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  • 1
    ISSN: 1432-0851
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The generation of lymphokine activated killer (LAK) cells by recombinant IL2 (rIL2) in collaboration with interferonγ (IFNγ) was examined in peripheral blood mononuclear cells (PBMC) from patients with malignant tumors of the digestive organs and breast cancer. LAK cytotoxicity could be induced by rIL2 at 10 units/ml in 10 of 12 patients and 20 of 37 using fresh autologous tumor cells and PK-1, an established solid tumor cell line as a target, respectively. Among 34 patients, in which titers of IFNγ produced were assayed, 12 showed no IFNγ production. All of these 12 patients had no or extremely low LAK activity, suggesting the correlation of LAK generation with the production of IFNγ in response to rIL2. LAK induction by rIL2 in PBMC of cancer patients was almost completely inhibited by addition of anti-IFNγ serum. Depressed LAK generation, which was accompanied by no or low levels of IFNγ production, was partially restored by addition of exogenous recombinant IFNγ. These results indicate that LAK induction by rIL2 in cancer patients involves the production of IFNγ and its interaction with rIL2. The results also suggested the presence of a factor(s) suppressing LAK induction by rIL2 in the serum of cancer patients. Based on these results, the cancer patients could be divided into the following three groups. Group 1, in which the serum suppressor activity was undetectable, had the same level of LAK cytotoxicity in PBMC as healthy controls. Group 2 showed the serum suppressor factor and had the lower level of cytotoxicity in PBMC when cultivated in autologous serum (AS) compared to healthy controls. The depressed LAK induction in AS medium was restored to a normal level in culture with fetal calf serum (FCS) plus rIL2, or by addition of rIFNγ, or high concentrations of rIL2 in AS medium. The last group (group 3), in which the serum suppressor factor was also found, had the lowest level of cytotoxicity compared to healthy controls. The LAK induction in these patients could not be restored to a normal level by culture in FCS medium, addition of exogenous rIFNγ or high concentrations of rIL2, suggesting the possibility that the deficit of LAK generation in this group might involve the dysfunction or the lack of IL2 responder cells, in addition to the presence of a serum suppressor factor(s).
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-1211
    Keywords: Mouse T cell T-cell receptor repertoire Adaptor ligation PCR Microplate hybridization assay Major histocompatibility complex
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. We developed an adaptor ligation PCR-based microplate hybridization assay (MHA) to analyze the repertoires of mouse T-cell receptor (TCR) α- and β-chain variable regions (TCRAV and TCRBV). RNA is transcribed to cDNA and an adaptor is ligated to the 5′ end of the cDNA, which is then used as a template for PCR with an adaptor-specific 3′ primer and a constant region-specific 5′ primer. After hybridization of PCR products with TCRAV- and TCRBV-specific probes on the microplate, quantitative ELISA was carried out.The entire TCRAV or TCRBV repertoires could be analyzed using a single 96-well plate in triplicate and completed in less than 4 h. The assay results demonstrated the high level of specificity and reproducibility of this method. Furthermore, MHA results correlated well with those of fluorescence-activated cell sorting. This method may provide important information about various T-cell-associated diseases including autoimmune disease. The influence of the MHC on mouse TCR repertoires was next studied using the newly developed mouse TCRAV and TCRBV repertoire assay. The analysis in six strains showed no significant correlation between MHC haplotypes and TCRAV and TCRBV repertoires. However, large differences among strains was observed in TCRBV, but not in TCRAV repertoires. There were also large differences within same strain in TCRBV, but not in TCRAV repertoires, indicating differences in individuals independent of genetic factors. These data suggest that TCRBV repertoires are more susceptible than TCRAV repertoires not only to genetic factors but also some environmental factors.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-7276
