[show abstract][hide abstract] ABSTRACT: Dogs given nonmyeloablative conditioning and marrow grafts from 2 dog leukocyte antigen (DLA)-identical littermate donors developed stable trichimerism and stably accepted a subsequent kidney graft from one of the marrow donors without the need for immunosuppression. In this study, we used trichimeras to evaluate strategies for adoptive immunotherapy to solid tumors, using the kidney as a tumor surrogate. Three DLA-identical trichimeric recipients were established by simultaneously infusing marrow from 2 DLA-identical donor dogs into a DLA-identical recipient conditioned with 2 Gy of total body irradiation (TBI) and given a short course of postgraft immunosuppression. After stable hematopoietic engraftment was confirmed, a kidney was transplanted from 1 of the 2 marrow donors into each respective trichimeric recipient. Peripheral blood lymphocytes from each kidney donor were then used to sensitize the alternate marrow donor. The trichimeric recipients were given donor lymphocyte infusions (DLIs) from the sensitized dogs and monitored for chimerism, graft-versus-host disease (GVHD), and kidney rejection. After DLI, we observed both prompt rejection of the transplanted marrow and donor kidney and disappearance of corresponding hematopoietic chimerism. Presumably due to shared minor histocompatibility antigens, host chimerism also disappeared, and GVHD in skin, gut, and liver developed. The native kidneys, although exhibiting lymphocytic infiltration, remained functionally normal. This study demonstrates that under certain experimental conditions, the kidney--an organ ordinarily not involved in graft-versus-host reactions--can be targeted by sensitized donor lymphocytes.
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 12/2008; 14(11):1201-8. · 3.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Canine embryonic stem (cES) cell lines were generated to establish a large-animal preclinical model for testing the safety and efficacy of embryonic stem (ES) cell-derived tissue replacement therapy. Putative cES cell lines were initiated from canine blastocysts harvested from natural matings. Times of harvest were estimated as 12-16 days after the presumed surge in circulating levels of luteinizing hormone. Four lines established from blastocysts harvested at days 13-14 postsurge satisfied most of the criteria for embryonic stem cells, whereas lines established after day 14 did not. One line, Fred Hutchinson dog (FHDO)-7, has been maintained through 34 passages and is presented here. FHDO-7 cells are alkaline phosphatase-positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and telomerase but do not express message for Cdx2, which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency-associated microRNAs (miRs) (miR-302b, miR-302c, and miR-367) characteristic of human and mouse ES cells. The FHDO-7 cells grow on feeder layers of modified mouse embryonic fibroblasts as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders, the cells form embryoid bodies (EBs). Under various culture conditions, the EBs give rise to ectoderm-derived neuronal cells expressing gamma-enolase and beta 3-tubulin; mesoderm-derived cells producing collagen IIA1, cartilage, and bone; and endoderm-derived cells expressing alpha-fetoprotein or Clara cell-specific protein.
[show abstract][hide abstract] ABSTRACT: Gene therapy for hemoglobinopathies requires efficient gene transfer into hematopoietic stem cells and high-level erythroid-specific gene expression. Toward this goal, we constructed a helper-dependent adenovirus vector carrying the beta-globin locus control region (LCR) to drive green fluorescent protein (GFP) expression, whereby the LCR-GFP cassette is flanked by adeno-associated virus (AAV) inverted terminal repeats (Ad.LCR-beta-GFP). This vector possesses the adenovirus type 35 fiber knob that allows efficient infection of hematopoietic cells. Transduction and vector integration studies were performed in MO7e cells, a growth factor-dependent CD34(+) erythroleukemic cell line, and in cord blood-derived human CD34(+) cells. Stable transduction of MO7e cells with Ad.LCR-beta-GFP was more efficient and less subject to position effects and silencing than transduction with a vector that did not contain the beta-globin LCR. Analysis of integration sites indicated that Ad.LCR-beta-GFP integration in MO7e cells was not random but tethered to chromosome 11, specifically to the globin LCR. More than 10% of analyzed integration sites were within the chromosomal beta-globin LCR. None of the Ad.LCR-beta-GFP integrations occurred in exons. The integration pattern of a helper-dependent vector that contained X-chromosomal stuffer DNA was different from that of the beta-globin LCR-containing vector. Infection of primary CD34(+) cells with Ad.LCR-beta-GFP did not affect the clonogenic capacity of CD34(+) cells. Transduction of CD34(+) cells with Ad.LCR-beta-GFP resulted in vector integration and erythroid lineage-specific GFP expression.
Journal of Virology 10/2005; 79(17):10999-1013. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Methods for regulating the growth of transplanted cells may have many applications in gene and cell therapy. One such method uses conditional signaling molecules that can be activated in response to artificial ligands called chemical inducers of dimerization (CIDs). Here we examine the response of human cord blood cells to a CID-triggered growth signal, in vivo. CD34+ cells transduced with a lentivirus vector encoding a derivative of the thrombopoietin receptor (F36VMpl) and green fluorescent protein (GFP) were transplanted into immune deficient mice. CID treatment was associated with a >12 – fold expansion of GFP+ CD71+ erythroid cells that were localized mainly in the marrow, and a unique subset of multipotential and erythroid progenitors that were confined to the spleen. Both the CD71+ cells and the progenitor colonies were intensely GFP+. The CID response was oligoclonal, with most colonies containing a single transgene copy. Despite expressing F36Vmpl, GFP+ B lymphoid and myeloid cells showed no response. CID-responsive progenitors and CD71+ cells were detectable for up to 5 weeks following the end of CID; however these effects were not sustained in secondary transplant recipients. These findings establish CIDs as growth factors for genetically modified human hematopoietic cells.The University of Washington has submitted a patent application for this application. Any royalties accruing from this application may lead to income for Dr. Blau.
[show abstract][hide abstract] ABSTRACT: Gab proteins are intracellular scaffolding and docking molecules involved in signaling pathways mediated by various growth factor, cytokine, or antigen receptors. Gab3 has been shown to act downstream of the macrophage colony-stimulating factor receptor, c-Fms, and to be important for macrophage differentiation. To analyze the physiological role of Gab3, we used homologous recombination to generate mice deficient in Gab3. Gab3(-/-) mice develop normally, are visually indistinguishable from their wild-type littermates, and are healthy and fertile. To obtain a detailed expression pattern of Gab3, we generated Gab3-specific monoclonal antibodies. Immunoblotting revealed a predominant expression of Gab3 in lymphocytes and bone marrow-derived macrophages. However, detailed analysis demonstrated that hematopoiesis in mice lacking Gab3 is not impaired and that macrophages develop in normal numbers and exhibit normal function. The lack of Gab3 expression during macrophage differentiation is not compensated for by increased levels of Gab1 or Gab2 mRNA. Furthermore, Gab3-deficient mice have no major immune deficiency in T- and B-lymphocyte responses to protein antigens or during viral infection. In addition, allergic responses in Gab3-deficient mice appeared to be normal. Together, these data demonstrate that loss of Gab3 does not result in detectable defects in normal mouse development, hematopoiesis, or immune system function.
Molecular and Cellular Biology 05/2003; 23(7):2415-24. · 5.37 Impact Factor