scholarly journals The Role of 5-HT on Proplatelet Formation and Thrombopoietin Production

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-14
Author(s):  
Mo Yang ◽  
Enyu Liang ◽  
Jieyu Ye ◽  
Beng H Chong ◽  
Liang Li
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Traumatic Stress ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Mice Model ◽  
Synthesis Inhibitor ◽  
Rhodamine Phalloidin ◽  
Proplatelet Formation ◽  
Group 5

Background: Our previous work confirmed that serotonin (5-HT) promotes the proliferation of hemopoietic stem cells and megakaryocytes (Yang M et al, Stem Cells, 2007; 2014). However, the mechanisms remain indefinite. Methods: Q-PCR, Flow Cytometry, Western Blot, or Immunofluorescence microscope were used in the receptor and TPO study. MTT/CCK-8, Proplatelet assay, and Flow Cytometry were also used in cell proliferation and apoptosis study. The relationship between 5-HT and TPO was studied in a traumatic stress mice model. Results: In-vitro study, there was a stimulating effect of 5-HT on proplatelet formation in human bone marrow megakaryocytes. Human BM MK progenitors cultured in serum-free medium with either 5-HT (200nM) or TPO (100 ng/ml) had more proplatelet bearing MKs than the control group (5-HT (12.3 ± 5.0)% vs. Control (6.2 ± 3.5)%, P=0.025; TPO (15.6 ± 2.5)% vs. Control, P=0.04; n=4). The 5-HT treatment group showed more mature and more in the final stage MK cells as compared to the TPO group. 5-HT2A, 2B, 2C receptors were detected in the surface of megakaryocytes. The effect of 5-HT on proplatelet formation in MK cells was via 5-HT2 receptors and this effect was reduced by 5-HT2 receptor inhibitor ketanserin. 5-HT acted on cytoskeleton reorganization in MKs via 5-HT2 receptors and ERK1/2 pathway. Using an immunofluorescence microscope with F-actin specific binder rhodamine-phalloidin staining, the polymerized actin level was lower in the control group than the 5-HT group and actin distributed diffusely throughout the cytoplasm. In contrast, the polymerization actin level was higher in the 5-HT group. Adding ketanserin and ERK1/2 inhibitor PD98059 to 5-HT treatment, the fluorescence intensity was correspondingly reduced. Our data also demonstrated that ERK1/2 was activated in MKs treated with 5-HT for 30 minutes. In a traumatic stress mice model, both of 5-HT and TPO were increased, but the increasing of TPO is posterior to 5-HT. After added LX1606, the synthesis inhibitor of 5-HT, 5-HT was reduced markedly, as well as TPO. The expression of TPO mRNA and the production of TPO protein were increased as compared with the control in this model. Conclusions: This study suggests that 5-HT promotes thrombopoiesis from two aspects: one is the direct effect on megakaryocytes. 5-HT could promote the proplatelet formation from megakaryocytes. The second is the indirect effect by promoting the production of TPO, which is a paracrine secretion to influence thrombopoiesis. Disclosures No relevant conflicts of interest to declare.

Effects of Serotonin on Proplatelet Formation and F-actin Reorganization in Human Megakaryocytes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2108-2108
Author(s):  
Yuan Shan Cheng ◽  
Yuan Sheng Liu ◽  
Godfrey ChiFung Chan ◽  
Jie Yu Ye ◽  
Yu Lung Lau ◽  
...  
Keyword(s):  
Stem Cells ◽  
Control Group ◽  
Rhodamine Phalloidin ◽  
Hematopoietic Stem ◽  
Actin Reorganization ◽  
Serum Free ◽  
Western Blot Method ◽  
Cytoskeleton Reorganization ◽  
Proplatelet Formation ◽  
Group 5

