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Query: UMLS:C0751781 (NOD)
 6,696 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Stable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34(+) cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45(+) cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions.
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PMID:High efficiency gene transfer to human hematopoietic SCID-repopulating cells under serum-free conditions. 978 52

Using a new adenoviral vector (Ad) construct, we expressed human thrombopoietin (TPO) cDNA (AdTPO) in mice with various inherited immune deficiency syndromes such as nude, SCID and NOD-SCID mice. Immune normal Balb/c mice and a vector construct without TPOcDNA (AdNull), respectively, were used for controls. All animals (3 per group) were treated with a single application of 10(9) PFU (plaque forming unit) of Ad (AdTPO or AdNull) intraperitoneally on day 0. Four to 5 weeks following AdTPO administration, SCID and NOD-SCID mice demonstrated peak concentration of PLT of 12- to 14-fold normal value simultaneously with maximum concentration of PMNs (10- to 12-fold normal value). Later on these animals had a chronic thrombocytosis. In contrast, Balb/c mice and nude mice experienced PLT peak concentration of 4- to 6-fold normal value without granulocytosis 1 to 2 weeks following AdTPO treatment. Only nude mice had chronically elevated PLTs. In contrast, Balb/c mice developed thrombocytopenia due to cross-reacting anti-TPO antibodies. Animals with chronic thrombocytosis revealed increased content of CFU-G/GM, CFU-GEMM and CFU-Meg in bone marrow compared with controls. In contrast, Balb/c mice showed decreased content of CFUs if anti-TPO-antibodies were present. Histologically, only SCID mice developed severe osteomyelofibrosis and osteomyelosclerosis, hepato-splenomegaly, extramedullary hematopoiesis in liver and lung and ultimately suffered of progressive pancytopenia, anisocytosis, fragmentocytosis and a lethal wasting syndrome. In contrast, NOD-SCID mice which demonstrated similar extent of TPO overexpression and in addition to the B- and T-cellular immune deficiency harbour defective monocytes and macrophages, did not develop fibrotic changes of the bone marrow. From these results, we conclude (1) chronic TPO overexpression in vivo may lead to thrombocytosis and granulocytosis with expansion of CFU-GM, -GEMM and -Meg; (2) in vivo expression of adenovirally mediated TPOcDNA depends on immune competency of the host; (3) functionally normal monocytes and macrophages are indispensable for development of secondary osteomyelofibrosis and (4) adenovirally mediated expression of xenogeneic transgenes may brake immune tolerance for the respective self protein leading to autoimmune phenomena. Our in vivo model might provide further insights into the pathophysiology of secondary osteomyelofibrosis and may prove useful in designing new strategies for immune therapies of cancer.
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PMID:[Adenovirus long-term expression of thrombopoietin in vivo: a new model for myeloproliferative syndrome and osteomyelofibrosis]. 982 87

We have previously demonstrated that high levels of allogeneic, donor-derived mouse haemopoietic progenitor cells engraft following in utero transplantation in NOD/SCID mice. To evaluate whether the fetal NOD/SCID haemopoietic microenvironment supports the growth and development of human fetal haemopoietic progenitor cells, we injected fetal liver mononuclear cells (FL) or fetal bone marrow (FBM) derived CD34+ cells into NOD/SCID mice on day 13/14 of gestation. At 8 weeks of age 12% of FBM recipients and 10% of FL recipients were found to have been successfully engrafted with CD45+ human cells. CD45+ cells were present in the BM of all chimaeric animals; 5/6 recipients showed engraftment of the spleen, and 4/6 recipients had circulating human cells in the peripheral blood (PB). The highest levels of donor cells were found in the BM, with up to 15% of the nucleated cells expressing human specific antigens. Multilineage human haemopoietic engraftment, including B cells (CD19), myelomonocytic cells (CD13/33) and haemopoietic progenitor cells (CD34), was detected in the BM of chimaeric mice. In contrast, no human CD3+ cells were detected in any of the tissues evaluated. When the absolute number of engrafted human cells in the PB, BM and spleens of chimaeric mice was determined, a mean 16-fold expansion of human donor cells was observed. Although multilineage engraftment occurs in these fetal recipients, both the frequency and the levels of engraftment are lower than those previously reported when human cells are transplanted into adult NOD/SCID recipients.
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PMID:In utero transplantation of human fetal haemopoietic cells in NOD/SCID mice. 982 1

