Regarding the association with prognostic markers, the Bcl2/Bax ratio showed a negative correlation to lymphocyte doubling time (r=−0.307; p=0.0451), while high-level Bax expression was associated with LPL-positive status (p=0.035). Both the expression of Bax and Bcl2/Bax ratio were higher in patients with unmutated vs. mutated IGHV rearrangements, but this difference did not reach statistical significance. Conclusions: Our results suggest that dysregulated expression of Bcl2 and Bax, which leads to a high Bcl2/Bax ratio in leukemic cells, contributes to the pathogenesis and clinical course of CLL.

Gene Expression Profile Reveals a Unique Pattern of Protein Kinases in Chronic Lymphocytic Leukemia (CLL): Potential Role of the Second Generation Protein Kinase Inhibitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3117-3117
Author(s):  
Simona Tavolaro ◽  
Sabina Chiaretti ◽  
Monica Messina ◽  
Nadia Peragine ◽  
Roberta Maggio ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kinase Inhibitors ◽  
Lymphocytic Leukemia ◽  
Pcr Analysis ◽  
Mutational Status ◽  
Healthy Donors ◽  
Igvh Mutational Status ◽  
B Lineage

Abstract Purpose. CLL is a malignancy of mature B cells with an heterogeneous clinical course. In order to identify novel therapeutic targets and given the role of protein kinase (PK) deregulation in cancer development and the availability of specific PK inhibitors, oligonucleotide arrays were used to determine if CLL patients exhibit a specific PK pattern. The results were validated by Q-PCR, and the in vitro efficacy of a dual specific inhibitor, dasatinib (Bristol-Myers Squibb, Princeton, NJ), was tested. Methods. The gene expression profile of 44 CLL and 137 acute lymphocytic leukemia (ALL) patients was evaluated using the HG U133 Plus 2.0 Affymetrix arrays. Two additional sets of CLL (49 cases) were utilized as test sets: 505 PK genes were used for all analyses, which included unsupervised clustering, Analysis of Variance (ANOVA) and t-test analysis. To validate the gene expression data, a Q-PCR approach was used on 14 CLL, 6 B-lineage ALL, 3 T-ALL and CD19+ enriched normal B cells, isolated from peripheral blood of 3 healthy donors. Finally, to evaluate the in vitro effects of dasatinib on primary CLL cells, the MTT assay was performed on 21 untreated CLL samples following 72 and 96 hours of drug exposure. Results. Unsupervised analysis on CLL samples and different ALL subgroups showed a very homogeneous PK gene profile in CLL. ANOVA corroborated these results. Among the PK that proved overexpressed in CLL, we identified 16 PK genes highly expressed in all 3 CLL sets analyzed. For these genes, Q-PCR analysis showed that CLL cases display a significantly higher expression level, when compared to B-lineage ALL, T-ALL and, more importantly, to CD19+ cells from healthy controls. PK expression was also analyzed within different CLL subclasses, subdivided on the basis of the IgVH mutational status, as well as CD38 and ZAP-70 expression. The comparison among these groups did not show a specific PK signature associated with biologic features; similar results were obtained by Q-PCR analysis. To validate the gene expression profiling and Q-PCR findings, and to test the efficacy of dasatinib, functional in vitro experiments were performed on primary CLL cells. Treatment with 1μM dasatinib induced an overall viability reduction of 40% after 96 hours in the CLL samples analyzed; no significant differences were associated with the IgVH mutational status. These findings indicate that this inhibitor is functionally active on CLL cells, without however reaching marked levels of cytotoxicity when used as a single agent. Conclusions. Our results highlight a very homogeneous PK signature in CLL, independently of specific biologically-defined prognostic subgroups. A set of these PKs is more highly expressed in CLL cells than in CD19+ cells from healthy donors. Dasatinib treatment induces a ∼40% viability reduction after 96 hours exposure. Overall, these results suggest that PK inhibitors, namely dual kinase inhibitors, may have a role in the management of CLL patients particularly in combination with other therapeutic agents. * ST and SC equally contributed to the study

Lapdesf 1 a Compound Hybrid of Hydroxyrea and Thalidomide Increases Gamma Globin Expression in the Human CD34+ Cultures and Reduces Chemotactic Activity in Neutrophils