Abstract We previously reported that serotonin (5-HT) is a growth factor for hematopoietic stem cells and megakaryocytic progenitor (Yang et al, Stem Cells, 2007). We further proposed a possible role of serotonin on megakaryocyte differentiation and platelet formation. The effect of serotonin on proplatelet formation and F-actin reorganization in human megakaryocytes (MKs) was investigated in this study. Our results showed that: There was a stimulating effect of serotonin on proplatelet formation in human bone marrow megakaryocytes. Human BM MK progenitors cultured in serum free medium with either 5-HT (200nM) or TPO (50 ng/ml) had more proplatelet bearing MKs than the control group (5-HT(11.33% ± 4.93) vs. control (6% ± 3.60), P=0.026; TPO (14.66% ± 1.53) vs. control, P=0.043; n=3). The 5-HT treatment group showed more mature and more in the final stage MK cells as compared to TPO group; The effect of serotonin on proplatelet formation in Meg-01 cells were via 5-HT2 receptors. Meg-01 cells strongly expressed 5-HT 2A, 2B, 2C receptors by using western blot method. 5-HT also promoted proplatelet formation in these cells and this effect was reduced by 5-HT2 receptor inhibitor ketanserin (KE); and Serotonin acted on cytoskeleton reorganization in human megakaryocytes via 5-HT2 receptors and ERK1/2 pathway. Using an immunofluorescence microscope with F-actin specific binder rhodamine-phalloidin staining, the polymerized actin level was lower in the control group (serum free) than the 5-HT group and actin distributed diffusely throughout the cytoplasm. In contrast, polymerization actin level was higher in 5-HT group. Adding ketanserin and ERK1/2 inhibitor PD98059 to 5-HT treatment, the fluorescence intensity was correspondingly reduced (5HT vs. Control, P=0.006; 5-HT vs.5-HT plus KE, P=0.014; n=6). Our data also demonstrated that ERK1/2 was activated in MK cells treated with 5-HT for 30 minutes (21.76% ± 7.42). Our studies showed that serotonin had a stimulating effect on proplatelet formation and F-actin reorganization in human megakaryocytes and this effect involved the 5-HT2 receptors and the activation of ERK1/2 pathway.

scholarly journals Effect of Placental Extract on Omentum Mesenchymal Stem Cells Differentiation in NMRI Mice

2017 ◽  
Vol 7 (1) ◽  
pp. 176
Author(s):  
Maryam Sadat Nezhadfazel ◽  
Kazem Parivar ◽  
Nasim Hayati Roodbari ◽  
Mitra Heydari Nasrabadi
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Control Group ◽  
Fat Cell ◽  
Nmri Mice ◽  
Differentiated Cells ◽  
Placental Extract

Omentum mesenchymal stem cells (OMSCs) could be induced to differentiate into cell varieties under certain conditions. We studied differentiation of OMSCs induced by using placenta extract in NMRI mice. Mesenchymal stem cells (MSCs) were isolated from omentum and cultured with mice placenta extract. MSCs, were assessed after three passages by flow cytometry for CD90, CD44, CD73, CD105, CD34 markers and were recognized their ability to differentiate into bone and fat cell lines. Placenta extract dose was determined with IC50 test then OMSCs were cultured in DMEM and 20% placenta extract.The cell cycle was checked. OMSCs were assayed on 21 days after culture and differentiated cells were determined by flow cytometry and again processed for flow cytometry. CD90, CD44, CD73, CD105 markers were not expressed, only CD34 was their marker. OMSCs were morphologically observed. Differentiated cells are similar to the endothelial cells. Therefore, to identify differentiated cells, CD31 and FLK1 expression were measured. This was confirmed by its expression. G1 phase of the cell cycle shows that OMSCs compared to the control group, were in the differentiation phase. The reason for the differentiation of MSCs into endothelial cells was the sign of presence of VEGF factor in the medium too high value of as a VEGF secreting source.

scholarly journals Effects of doxorubicin associated with amniotic membrane stem cells in the treatment of canine inflammatory breast carcinoma (IPC-366) cells

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Jéssica Borghesi ◽  
Sara Caceres ◽  
Lara Carolina Mario ◽  
Angela Alonso-Diez ◽  
Ana Carolina Silveira Rabelo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Amniotic Membrane ◽  
Triple Negative ◽  
Cellular Growth ◽  
Control Group ◽  
Group Iii ◽  
Chemotherapy Drugs ◽  
Group I ◽  
Group Ii