The AC133 antigen is a novel antigen selectively expressed on a subset of CD34+ cells in human fetal liver, bone marrow, and blood as demonstrated by flow cytometric analyses. In this study, we have further assessed the expression of AC133 on CD34+ cells in hemopoietic samples and found that there was a highly significant difference between normal bone marrow and cord blood versus aphereses (p <0.0001) but not between bone marrow and cord blood. Most of the clonogenic cells (67%) were contained in the CD34+AC133+ fraction. Compared with cultures of the CD34+AC133- cells, generation of progenitor cells in long-term culture on bone marrow stroma was consistently 10- to 100-fold higher in cultures initiated with CD34+AC133+ cells and was maintained for the 8-10 weeks of culture. Only the CD34+AC133+ cells were capable of repopulating NOD/SCID mice. Human cells were detectable as early as day 20, with increased levels (67%) apparent 40 days post-transplantation. Five thousand CD34+AC133+ cells engrafted about 20% of the mice, while no engraftment was observed in animals transplanted with up to 1.2 x 10(5) CD34+AC133- cells. The CD34+AC133+ population was also enriched (seven-fold) in dendritic cell precursors, and the dendritic cells generated were functionally active in a mixed lymphocyte reaction assay. AC133+ cells should be useful in the study of cellular and molecular mechanisms regulating primitive hemopoietic cells.
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PMID:CD34+AC133+ cells isolated from cord blood are highly enriched in long-term culture-initiating cells, NOD/SCID-repopulating cells and dendritic cell progenitors. 983 64

Purified CD34(+) and CD34(+)CD38(-) human umbilical cord blood (UCB) cells were transduced with the recombinant variant of Moloney murine leukemia virus (MoMLV) MFG-EGFP or with SF-EGFP, in which EGFP expression is driven by a hybrid promoter of the spleen focus-forming virus (SFFV) and the murine embryonic stem cell virus (MESV). Infectious MFG-EGFP virus was produced by an amphotropic virus producer cell line (GP+envAm12). SF-EGFP was produced in the PG13 cell line pseudotyped for the gibbon ape leukemia virus (GaLV) envelope proteins. Using a 2-day growth factor prestimulation, followed by a 2-day, fibronectin fragment CH-296-supported transduction, CD34(+) and CD34(+)CD38(-) UCB subsets were efficiently transduced using either vector. The use of the SF-EGFP/PG13 retroviral packaging cell combination consistently resulted in twofold higher levels of EGFP-expressing cells than the MFG-EGFP/Am12 combination. Transplantation of 10(5) input equivalent transduced CD34(+) or 5 x 10(3) input equivalent CD34(+)CD38(-) UCB cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in median engraftment percentages of 8% and 5%, respectively, which showed that the in vivo repopulating ability of the cells had been retained. In addition, mice engrafted after transplantation of transduced CD34(+) cells using the MFG-EGFP/Am12 or the SF-EGFP/PG13 combination expressed EGFP with median values of 2% and 23% of human CD45(+) cells, respectively, which showed that the NOD/SCID repopulating cells were successfully transduced. EGFP+ cells were found in all human hematopoietic lineages produced in NOD/SCID mice including human progenitors with in vitro clonogenic ability. EGFP-expressing cells were also detected in the human cobblestone area-forming cell (CAFC) assay at 2 to 6 weeks of culture on the murine stromal cell line FBMD-1. During the transduction procedure the absolute numbers of CAFC week 6 increased 5- to 10-fold. The transduction efficiency of this progenitor cell subset was similar to the fraction of EGFP+ human cells in the bone marrow of the NOD/SCID mice transplanted with MFG-EGFP/Am12 or SF-EGFP/PG13 transduced CD34(+) cells, ie, 6% and 27%, respectively. The study thus shows that purified CD34(+) and highly purified CD34(+)CD38(-) UCB cells can be transduced efficiently with preservation of repopulating ability. The SF-EGFP/PG13 vector/packaging cell combination was much more effective in transducing repopulating cells than the MFG-EGFP/Am12 combination.
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PMID:Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long-term cultures and multilineage engraftment of immunodeficient mice. 983 3