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2124-2124
Author(s):  
Sheley Gambero ◽  
Carolina Lanaro ◽  
Flavia Rubia Pallis ◽  
Carla Fernanda Franco-Penteado ◽  
Lidia Moreira Lima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Cell Cultures ◽  
Blood Cells ◽  
Mononuclear Cells ◽  
Globin Gene ◽  
Chemotactic Activity ◽  
Significant Difference ◽  
Globin Gene Expression

Abstract Abstract 2124 Sickle cell anemia (SCA), a disorder in which the inheritance of the gene codifies abnormal hemoglobin S (HbS), leads to Hb polymerization, causing a series of cellular alterations in the red cell. Fetal hemoglobin (HbF) is a modulator of clinical and hematologic features. Higher HbF levels are associated with a reduced rate of acute painful episodes, less frequent acute chest syndromes and protection against morbidity and mortality. Previous results showed that Lapdesf1(2-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]ethyl nitrate, a novel compound that links hydroxyurea and thalidomide's phtalimide, increases HbF and has an anti-inflammatory effect in sickle mice. The aim of this study to evaluate whether Lapdesf1 induces HbF gene expression in CD34+ cell cultures. We also evaluated the chemotaxis and production of reactive oxygen species (ROS) in neutrophils treated in vitro with Lapdesf1.CD34+cell cultures from 8 healthy volunteers were treated with Lapdesf1 on day 9 and the gama-globin (γ-globin) gene expression was evaluated on day 13 by Real Time PCR. We used, as a positive control, Hydroxyurea (HU), thalidomide and both together. Neutrophils, platelets, mononuclear cells and red blood cells were isolated from peripheral blood samples of healthy controls and patients with SCA (Transfusion-independent patients, not on hydroxyurea treatment). ROS measurement was performed by the incubation with 2'-7'-dichlorofluorecin diacetate (DCFH) and analyzed by flow cytometry. Spontaneous and IL-8-induced neutrophils chemotaxis were assessed using a 96-well chemotaxis chamber assay (ChemoTXNeuroprobe). Data were analyzed statistically using ANOVA followed by Dunnett's test, where a P value of less than 0.05 was considered to be significant. The study was approved by the Research Ethics Committee of the Faculty of Medical Sciences of University of Campinas (UNICAMP).Lapdesf1 (5μM) increased γ-globin gene expression, compared with that of the control (1.85 ± 0.54 vs 0.66 ± 0.16, P<0.05, n=8). HU and thalidomide also increased at 100μM (1.81 ± 0.13; 1.85 ± 0.06, P<0.05, n=3), however treatment with HU and thalidomide together did not increase γ-globin gene expression (0.99 ± 0.04, P<0.05, n=3). There was no significant difference in the ROS production in platelets, red blood cells, mononuclear cells and neutrophils, treated with Lapdesf1. In addition, no significant differences were observed in the chemotaxis of controls and SCA neutrophils. For IL-8-induced chemotaxis, treatment with Lapdesf1 reduced the chemotactic activity at 300 and 600μM (3.94 ± 1.17; 3.86 ± 1.06 respectivelyvs 21.76 ± 6.06, P<0.05, n=3) in controls and 300μM (4.96 ± 0.59 vs 17.54 ± 7.12, P<0.05, n=3) in SCA compared with IL-8 induced control. Our results showed that the Lapdesf1 is capable of inducing CD34+ cell γ-globin gene expression at a low concentration and reducing chemotactic activity. Even though further studies are needed, these results suggest that Lapdesf1 may be a promising drug candidate that may provide multiple beneficial actions in the treatment of sickle cell disease symptoms and offering an alternative drug therapy. This work was supported by FAPESP, INCT and CNPq. Disclosures: No relevant conflicts of interest to declare.

