The purpose of this project is to identify abnormalities in the hematopoietic microenvironment (ME) that are associated with aplastic anemia (AA). Both AA and normal control ME will be established in vitro as long-term marrow cultures (LTMC) and a comparative analysis of their cellular composition performed using immune cytochemistry of marrow biopsies to establish an in vitro/in vivo correlate. Growth factor gene transcription in LTMC will be analyzed using RNAse protection assays and/or polymerase chain reactions as required. When possible the actual translation and secretion of bioactive cytokines will be detected by bioassays and ELISA. The possibility that patient lymphocytes may interfere with ME function will be addressed by determining whether the addition of lymphocytes to the LTMC interferes with the production of growth factors. The relative ability of AA-LTMC to support the growth of normal progenitors will be determined by adding isolated populations of """"""""stem cells"""""""" to the cultures. These cells will be obtained from normal marrow donors, sex-mismatched with the AA-LTMC to provide markers, and isolated on the basis of CD34 expression and low-intensity staining with rhodamine-123 to eliminate more mature cells that could contribute to the ME. In conjunction with Project 1, patients will be studied before and after treatment to determine if ME defects are altered by therapy, and whether such changes correlate with clinical response.
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