The Study Of The Effect and Mechanism Of Chemotherapy Plus Granulocyte Colony Stimulating Factor (DAG) for Refractory Paroxysmal Nocturnal Hemoglobinuria In Vitro

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4717-4717
Author(s):  
Zonghong Shao ◽  
Xifeng Dong ◽  
Rong Fu
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Death Rate ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Cell Cycle Kinetics ◽  
Apoptosis Rate ◽  
Significant Difference ◽  
Vitro Effect

Objective To compare the response of GPI-AP negative or positive bone marrow mononuelear cells(BMMNCs) from PNH patients to DAG in vitro and explore the related mechanism. Methods Seventeen PNH patients as well as fourteen normal controls were enrolled. CD59-/CD59+ cells were sorted by magnetic activated cell-sorting system. Then the cells were incubated in IMDM medium containing several hemapoitic growth factors with DAG or G-CSF for 48h in vitro. The cell cycle kinetics and apoptosis of these cells were detected by flow cytometry(FCM). The expressions of CD114 on CD34+CD59- and CD34+CD59+ bone marrow cells(BMC) after incubated with G-CSF were measured by FCM. And another 14 PNH peripheral blood samples were obtained, the expression of CD44/CD49d on CD59- and CD59+ cells were analyzed by FCM respectively. The mRNA of CD114 and CD44/CD49d was also tested in 22 PNH patients vs 14 controls and CD59- vs CD59+ cells from 14 PNH patients by Q-PCR. Results After incubated with DAG for 48h in vitro, the death rate and apoptosis rate for GPI-AP negative and positive cells(CD59-/CD59+ BMMNCs cells): for CD59- BMMNCs, compared with control group, the death rate of DAG group increased (27.29±22.04% vs 19.10±20.93%), apoptosis rate also increased(10.55±12.34% vs 7.2±6.76%), there was no significant difference for them; for CD59+ BMMNCs, compared with control, the death rate of cells from DAG group increased significantly (31.89±26.75% vs 12.83±18.92%)(P<0.05), whereas there was no significant difference for the apoptosis rate (9.66±7.96% vs 6.31±1.32%); for the CD59- and CD59+ BMMNCs, the death rate was significant higher than apoptosis rate respectively(P<0.05). For cell cycle kinetics, there was no significant difference between the two kinds of BMMNCs. As to the percentage of CD114, compared with control group, it increased significantly in CD34+CD59+ BMMNCs from G-CSF group (48.12±41.20% vs 12.84±15.32%) (P<0.05), whereas there was no significant difference for CD34+CD59- BMMNCs (41.76±44.62% vs 26.79±41.62%). And the variation of CD114 for CD34+CD59+ BMMNCs was higher than that for CD34+CD59- BMMNCs(33.97±36.03% vs 14.88±27.02%)(P<0.05). The expression of CD44/CD49d protein: the expression of CD44 for CD59+was higher than that for CD59- cells(97.66±4.21% vs 93.46±9.52%, P<0.05); and there was no significant difference for CD49d expression in CD59- and CD59+ cells(38.46±27.37% vs 43.79±24.77%). The mRNA expressions of CD114, CD44 and CD49d in 22 PNH patients compared with 15 control and CD59- cells compared with CD59+ cells from 14 PNH patients : for CD114, its mRNA expression was higer for CD59+ cells compared with that for CD59- cells(2.78±2.52 vs 1.69±2.34, P<0.05), but there was no significant difference for CD114 in PNH patients and controls; for CD44, the significant difference exited for PNH patients compared with controls and CD59- cells compared with CD59+ cells(1.73±2.20 vs 3.80±3.87, P<0.05; 0.82±0.75 vs 2.38±2.42, P<0.05); for CD49d, no significant difference exited for PNH patients compared with controls and CD59- cells compared with CD59+ cells(2.83±2.62 vs 2.56±3.04; 1.74±2.60 vs 1.94±3.02). Conclusions In vitro, effect of DAG was similar on CD59- and CD59+ BMMNCs, the style of death was necrosis not apotosis and the cell cycle was not influenced by DAG. The variation of CD114 for CD34+CD59- after G-CSF stimulation was less than that in CD34+CD59+ cells, and mRNA of CD114 was lower in CD59- cells compared with CD59+ cells, which may indicating the mechanism for the remission of PNH patients after DAG chemotherapy. The protein and mRNA of CD44 was lower in PNH patients and CD59- cells compared with control and CD59+ cells respectively, which may explain the inferior growth of PNH cells, because they can not fully use the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