Analysis of T-helper Responses and FOXP3 Gene Expression in Patients with Japanese Cedar Pollinosis

2008 ◽  
Vol 22 (6) ◽  
pp. 582-588 ◽  
Author(s):  
Kazuaki Chikamatsu ◽  
Koichi Sakakura ◽  
Tomokazu Matsuoka ◽  
Shuichiro Endo ◽  
Goro Takahashi ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Immune Responses ◽  
Mononuclear Cells ◽  
Japanese Cedar ◽  
T Helper ◽  
Foxp3 Gene ◽  
Healthy Donors ◽  
Japanese Cedar Pollinosis

Background Evidence has been accumulated indicating that regulatory T (T-reg) cells play a crucial role in the maintenance of peripheral T-cell tolerance to allergens. To explore the role of FOXP3, which is required for the development of T-reg cells, in allergen-specific immune responses, we examined the relationship between the alteration of FOXP3 gene expression and in vitro immune responses against allergens. Methods Peripheral blood mononuclear cells obtained from 19 human histocompatibility leukocyte antigens (HLA)-DPB1*0501 donors, including patients with Japanese cedar pollinosis and nonallergic healthy donors, were stimulated with Cry j 1 p61-75 peptide. On day 7, T cells were tested for peptide-specific reactivity in IFN-γ and interleukin (IL)-5 enzyme-linked immunospot (ELISPOT) assays. Real-time quantitative RT-PCR was performed to assess relative change of FOXP3 gene expression before and after in vitro stimulation. Neutralization assays using anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) and anti-IL-10 monoclonal antibody were also performed. Results Of 14 patients with allergic pollinosis tested, 10 responders displayed T-helper type 2 (Th2)-polarized reactivity to Cry j 1 p61-75, and 2 donors showed Th0 responses. Notably, the change of FOXP3 gene expression in donors showing peptide-specific T-helper responses was significantly lower than that in nonresponders, regardless of allergic pollinosis. Conclusion Our data indicate that FOXP3 is functional in nonallergic healthy donors as well as allergic patients, and FOXP3-expressing T cells may be responsible for the down-regulation of allergen-specific T-helper responses in individuals. A better understanding of the nature and specificity of FOXP3-expressing T cells in a suppressive mechanism is necessary to develop new immunotherapies against allergic rhinitis.

scholarly journals The Cell Surface NADH Oxidase ENOX2 Is Highly Expressed in Chronic Myeloid Leukemia (CML): a Redox Protein As a Secreted Surrogate Biomarker and Druggable Target

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3059-3059
Author(s):  
Seda Baykal ◽  
Maud Voldoire ◽  
Christophe Desterke ◽  
Nathalie Sorel ◽  
Hyacinthe Atchroue Johnson Ansah ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Pattern Matching ◽  
Nicotinamide Adenine Dinucleotide ◽  
Drug Target ◽  
Chronic Phase ◽  
P Value ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Nadh Ratio

