A deletion of telomerase RNA component (Terc−/−) in C57BL/6 (B6) mice resulted in hematopoietic lineage skewing with increased neutrophils and CD11b+ myeloid cells and decreased red blood cells and CD45R+ B lymphocytes when animals reach ages older than 12 months. There was no decline in bone marrow (BM) c-Kit+Sca-1+Lin− (KSL) cells in old Terc−/− mice, and the lineage skewing phenomenon was not transferred when BM cells from old Terc−/− donors were transplanted into young B6 recipients. Necropsy and histological examinations found minimal to no change in the lung, spleen and liver but detected severe epithelia degeneration, ulceration and infection in small and large intestines, leading to enteritis, typhlitis and colitis in old Terc−/− mice. In a mouse model of dextran-sulfate-sodium-induced typhlitis and colitis, development of intestinal pathology was associated with increases in neutrophils and CD11b+ myeloid cells and a decrease in CD45R+ B cells, similar to those observed in old Terc−/− mice. Treatment of 11–13 month old Terc−/− mice with antibiotic trimethoprim-sulfa water reduced neutrophils and myeloid cells and increased B lymphocytes in the blood, indicating that mitigation of intestinal infection and inflammation could alleviate hematological abnormalities in old Terc−/− animals.

516 We have previously shown that sex hormones up-regulate telomerase gene expression and telomerase activity in cultured human peripheral blood leukocytes and bone marrow (BM) CD34 cells (Calado et al. Blood 2009). In the current study, we tested the effects of androgens in the preservation of telomeres in vivo using mouse models. We first confirmed that mouse BM cells in vitro responded to 2–5mM testosterone, a primary androgen, with a 1.3–3.8 fold increase (P<0.05) in telomerase RNA expression in BM cells from mice heterozygous for the telomerase RNA gene deletion (Terc+/−) and in wild type controls but had no effect on BM cells from Terc−/− knockout mice. We then treated normal B6, Terc−/−, and Terc+/− mice with testosterone (in corn oil emulsion, 350 micrograms/mouse once weekly by subcutaneous injection) and found 1.3 – 2.1 fold up-regulation in telomerase mRNA expression in blood leukocytes from Terc+/+ and Terc+/− but not Terc−/− mice, suggesting that androgens can potentially up-regulate telomerase activity in vivo. To directly test androgen effect on telomere repair, we injected testosterone (350 micrograms/mouse/week) or corn oil into B6, Terc+/− and Terc−/− mice; there was no testosterone effect on telomere length in either B6 or Terc−/− mice at steady state after six months of treatment. In Terc+/− mice, however, testosterone treatment produced gain of telomere length relative to corn oil controls at four (1.94 ± 1.18 vs 0.49 ± 1.32 Kbs, P<0.4247) and six (2.08 ± 0.52 vs 0.18 ± 0.58 Kbs, P<0.0256) months, indicating that androgen treatment can extend telomeres in vivo under specific conditions. As telomere erosion is minimal under regular conditions in wild-type mice, especially during a short period of time, we hypothesized that androgen effects might be more evident under circumstances of hematopoietic stress and the requirement for accelerated cell replication. Thus, we tested the effect of testosterone on telomere maintenance in three different circumstances. First, we used a BM transplantation model in which we transplanted limiting numbers (5 × 105) of BM cells from Terc+/+, Terc+/−, or Terc−/− donors into lethally-irradiated B6-CD45.1 recipients, and then exposed recipients to weekly injections of testosterone or corn oil for four months. There was no effect of testosterone treatment in recipients of Terc+/+ and Terc−/− BM cells. However, testosterone injection led to telomere gain in recipients of Terc+/− BM cells at three (2.90 ± 0.84 vs 0.79 ± 0.89 Kbs, P<0.0947) and four (2.00 ± 0.61 vs 0.15 ± 0.65 Kbs, P<0.0446) months, respectively (Fig 1A). Second, we used a chemical stress model in which Terc+/− mice received three monthly intraperitoneal injections of 5-fluorouracil at 100 micrograms/gram of body weight; animals were divided into testosterone or corn oil treatment groups. At four months, testosterone treatment resulted in gain of telomere length (1.39 ± 0.60, 0.73 ± 0.50, 1.51 ± 0.62 and 0.97 ± 0.54 Kbs) while telomere attrition occurred in corn oil control animals (−0.03 ± 0.68, −0.93 ± 0.58, −0.37 ± 0.60 and 0.11 ± 0.62 Kbs; Fig 1B), showing a significant overall testosterone effect (P<0.0007) on telomere maintenance. Third, we used repeated irradiation in which Terc+/− mice received three monthly sub-lethal (6 Gys) doses of radiation accompanied by testosterone or corn oil injections. Again, corn oil control mice showed marked telomere attrition (−0.99 ± 0.28, −1.55 ± 0.43, −1.16 ± 0.38 and −0.23 ± 0.36 Kbs) whereas mice that received testosterone had gain or reduced attrition of telomere length (0.46 ± 0.32, −0.51 ± 0.50, −0.03 ± 0.43 and 0.83 ± 0.41 Kbs; P<0.0019; Fig 1C). When the same repeated irradiation protocol was applied to a small group of old Terc+/− mice at 21–26 months of age, telomere attrition was apparent in corn oil control animals at two to four months (−1.50 ± 1.17, −2.68 ± 0.62 and −1.75 ± 1.27 Kbs) but testosterone prevented telomere loss (1.27 ± 0.83, 0.20 ± 0.44 and1.21 ± 1.04 Kbs; P<0.0082; Fig 1D). We conclude that androgens are relatively inactive under normal, steady state conditions, but testosterone at pharmacologic doses can protect telomeres under conditions associated with hematopoietic stress. One implication of these results is the potential utility of sex hormone replacement or pharmacologic administration in patients at risk of secondary hematologic malignancies after chemo- and radiation therapies. Disclosures: No relevant conflicts of interest to declare.