IL-1β Plays An Important Role On Thrombocytosis In An Immune Vasculitis Model Via Promoting Megakaryopoiesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2325-2325
Author(s):  
Mo Yang ◽  
Min Zhou ◽  
Su yi Li ◽  
Beng Chong ◽  
Xiao jing Li
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Platelet Aggregation ◽  
Conflicts Of Interest ◽  
Serum Levels ◽  
Control Group ◽  
Type I ◽  
Anti Inflammation ◽  
Immune Vasculitis

Abstract Thrombocytosis and inflammation cytokines may be involved in the pathogenesis of vasculitis. Our previous study have showed that major inflammation cytokine IL-1β play an important role on in-vitro megakaryopoiesis (Yang M et al, Br J Haematol 2000). In this study, we investigated the changes of IL-1β and megakaryopoiesis and the effect of aspirin in an immune vasculitis model. Rabbit immune vasculitis model was established by intravenous injection of bovine serum albumin. In this model, platelet number and function of periphery blood, megakaryocyte number and the CFU-MK formation of the bone marrow, and serum levels of inflammatory cytokines were investigated. After treatment with BSA for 7 days, the platelet count, platelet aggregation and the expression of AnnexinⅤ were significantly increased in this vasculitis model group compared with normal control group (n=6). The serum levels of inflammatory cytokine IL-1β was also significantly higher in vasculitis model. There were positive correlations between platelet count and IL-1β levels (R=0.55), platelet aggregation and IL-1β levels (R=0.603). Treatment with aspirin (100 mg/kg/d) significantly decreased all these parameters, showing aspirin had anti-platelets and anti-inflammation effects. Our results also demonstrated that megakaryocyte number and the formation of CFU-MK were significantly increased in vasculitis group as compared to those in normal group. Treatment with aspirin significantly reduced the number of megakaryocytes and the formations of CFU-MK in bone marrow in this immune vasculitis model. Our study further demonstrated that IL-1β alone or in combination with TPO induced in-vitro CFU-MK formation. Using RT-PCR techniques, the mRNA of of IL-1 type I and type II receptors (IL-1 RI and RII) were detected in cultured CD61+ CD41+ cells and four megakaryocytic cell lines. The expression of IL-1 RI and RII was also confirmed by flow cytometry and immunofluorescence staining in bone marrow megakaryocytes. Moreover, the IL-1R bloker can reduced IL-1β induced megakaryopoiesis. This sudy showed that IL-1β may play an important role in the pathogenesis of immune vasculitis. Aspirin has anti-inflammation effects in this model which may be mediated via inhibiting megakaryopoiesis and platelet formation. Disclosures: No relevant conflicts of interest to declare.

Utility of Telomere in Diagnosis of Bone Marrow Failure Syndromes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4793-4793
Author(s):  
Hasan Ahmed Abdel-ghaffar ◽  
Hosam Zaghloul ◽  
Ahmed El-Waseef ◽  
Mohamed El-Naggar ◽  
Mohamed Mabed ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Telomere Length ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Quantitative Polymerase Chain Reaction ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Relative Telomere Length ◽  
Bone Marrow Failure Syndromes ◽  
Significant Difference

Abstract Background and aim of the work: Bone marrow failure syndromes (BMFS) includes inherited and acquired conditions. Inherited bone marrow failure includes a number of syndromes; with Fanconi anemia (FA) being the most common one of them. Telomeres are eroded with cell division, but in hematopoietic stem cell, maintenance of their length is mediated by telomerase. Short telomeres can result in instability of cell function where diseases occur. Bone Marrow Failure might be developed due to low telomerase activity or short telomeres. Our study is aiming to evaluate the utility of Real Time Quantitative-Polymerase Chain Reaction (RT-qPCR) in measuring the relative telomere length and its significance in diagnosis and prognosis of patients with BMFS. Materials and methods: The study includes 3 groups: A group of congenital BMF (29 patients), a group of acquired BMF (10 patients) and a third control group (15 cases). The relative telomere length is evaluated for them using RT-qPCR. Results: We have found that there is a significant difference in relative telomere length between congenital group and controls (p=0.001), also a significant difference between acquired group and controls (p= 0.029). However, there is no significant difference between congenital and acquired groups (p= 0.479). There is no significant correlation between the telomere length and the overall survival or prognosis of the patients of BMFS. Conclusion: We conclude that the telomere length is significantly altered in patients with BMFS whether being congenital or acquired compared to the control group. Disclosures No relevant conflicts of interest to declare.

