Notch signaling is critical for osteoblastic differentiation; however, the specific contribution of individual Notch ligands is unknown. Parathyroid hormone (PTH) regulates the Notch ligand Jagged1 in osteoblastic cells. To determine if osteolineage Jagged1 contributes to bone homeostasis, selective deletion of Jagged1 in osteolineage cells was achieved through the presence of Prx1 promoter-driven Cre recombinase expression, targeting mesenchymal stem cells (MSCs) and their progeny (PJag1 mice). PJag1 mice were viable and fertile and did not exhibit any skeletal abnormalities at 2 weeks of age. At 2 months of age, however, PJag1 mice had increased trabecular bone mass compared to wild-type (WT) littermates. Dynamic histomorphometric analysis showed increased osteoblastic activity and increased mineral apposition rate. Immunohistochemical analysis showed increased numbers of osteocalcin-positive mature osteoblasts in PJag1 mice. Also increased phenotypically defined Lin−/CD45−/CD31−/Sca1−/CD51+ osteoblastic cells were measured by flow cytometric analysis. Surprisingly, phenotypically defined Lin−/CD45−/CD31−/Sca1+/CD51+ MSCs were unchanged in PJag1 mice as measured by flow cytometric analysis. However, functional osteoprogenitor (OP) cell frequency, measured by Von Kossa+ colony formation, was decreased, suggesting that osteolineage Jagged1 contributes to maintenance of the OP pool. The trabecular bone increases were not due to osteoclastic defects, because PJag1 mice had increased bone resorption. Because PTH increases osteoblastic Jagged1, we sought to understand if osteolineage Jagged1 modulates PTH-mediated bone anabolism. Intermittent PTH treatment resulted in a significantly greater increase in BV/TV in PJag1 hind limbs compared to WT. These findings demonstrate a critical role of osteolineage Jagged1 in bone homeostasis, where Jagged1 maintains the transition of OP to maturing osteoblasts. This novel role of Jagged1 not only identifies a regulatory loop maintaining appropriate populations of osteolineage cells, but also provides a novel approach to increase trabecular bone mass, particularly in combination with PTH, through modulation of Jagged1.

Fatal disseminated Toxoplasma gondii infection was diagnosed in 2 captive magpie geese (Anseranas semipalmata) from a zoo in Texas. Both geese died suddenly, without apparent clinical signs. Lesions associated with T. gondii tachyzoites were seen in lungs, pancreas, liver, adrenals, bursa of Fabricius, spleen, brain, and kidneys. Toxoplasmic pneumonia and hepatitis were considered to be the primary cause of death. An unusual feature was the presence of numerous tissue cysts in hepatocytes of both geese. The diagnosis was confirmed immunohistochemically. Antibodies to T. gondii were found in 2 of 11 other geese from the zoo examined using the modified agglutination test. This is the first report of T. gondii infection in magpie geese (Anseranas semipalmata).