    Keywords: experimental tumor metastasis ; gelatinase ; knockout mice ; matrix metalloproteinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Matrix metalloproteinases (MMPs) are thought to play a key role in tumor invasion and metastasis. The role of MMP-9 (gelatinase B) in tumor metastasis was examined in MMP-9-deficient mice produced by gene targeting using embryonic stem cells. MMP-9-deficient mice develop normally and are fertile. In these mice, the number of metastatic colonies of B16-BL6 melanoma cells or Lewis lung carcinoma cells that were implanted intravenously fell by 45% for B16-BL6 melanoma and 59% for Lewis lung carcinoma (p=0.03 and p=0.0043, respectively). Gelatin zymography showed that both tumor cell lines did not secrete MMP-9 by themselves but the host cells surrounding the tumor cells secrete MMP-9 in vivo. These results indicated that host-derived MMP-9 plays an important role in the process of tumor metastasis.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Although the fetal liver has been thought to be the main hemopoietic organ in the embryonal period, whether or not hepatocytes play a major role in hemopoiesis remains obscure. We have established an epithelial cell line from the murine fetal liver, which can support hemopoiesis in vitro. The proliferation of the epithelial cells was promoted synergistically by both epidermal growth factor (EGF) and insulin. The cells were identified as epithelial cells by the presence of desmosomes and tight junctions. Cytoplasmic organelles including small mitochondria and dilated Golgi apparati as well as intercellular canalicular structures similar to bile canaliculus also helped in confirming the hepatic origin of the cell line (designated as FHC). The cells in the primary culture were positive for both α -fetoprotein and albumin, indicating the hepatocytic nature of the cell line. Cloned FHC cells were demonstrated to have the ability to maintain hemopoietic progenitors in fetal liver and adult bone marrow in the coculture, and among them, FHC-4D2 clone displayed the greatest activity. Hemopoiesis-supporting function could also be seen even when bone marrow cells were separated from FHC-4D2 cells by nitrocellulose membrane. Column chromatography revealed three distinct peaks of hemopoietic activities with different molecular sizes in the supernatant of FHC-4D2. Neutralization test with antibodies and proliferative response to interleukin-3 (IL-3)/granulocyte-macrophage colony stimulating factor (GM-CSF)-IC2 cells demonstrated that the hemopoietic activities were attributed to GM-CSF and macrophage colony stimulating factor (M-CSF). Transcripts of GM-CSF and M-CSF were readily detectable in Northern blot analysis, whereas no messages for IL-3, IL-6, CSF for granulocytes (G-CSF) or erythropoietin (EPO) were identified. Therefore, this is the first report on the fetal hepatocyte cell line capable of supporting hemopoiesis. © 1993 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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  • 5
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have previously shown that a fetal liver-derived epithelial cell clone, FHC-4D2, could support hematopoiesis in vitro through its colony-stimulating factor (CSF) activities in a short-term culture. In this study, since FHC-4D2 cells were found capable of maintaining hematopoietic progenitors in the coculture for a long time, we examined how FHC-4D2 could exert hematopoietic supporting activity in a long-term culture by coculturing adult bone marrow (BM) cells or fetal liver (FL) cells on a monolayer of FHC-4D2 cells. This clone could maintain the colony-forming unit of granulocytes and macrophages (CFU-GM) of BM for ≥ 12 weeks under the coculture condition, but the fibroblastic cell clone from the fetal liver, FHC-4A3, could not support the survival of CFU-GM, even for 1 week. In addition to BM CFU-GM, the FHC-4D2 clone also supported the survival of FL CFU-GM, burst-forming unit of erythroid cells (BFUe), and colony-forming unit of mixed progenitors (CFU-Mix) for longer than 4 weeks. When BM cells were separated by a membrane filter from the FHC-4D2 cells in the coculture, the comparable number of CFU-GM was maintained at day 3, but virtually no hematopoietic progenitors were detected at the end of the first week. CFU-GM were present in both nonadherent and adherent cells to the FHC-4D2 cells at day 3 of the coculture, but at day 7, the adherent population contained greater number of CFU-GM. CFU-GM derived from the adherent cells formed larger colonies and contained more bipotential CFU-GM than the nonadherent population. When BM cells from mice given 5-fluorouracil were cocultured with FHC-4D2 cells under the limiting dilution condition, interleukin-3 (IL-3)-responsive CFU-GM were induced from immature hematopoietic progenitor cells that were otherwise unresponsive to IL-3. From these data we conclude that the FHC-4D2 clone could generate and maintain IL-3-responsive hematopoietic progenitors via close contact and that, in the fetal liver, the contact between hepatocytes and hematopoietic cells may be critically important in inducing the differentiation of resting, IL-3-unresponsive immature hematopoietic cells into CFU-GM (progenitors responsive to IL-3) and in triggering the self-renewal of CFU-GM. © 1994 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
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