Autophagy Regulation and Dysfunction in Bone Marrow Malignancies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4623-4623
Author(s):  
Fernando V Pericole ◽  
Mariana Lazarini ◽  
Adriana S. S. Duarte ◽  
João Machado-Neto ◽  
Sara T. Olalla Saad
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Low Risk ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Combination Treatments ◽  
Molecular Machinery ◽  
Total Bone Marrow

Abstract Abstract 4623 Introduction: Autophagy is a catabolic pathway by which cytoplasmic materials are degraded into the lysosome and it is also a quality control system for proteins and organelles. Autophagy plays an important role in cell adaptation to starvation, hypoxia, cell survival and cancer. Its core molecular machinery is tightly linked to metabolic pathways, such as LKB1/AMPK and mTORC1. Autophagy has been shown to play several important roles in cancer. Indeed, multiple autophagy genes have been characterized as tumor suppressor genes. In hematopoietic system, autophagy is required during myeloid and lymphoid differentiation, terminal erythroid mitochondrial clearance, production of proplatelets and also differentiation of monocytes into macrophages. Interestingly, autophagy seems disturbed in most bone marrow malignancies. Evidence in mice suggests that autophagy suppression (ATG7 or ATG5 knockdown models) in hematopoietic stem cells may be implicated in Acute Myeloid Leukemia (AML) pathogenesis. In Multiple Myeloma (MM), in vitro studies using cell lines showed autophagy activation and lysosome inhibitors (such as chloroquine) are currently been used in various combination treatments in clinical trials. Aim: The aim was to characterize the expression of autophagy machinery key genes (BECN1, MAP1LC3A, SQSTM1), as well as hypoxia master regulator (HIF1A) in total bone marrow cells from bone marrow malignancies: myelodysplasia (MDS), MM and AML patients, excluding acute promyelocytic leukemia. Methods: BECN1, MAP1LC3A, SQSTM1 and HIF1A levels were verified, by q-PCR, in diagnostic (or without any treatment) BM aspirates from 22 normal donors, 30 MDS (17 low-risk and 13 high-risk, according 2008 WHO classification), 43 AML and 11 MM patients. Results: BECN1 gene expression was increased in MM, compared with control group. All other groups did not differ from the control group. Comparing diseases amongst each other, AML had a lower BECN1 expression, compared with low-risk MDS and with MM (Figure 1A). Disclosures: No relevant conflicts of interest to declare.

Study on Telomerase Activity of Human Bone Marrow Mesenchymal Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4255-4255
Author(s):  
Jingyuan Li ◽  
Xiaoyu Lai ◽  
Huang He
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Telomerase Activity ◽  
Negative Control ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Significant Difference

Abstract Human mesenchymal stem cells(hMSCs) have multiple differentiate potential, and it can differentiate into adipocytes, osteogenic cells, chondrocyte and neural cells et al. It has been reported that telomerase activity in hMSCs is negative, but it is still controversial and telomerase activity in hMSCs-derived adipocytes has not been reported. We investigate the telomerase activity in hMSCs before and after their committed differentiation into adipocytes in vitro and cryopreservation. hMSCs were isolated from normal human bone marrow fellowed by cell culture in DMEM with low glucose containing 10% FBS. The FACS was performed to examine the expression of cell surface molecules and analyze cell cycle of primary hMSCs.Then some of hMSCs were induced to differentiate into adipocytes in vitro by being treated with adipocytic medium fellowed by being stained with oil red O, and the others were cryopreserved in liguid nitrogon for three months. TRAP assay(telomerase repeat amplification protocol assay)was employed to detect telomerase activity in those hMSCs. T293 cells and α-Interferon were analyzed with each test as an additional positive control and negative control respectively. Telomerase activity was expressed as a percentage of the relative telomerase activity (RTA) of the hMSCs relative to the RTA of T293 cells. The results indicated the cells were positive for SH2, SH3, CD90 and negative for CD34, CD45, AC133. It was showed that the majority of primary hMSCs(85%) was at cell cycle of G0/G1 phase and the minority of hMSCs was at S, G2 or M phase. 80% hMSCs was orange adipocytes after they were treated with adipocytic medium for 3–4weeks. Telomerase activity was negative in hMSCs both at the beginning of culture and at the later stages during cell expansion,telomerase activity in hMSCs-passage 1–3(n=10) and hMSCs-passage 4–7(n=9) made no significant difference(1.46±0.83% vs 1.46±0.67%, p=0.99). Cryopreservation did not affect the telomerase activity in hMSCs. Telomerase activity in fresh hMSCs(n=13) and frozen hMSCs(n=6) made no significant difference(1.41±0.44% vs 1.51±1.07%, p=0.64). Telomerase activity in hMSCs-derived adipocytes(n=3) was significantly higher than in hMSCs(n=19)( 11.8±2.52% vs 1.46. ±0.67%, p&lt;0.00001). It is concluded that hMSCs are telomerase-negative, and the stage of culture or cryopreservation does not affect their telomerase activity. After being induced to differentiated into adipocytes, hMSCs telomerase activity is upregulated.

