We have previously shown that the keys to high-level nontoxic chimerism in syngeneic models are stem cell toxic, nonmyelotoxic host treatment as provided by 100-cGy whole-body irradiation and relatively high levels of marrow stem cells. This approach was unsuccessful in H-2 mismatched B6.SJL to BALB/c marrow transplants, but with tolerization, stable multilineage chimerism was obtained. Ten million B6.SJL spleen cells were infused intravenously into BALB/c hosts on day ؊10 and (MR-1) anti-CD40 ligand monoclonal antibody (mAb) injected intraperitoneally at varying levels on days ؊10, ؊7, ؊3, 0, and ؉3 and the BALB/c mice irradiated (100 cGy) and infused with 40 million B6.SJL/H-2 mismatched marrow cells on day 0. Stable multilineage chimerism at levels between 30% to 40% was achieved in the great majority of mice at 1.6 mg anti-CD40 ligand mAb per injection out to 64 weeks after transplantation, without graft-versus-host disease. The transplanted mice were also tolerant of donor B6.SJL, but not third-party CBA/J skin grafts at 8 to 9 and 39 to 43 weeks after marrow transplantation. These data provide a unique model for obtaining stable partial chimerism in H-2 mismatched mice, which can be applied to various clinical diseases of man such as sickle cell anemia, thalassemia, and autoimmune disorders. (Blood. 2001;97:557-564)
We have examined the effects of heat shock on glucocorticoid receptor (GR)-mediated gene transcription in an L929 cell line derivative (LMCAT2) stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter plasmid. Exposure of the LMCAT2 cells to heat or chemical shock resulted in a large increase in dexamethasone (Dex)-induced expression of CAT enzyme activity. This potentiation of hormone-induced MMTV-CAT expression was dependent on the magnitude of the stress event and on the Dex concentration, with maximal increases observed for 1 microM Dex after 2 h at 43 C or 2 h at 200 microM sodium arsenite. Heat shock potentiation of MMTV-CAT expression was not seen in an L929 cell derivative devoid of GR or in LMCAT2 cells treated with RU486 antagonist, suggesting that this effect of stress on CAT gene expression was mediated by the GR. Using a quantitative Western blot procedure, the amount of GR protein in the nucleus of cells subjected to combined heat shock and Dex treatment was no greater than the amount of nuclear GR in cells treated with hormone alone, indicating that the stress potentiation effect was not the result of increased nuclear translocation or retention by the GR. In addition, equally strong potentiations of MMTV-CAT expression were observed for cells subjected to heat shock either before or after Dex-mediated translocation of the GR to the nucleus. Thus, the major effect of stress on GR transcription enhancement activity appears to occur after the GR is bound to its high affinity nuclear acceptor sites. We have used a series of MMTV-CAT reporter constructs containing varying portions of the long terminal repeat regulatory region to show that a putative heat shock transcription factor-binding sequence at position -437 of the long terminal repeat is not required for this effect of heat shock on MMTV-CAT expression. A stress-induced increase in hormone-mediated CAT gene expression was observed for a minimal CAT reporter controlled by two synthetic glucocorticoid response elements and a TATA box sequence. Thus, it is unlikely that any DNA-binding transcription factor, other than GR, is required for this effect of stress on transcription by the hormone-bound GR. Based on these results, a model of heat shock enhancement of GR-mediated gene expression is developed in which stress acts on the DNA-bound GR, on a putative heat shock-activated adaptor, or on components of the RNA-polymerase-II complex.
Betafectin® PGG-Glucan, a novel β-(1,6) branched β-(1,3) glucan purified from the cell walls of Saccharomyces cerevisiae, has been shown to synergize with myeloid growth factors in vitro and to enhance hematopoietic recovery in myelosuppressed mice and primates. Here we report that PGG-Glucan is also capable of mobilizing peripheral blood progenitor cells (PBPC). PGG-Glucan (0.5 mg/kg to 16 mg/kg) was administered intravenously to C3H/HeN male mice and blood collected at times ranging from 30 min to seven days after injection. Based on granulocyte-macrophage colony-forming cell (GM-CFC) levels, peak mobilization occurred 30 min after a 2 mg/kg PGG-Glucan dose.
High levels of chimerism in syngeneic BALB/c transplants were reported when hosts were exposed to 1 Gy (100 cGy) whole body irradiation (WBI) and infused with 40 ؋ 10 6 marrow cells. The recovery of host stem cells and alterations of enhanced host engraftability at varying times after 1 Gy WBI have now been evaluated in this study. Male BALB/c marrow (40 ؋ 10 6 cells) was infused into female BALB/c hosts immediately or at 6, 12, and 24 weeks after 1 Gy WBI of host female BALB/c mice; engraftment percentages 8 weeks after cell injection at week 0, 6, 12, or 24 were 68% ؎ 12%, 45% ؎ 15%, 51% ؎ 12%, or 20% ؎ 8%, respectively. Eight-week engraftment levels in nonirradiated hosts average 7.7%. Conversely, engraftable stem cells measured at 8 weeks postengraftment in 1 Gyexposed hosts were reduced to 8.6% ؎ 3% of nonirradiated mice at time 0, 35% ؎ 12% 6 weeks later, 49% ؎ 10% at 3 months, and 21% ؎ 7% at 6 months. Engraftment was still increased and stem cell decreased 1 year after 1 Gy. Furthermore, the primary cells transplanted into 1 Gy hosts can be serially transplanted, and the predominant effect of 1 Gy is directly on engrafting stem cells and not through accessory cells. These data show that transplantation in 1 Gy mice may be delayed until recovery of hematopoiesis, suggesting strategies in allogeneic transplantation to avoid the adverse effects of cytokine storm.
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