scholarly journals Stanozolol and Danazol Have Different Effects on Hematopoiesis in the Murine Model of Immune-Mediated Bone Marrow Failure

2021 ◽  
Vol 8 ◽  
Author(s):  
Hongmin Li ◽  
Zhangbiao Long ◽  
Tao Wang ◽  
Bing Han
Keyword(s):  
Bone Marrow ◽  
Cyclosporin A ◽  
Aplastic Anemia ◽  
Mononuclear Cells ◽  
Bone Marrow Failure ◽  
Erythropoietin Receptor ◽  
Bone Marrow Mononuclear Cells ◽  
Colony Forming Units ◽  
Colony Assays

Background: Stanozolol and danazol are widely used in the treatment of aplastic anemia; however, their mechanisms of action are unclear.Methods: Bone marrow mononuclear cells from 10 patients newly diagnosed with aplastic anemia and 10 healthy volunteers were collected and cultured together with stanozolol, danazol, or blank control separately for marrow colony assays. K562 cell lines that had been incubated with stanozolol, danazol, or blank control were tested for erythroid or megakaryocytic differentiation. Meanwhile, CB6F1/Crl mice were injected with 1 × 106 C57BL/6 donor-originated lymphocytes after irradiation with 5 Gy total body irradiation to establish a model for immune-mediated bone marrow failure (aplastic anemia mouse model). Mice with aplastic anemia were treated with cyclosporin A monotherapy, cyclosporin A in combination with stanozolol, and cyclosporin A in combination with danazol for 30 days. Peripheral blood cell counts once a week and bone marrow colony assays at the end of 1 month were performed. The proportion of T cell subsets, level of inflammatory factors, erythropoietin, and thrombopoietin were detected before and after treatment. The levels of erythropoietin receptors on bone marrow mononuclear cells after treatment were tested using western blotting.Results: In the ex vivo experiments, the number of burst-forming units-erythroid; colony-forming units-granulocyte and macrophage; and colony-forming units-granulocyte, erythrocyte, monocyte, and megakaryocyte in the patients with aplastic anemia were significantly lower than that in the normal controls (P < 0.05). However, the number of colonies and mean fluorescence intensity of CD235a or CD41 expression in the harvested cultured cells were not significantly different among the different treatment groups in the patients with aplastic anemia, normal controls, and K562 cell lines. These results show that stanozolol and danazol produce no direct hematopoiesis-stimulating effects on progenitor cells. In the in vivo experiment, the mice with aplastic anemia treated with cyclosporin A and danazol exhibited the most rapid recovery of platelet; the platelet count returned to normal levels after 3 weeks of treatment, which was at least 1 week earlier than in the other groups. In contrast, mice treated with cyclosporin A and stanozolol exhibited the highest hemoglobin level at the end of treatment (P < 0.05). Bone marrow colony assays at 30 days showed that the number of burst-forming units-erythroid was the highest in mice treated with cyclosporin A and stanozolol, while the number of colony-forming units-granulocyte and macrophage was the highest in those treated with cyclosporin A and danazol. Compared to cyclosporin A monotherapy, additional stanozolol and danazol can both increase the level of regulatory T cells and upregulate interleukin-10, inhibiting the expression of tumor necrosis factor-α (P < 0.05). However, IL-2 was more effectively reduced by danazol than by stanozolol (P < 0.05). The cyclosporin A- and stanozolol-treated mice showed higher serum erythropoietin (corrected by hemoglobin level) and higher erythropoietin receptor levels in bone marrow mononuclear cells than the other groups (P < 0.05).Conclusions: Neither stanozolol nor danazol directly stimulated hematopoiesis in vitro. However, in vivo, stanozolol may exhibit an advantage in improving erythropoiesis, while danazol may induce stronger effects on platelets. Both danazol and stanozolol exhibited immunosuppressive roles. Stanozolol could enhance the secretion of erythropoietin and expression of erythropoietin receptor in bone marrow mononuclear cells.

scholarly journals Interferon-gamma gene expression in unstimulated bone marrow mononuclear cells predicts a good response to cyclosporine therapy in aplastic anemia