Abstract Background Tumours in mammary glands represent the most common neoplasia in bitches, as in humans. This high incidence results in part from the stimulation of sex hormones on these glands. Among mammary tumours, inflammatory carcinoma is the most aggressive, presenting a poor prognosis to surgical treatment and chemotherapy. One of the most widely used chemotherapy drugs for breast cancer treatment is doxorubicin (DOXO). Alternative therapies have been introduced in order to assist in these treatments; studies on treatments using stem cells have emerged, since they have anti-inflammatory and immunomodulatory properties. The aim of this study was to evaluate the effects of DOXO and canine amniotic membrane stem cells (AMCs) on the triple-negative canine inflammatory mammary carcinoma cell line IPC-366. Methods Four experimental groups were analysed: a control group without treatment; Group I with DOXO, Group II with AMC and Group III with an association of DOXO and AMCs. We performed the MTT assay with DOXO in order to select the best concentration for the experiments. The growth curve was performed with all groups (I-III) in order to verify the potential of treatments to reduce the growth of IPC-366. For the cell cycle, all groups (I-III) were tested using propidium iodide. While in the flow cytometry, antibodies to progesterone receptor (PR), estrogen receptor (ER), PCNA, VEGF, IL-10 and TGF-β1 were used. For steroidogenic pathway hormones, an ELISA assay was performed. Results The results showed that cells treated with 10 µg/mL DOXO showed a 71.64% reduction in cellular growth after 72 h of treatment. Reductions in the expression of VEGF and PCNA-3 were observed by flow cytometry in all treatments when compared to the control. The intracellular levels of ERs were also significantly increased in Group III (4.67% vs. 27.1%). Regarding to the levels of steroid hormones, significant increases in the levels of estradiol (E2) and estrone sulphate (S04E1) were observed in Groups I and III. On the other hand, Group II did not show differences in steroid hormone levels in relation to the control. We conclude that the association of DOXO with AMCs (Group III) promoted a reduction in cell growth and in the expression of proteins related to proliferation and angiogenesis in IPC-366 triple-negative cells. Conclusions This treatment promoted ER positive expression, suggesting that the accumulated oestrogen conducted these cells to a synergistic state, rendering these tumour cells responsive to ERs and susceptible to new hormonal cancer therapies.

135 Strategies for transfection of bovine mesenchymal stem cells with pBC1-anti-CD3 vector

2020 ◽  
Vol 32 (2) ◽  
pp. 194
Author(s):  
F. B. Duarte ◽  
S. N. Báo ◽  
M. Brígido ◽  
J. M. Araújo ◽  
E. d. O. Melo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Calf Serum ◽  
Genetic Material ◽  
Cell Receptor ◽  
Wharton’S Jelly ◽  
Control Group ◽  
Wharton's Jelly ◽  
Transgenic Cells

Cells from different origins behave differently regarding the incorporation of exogenous genetic material and the formation of transgenic cells. In this context, the objective of this study was to verify the potential of transfection of bovine mesenchymal stem cells from Wharton's jelly and adipose tissue, comparing two transfection protocols, using Lipofectamine LTX and Plus or Xfect reagents, with the integration of humanized anti-CD3. Skin fibroblasts were used as a control group. Humanized anti-CD3 is a monoclonal antibody that interacts with the CD3 molecule of the T-cell receptor, leading to the suppression of T-cells. This antibody is considered an option in the treatment of human autoimmune diseases and against the rejection of transplanted organs. Humanized anti-CD3 was used in this work for the production of bovine transgenic cells that, in the future, will be used in the development of bioreactor animals. In all steps of this study, cell types were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics, in an incubator at 39°C with 5% CO2 in air with saturated humidity. All cells were plated at 5×105 into 24-well culture dishes and co-transfected with vector pBC1-anti-CD3-IRES-FEO and pEF-NEO-GFP using Lipofectamine LTX with reagent Plus or Xfect. Forty-eight hours after transfection, neomycin was added in each treatment and cells were cultured for 2 weeks. Treated cells were submitted to fluorescence microscopy, flow cytometry, and PCR evaluations. Wharton's jelly cells were sensitive to treatments and started necrosis. In the flow cytometry assay, the median fluorescence was higher in adipocytes than in fibroblasts, for both the Xfect reagent (20.057±1.620.7 and 10.601±702.86, respectively, P<0.05) and for LTX (19.590±113.84 and 10.518±442.65 respectively, P<0.05). These results, associated with the evaluation of epifluorescence, demonstrated that adipocytes presented a better response to transfection than did other cells, independent of the kit used. Performing PCR on co-transfected adipocytes and fibroblasts demonstrated the presence of anti-CD3, making this approach feasible in future experiments. Southern blotting analysis is being performed to confirm DNA integration. Financial support was provided by Fundação de Amparo à Pesquisa do Distrito Federal (FAPDF); Embrapa MP1.