ROCK1 Functions as a Suppressor of Inflammatory Cell Migration by Regulating PTEN Phosphorylation and Stability.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 240-240
Author(s):  
Sasidhar Vemula ◽  
Jianjian Shi ◽  
Philip Hanneman ◽  
Lei Wei ◽  
Reuben Kapur
Keyword(s):  
Bone Marrow ◽  
Acute Inflammation ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
Phagocytic Index ◽  
In Vivo Model ◽  
Significant Difference ◽  
Rho Kinases

Abstract Abstract 240 Neutrophils and macrophages are major cellular components of the innate immune response and are recruited rapidly in large numbers to sites of infection. The small family of Rho GTPases and its downstream effectors, Rho kinases (Rho-associated, coiled-coil containing protein kinase) have been implicated in regulating various cellular functions including actin cytoskeleton organization, cell adhesion, and cell motility in non-hematopoietic cells. Rho kinases (ROCK1 and ROCK2) belong to a family of serine/threonine kinases whose role in inflammation is not known. Here we show that deficiency of ROCK1 but not ROCK2 results in increased recruitment of macrophages (3.2 fold, n=8, *p<0.01) and neutrophils (3.4 fold, n=5 *p<0.05) compared to WT controls in an in vivo model of aseptic peritonitis. In vitro, deficiency of ROCK1 in bone marrow derived macrophages shows a significant increase in haptotactic transwell migration in response to M-CSF as well as MCP-1 on fibronectin as well as an increase in migration towards the wounded area in a wound healing assay compared to controls (∼3 fold, n=3, *p<0.005). Consistently, deficiency of ROCK1 in bone marrow derived neutrophils also shows a ∼2.63 fold increase in migration in response to fMLP compared to WT bone marrow derived neutrophils (BMNs) in a chemotactic migration assay. ROCK1 deficient macrophages also demonstrate a ∼2.5 fold increase in adhesion on fibronectin (n=3, *p<0.002). The enhanced migration and adhesion in ROCK1−/− macrophages was observed in spite of comparable expression of F4/80 (WT; 85.63% vs. ROCK1−/−; 88.68%, n=4), α4β1 and α5β1 integrins (WT; 67.49% & 88.2% vs. ROCK1−/−; 71.82 % & 87.09%, n=4), while no significant difference in the phagocytosis of sheep red blood cells was observed between WT and ROCK1−/− macrophages (Phagocytic index: WT; 98% vs. ROCK1−/− 97%, n=3, p>.05). Close examination of the cytoskeleton of ROCK1 deficient macrophages using confocal microscopy revealed more F-actin content on the entire cell surface compared to wildtype controls. Consistently, flow cytometric analysis using Alexa 488-phalloidin staining revealed abundance of F-actin in ROCK1−/− macrophages compared to WT controls (WT; 46.19% vs. ROCK1−/−; 65.23%, n=3, *p<0.05). Furthermore, immunofluorescence imaging of podosomes carried out using anti-vinculin antibody revealed more pronounced and increased podosomes in ROCK1 deficient macrophages compared to WT controls (n=3, *p<0.05). Biochemical analysis of ROCK1−/− macrophages revealed that the enhanced recruitment of ROCK1 deficient macrophages and neutrophils was apparent in spite of normal expression of ROCK2 in ROCK1−/− cells and a 60% reduction in overall ROCK activity. Interestingly, although both ROCK1 and ROCK2 co-immunoprecipitate with PTEN in response to cytokine induced stimulation, only ROCK1 appeared to be essential for PTEN phosphorylation, activation and stability. In the absence of ROCK1, PTEN phosphorylation, its activity and stability were significantly impaired in spite of the presence of ROCK2 (n=3, *p<0.05). Consequently, an increase in the activation of downstream targets of PTEN including AKT, GSK-3β and cyclinD1 was observed in ROCK1 deficient macrophages relative to controls (n=3). Taken together, these studies reveal a biochemical pathway involving ROCK1 and PTEN which is involved in the recruitment of macrophages and neutrophils during acute inflammation. Thus, ROCK1 likely functions as a physiologic regulator of PTEN whose function is to repress excessive recruitment of macrophages and neutrophils during acute inflammation. Disclosures: No relevant conflicts of interest to declare.