Abstract Ecto-nicotinamide Adenine Dinucleotide Oxidase 2 (ENOX2) or Tumor-Associated Nicotinamide Adenine Dinucleotide Oxidase (tNOX), plays a major role as a membrane hydroxyquinone oxidase catalyzing the conversion of reduced NADH to the oxidized NAD+ .Cellular NAD+/NADH ratio plays a major role in the regulation of several metabolic pathways such cell cycle and apoptosis. The membrane NAD+/NADH ratio has been shown to be involved membrane lipid and sphingomyelin metabolism. Several lines of experimental data indicate that ENOX2, as compared to ENOX1 isoform, is preferentially expressed in cancer cells and ENOX2 is also secreted in the serum of patients with cancer and representing a surrogate marker for cancer progression as well as a drug target. The expression of ENOX2 in CML has not been studied so far. To this end, we have analyzed the expression of ENOX2 by Western blots in 40 patients with CML at diagnosis and compared this expression to that of control cells from healthy donors (n= 30). ENOX2 expression was found to be highly increased ( x 6.6 fold) in primary leukemic cells and this was highly significant ( p= 0.0081). To determine if ENOX2 expression is related directly to BCR-ABL expression, we have analyzed the expression of ENOX2 in UT7 cell line and its BCR-ABL-expressing counterparts UT7-11. As compared to parental UT7, ENOX2 expression was highly increased in UT7 cells expressing either native or T315I-mutated BCR-ABL. This increase was also documented in DOX- inducible cell line BaF/p210 sin1.55 in which activation of BCR-ABL expression correlated with ENOX2 expression. The expression of ENOX2 in CML cells was a tyrosine kinase-dependent event as demonstrated by Western blot experiments showing that ENOX2 expression was reduced UT7/11 cell line treated with Imatinib mesylate (1 microM) for 6, 18 and 24 hours whereas there was no change in ENOX2 expression in similarly treated parental UT7 cell line. As ENOX2 is a protein secreted from tumor cells, we have analyzed the levels of ENOX2 in the plasma of CML patients at diagnosis as compared to controls. A series of 41 patients with CML as compared to plasma from 28 healthy donors were analyzed by ELISA. This analysis showed a highly significant increase of ENOX2 protein levels in the plasma of patients with CML (mean levels of 800 pg/ml in CML versus 500 pg/ml, Mann-Whitney U-Test, p < 0.0001). There was no correlation between ENOX2 levels and leukocyte numbers at diagnosis. In order to determine the potential clinical value of ENOX2 expression in different phases of the disease, ENOX2 expression in CML CD34+ cells was compared to healthy donor samples in microarray dataset GSE 4170. CML patients in chronic phase overexpressed ENOX2 in CD34+ cells as compared to control (two-sided test with Welch correction p-value=0.00054). Gene expression pattern matching correlating ENOX2 in CML CD34+ cells was determined in three phases of the disease. Pavlidis template matching algorithm used with ENOX2 as predictor allowed to discover 301 genes correlated with CML in chronic phase. Unsupervised principal component analysis performed with ENOX2 pattern matching gene expression profile allowed us to discriminate the 3 phases of CML in CD34+ cell compartment in a highly significant manner (p-value 6.75E-15). Functional enrichment performed with ENOX2 pattern matching on Gene Ontology Biological Process database revealed implication of different pathways of cell signaling such as: Rho GTPase, MAPKs, GPCR, RAS and NOTCH pathways, the latter being connected to stem cell biology. This analysis also showed implication of metabolic functions such as carbohydrate homeostasis. Other functionalities that could act on hematopoiesis have been also highlighted by this analysis such as proliferation, integrin-mediated signaling, and circadian rhythm. Finally genes related to angiogenesis have been also found to be implicated in ENOX2 signalling such as placental growth factor (PLGF) also EPHB3 which is a receptor tyrosine kinase implicated in cell migration. Overall these results suggest that ENOX2 pathway plays a major role in the pathogenesis of CML and represents, to the best of our knowledge, the first surrogate secreted tumor marker in CML. ENOX2 expression correlates with disease progression and experiments are underway to determine the use of ENOX2 as a drug target in CML and T315I-mutated CML stem cells. Figure Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 958-970 ◽  
Author(s):  
CE Sandstrom ◽  
JG Bender ◽  
ET Papoutsakis ◽  
WM Miller
Keyword(s):  
Cell Cultures ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Similar Distribution ◽  
Peripheral Blood Mononuclear ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells

Ex vivo expansion of peripheral blood mononuclear cells (MNCs), cultured both directly and after selection for CD34+ cells, was compared in static and continuously perfused cultures containing interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G- CSF), and stem cell factor (SCF). Cultures inoculated with either MNCs or CD34+ cells produced cells that were remarkably similar after 10 days of culture, as evidence by cell morphology, expression of CD34, CD33, CD15, and CD11b, and the fractions of cells giving rise to colony- forming units granulocyte-monocyte (CFU-GM) and long-term culture- initiating cells (LTC-IC). Static and perfusion cultures gave similar average total cells and CFU-GM expansions for both MNC and CD34+ cell cultures. However, those samples that performed poorly in static culture performed at near-normal levels in perfusion. In addition, perfusion supported higher LTC-IC numbers for both MNC and CD34+ cell cultures. While total cell expansion was about ten times greater in CD34+ cell cultures (approximately 100-fold), CFU-GM expansion (approximately 20-fold) was similar for both MNC and CD34+ cell cultures. The similar distribution of cell types produced in MNC and CD34+ cell cultures allows direct comparison of total and colony- forming cell production. After 15 days in perfusion, MNC cultures produced 1.5-, 2.6-, and 2.1-fold more total cells, CFU-GM, and LTC-IC, respectively, than the same sample selected and cultured as CD34+ cells. Even if the CD34+ selection process was 100% efficient, CFU-GM production would be 1.5-fold greater for MNCs than for CD34+ cells.

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