Improved Immune Recovery after Transplantation of TCRαβ/CD19 Depleted Allografts from Haploidentical Donors in Pediatric Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 852-852
Author(s):  
Peter Lang ◽  
Tobias Feuchtinger ◽  
Heiko-Manuel Teltschik ◽  
Wolfgang Schwinger ◽  
Patrick Schlegel ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Lymphocytes ◽  
Nk Cells ◽  
Pediatric Patients ◽  
Conflicts Of Interest ◽  
Γδ T Cells ◽  
Control Group ◽  
Unrelated Donor ◽  
Immune Recovery

Abstract Transplantation of haploidentical stem cells has become an accepted option for pediatric patients and adults with high risk malignancies who lack a matched related or unrelated donor. In recent years, the majority of pediatric transplant centers chose the CD34 positive selection of peripheral stem cells, which allowed minimizing GvHD by effective reduction of T cells in the graft. However, infectious complications caused by delayed immune recovery were a major reason for transplant related mortality (TRM). In order to improve the immune recovery, we have established a new T-cell depletion method which removes αβ+ T-lymphocytes via a biotinylated anti-TcRαβ antibody followed by an anti-biotin antibody conjugated to magnetic microbeads while retaining γδ+ T-lymphocytes, natural killer (NK) cells and other cells in the graft. In addition, CD19+ B-lymphocytes were concomitantly depleted for the prevention of post-transplant EBV-associated lymphoproliferative disease. Immune recovery was retrospectively analyzed in a cohort of 41 patients with acute leukemia, MDS and non-malignant diseases, who received αβ T and B cell depleted allografts from haploidentical family donors. Conditioning regimens consisted of fludarabine or clofarabine, thiotepa, melphalan and serotherapy with OKT3 or ATG-Fresenius®. Graft manipulation was carried out with anti TCRαβ and anti CD19 antibodies and immunomagnetic microbeads. γδ T cells and NK cells remained in the grafts. Primary engraftment occurred in 88%, acute graft versus host disease (aGvHD) grade II and III-IV occurred in 10% and 15%. Immune recovery data were available in 26 patients and comparable after OKT3 (n=7) or ATG-F® (n=19). Median time to reach > 100 CD3+/µl, > 200 CD19+ cells/µl and > 200 CD56+ cells/µl for the whole group was 13, 127 and 12.5 days. Compared to a historical control group of patients with CD34 positive selected grafts, significantly higher cell numbers were found for CD3+ at days +30 and +90 (267 vs. 27 and 397 vs. 163 cells/µl), for CD3+4+ at day +30 (58 vs. 11 cells/µl) and for CD56+ at day +14 (622 vs. 27 cells/µl). The clinical impact of this accelerated immune recovery will be evaluated in an ongoing prospective multi-center trial. Disclosures No relevant conflicts of interest to declare.

scholarly journals Stem Cell Graft Cellular Composition, Hematological and Immune Recovery in NHL Patients Mobilized with or without Plerixafor: A Prospective Comparison