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2532-2535 ◽  
Author(s):  
S Nakao ◽  
M Yamaguchi ◽  
S Shiobara ◽  
T Yokoi ◽  
T Miyawaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Aplastic Anemia ◽  
Interferon Gamma ◽  
Messenger Rna ◽  
Mononuclear Cells ◽  
Bone Marrow Mononuclear Cells ◽  
Ifn Gamma ◽  
Exact Test ◽  
Transfusion Independence

Cyclosporine (CyA) therapy has been shown to be effective in some patients with aplastic anemia. In an attempt to characterize aplastic patients likely to benefit from CyA therapy, we examined bone marrow mononuclear cells (BMMC) obtained before therapy from 23 patients with aplastic anemia, who were treated with CyA alone. Expression of four myelosuppressive cytokines, including tumor necrosis factor (TNF), lymphotoxin, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and interferon-gamma (IFN-gamma) was examined using polymerase chain reaction (PCR)-assisted messenger RNA (mRNA) amplification. mRNA for TNF, lymphotoxin, and MIP-1 alpha was readily detectable at variable levels in BMMC from normal and transfused controls as well as in BMMC from aplastic patients. In contrast, IFN-gamma mRNA was only demonstrable in BMMC from some patients with aplastic anemia, irrespective of a history of transfusions. Of 13 patients who responded to CyA therapy and achieved transfusion-independence, IFN-gamma mRNA was detected in 12 patients, whereas the mRNA was only detectable in 3 of 10 patients refractory to CyA therapy (P = .003, Fisher's exact test). Follow-up examination of BMMC obtained from seven CyA-responding patients after hematologic remission showed disappearance of IFN-gamma mRNA in four patients. These results suggest that detection of IFN- gamma gene expression in pretreatment BMMC from aplastic patients using PCR may be helpful in predicting a good response to CyA therapy.

Hematopoietic stem cells with controllable tEpoR transgenes have a competitive advantage in bone marrow transplantation

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3710-3715 ◽  
Author(s):  
Suzanne Kirby ◽  
William Walton ◽  
Oliver Smithies
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Erythropoietin Receptor ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Colony Forming Units

Abstract In a previous study, it was found that a truncated erythropoietin receptor transgene (tEpoR tg) enables multilineage hematopoietic progenitor amplification after treatment with erythropoietin (epo) in vitro and in vivo. This study used competitive bone marrow (BM) repopulation to show that tEpoR tg facilitates transplantation by hematopoietic stem cells (HSC). Individual multilineage colonies, committed myeloid progenitor colonies, and lymphoid colonies (pre-B colony-forming units) were grown from the marrow of animals 6 months after they received a 50/50 mixture of transgene and wild-type BM cells. In epo-treated recipients, the transgene-bearing cells significantly outcompeted the wild-type cells (84%-100% versus 16%-0%, respectively). In recipients treated with phosphate-buffered saline, the repopulation was minimally different from the donor mixture (49%-64% transgene versus 51%-36% wild-type). The epo-induced repopulation advantage is maintained in secondary transplants. In addition, neither accelerated HSC depletion nor uncontrollable proliferation occurred during epo-stimulated serial transplants of transgene-containing BM. Thus, the tEpoR tg functions in a benign fashion in HSC and allows for a significant and controllable repopulation advantage in vivo without excessive HSC depletion relative to wild-type BM.

PM373 In Vivo Assessment of Biodistribution and Efficacy of Bone Marrow Mononuclear Cells for Ischaemia-mediated Neovascularisation

Global Heart ◽  
2014 ◽  
Vol 9 (1) ◽  
pp. e137
Author(s):  
Sui Ching G. Yuen ◽  
Zoe Clayton ◽  
Ashanti Dantanarayana ◽  
Laura Lecce ◽  
Louise Dunn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Bone Marrow Mononuclear Cells ◽  
In Vivo Assessment

scholarly journals Characterization of Bone Marrow Mononuclear Cells on Biomaterials for Bone Tissue EngineeringIn Vitro

