Macrophage heterogeneity and Ir-gene control as factors involved in the immune response of guinea pigs to infection with Leishmania enrietti.
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Biomedical subjects
Publications and source records attributed to G B Price.
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A cell--cell hybrid of a Chinese hamster ovary (CHO) cell line and human peripheral blood lymphoblasts from a patient with B-cell chronic lymphocytic leukemia produces a surface antigen detectable by a monoclonal antibody. This surface antigen can be detected at significant levels on a fraction of chronic lymphocytic leukemia cells, but not on normal human lymphocytes from peripheral blood or bone marrow. Two different clones capable of transforming mouse cells to produce the surface antigen were isolated from a gene library of the hybrid cell by identification of small portions of the library containing functional genes, followed by detection of clones with human-specific reiterated sequences. Both clones also contain CHO-specific reiterated sequences and are therefore human--CHO recombinants. From the frequency of antigen-producing clones in the library, we estimate that they were present in the hybrid cell genome at a copy number of 100 to 1000.
A microculture method in methylcellulose has been developed for the study of human T cell colony formation. The technique is simple, reliable, does not require preincubation with lectin and requires small numbers of cells. Colony formation was dependent on the presence of phytohemagglutin-conditioned medium, a T colony precursor cell (TCPC), and a "helper" or accessory T cell. Plating efficiency was increased 10-fold in the presence of irradiated feeder cells. Progenitors of the T colony cells were identified in peripheral blood, tonsil, and spleen but not in thymus or thoracic duct. They were isolated in the E-rosetting, theophylline-resistant, Fc-IgG-negative cell populations. In peripheral blood the frequency of TCPC and accessory cells, the T colony forming unit, was estimated to be 8 X 10(-3). Colony cells proliferated in response to lectins and allogeneic cells. Forty to 80% of the cells were Ia-positive and stimulated both autologous and allogeneic mixed lymphocyte responses. They were incapable of mediating antibody-dependent cytotoxicity. In contrast, they were effective in assays of spontaneous cytotoxicity but only against certain target cells. This method for the analysis of T colony formation should prove valuable in the functional analysis of T cell subsets in immunodeficiency states or the transplant recipient.
We have developed hardware components for our flow cytometer cell sorter which can perform real-time calculations of either fluorescence polarization (FP) or anisotropy on a cell-by-cell basis and which can provide gating signals for the sorting of these cells into two populations. Artificial FP measurements, using electronic pulses varied over a 40dB range, were used to evaluate the computer's useful dynamic range. Real FP measurements, using human peripheral blood lymphocytes stimulated with phytohemagglutinin and labeled with fluorescein diacetate in saline, are presented to allow comparison with earlier work.
Levamisole, which has immunostimulant activity, is now being used to treat some forms of cancer. We report that the drug enhances granulocyte colony formation. The mechanism of action appears to be partly through modulation of molecules on cell membranes. The molecular content of colony-stimulating activity (CSA) released into leucocyte-conditioned medium by cells of leukaemic and some preleukaemic patients can be quantitatively altered by levamisole, both in vitro and in vivo, but the CSA produced is qualitatively similar to that released by normal cells. The demonstrated levamisole enhancement of colony formation, and altered CSA types detected in leucocyte-conditioned medium, makes this drug a promising candidate for treatment of selected leukaemic states and in preleukaemia.
Cells in T cell colonies grown in vitro from normal human peripheral blood leukocytes can kill a variety of different target cells against which the blood donor has not knowingly been previously sensitized, i.e., the colony cells exhibit spontaneous cytotoxicity. Tumor target cells are preferentially killed. The ability to kill two different targets segregates independently in different colonies, which implies that the effector cells are specificity restricted. A majority (perhaps all) of the effector cells possess receptors for sheep erythrocytes and do not possess receptors for the Fc end of IgG, making it unlikely that they are either "natural killer" (NK) cells of cells mediating antibody-dependent cellular cytotoxicity. They may, however, be cytotoxic T lymphocytes.