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1193-1193
Author(s):  
Jaakko Valtola ◽  
Ville Varmavuo ◽  
Antti Ropponen ◽  
Marja Pyörälä ◽  
Anne Nihtinen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Prospective Study ◽  
Nk Cells ◽  
Immune Reconstitution ◽  
Control Group ◽  
Immune Recovery ◽  
Cellular Composition ◽  
Cd34 Cells ◽  
Significant Difference

Abstract Introduction: Plerixafor, a reversible CXCR4 antagonist, may be used to enhance mobilization of CD34+ cells after G-CSF or chemotherapy plus G-CSF. There is a paucity of prospective data regarding cellular composition of grafts collected after plerixafor in chemomobilized patients. Also the data concerning hematopoietic or immune recovery after high-dose chemotherapy in plerixafor treated lymphoma patients is limited. Patients and methods: Thirty-one patients with NHL were included into this prospective study. There were 14 males and 17 females with a median age of 62 years (range 19 - 73). Altogether fourteen patients received plerixafor (plerixafor group) for poor or late mobilization whereas 17 did not (control group). All patients were mobilized with chemotherapy plus G-CSF. Cryopreserved graft samples were analyzed with flow cytometry for T and B cells (CD3/CD8/CD45/CD19) as well as for NK cells (CD3/CD16+CD56). Also CD34+ cell subclasses were analyzed (CD34/CD38/CD133). Complete blood counts were evaluated at +15 days, 1, 3, 6 and 12 months post-transplant. To evaluate immune reconstitution, flow cytometry of lymphocyte subsets (T, B, NK) was performed at 1, 3 and 6 months after the graft infusion with the same antibody panel as for graft analysis. Results: The median number of infused viable CD34+ cells was higher in the control group (3.1 x 106/kg vs. 2.0 x 106/kg, p = 0.036) (Table 1). The median percentage of the most primitive stem cells (CD34+CD133+CD38-) in the grafts was higher in the plerixafor group (3.5 % vs 1.2 %, p = 0.001), but there was no significant difference in the absolute counts (0.07 x 106/kg vs 0.05 x 106/kg, p = 0.620). The median amounts of CD3+CD4+ and CD3+CD8+ T cell subsets, CD3+ and NK (CD3-CD16/56+) cells were all significantly higher in the plerixafor group (Table 1). The neutrophil counts at +15 days after the graft infusion were lower in the plerixafor group (2.1 x 109/l vs. 4.8 x 109/l, p = 0.013). Otherwise there was no significant difference in the hematological reconstitution between the groups. The immune reconstitution was comparable except for the higher number of NK cells in the plerixafor group at one month (0.4 x 109/l vs. 0.1 x 109/l, p = 0.001). Also a trend towards faster recovery of blood CD4+ T cells was observed after one month in the plerixafor group (0.2 x 109/l vs. 0.1 x 109/l, p = 0.097). Conclusions: This prospective study evaluating cellular composition of grafts confirms that the apheresis products collected from plerixafor-treated NHL patients contain a greater proportion of the more primitive stem cells and a greater number of T lymphocytes and NK cells compared to patients mobilized without plerixafor. Hematopoietic reconstitution was comparable between the groups except for slower neutrophil recovery in the plerixafor-group. Immune reconstitution was comparable but NK cell as well as CD4+ T cell recovery was faster in the plerixafor group. These results will be further evaluated in a larger set of patients. Also the possible effect of graft composition on the progression free and overall survival will be evaluated in the ongoing GOA (Graft and Outcome in Autologous transplantation) study. Table 1. Cellular composition of freezed grafts of NHL patients mobilized with or without plerixafor. Mobilization with plerixafor (n = 14) Mobilization without plerixafor (n = 17) P - value Blood graft analysis (x106/kg) CD34 w/a 7AADCD34 w 7AADCD34+CD38-Proportion of CD34+ CD133+CD38- cells from all CD34+ cells (%)CD3+CD4+CD8+CD19+NK 2.1 (0.8 – 5.3)2.0 (0.6 – 5.5)0.07 (0.01 – 0.17)3.5 (0.80 – 10.80)178.3 (49.2 – 454.4)82.1 (29.1 – 267.1)75.4 (16.5 – 279.1)0.0 (0.0 – 0.0)21.5 (0.4 – 39.5) 3.4 (1.9 – 7.2)3.1 (1.5 – 6.7)0.05 (0.11 – 0.18)1.2 (0.44 – 5.3)66.2 (16.6 – 415.4)35.6 (8.0 – 114.3)23.3 (8.4 – 301.8)0.0 (0.0 – 3.2)6.6 (0.6 – 20.7) 0,0070.0360.6200.0010.0010.0010.0030.1920.001 7AAD = 7-Aminoactinomycin D w/a = without w = with Disclosures Jantunen: Sanofi: Employment.