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Dirk Henrich ◽  
René Verboket ◽  
Alexander Schaible ◽  
Kerstin Kontradowitz ◽  
Elsie Oppermann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Tissue ◽  
Surface Coating ◽  
Mononuclear Cells ◽  
Demineralized Bone Matrix ◽  
Bone Matrix ◽  
Bone Marrow Mononuclear Cells ◽  
Beta Tricalcium Phosphate ◽  
Initial Adhesion

Bone marrow mononuclear cells (BMCs) are suitable for bone tissue engineering. Comparative data regarding the needs of BMC for the adhesion on biomaterials and biocompatibility to various biomaterials are lacking to a large extent. Therefore, we evaluated whether a surface coating would enhance BMC adhesion and analyze the biocompatibility of three different kinds of biomaterials. BMCs were purified from human bone marrow aspirate samples. Beta tricalcium phosphate (β-TCP, without coating or coated with fibronectin or human plasma), demineralized bone matrix (DBM), and bovine cancellous bone (BS) were assessed. Seeding efficacy onβ-TCP was 95% regardless of the surface coating. BMC demonstrated a significantly increased initial adhesion on DBM andβ-TCP compared to BS. On day 14, metabolic activity was significantly increased in BMC seeded on DBM in comparison to BMC seeded on BS. Likewise increased VEGF-synthesis was observed on day 2 in BMC seeded on DBM when compared to BMC seeded on BS. The seeding efficacy of BMC on uncoated biomaterials is generally high although there are differences between these biomaterials. Beta-TCP and DBM were similar and both superior to BS, suggesting either as suitable materials for spatial restriction of BMC used for regenerative medicine purposesin vivo.

scholarly journals Stanozolol and Danazol Have Different Effects on Hematopoiesis in Murine Model of Immune-Mediated Bone Marrow Failure

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2506-2506
Author(s):  
Hongmin Li ◽  
Zhangbiao Long ◽  
Bing Han ◽  
Zhao Wang
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Failure ◽  
Precursor Cells ◽  
Hematopoietic Precursor Cells ◽  
Immune Mediated ◽  
Hematopoietic Precursor ◽  
Colony Assays ◽  
Normal Controls

Background: Stanozolol and danazol are widely used in the treatment of aplastic anemia (AA), however, they may have different effects on the recovery of hematopoiesis with the detail mechanism unclear. Methods: Bone marrow mononuclear cells from 5 newly diagnosed AA patients and 5 healthy volunteers were collected for marrow colony assays and cultured together with stanozolol, danazol or blank control, separately. After incubated for 14 days, colonies of different lineage were calculated, and erythroid or megakaryocytic differentiation was also identified by the mean fluorescence intensity (MFI) of CD235a or CD41 expressed on the harvest cells. Meanwhile, CB6F1/Crl mice were injected with 1×106 C57BL/6 donor originated lymphocytes after irradiated with 5Gy total body irradiation to setup a model for immune-mediated bone marrow failure (AA mice model). AA mice were treated with CsA monotherapy, CsA combined with stanozolol, CsA combined with danazol for 30 days, respectively. Peripheral blood cell counts once a week, bone marrow colony assays at the end of one month were performed. Proportion of T cell subsets, level of inflammatory factors, EPO and TPO were detected before and after treatment. Level of EPO receptor on the progenitor cells after treatment were tested by western blot. Results: For ex vivo experiment, although the number of BFU-E, CFU-GM and CFU-GEMM colonies of AA patients were significantly lower than that of the normal controls (P<0.05), the number of colonies and MFI of CD235a or CD41 expression of the harvested cultured cells had no significant difference among different treatment groups, either in AA patients or in normal controls, showing no direct hematopoietic stimulating effects of stanozolol and danazol on progenitor cells. However, in the in vivo experiment, AA mice treated with CsA and danazol showed the most rapid recovery of megakaryopoiesis, with the platelet count returned to normal level after three weeks' treatment, at least one week earlier than the other groups. Whereas mice treated with CsA and stanozolol had the best hemoglobin level at the end of treatment (P<0.05). Bone marrow colony assays at the 30 days showed that the number of BFU-E was the highest in mice treated with CsA and stanozolol while the number of CFU-GM was the highest in those with CsA and danazol. Compared to CsA monotherapy, additional stanozolol and danazol can both increase the level of regulatory T cells and up-regulate interleukin-10 (P<0.05). But interferon-α and tumor necrosis factor-α were more effectively reduced by danazol than stanozolol (P<0.05). CsA and stanozolol- treated mice showed higher serum EPO (corrected by HGB level)and higher EPO receptor(EPOR) level on the hematopoietic precursor cells compared with other groups (P<0.05). Conclusions: Neither stanozolol or danazol directly stimulated hematopoiesis in vitro. But in vivo, stanozolol may have advantage in improving erythropoiesis while danazol may has more effects on white cells or platelet. Danazol had more comprehensive immunosuppressive roles compared with stanozolol. Stanozolol can enhance the expression of EPO, probably by increasing EPOR expression on hematopoietic precursor cells. Disclosures No relevant conflicts of interest to declare.

