Inflammation Caused by Allogeneic Lymphocytes Results in Aneuploidy in Bone Marrow Mononuclear Cells (BMMNC)

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Patients with aplastic anemia and other autoimmune diseases such as rheumatoid arthritis are more likely to develop myelodyplasia when compared to healthy controls. Inflamed tissue from Barrett's esophagus, areas of bronchitis, healing burn tissue, and bowel with ulcerative colitis commonly show increased numbers of tetraploid and aneuploid cells, suggesting that inflammation may have a role in promoting genomic instability. We recently demonstrated the common occurrence of aneuploid cells in the 15 buccal smears of transplant patients with graft versus host disease involving the oral mucosa but not in transplant patients without GVSH when these cells were examined by fluorescence in situ hybridization (FISH). Aneuploidy could be reproduced in 5 normal keratinocyte cultures following incubation with allogeneic HLA-mismatched lymphocytes. Oxidative stress and repeated cell division with telomere shortening may play a role in producing karyotypic abnormalities. Alternatively, in the AA bone marrow factors such as increased cytokine expression and the presence of cytotoxic T cells in marrow may lead to a relatively improved survival of cells with certain chromosomal abnormalities. In this respect, we previously demonstrated survival advantages for trisomy 8 and monosomy 7 relative to diploid cells (Proc Natl Acad Sci 2006; 10314483: Blood . 2007; 109(6):2399). In order to more thoroughly assess the role played by inflammation in producing aneuploidy, we examined five samples of normal bone marrow co-cultured for two weeks with allogeneic, autologous lymphocytes (effector to target ratio of 2:1), or with interferon alone (1000 u/mL); media was changed after 24 hours and FISH was performed using probes to chromosomes 7,8 and 20 after two weeks. Slides were read by three different individuals blinded to the identity of the slides. All bone marrow samples with allogeneic lymphocyte and with interferon but not those cultured with autologous lymphocytes showed significant numbers of aneuploid cells; Aneuploidy was most prominent in bone marrow treated with allogeneic lymphocytes (monosomy 7 mean control=1%±1%; treated=11±2%; p=0.01; trisomy 8 control=0±0%; treated=6±1%; p=0.06; deletion 20 control=1±1%; treated=14%; p=0.01; N=5). In order to ensure that these changes were not secondary to apoptosis-related DNA disintegration and to determine if aneuploidy occurred in cells capable of self renewal, bone marrow cells were exposed to allogeneic lymphocytes and to interferon g (1000 units/mL) in short-term methylcellulose culture for two weeks; we subsequently picked cells from hematopoietic colonies, pooled these cells, and stained them with annexin-FITC. Cells were sorted by flow cytometry and annexin-negative cells were subjected to FISH using centromeric probes to chromosome 8, 7 and 20. These isolated cells, derived from single progenitors, still demonstrated significant numbers of trisomy 8, monosomy 7, and deletion (20) by FISH (Fig. 1). Spectral karyotyping (SKY) analysis demonstrated chromosomal abnormalities in 13/25 (52%) of cells and prominent telomeric fusion in many metaphase preparations (Fig II). In order to determine whether telomeric shortening could play a role in fusion of chromosomes and subsequently aneuploidy, we measured telomere length by Southern hybridization: bone marrow cells cultured with allogeneic lymphocytes showed a 20% decline in telomere length relative to control populations. These in vitro data suggest that inflammatory changes may be associated with genomic instability, possibly secondary to accelerated telomere attrition in response to regeneration. [Display omitted] [Display omitted]