scholarly journals ATRA Could Correct the Defective S1P-Mediated Cytoskeletal Reorganization in Proplatelet Formation of ITP

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 218-218 ◽  
Author(s):  
Qiu-Sha Huang ◽  
Jing Xue ◽  
Chen-Cong Wang ◽  
Ya-Zhen Qin ◽  
Lan-Ping Xu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rho Gtpases ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Therapeutic Effects ◽  
Cytoskeletal Reorganization ◽  
Mice Model ◽  
Mrna Sequencing ◽  
Healthy Donors ◽  
Proplatelet Formation

Introduction Sphingosine-1-phosphate (S1P) is now emerging as a vital lipid mediator. Activation of sphingosine kinase (SphK) produces intracellular S1P, which in turn can be secreted out of the cell and act extracellularly by binding to S1P receptors (S1PR). Recent studies suggest that the "inside-out" signaling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) (Blood, 2013; J EXP MED, 2012). PPF requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases which can be activated by S1P, including Rac1 and Cdc42, have been shown to be master regulators of cytoskeletal rearrangements. The pathogenesis mechanisms of immune thrombocytopenia (ITP) are not entirely understood. Our previous data indicated that impaired PPF contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that S1P-mediated microtubule reorganization is defective in PPF of ITP. All-trans retinoic acid (ATRA), which has demonstrated to be a promising option for ITP patients in our previous study (Lancet Haematology, 2017), could correct the altered microtubule reorganization and promote PPF. Methods Thirty consecutive patients with primary ITP and 20 healthy donors were enrolled in our study. MKs were isolated from bone marrow samples, and they were collected again after ITP patients received ATRA therapy. MK mRNA sequencing by microarray was used to assess the difference of gene expression between ITP and controls. Microtubule regrowth assay was performed to observe microtubule dynamic behavior. In this assay nocodazole was first used to induce complete depolymerization of microtubule network, followed by drug washout to allow microtubule regrowth over time. ATRA was added to the culture medium of MKs to determine the mechanism of ATRA in correcting impaired PPF. Additionally, ITP mice model was established to observe the therapeutic effects of ATRA in PPF. Pf4-Cre/loxP system was used to specifically knock down gene of MKs. Results S1P concentration in bone marrow from ITP patients was lower compared to healthy donors. MKs mRNA sequencing demonstrated that S1P synthetase SphK2 and S1P receptor S1PR1 gene were downregulated while S1P lyase (SPL) gene was upregulated in ITP patients, which caused abnormal S1P signaling. Furthermore, we observed that PPF capacity of MKs in patients with ITP was reduced. Pharmacological disruption of S1PR1 blocked PPF, exogenous S1P corrected impaired PPF. Collectively, deregulation of S1P signaling was associated with impaired PPF in ITP. To verify the downstream role of S1P in regulating PPF, the Rho GTPases detection of MKs revealed a decrease in Cdc42 and Rac1 levels from ITP patients. Immunofluorescence of the differentiated MKs showed that the expression and distribution of β1 tubulin were abnormal from ITP patients. Early PPs from MKs of healthy donors displayed a well-organized tubulin bundles resembling bunches of grapes. In contrast, in MKs from ITP patients, tubulin was disorganized in thick bundles. In addition, TEM analysis of the MKs showed an irregular distribution of granules, tortuous membranes and impaired proplatelet structure. In microtubule regrowth assay, MKs from ITP patients had significantly lower microtubule regrowth at 10 min post-nocodazole washout compared with controls. Together, microtubule alteration resulted in impaired PPF in ITP. We tested whether S1P pathway were required for microtubule reorganization, both SphK2-/- and S1PR1-/- mice displayed significantly reduced S1P, Cdc42 and Rac1, altered microtubule architecture and defective PPF. Taken together, abnormal S1P pathway accounted for impaired microtubule reorganization in ITP. Next, we explored the effect of ATRA on microtubules reorganization in ITP patients, our data showed that in vitro treatment with ATRA restored microtubules structure by upregulating S1P and activating Rho GTPases. In vivo studies showed that ARTA could rescue the impaired PPF in both patients and mice model with ITP. Conclusions The MKs of ITP patients displayed defective cytoskeletal reorganization regulated by S1P pathway. ATRA restored cytoskeletal structure and corrected impaired PPF by upregulating S1P and activating Rho GTPases. It sheds light on a novel mechanism of ITP pathogenesis and provides a basis for the therapeutic potential of ARTA in ITP patients. Disclosures No relevant conflicts of interest to declare.