Engineering Biomimetic Culture Systems: Impact On Human Bone Marrow-Derived Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3628-3628
Author(s):  
Marina Prewitz ◽  
Friedrich Philipp Seib ◽  
Martin Bornhaeuser ◽  
Carsten Werner
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Human Peripheral Blood ◽  
Cell Lysis ◽  
Published Data ◽  
Differentiation Potential ◽  
Significant Difference ◽  
Defined Culture

Abstract Abstract 3628 Poster Board III-564 The bone marrow (BM) harbours haematopoietic stem/progenitor cells (HSCs) in anatomically distinct sites (niches) where HSCs are subjected to regulatory cues such as cytokines, cell-cell contacts and extra-cellular matrix (ECM) all of which control stem cell fate. In particular mesenchymal stromal cells (MSCs) are an integral part of the bone marrow and are known to be key regulators of the HSC niche. We have previously shown that bio-artificial scaffolds can have a significant impact on the in vitro behaviour of MSCs. Here, we are therefore focussing on the role of (native) ECM within the MSC-HSC microenvironment by building on our previous findings and published data (Seib et al.,Tissue Eng Part A., 2009 in press). Thus the aim of the current study is (a) to identify niche-specific ECM components and (b) the use of such ECMs for in vitro culture of BM-derived stem cells. To mimic the natural ECM composition of the BM, different ECM types were generated from BM-derived cells using (a) Dexter cultures, (b) standard MSC cultures, (c) MSCs subjected to osteogenic differentiation. After 10 days of culture those MSC-derived ECMs were decellularised using 0.5% Triton-X and 20mM NH4OH leaving only the ECM behind (verified by scanning electron microscopy). Those ECMs were used as a substrate for a second culture of MSCs, which were analysed for their proliferation and differentiation potential. Cell-free ECM from standard MSC cultures improved MSC proliferation compared to cells grown on regular tissue culture plastic (TCP) over the period of 8 days. Most notably, all cell-free ECM preparations lead to a significant difference in the cytoskeletal arrangement of MSCs during the first 2 days of culture compared to TCP controls. Cultivation of MSCs on native ECM provided a guiding structure for those cells to grow into, and helped to maintain an elongated cell shape compared to substantial cell spreading on TCP (roundness 0.2 versus 0.5 and cell area of 2.2 versus 8.2mm2, respectively, p<0.001, n=60. A factor of 1 was set to equate to a perfect circle). Next, we investigate if native ECM could either directly improve HSC cultures or maximise MSC feeder characteristics. For the latter set of studies MSCs were initially cultured for 7 days on cell-free ECM (from standard MSC cultures) and subsequently co-cultured with human peripheral blood CD34+ HSCs in serum free medium supplemented with cytokines (Tpo, Flt3, and SCF at 10ng/ml). Following a 14 day culture period up to 3.5-fold more CD34+ cells were present in ECM co-cultures compared to TCP co-cultures that was accompanied with an overall expansion of CD45+ cells of 109-fold versus 35-fold, respectively. Our data suggest that ECM preparations derived from MSCs might be useful to accomplish better expansion of HSCs under defined culture conditions. In addition, this system permits the identification of bimolecular key components that can be utilized in the future design of simple and robust carrier systems for improved HSC maintenance in vitro. Figure HSC-MSC co-culture on preformed ECM substrates. (A) MSC-derived ECM (from standard MSC culture) following cell lysis (complete absence of cells). (B) Growth of a new set of MSCs on ECM substrates as shown in (A). (C) HSC-MSC co-culture on ECM substrates. Scale bars at 2μm. Arrow heads point out ECM structures. Figure HSC-MSC co-culture on preformed ECM substrates. (A) MSC-derived ECM (from standard MSC culture) following cell lysis (complete absence of cells). (B) Growth of a new set of MSCs on ECM substrates as shown in (A). (C) HSC-MSC co-culture on ECM substrates. Scale bars at 2μm. Arrow heads point out ECM structures. Disclosures: No relevant conflicts of interest to declare.

