Androgens Androgens: Androgens are natural male hormones that can cause your bone marrow to make more red blood cells. This can improve anemia. Androgens are sometimes used to treat aplastic anemia and PNH. Androgens that may be used include: Danazol (Danacrine®) Fluoxymestrone (Halotestin®) Oxymetholone … have been used alone or in combination with other immunosuppressive therapy immunosuppressive therapy: Immunosuppressive drug therapy lowers your body's immune response. This prevents your immune system from attacking your bone marrow, allowing bone marrow stem cells to grow, which raises blood counts. For older patients with acquired aplastic anemia, immunosuppressive drug therapy is the… in aplastic anemia aplastic anemia: (ay-PLASS-tik uh-NEE_mee-uh) A rare and serious condition in which the bone marrow fails to make enough blood cells - red blood cells, white blood cells, and platelets. The term aplastic is a Greek word meaning not to form. Anemia is a condition that happens when red blood cell count is low. Most… with inconsistent responses being reported. However, the exact mechanism of action of androgens is not known. Recent studies have shown that it tends to protect from telomere telomere: A telomere is the end of a chromosome. Telomeres are made of repetitive sequences of non-coding DNA that protect the chromosome from damage. Each time a cell divides, the telomeres become shorter. EVvntually, the telomeres become so short that the cell can no longer divide. shortening in haematopoietic tissues by increasing telomerase activity. In addition, androgens exert various biological effects on both haematopoietic cells and stromal cells. Androgen receptors are found to be expressed on stromal cells, macrophages, endothelial cells, myeloblasts, myelocytes, neutrophils, and megakaryocytes.
Androgens also have a suppressive effect on the immune system. Shortened telomeres in aplastic anemia were initially attributed to stress factors but was later recognized that telomere maintenance was regulated by a network of genes. Mutations in these genes results in short telomeres which accelerate senescence and
apoptosis
apoptosis:
Programmed cell death.
. Recent studies have shown that androgens amplify telomerase activity in hematopoietic progenitor cells, even in individuals who had reduced telomerase activity caused by TERT mutations.
This project aims to answer two important. clinically relevant questions. The answers to these questions will aid in improving the treatment outcomes of many patients with aplastic anemia in developing countries.
Androgens Androgens: Androgens are natural male hormones that can cause your bone marrow to make more red blood cells. This can improve anemia. Androgens are sometimes used to treat aplastic anemia and PNH. Androgens that may be used include: Danazol (Danacrine®) Fluoxymestrone (Halotestin®) Oxymetholone … improve hematopoiesis hematopoiesis: (hi-mat-uh-poy-EE-suss) The process of making blood cells in the bone marrow. in aplastic anemia aplastic anemia: (ay-PLASS-tik uh-NEE_mee-uh) A rare and serious condition in which the bone marrow fails to make enough blood cells - red blood cells, white blood cells, and platelets. The term aplastic is a Greek word meaning not to form. Anemia is a condition that happens when red blood cell count is low. Most… subjects by known and unknown mechanisms. We aim to study the effect of androgens on hematopoietic stem cells stem cells: Cells in the body that develop into other cells. There are two main sources of stem cells. Embryonic stem cells come from human embryos and are used in medical research. Adult stem cells in the body repair and maintain the organ or tissue in which they are found. Blood-forming (hemapoietic) stem… and stromal cells by colony assays, transcriptome analysis and telomere telomere: A telomere is the end of a chromosome. Telomeres are made of repetitive sequences of non-coding DNA that protect the chromosome from damage. Each time a cell divides, the telomeres become shorter. EVvntually, the telomeres become so short that the cell can no longer divide. biology.
The source of haematopoietic cells (HSC) were from donors of stem cell transplantation. HSCs were isolated from the peripheral blood mononuclear cells using EasySep Human CD34Positive Selection kit from Stem cell technologies. The isolation was carried out according to manufacturer’s protocol protocol: An action plan that describes what will be done in a clinical trial and how it will be carried out. This plan is reviewed and approved by a committee at each place doing the clinical trial. This committee is known as the Institutional Review Board. . The CD34 isolated cells were cultured in Stemspan SFEM medium in a 24 well plate with expansion supplement supplement: see dietary supplement . Approximately 104/well were cultured. The cells were incubated at 370C at 5% CO2 for 7 days. We have now done five normal HSC culture. Viability was checked at day7 using 7-AAD. DNA and RNA were extracted from the cultured cells. The next set of analysis will now include androgen treatment and further analysis will be carried out.
The relative telomere length assay was standardized using a relative quantitation method (Cowthan, 2002; O’Callaghan, 2011). The telomere length measurement will be done for the normal CD34 cells in culture and also after androgen treatment. Moreover patients on these drugs will also be assessed serially for telomere length.
The response to androgen treatment is varied among patients. Metabolism of androgens and clearance can differ between individuals. Immune regulation can be modulated by androgens. This study aims to analyze these parameters in responders and non-responders and elucidate determinants of response
Sixty seven patients who have taken androgens have been recruited so far. Males were slightly higher than females (n=37 vs n=30). Majority of them were severe and very severe aplastic anemia (42%) and 28% of them were non-severe AA. Three cases with Fanconi anemia Fanconi anemia: A rare inherited disorder that happens when the bone marrow does not make enough blood cells (red cells, white cells, and platelets). Fanconi anemia is diagnosed early in life. People with Fanconi anemia have a high likelihood of developing cancer. Genetic testing is used to diagnose Fanconi anemia. and eight cases with MDS were also included. Peripheral blood/ Bone marrow Bone marrow: The soft, spongy tissue inside most bones. Blood cells are formed in the bone marrow. aspirate was collected at diagnosis. Serum was separated and is stored at -80°C for further analysis.
Pure compounds of Stanazolol and Danazol were commercially obtained from Sigma Aldrich. A stock solution of 1mg/ml was prepared using acetonitrile. An intermediate stock solution (100ug/ml) was prepared by 1 in 10 dilution of the stock. A serial dilution (1 in 2) was made to different concentrations ranging from 100ug/ml to 6.25ug/ml.
An HPLC based assay was standardized using these set of standards. A C-18 column was used with acetonitrile as the mobile phase. The detection was using a diode array detector at 230nm for and 280nm for Stanozolol and Danazol respectively. A linear good fit standard curve was obtained and now the serum samples need to be processed. Since the method is now established, we will now analyze the patients’ samples for drug levels and correlate them with response.