Effect of rat bone marrow derived-mesenchymal stem cells on granulocyte differentiation of mononuclear cells as preclinical agent in cell-based therapy

2021 ◽  
Vol 21 ◽  
Author(s):  
Ezzatollah Fathi ◽  
Sheyda Azarbad ◽  
Raheleh Farahzadi ◽  
Sara Javanmardi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Growth Factors ◽  
Mononuclear Cells ◽  
Control Group ◽  
Flow Cytometry Analysis ◽  
Cell Lineages ◽  
Experimental Group

Background: Bone marrow mononuclear cells (BM-MNCs), as a collection of hematopoietic and mesenchymal stem cells (MSCs), are capable of producing all blood cell lineages. The use of cytokines, growth factors, or cells capable of secreting these factors will help in stimulating the proliferation and differentiation of these cells into mature cell lines. On the other hand, MSCs are multipotent stromal cells that can be differentiated into various cell lineages. Moreover, these cells can control the process of hematopoiesis by secreting cytokines and growth factors. The present study aimed to investigate the effect of BM-derived MSCs on the differentiation of MNCs based on the assessment of cell surface markers by flow cytometry analysis. Methods: For this purpose, the MNCs were purified from rat BM using density gradient centrifugation. After that, they were cultured, expanded, and characterized. Next, BM-derivedMSCs were co-cultured with MNCs and then were either cultured with MNCs alone (control group) or co-cultured MNCs with BM derived-MSCs (experimental group). Finally, they were collected on day 7 and subjected to flow cytometry analysis for granulocyte markers and ERK protein’s investigation. Results: It was found that the expression levels of CD34, CD16, CD11b, and CD18 granulocyte markers, as well as protein expression of ERK, have significantly increased in the experimental group compared to the control group. Conclusion: Therefore, it can be concluded that MSCs could affect the granulocyte differentiation of MNCs via ERK protein expression, which is a key component of the ERK signaling pathway.

Effects of 5-Bromotetrandrine and Daunorubicin on the Expression of Survivin In K562/AO2 Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4947-4947
Author(s):  
Bao-An Chen ◽  
Xiao-hui Cai ◽  
Jun Wang ◽  
Chong Gao ◽  
Jia-hua Ding ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mtt Assay ◽  
Conflicts Of Interest ◽  
Acquired Resistance ◽  
Leukemic Cells ◽  
Control Group ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Typical Experiment