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Sirolimus is an approved drug to prevent solid organ transplant rejection and to treat lymphangioleiomyomatosis. Sirolimus also has been employed in a broad range of clinical circumstances, including induction of tolerance in nonmyeloablative HLA-mismatched bone marrow (BM) transplantation and treatment of graft-versus-host disease following allogeneic stem cell transplantation. Sirolimus suppresses mammalian target of rapamycin (mTOR), a serine-threonine protein kinase that responds to multiple signals to regulate cellular metabolism, survival, and growth. We recently reported the application of sirolimus on murine models of immune-mediated BM failure (Feng X, Haematologica, 2017;102:1691-1703) in which sirolimus alleviated pancytopenia and attenuated BM destruction by depleting active cytotoxic T cells and expanding regulatory T cells. Controls for these experiments suggested that sirolimus might also act directly to preserve hematopoietic stem and progenitor cells (HSPCs) in non-immune BM damage and hematopoietic cell injury. In the current study, we systematically tested the effects of sirolimus on HSPC regeneration following irradiation and exposure to cytotoxic drugs. B6 mice subjected to 5 Gys total body irradiation (TBI) showed large declines in HSPC numbers, which were abrogated by sirolimus at 2 mg/Kg/day by intraperitoneal (i.p.) injection for 13 days: we observed significant increases (2-4-fold) in the proportions and absolute numbers of HSPCs relative to TBI controls when animals were evaluated at 14 days (Fig. 1A). Sirolimus did not alter peripheral blood cell counts or BM cellularity. Functionally, BM cells from sirolimus-treated TBI mice contained 2-3-fold more colonies than did marrow of TBI controls. In a competitive repopulation stem cell assay, recipients of sirolimus-treated TBI donor BM cells had enhanced donor engraftment, as measured in recipient animals' peripheral blood and BM. There was also an increased recovery of donor HSPCs in recipient BM, relative to recipients of TBI-only donor BM cells, evaluated at 7 months after donor cell infusion. In other experiments, sirolimus at the same dose following busulfan (10 mg/kg i.p. three times weekly for 10 injections; Fig. 1B) or 5-fluorouracil (150 mg/Kg on days 0, 15, and 29 respectively; Fig. 1C) induced similar improved HSPC regeneration. Sirolimus-mediated HSPC regeneration was associated with c-Kitup-regulation in BM cells. In a PCR-based transcriptome assay of hematopoiesis genes performed on sorted Lin-CD150+ BM cells, sirolimus-treated TBI mice showed a 90% increase in c-Kit mRNA relative to TBI controls. Expression of other stem cell and transcription factor genes, including Cd34, Gata2,Angpt1, and Etv6, was also increased, while expression of T cell differentiation and Notch signaling genes, such as Cd2, Cd4, Cd8a, Tnfsf11, Jag1a, and Il-1a, was decreased in sirolimus-treated TBI mice relative to TBI controls. In parallel experiments, sirolimus had no effect on HSPCs in steady-state B6 mice that were not subjected to hematopoietic injury. Sirolimus stimulated HSPC expansion in mice carrying the Wv mutation in the c-Kit locus (Fig. 1D). To test the potential utility of sirolimus in humans, we pre-irradiated human BM CD34+ cells with 5 Gys to induce injury, then transplanted the radiated cells into immunodeficient NSG mice, followed by sirolimus treatment (2 mg/Kg/day i.p. for 4 injections). Sirolimus significantly increased engraftment of human CD45+ cells in recipient blood and BM at 5 weeks relative to the mice without sirolimus treatment (Fig. 1E). In summary, sirolimus augments c-Kit expression and HSPC recovery in response to hematopoietic stress. This novel functional property detected in murine models and xenotransplantation of human cells suggests potential clinical applicability of sirolimus to ameliorate hematopoietic cell injury and accelerate HSPC regeneration after BM hematopoietic injury. Disclosures Young: CRADA with Novartis: Research Funding; GlaxoSmithKline: Research Funding; National Institute of Health: Research Funding.

The role of mammalian target of rapamycin and its suppressor sirolimus in the regulation of hematopoietic stem and progenitor cells (HSPCs) is controversial. We show here that sirolimus enhanced regeneration of HSPCs in mice exposed to sublethal total body irradiation (TBI) and other regenerative stressors. Sorted Lin−CD150+ bone marrow cells from sirolimus-treated TBI mice had increased expression of c-Kit and other hematopoietic genes. HSPCs from sirolimus-treated TBI mice were functionally competent when tested by competitive engraftment in vivo. Postradiation regeneration of HSPCs in mice treated with sirolimus was accompanied by decreased γ-H2AX levels detected by flow cytometry and increased expression of DNA repair genes by quantitative polymerase chain reaction. Reduction of cell death and DNA damage post-radiation by sirolimus was associated with enhanced clearance of cellular reactive oxygen species (ROS) in HSPCs. Increased HSPC recovery with sirolimus was also observed in mice injected with hematoxic agents, busulfan and 5-fluorouracil. In contrast, sirolimus showed no effect on HSPCs in normal mice at steady state, but stimulated HSPC expansion in mice carrying the Wv mutation at the c-Kit locus. In human to mouse xenotransplantation, sirolimus enhanced engraftment of irradiated human CD34+ cells. In summary, our results are consistent with sirolimus' acceleration of HSPC recovery in response to hematopoietic stress, associated with reduced DNA damage and ROS. Sirolimus might have clinical application for the treatment and prevention of hematopoietic injury.