1244 Mice carrying germline deletion of the telomerase RNA component (Terc−/−) have functional alterations in various tissues but can breed and live normally for six generations. We acquired Terc−/− mice at G3 generation under the C57BL/6 (B6) background, backcrossed to normal inbred B6 mice, interbred between Terc+/− offspring to generate Terc−/−, Terc+/− and Terc+/+ mice at later generations, and analyzed cytological changes in hematological tissues using animals at young (3–6 months), middle (12–18 months) and old (20–26 months) ages. Cellular composition was relatively normal in peripheral blood (PB), bone marrow (BM) and spleen in young Terc−/− and Terc+/− mice relative to Terc+/+ littermates. However, middle and old Terc−/− mice had significantly lower levels of red blood cells, hemoglobin and hematocrit, and significantly higher percentage red cell distribution width and neutrophil counts in comparison to old Terc+/+ mice and young B6 controls. Middle and old Terc−/− mice also showed typical signs of lineage bias with significantly higher CD11b cell percentage and significantly lower CD45R cell percentage in BM, PB and spleen. Interestingly, BM cells from middle and old Terc−/− mice contained higher percentage and total number of Lin−Sca1+Kit+CD150+ hematopoetic stem and progenitor cells than those from young Terc−/− and young B6 controls. Transplantation of old Terc−/− BM cells into lethally irradiated young B6 recipients resulted in effective donor cell engraftment with relatively normal levels of blood cell counts and normal CD11b and CD45R cell percentages at four to twelve months after transplantation, indicating that the anemia, neutrocytosis and hematopoietic lineage bias seen in aged Terc−/− mice were not intrinsic to hematopoietic stem or progenitor cells. We therefore hypothesize that the age-related hematological abnormalities in Terc−/− mice were responses secondary to inflammatory stresses, or were results of BM stromal dysfunction, or both. Since hematological abnormalities were easily detectable in middle Terc−/− (12–18 months) mice, we used animals of this age group in our future studies. Middle Terc−/− mice were smaller in size compared to littermates, lethargic, in a hunched posture, and produced a soft stool instead of fecal pellets. At necropsy, the walls of the ileum, cecum and colon were thickened; the ceca were diminished in size, and the cecal and colonic digesta was soft and pasty. Histologic changes in the small intestine, cecum and colon consisted of dysplastic mucosal epithelium with anisocytosis, cytomegaly, and karyomegaly. The small intestinal villi were blunted and fused. Mucosal crypts were lost in the small intestine, cecum and colon along with mucosal ulceration, epithelial attenuation and bacterial colonization of ulcerated areas. There was marked suppurative intestinal inflammation along with severe enteritis, typhlitis and colitis (Figure 1, A & B). In these same animals, BM hematopoiesis was dominated by myeloid precursors with a sharp decline in the presence of erythroid precursors (Figure 1, C & D). In the skin sections, the vast majority of hair follicles were in telogen, a resting phase, and were located close to the epidermis with few sebaceous glands around the hair follicle. Liver, heart, lung and kidney were relatively normal based on microscopic examinations. We analyzed plasma concentrations of selected hematopoietic cytokines and found insignificant declines in GM-CSF and IL-3 and significant increases in G-CSF and IL-6 in middle Terc−/− mice. Flow cytometry analyses revealed a lower percentage of CD3−CD11b−CD45R−CD44+Sca1+ stromal cells in the BM of middle Terc−/− animals. Culturing middle Terc−/− BM cells in vitro in alpha-modified eagle media at 33 degrees for two weeks resulted in the development of stromal feeder layers with normal morphological appearance but reduced functional efficacy to support cobblestone colony formation from freshly-overlaid normal B6 BM cells. We conclude that constitutional Terc deficiency in middle and old Terc−/− mice causes obvious intestinal epithelia degeneration and severe intestinal inflammation that adversely affects normal hematopoiesis to result in neutrocytosis, anemia and lineage bias disfavoring B cells. Dysfunction of BM stromal cells might also contribute to hematological abnormalities in aged Terc-deficient animals. Disclosures: No relevant conflicts of interest to declare.