Acute myeloid leukemia (AML) occurs as the result of malignant transformation in a hematopoietic progenitor cell, which proliferates to form an accumulation of AML blasts. Only a minority of these AML cells are capable of proliferation in vitro, suggesting that AML cells may be organized in a hierarchy, with only the most primitive of these cells capable of maintaining the leukemic clone. To further investigate this hypothesis, we have evaluated a strategy for purifying these primitive cells based on surface antigen expression. As an in vitro endpoint, we have determined the phenotype of AML progenitor cells which are capable of producing AML colony-forming cells (CFU) for up to 8 weeks in suspension culture (SC) and compared the phenotype with that of cells which reproduce AML in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. AML cells were fluorescence-activated cell sorted (FACS) for coexpression of CD34 and CD71, CD38, and/or HLA-DR and the subfractions were assayed in vitro and in vivo at various cell doses to estimate purification. While the majority of primary AML CFU lacked expression of CD34, most cells capable of producing CFU after 2 to 8 weeks in SC were CD34(+)/CD71(-). HLA-DR expression was heterogeneous on cells producing CFU after 2 to 4 weeks. However, after 6 to 8 weeks in SC, the majority of CFU were derived from CD34(+)/HLA-DR- cells. Similarly, the majority of cells capable of long-term CFU production from SC were CD34(+)/CD38(-). Most cells that were capable of engrafting NOD/SCID mice were also CD34(+)/CD71(-) and CD34(+)/HLA-DR-. Engraftment was not achieved with CD34(+)/CD71(+) or HLA-DR+ subfractions, however, in two patients, both the CD34(+) and CD34(-) subfractions were capable of engrafting the NOD/SCID mice. A three-color sorting strategy combining these antigens allowed approximately a 2-log purification of these NOD/SCID leukemia initiating cells, with engraftment achieved using as few as 400 cells in one experiment. Phenotyping studies suggest even higher purification could be achieved by combining lack of CD38 expression with the CD34(+)/CD71(-) or CD34(+)/HLA DR- phenotype. These results suggest that most AML cells capable of long-term proliferation in vitro and in vivo share the CD34(+)/CD71(-)/HLA-DR- phenotype with normal stem cells. Our data suggests that in this group of patients the leukemic transformation has occurred in a primitive progenitor, as defined by phenotype, with some degree of subsequent differentiation as defined by functional assays.
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PMID:Most acute myeloid leukemia progenitor cells with long-term proliferative ability in vitro and in vivo have the phenotype CD34(+)/CD71(-)/HLA-DR-. 983 39

While it is known that mice with genetic immune defects are useful for establishing durable engraftment of human tumor xenografts, the relative role of components of host innate and adoptive immunity in engraftment has not been determined. We directly compared the ability of four strains of genetically immunodeficient mice (NOD/SCID, SCID, Nude and Rag-1-deficient) to successfully engraft and support the human cell lines Daudi, Raji, Namalwa and Molt-4 as subcutaneous tumors. We additionally examined the effect of further immunosuppression of the mice by whole body irradiation at a dose of 600 cGy for Nude and Rag-1 and 300 cGy for SCID mice and by administration of anti-natural killer (asialo-GM1) antibody on tumor growth. Mice with each of the defects supported xenografts to varying degrees. We found differences in growth characteristics in the cell lines tested, with Namalwa consistently producing the largest tumors. With all cell lines studied, optimal growth was achieved using NOD/SCID mice. Overall, tumor growth was somewhat enhanced by pretreatment with radiation with little additional benefit from the addition of anti-asialo-GM1 antibody. The importance of multiple components of the innate and adoptive immune system in xenotransplantation were best demonstrated when results in untreated NOD/SCID mice were compared to SCID, nude and RAG-1-deficient mice. The NOD/SCID mouse with or without additional immunosuppression provides the optimal model for the study of the biology and treatment of human leukemias and lymphomas.
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PMID:Xenotransplantation of human lymphoid malignancies is optimized in mice with multiple immunologic defects. 984 34