Natalizumab Increases Circulating CD34+ Cells with An Impaired Functional Potential In Patients with Multiple Sclerosis without Affecting the Bone Marrow.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2620-2620
Author(s):  
Christian Saure ◽  
Fabian Zohren ◽  
Thomas Schroeder ◽  
Ingmar Bruns ◽  
Ron Patrick Cadeddu ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
T Cell Subsets ◽  
Cell Counts ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Long Term Treatment ◽  
Significant Difference

Abstract Abstract 2620 Introduction: In our previous report (Zohren et al., Blood 2008) we could show that the blockade of the heterodimer VLA-4 by the monoclonal IgG4 antibody natalizumab leads to a significant increase in circulating CD34+ cells in patients with multiple sclerosis (MS). We now extend our analysis on the influence of natalizumab on CD34+ cells comparing bone marrow (BM) and peripheral blood (PB) derived CD34+ cells of natalizumab patients with those from healthy donors. Methods: A total of 83 patients with MS receiving natalizumab were included. In vitro adhesion, migration and apoptosis assays as well as LTC-IC of immunomagnetically enriched CD34+ cells were conducted. Flow cytometric analyses were performed to assess phenotype and composition of the CD34+ subsets. Results: The median concentration of circulating CD34+ cells was significantly greater compared to normal donors (7.7/μL vs. 1.8/μ L; p= 0.0001) and remained relatively stable during a one year treatment with natalizumab. Leukocyte cell counts, the number of T cell subsets as well as the number of CD19+ B cells and CD56+ natural killer cells were in normal range in PB and BM after short- and long-term treatment with natalizumab. However, we found significantly reduced adhesion and migration abilities of circulating CD34+ cells under natalizumab treatment in comparison to G-CSF mobilized CD34+ cells of healthy donors. Moreover, the self-renewal capacity of these cells was poor. In contrast, no significant difference was seen between the BM of natalizumab patients and the BM of healthy donors with regard to cellularity and proportion of CD34+ cells. In addition, neither co-expression of CD49d nor the adhesion ability of the BM derived CD34+ cells revealed a significant difference between the two collective. Conclusions: Our data indicate that natalizumab mediates an increase in circulating CD34+ cells by impaired homing. These findings argue against the use of natalizumab-exposed PB CD34+ cells for transplantation. Disclosures: No relevant conflicts of interest to declare.

Pro- and Antiapoptotic Proteins Expression on Bone Marrow and Peripheral Blood CD34+Cells in Acute Leukemia (AL) Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4996-4996
Author(s):  
Elena E. Khodunova ◽  
Elena N Parovichnikova ◽  
Irina V. Galtzeva ◽  
Sergey M. Kulikov ◽  
Valeri G Savchenko
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Expression Level ◽  
Leukemia Cells ◽  
Control Group ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Blast Cells

