Oral cyclosporin-A is effective treatment for untreated and also for previously immunosuppressed patients with severe bone marrow failure*

Abstract Aplastic anemia(AA) is an unusual hematologic disease and the paradigm of the human bone marrow failure syndromes. Abnormal immunological responses have been found in most aplastic patients. Activated type 1 cytotoxic T cells may be the main contributing factor of pathogenesis. IDO is a rate-limiting enzyme in Tryptophan(Trp) metabolism. The proliferation of Th1 cells is specifically inhibited by the over-expression of IDO, which can degrade the Trp in local environment, hamper the conductioin of the activating signal in T cell and induce immune tolerance. 3,4-DAA is an IDO agonist and can activate IDO. In our experiment, Balb/c mice were irradiated (5.0Gy of 60Co), and then infused with 5×106 lymph node(LN) cells from DBA/2 mice in 4 hours. Blood count was monitored and marrow damage was assessed by histogical study. Concentrations of serum IFN-γ were measured by ELISA. The levels of Tryptophan and kynurenine were evaluated by high performance liquid chromatography (HPLC). CD4+CD25+ T cells in spleen were analyzed by flow cytometry. The level of Foxp3 in CD4+CD25+ T cell was measured by RT-PCR. Irradiation and infusion of LN cells led to rapid development of severe pancytopenia and bone marrow hypoplasia. Bone marrow of affected mice showed lymphocyte infiltration. Serum IFN-γ concentration increased 3.7 fold at d6 post infusion. The recipient mice were divided into 4 different treating groups as follow: 0.9% Sodium Chloride as control; Cyclosporin A (CsA) (50ug/g/d ×5d, peritoneal injection); 3,4-DAA(5mg/d, orally daily); the combination of CsA and 3,4-DAA. The effects of CsA, IDO agonist and CsA combined IDO agonist were analyzed at day 6,10,14,21,24 and 28 after LN cells infusion. The white cell and the platelet recovered to near normal, respectively (4.2±0.32)×109, (937±190.47)×1012 at d21 in the combination group. Early stage treatment with CsA can improve periphery blood cells and BM nucleated cells, but long term effect was not remarkable. In contrast, the 3,4-DAA group exhibited slow and gradually enhanced role. Periphery blood cells and BM nucleated cells were improved remarkably in the combination group. The number and function of CD4+CD25+T cells also increased remarkably. In the treatment of AA, abnormal immune response in bone marrow was inhibited by CsA, meanwhile immune tolerance was induced through up-regulating the regulatory T cells(Treg) by 3,4-DAA. In this way, the balance of immune in bone marrow could be reestablished quickly. CsA combined IDO agonist could provide a new strategy for the management of AA.

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The DNA Synthesis Inhibitor, Hydroxyurea, as a Potential Novel Therapeutic Approach for Fanconi Anemia: Further Studies in Complementation Group D1.

Blood ◽

10.1182/blood.v114.22.4214.4214 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4214-4214

Author(s):

W. Clark Lambert ◽

Monique M Brown

Keyword(s):

Bone Marrow ◽

Dna Synthesis ◽

Effective Treatment ◽

Sickle Cell ◽

Fanconi Anemia ◽

Sickle Cell Anemia ◽

Bone Marrow Failure ◽

Complementation Group ◽

Lymphoblastoid Cells ◽

Complementation Groups

Abstract Abstract 4214 Fanconi anemia (FA) is an inherited, cancer-prone bone marrow failure disease. FA is heterogeneous, with 13 complementation groups, but all groups have in common hypersensitivity to agents that produce DNA interstrand cross links (DISCLs), with associated increased clastogenicity, as a diagnostic hallmark. Although progress has been made in treating FA, particularly using bone marrow transplantation (BMT) to prevent bone marrow failure and leukemogenesis, BMT is not a trivial procedure, and treatment remains challenging. Head and neck cancers, which occur in high frequency in FA, are a particular problem that is not well remedied by BMT. Lymphoblastoid cells from normal subjects and from patients with FA were treated in culture with psoralen plus ultraviolet A radiation (PUVA) in a regimen shown to produce DISCLs. Following this, cells were treated with hydroxyurea, 5-fluorouracil, or high dose thymidine, in doses we have shown to produce a marked decrease in rate of DNA synthesis, for 24 hours. We have previously shown that clastogenicity and cytotoxicity, measured as trypan blue exclusion as well as colony forming ability (CFA), are markedly increased in FA cells, complementation groups A, B, C, and E, associated with deficiencies in their corresponding FA core proteins, but these increases are not observed in these FA cells subsequently treated with any of these other, DNA synthesis retarding agents, which effectively correct the FA phenotype in culture. FA A and C cells genetically corrected for the FANC A and G gene, respectively, display normal clastogenicity and cytotoxicity following PUVA, and do not show this correction following subsequent treatment with hydroxyurea, 5-fluorouracil, or high dose thymidine. We now report similar results for short term cell viability, and similar, although less marked, results for clastogenicity in FA complementation group D1 cells, associated with a deficiency in BRCA2. When all drugs were removed after these treatments and the cells cultured for 10 days without any drug in CFA assays, the FA group D1 cells resembled normals, however, and did not show this correction. We propose that the mechanism in FA A, B, C and G cells is related to a decrease in the rate of DNA synthesis, which we have shown occurs in normal but not FA cells following PUVA, and which is also produced by these other agents in the concentrations used here. The partial correction observed in FA group D1 cells may be due to this or a different mechanism. Partial or complete correction appears to apply to multiple FA complementation groups. Hydroxyurea has been used for many years as a safe and effective treatment for sickle cell anemia and other diseases. It is now proposed as a possible treatment for FA to delay or even prevent development of bone marrow failure and/or other complications, including leukemogenesis and carcinogenesis, with or without prior BMT. In some cases it may serve as a viable alternative where BMT is not fungible. Alternatively it may obviate the need for BMT altogether in responsive patients, or be effectively used in combination with other modalities. Complementation group may be important in determining which patients may be less responsive or require modified regimens. Disclosures: Off Label Use: We have obtained laboratory results which show partial or complete restoration of cytotoxicity and clastogenicity, as well as colony forming ability in the absence of drug in FA A, B, C, and G but not D1 cells, following treatment with a DNA cross-linking agent, in Fanconi anemia lymphoblastoid cells, by subsequent application of hydroxyurea, to normal levels. Hydroxyurea has been used for many years as a safe and effective treatment for sickle cell anemia. It is now proposed as a possible treatment for Fanconi anemia to delay or even prevent development of bone marrow failure and/or other complications, including leukemogenesis and carcinogenesis. It may be less effective in FA complementation group D1. Disclosures: Off Label Use: We have obtained laboratory results which show partial or complete restoration of cytotoxicity and clastogenicity, as well as colony forming ability in the absence of drug, following treatment with a DNA cross-linking agent, in Fanconi anemia lymphoblastoid cells, by subsequent application of hydroxyurea, to normal levels. Hydroxyurea has been used for many years as a safe and effective treatment for sickle cell anemia. It is now proposed as a possible treatment for Fanconi anemia to delay or even prevent development of bone marrow failure and/or other complications, including leukemogenesis and carcinogenesis..

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

Frontiers in Medicine ◽

10.3389/fmed.2021.615195 ◽

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.

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