Cell sorting has been used as an approach to describing surface markers and functional receptors of hemopoietic stem cells and progenitors. Significant enrichment of CFU-S was obtained with fluorescent cell sorting of human antihuman sperm-labelled bone marrow cells; significant enrichment of human CFU-C was obtained by cell sorting of hybridoma (KGP1)-labelled human bone marrow cells. Significant changes in human bone marrow CFU-C, but not CFU-E were observed by fluorescence polarization after exposure to a putative stimulator of CFU-C. A description of preliminary investigations of a hybrid clone from Dexter culture cells x Chinese hamster ovary and fluorescence polarization measurements after exposure to purified mouse-lung CSF are also presented.
We have examined the properties of hybrid cells formed by polyethylene glycol-mediated fusion of the GRC+L-73 line of Chinese hamster ovary (CHO) cells with peripheral blood cells from patients with chronic lymphocytic leukemia (CLL) or with bone marrow cells from patients with malignant lymphoma. The results indicate that hybrid cells can be detected by their ability to form "foci" of characteristic morphology in the presence of a monolayer of parental CHO cells and that clones isolated from such foci express aspects of the differentiation status, as detected by immunologic markers, of the human parental cells.
Cell sorting has been used as a method for characterizing hemopoietic stem cells and progenitors. Fluorescent antibody-surface labels and changes in fluorescence polarization induced by in vitro stimulation with potential hemopoietic regulators were used. As detected by significant enrichment of CFU-S (pluripotent stem cells) in fluorescence-activated cell sorting, some CFU-S bear 'unique antigens' recognized by rabbit anti-human brain sera, human anti-human sperm sera, and 129 anti-F9 serum, but not A . TH anti-A . TL (Ia) ascites. Significant changes in fluorescence polarization induced by in vitro stimulation of mouse bone marrow with potential hemopoietic regulators were also observed; further, progenitors of human T-lymphocyte colonies were observed to exhibit a significantly decreased mean polarization value after short-term stimulation with PHA-LCM (phytohemagglutinin-stimulated leukocyte conditioned medium).
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Hemopoietic growth and immunological inductive factors from human peripheral leukocyte conditioned medium and mouse macrophage culture supernatant fluids were compared. Factors derived from human peripheral leukocyte conditioned medium were found to substitute for those of mouse macrophage derivation in immunological assays, namely, induction of a cytotoxic T-lymphocyte response in macrophage-depleted mouse spleen cultures. Association of macrophage-derived factors with B2-microglobulin (B2m) antigen determinants was observed by inhibition with great antihuman B2m, affinity chromatography, and direct replacement of mouse macrophage-derived factors with human urinary B2m.
The genetic restriction involved in the reconstitution of immune responses in macrophage-depleted mouse spleen cultures, or the induction of colony formation in bone marrow cultures, by different molecular-weight species of lymphostimulatory molecules derived from mouse peritoneal cell suspensions is reported. The data suggest little evidence for genetic restriction in the ability of any of the factors to stimulate bone marrow colony formation in vitro. However, when immunological stimulation was investigated, a restriction coded for by genes in the K/D end of the MHC (70-90K factor) or in the I region of the MHC (30-45K factor) was observed. A third species of lymphostimulatory molecule (15K) showed no such restriction. Further evidence is presented to suggest that the active moiety in the 70-90K molecule(s) is a 15K-like species (nonrestricted in its ability to reconstitute cells from different strains of mice.
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A fluorescence-activated cell sorter has been used to sort out enriched populations of murine hemopoietic stem cells from mouse bone marrow, based on fluorescence of cells labeled with various antisera. Significant positive enrichment for stem cells was obtained with rabbit anti-human brain, human anti-human sperm and mouse anti-mouse F9 sera but not with mouse anti-mouse Ia serum. These results support the hypothesis that the hemopoietic stem cell exhibits 'unique' surface antigens cross-reactive with sperm, brain and embryonic antigens.
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