Abstract Abstract 4947 Objective: The aim of this study was to investigate the expression of survivin and the apoptosis induced by DNR and BrTet in the leukemic cells K562/A02. Methods: In a typical experiment, the K562/AO2 cells were treated with daunorubicin (DNR), 5-bromotetrandrine (BrTet), or DNR and BrTet for 48 hours, and the cells treated without any drugs were used as control group. Cell proliferation was analyzed by MTT assay. Cells apoptosis and the concentration of DNR within the cells were measured by Flow cytometry (FCM). The expressions of mRNA and protein of survivin were determined by semi-quantitative reverse transcription PCR (RT-PCR) and Western blot, respectively. Results: The results of MTT assay indicated that DNR and BrTet were both able to inhibit the proliferation of K562/AO2 cells in dose-dependent manner. The fresh evidence from flow cytometry showed that a higher apoptosis rate could be induced and a higer concentration of DNR could be detected in K562/AO2 cells by DNR and BrTet as compared with those by DNR or BrTet in the same concentrations(P<0.01). RT-PCR revealed that the expression of survivin mRNA, a higer expression in K562/AO2 cells with acquired resistance to adriamycin than that in parent K-562 cells, decreased in the DNR and BrTet group (P<0.05), but there was no obvious change in other groups(P>0.05). Western bolt demonstrated that the expression of survivin protein was much lower in the DNR and BrTet group(P<0.05). Conclusion: BrTet could increase the concentration of DNR and reverse the multidrug resistance(MDR) in the K562/AO2 cells. Survivin may play an important role in apoptosis induced by DNR. Survivin could be a target for the treatment of MDR in haematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

A Recombinant Thrombomodulin Improves Haemostatic Disturbance and Inflammation in Setpic Patients with DIC

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2293-2293
Author(s):  
Noritaka Yada ◽  
Kenji Nishio ◽  
Hidetada Fukushima ◽  
Tadahiko Seki ◽  
Yasuyuki Urizono ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Clinical Course ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Fibrinogen Degradation Product ◽  
Soluble Thrombomodulin ◽  
Anti Inflammatory ◽  
Group 5 ◽  
Group Control Group ◽  
Inflammatory Effect

Abstract Abstract 2293 Background: Recently, a recombinant human soluble thrombomodulin (rTM) has become commercially available for patients with disseminated intravascular coagulation (DIC) in Japan, which is composed of the active, extracellular domain of thrombomodulin. However, its anti-thromobotic and anti-inflammatory effect during the clinical course in patients with DIC has not been reported. Aim: To investigate the effect of rTM on mortality, haemostatic disturbance, and inflammation in septic patients with DIC. Methods: The patients with sepsis who met the Japanese diagnostic criteria for acute DIC and showed a level of antithrombin (AT) lower than 70% were recruited and divided into two groups, one group (Control group) treated with AT products and Gabexate mesilate (GM), and the other group (TM group) treated with rTM (0.06 mg kg(-1) for 30 min, once daily) in addition to AT and GM. The effect of rTM during the clinical course were investigated from the differences between TM and Control groups in mortality, DIC scoring, haemostatic and inflammatory markers on days 0, 3, 5, 7 (day 0 is just before initiation of therapy). Results & Discussion: Eighteen patients were included in the TM group, and 16 patients in the Control group. There were no differences in APACHEII score at day 0, DIC score at day 0, or mortality at day 28 between the groups. The effects of the rTM were as follows; 1) Patients in the TM group showed earlier DIC resolution at day 5 than Control at day 7. 2) Hypercoagulable state expressed by the increased levels of soluble fibrin monomer (SF) or thrombin-antithrombin complex (TAT) was improved more quickly in TM group compared with Control group, suggesting that rTM decreased thrombin generation. 3) AT was increased much more at day 3 after AT product administration in TM group than in Control group, which also can be explained by suppression of thrombin generation by rTM. 4) Increased levels of the complex of α2PI/plasmin, D-dimer, and fibrin/fibrinogen degradation product were also improved earlier in TM group than Control group. 5) Plasmin-alfa2 plasmin inhibitor complex (PIC) was significantly lower at day 7 in TM group than Control group, suggesting anti-fibrinolytic effect of rTM. 6) Decreased level of ADAMTS13 increased more quickly in TM group. 5) TNF-α, IL-6, HMGB1 were decreased significantly at day 3 compared with day 1 only in TM group. These anti-inflammatory effect can be evoked through direct sequestration of HMGB1 by rTM or decreased thrombin generation by rTM, because thrombin is a strong pro-inflammatory molecule through PAR1. Conclusion: rTM may be beneficial in improving septic DIC via its anti-thrombotic and anti-inflammatory properties. Disclosures: No relevant conflicts of interest to declare.

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