Monosomy 7 Detected by FISH at Disease Presentation Is a Marker for Non-Response to Immunosuppression.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1697-1697 ◽  
Author(s):  
Elaine Sloand ◽  
Phillip Scheinberg ◽  
Edward Fenlon ◽  
Jan Blancato ◽  
Loretta Pfannes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Aplastic Anemia ◽  
Mononuclear Cells ◽  
Bone Marrow Failure ◽  
Immunosuppressive Treatment ◽  
Chromosome 8 ◽  
Monosomy 7 ◽  
Clonal Progression ◽  
Cell Disorder

Abstract About fifteen percent of patients with severe aplastic anemia (sAA) who undergo immunosuppression therapy (IST) develop clonal disease in the decade following treatment. Of 122 patients treated with horse ATG/CsA at NIH, 13 developed cytogenetic abnormalities (monosomy 7 in 9 patients, 7p deletion in one, and trisomy 8 in 2) (Rosenfeld et al: JAMA289:1130; 2003). Monosomy 7 usually occurs in patients with sAA who are unresponsive to IST. Factors responsible for clonal progression in bone marrow failure are still unclear. We and others have reported that monosomy 7 may be detected by fluorescent in situ hybridization (FISH) months before conventional cytogenetics and that high levels of GCSF foster preferential expansion of the monosomy 7 clone in vitro (Sloand E et al; Proc. Natl. Acad. Sci2006;103:14483). We hypothesized that a clone of monsomy 7 cells might indicate an underlying stem cell disorder unlikely to respond to immunosuppression or reflect more severe disease marked by higher endogenous GCSF levels. FISH was undertaken on 40 bone marrow samples obtained from aplastic anemia patients at presentation and before immunosuppressive treatment. Bone marrow mononuclear cells were hybridized with centromeric probes specific for chromosome 7 and chromosome 8 (to control for hybridization efficiency); samples were assessed on duplicate slides by three investigators who were unaware of the diagnoses and outcomes. Twenty-five healthy controls were tested concurrently. The upper limit of normal was set at 6% based on control data. Of twenty-one patients with &gt; 6% monosomy 7 cells, response to IST was observed in 12 (57%), while 6 of 19 (84%) with normal FISH responded to IST (p=0.08). Thirteen patients had monosomy 7 frequencies of &gt;10%. Of these 5 (38%) responded to IST, compared with 23/27 (85%) with ≤10% monosomy 7. The proportion of monosomy 7 cells in the bone marrow correlated with age (R2 =0.6, p&lt;0.001). Two of the 23 non-responding patients later developed clonal progression with monosomy 7 or 7q-; both had &gt;10% monosomy 7 by FISH at presentation, but normal cytogenetics. The two responding patients tested with &gt;6% monosomy 7 prior to IST demonstrated &lt;2% following successful IST, suggesting that the high endogenous levels of G-CSF present before IST response may have facilitated expansion of the clone. Significant monosmy 7 clones may reflect a stem cell disorder in patients not responding to IST.

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