In rheumatoid arthritis, T lymphocytes have been proposed to play a pivotal role in the disease process, but they have also been considered to simply represent an epiphenomenon in a primarily synoviocyte/macrophage-driven disease. To directly examine the contribution of CD4 T cells in synovitis, T cells were either depleted from or adoptively transferred into NOD-SCID mice engrafted with rheumatoid synovial tissue. Injection of anti-CD2 antibody resulted in the elimination of 80-90% of tissue-infiltrating T cells in the synovial grafts and was followed by a marked decline in the production of IL-1beta (loss of 70%), TNF-alpha (loss of 86%), and IL-15 (loss of 84%) mRNA. Also, transcription of MMP-1 and MMP-2 was reduced by 72% in anti-CD2-treated chimeras. Immunohistochemistry demonstrated that the cytokines and proteases derived mostly from CD68(+) synovial cells, which disappeared from the tissue upon T cell depletion. Adoptive transfer of autologous tissue-derived T cell lines and T cell clones into synovium-SCID mouse chimeras augmented the production of IFN-gamma as well as TNF-alpha in the synovial infiltrates. Administration of IFN-gamma in small doses to anti-CD2-treated chimeras restored the survival and the functional activity of CD68(+) synovial cells. In vitro studies confirmed the critical role of synovial T cells and IFN-gamma in the survival of synovial CD68(+) cells. These data demonstrate that the production of proinflammatory cytokines and of tissue-degrading enzymes in rheumatoid synovitis is T cell dependent and that CD4 T cells are primary regulatory cells in this disease.
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PMID:Production of cytokines and metalloproteinases in rheumatoid synovitis is T cell dependent. 988 54

The development of autoimmune diabetes in NOD mice results from selective destruction of beta-cells by a T-cell-dependent autoimmune process. However, the mechanisms that control the generation of beta-cell cytotoxic T-cells in vivo are poorly understood. We recently established 8.3-T-cell receptor (TCR)-beta transgenic NOD mice that show a selective acceleration of the recruitment of CD8+ T-cells into the islets of prediabetic animals, resulting in rapid beta-cell destruction and early onset of diabetes. This study was initiated to determine the role of macrophages in the development and activation of diabetogenic CD8+ T-cells in 8.3-TCR-beta transgenic NOD mice. Inactivation of macrophages in these transgenic mice resulted in the complete prevention of diabetes. When splenic T-cells from macrophage-depleted 8.3-TCR-beta transgenic NOD mice were transfused into severe combined immunodeficiency disease (NOD.scid) mice, none of the recipients developed diabetes up to 10 weeks after transfer, while most of the recipients of T-cells from age-matched control 8.3-TCR-beta transgenic NOD mice became diabetic. When intact NOD islets were transplanted under the renal capsule of macrophage-depleted 8.3-TCR-beta transgenic NOD mice, the majority of the grafted islets remained intact, while most of the islets grafted into age-matched, control 8.3-TCR-beta transgenic NOD mice were destroyed within 3 weeks after transplantation. The depletion of macrophages in these mice resulted in a decrease in the Th1 immune response along with an increase in the Th2 immune response because of significant decreases in the expression of macrophage-derived cytokines, particularly interleukin-12, and a decrease in beta-cell-specific T-cell activation, as shown by significant decreases in the expression of Fas ligand (FasL), CD40 ligand (CD40L), and perforin, as compared with control mice. We conclude that macrophages are absolutely required for the development and activation of beta-cell cytotoxic CD8+ T-cells in 8.3-TCR-beta transgenic NOD mice.
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PMID:Absolute requirement of macrophages for the development and activation of beta-cell cytotoxic CD8+ T-cells in T-cell receptor transgenic NOD mice. 989 20

Efficient gene transfer into human hematopoietic stem cells (HSCs) is an important goal in the study of the hematopoietic system as well as for gene therapy of hematopoietic disorders. A lentiviral vector based on the human immunodeficiency virus (HIV) was able to transduce human CD34+ cells capable of stable, long-term reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. High-efficiency transduction occurred in the absence of cytokine stimulation and resulted in transgene expression in multiple lineages of human hematopoietic cells for up to 22 weeks after transplantation.
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PMID:Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors. 992 27


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