Abstract Abstract 4996 It was shown that drug resistance, poor-risk cytogenetics and poor prognosis in AL is associated with high level of Bcl-2 expression and low Bax/Bcl-2 ratio (<0,3). Fas-antigen (CD95) as a protein triggering the extrinsic apoptotic pathway is differently expressed on hematopoietic precursors. More immature CD34+/CD38- AML blast cells have lower expression of Fas/Fas-L and lower Fas-induced apoptosis than CD34+/CD38+cells. CD34+/CD38− leukemia precursors also have a reduced sensitivity to daunorubicin in vitro and increased expression of multidrug resistance genes (mrp/lrp). CD34+ leukemia cells have not yet been properly characterized regarding the expression of angiotensin converting enzyme (ACE) which regulatory influence on hematopoiesis is now beeing extensively investigated. ACE expression on blast cells is high, but it's still unknown how CD34+ACE+ leukemia cells behave after chemotherapy. Recent publications indicate that CD34+ACE+ hematopoietic precursors transplanted into NOD/SCID mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+ACE- counterparts. We have studied the dynamics of Bcl-2, Bax, CD95 and ACE expression on CD34+ cells in peripheral blood (PB) and bone marrow (BM) in AL pts during treatment. PB and BM samples were collected before and on +36 day after chemotherapy. The antigens were detected by flow cytometry using monoclonal antibodies. We calculated 10 000 cells in each sample. 19 pts were included in the study: 10 - AML and 9 - ALL. The control group comprised 8 healthy donors. At time of diagnosis there were 40±5,7% of CD34+ cells in BM and 26±4,9% - in PB. There was no significant difference between AML and ALL. CD34+ cells in BM and PB of healthy donors constituted 1,6% and 0,27%, respectively. After induction therapy (+36 day) CD34+ cells decreased in BM to 6,1%±3,3 (p=0,0001), in PB to 3,7%± 2,7 (p=0,0008) in all pts. The data on antigens expression on CD34+ cells of BM and PB are presented in table 1 CD34+/Bcl-2+ CD34+/Bax+ CD34+/CD95+ CD34+/ACE+ BM PB BM PB BM PB BM PB AML pts n=10 0 day 38±11,6* 41±14 24,4±7,9 29,2±7,6* 16,4±8,5 23,2±7,8 21,7±9,5 20,8±8,7* 36 day 13,5±3,4** 23,7±5** 46,2±11,5 50,3±11 19,9±5,5 36,4±10 34±6,6 35±9,2** ALL pts n=9 0 day 36±11 33,7±12 46,2±9,4 37,4±3,7* 3,4±1,1* 7,1±2,5* 41±10,9 33,2±9,7* 36 day 18,4±5,8 26±8,9 38±11,8 40,5±10 26,2±9,1** 40,9±9,2** 34±10 62,8±10** Donors n=8 11,7±1,6 26,1±5,9 22,8±4 67,8±6,7 13,4±3,2 47,7±11,6 28±5,3 68,2±10,2 * − p<0.05 compare with donors ** − p<0.05 compare with day 0 CD34/Bcl-2 expression in BM in AML pts is significantly higher (p=0,04) at the diagnosis comparing with donors. CD34/Bcl-2 expression in PB in AML pts and in BM and PB in ALL pts is higher too, but not significantly. This expression level decreased substantially in BM and PB in AML pts on +36 day comparing with day 0 (p<0,05). We did not found significant changes in ALL pts. CD34/Bax expression in PB is significantly lower (p=0,003) both in AML and ALL pts in comparison with donors. In AML, not in ALL, chemotherapy caused augmentation of Bax expression in CD34+ BM and PB cells on +36 day. BM and PB CD34+ cells in donors had different expression characteristics of Bcl-2 and Bax, demonstrating much higher level of pro- and antiapoptotic markers in PB cells. On the contrast CD34+ leukemia cells in BM and PB had similar characteristics regarding CD34/Bcl-2 and CD34/Bax expression. This fact demonstrates the heterogeneity of donor CD34+cells in BM and PB and points that leukemia CD34+cells in BM and PB are rather similar. CD95 expression on CD34+ BM and PB before treatment is significantly lower (p=0,01, p=0,008) in ALL pts in comparison with donors, and this expression level increased after chemotherapy (p<0,05). CD34/CD95 expression in AML pts is similar with donors, and we didn't find changes after treatment. CD34/ACE coexpression in BM cells of leukemia pts and donors did not differ much at any time of evaluation. But CD34/ACE expression in PB cells of AML and ALL pts was much lower (p<0,05) than in donors and substantially increased on the day 36. So, our data demonstrate that Bcl-2, Bax, CD95 and ACE expression on CD34+ cells in AL pts and donors significantly differs. The chemotherapy provokes critical changes in CD34/CD95 expression in BM and PB in ALL pts, CD34/Bcl-2 expression in AML pts and ÑÂ34/ACE expression in PB in all AL pts. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Anti-Apoptotic Effect of Angelica Polysaccharide (APS) on Cryopreservation of Platelets

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3250-3250
Author(s):  
Mo Yang ◽  
Weiqing Su ◽  
Liuming Yang ◽  
Huimin Kong ◽  
Huiling Wei ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Control Group ◽  
Apoptotic Effect ◽  
Vitro Effect

Abstract Background: Angelica Polysaccharide (APS) is from the root of Radix Angelicae Sinensis (Danggui). Danggui has been used for centuries to treat blood-deficiency related diseases. The hematopoietic effect of Danggui may be related to its constituent, polysaccharide. The effects of angelica polysaccharide on cryopreservation of platelets and megakaryocytes have not been well studied. This study focused on anti-apoptotic effect of APS and TPO on cryopreservation of platelets and megakaryocytes and provided new methods for prolonging the preservation time of platelets in vitro. Methods: The expression of platelet membrane glycoprotein CD41 and CD61, as well as the platelet apoptotic rate, Caspase 3 expression and mitochondrial membrane potential (MMP) were detected by flow cytometry; the anti-apoptotic mechanism of APS by PI3K /AKT signaling pathway was analyzed by Western blot assay. CFU assays were used to determine the effects of APS on megakaryocytic progenitor cells. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were conducted in megakaryocytic cell line M-07e. The effects of APS on cells treated with Ly294002, PI3K inhibitor and the effect of APS on the p-AKT were also studied. Results: The platelets were divided into 4 group: control group (4 ℃ stored platelets), APS group (APS-treated platelets stored at 4 ℃), LY294002 group (LY294002-treated platelets stored at 4 ℃) and LY294002+APS group (LY294002+APS treated platelets stored at 4 ℃). The apoptotic rate of platelets in LY294002 group was obviously increased. Compared with control group, the expression of CD41 and CD61 gradually decreased along with the enhancement of LY294002 concentrations (r=-0.953). The apoptotic rate of platelets in LY294002 group was enhanced significantly (P&lt;0.05). While in LY294002+APS group, the apoptotic rate of platelets was significantly reduced (P&lt;0.05) as compare with LY294002 group, which suggest that APS has an anti-apoptotic effect on the cryopreserved platelets. APS decreased the expression of Caspase-3 and inhibited the reduction of mitochondrial membrane potential induced by LY294002. Moreover, APS increased the activation of PI3K /AKT pathway in Platelets . We further analyzed the in vitro effect of APS on CFU-MK formation. APS (50 ug/ml) enhanced TPO (50 ng/ml) -induced CFU-MK formation (p=0.06, n=4). APS also significantly enhanced PDGF, bFGF and VEGF-induced CFU-MK formation (n=4). Moreover, the anti-apoptotic effect of APS in M-07e cells was also demonstrated by Annexin-V, Caspase-3, and JC-1 assays. Adding LY294002 alone increased the percentage of cells undergoing apoptosis. However, additional of APS to LY294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of APS significantly increased the p-AKT. Conclusion: APS, like TPO, has an anti-apoptotic effect on the cryopreserved platelets and megakaryocytes through activating PI3K/AKT, decreasing the expression of Caspase-3 and inhibiting the reduction of MMP. Disclosures No relevant conflicts of interest to declare.

The Study of Effect and Mechanism by Vitamin K2 on Human MDS Cell Line MUTZ-1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4357-4357
Author(s):  
Bao-An Chen ◽  
Xin Xu ◽  
Ze-Ye Shao ◽  
Jia-Hua Ding ◽  
Guo-Hua Xia ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Caspase 3 ◽  
Cellular Proliferation ◽  
Vitamin K2 ◽  
Time Dependent ◽  
Control Group ◽  
Dependent Manner ◽  
Significant Difference

Abstract The myelodysplastic syndromes (MDS) are characterized by hemopoietic insufficiency associated with cytopenias leading to serious morbidity and the additional risk of leukemic transformation. Vitamin K2(VK2) is reported to induce apoptosis or differentiation of leukemic cell lines in vitro. For investigating the effects and mechanism of VK2 on human MDS cell line MUTZ-1 in vitro,we observed the changes of morphologic features of MUTZ-1 cells by exposing to VK2.The transmission electron microscope was used to observe the apoptosis of MUTZ-1 cells. Cellular proliferation was determined by the MTT assay. The flow cytometry was used to analysis apoptosis rate and the change of cell cycle. The expression of apoposis-related genes bcl-2, survivin and bax were detected by reverse transcriptase polymerase chain reaction(RT-PCR).The activity of caspase-3 was detected by chemiluminescence assay. After exposing to 10μmol L−1 and higher concentration of VK2, it could inhibit MUTZ-1 cells proliferation in a dose-and time-dependent manner(p&lt;0.05). At concentration of 5μmol/l VK2 treatment, it might accelerate cellular proliferation, but there’s no significant difference compared with control group. Apoptosis peak on FCM and positive Annexin-V FITC/PI on cell membrane showed that VK2 induced apoptosis of MUTZ-1 cells in a dose-and-time-dependent manner, G0/G1 cell cycle arrest, significantly dow-regulated the expression of bcl-2 and survivin, but had no effect on the expression of bax.The activities of caspase-3 were significantly increased. Low concentration of VK2 could facilitate cell proliferation. The higher concentration of VK2 could induce apoptosis of MUTZ-1 cells. These results indicate that VK2 induces MUTZ-1 cells apoptosis by activating caspase-3 pathway, the apoptosis related genes bcl-2, survivin down-regulated might play an important role in this process.

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