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C C Stewart

Publications and source records attributed to C C Stewart.

At least 91 records · Page 5Linked to original sources
PubMed1989

Flow cytometric analysis of oncogene expression in human neoplasias.

More than 40 oncogenes have been described and characterized. These oncogenes code for products intimately associated with cell proliferation. Their products include growth factors, growth factor receptors, transmembrane signaling molecules, second and third messengers, and nuclear proteins involved in gene regulation. Some oncogenes seem to be expressed in every malignancy, while others seem to be expressed only in certain malignancies. This expression pattern suggests some oncogenes may have tissue type specificity. Some oncogenes may also be expressed in only a fraction of the malignant cells, such as the proliferating cells. Flow cytometry has only recently been applied to the simultaneous measurement of oncoprotein levels and DNA content of malignant cells. As this application is developed, our understanding of oncogene expression in malignancy will be enhanced and its diagnostic importance will be realized.

Flow Cytometry↗
PubMed1988

Isolation and reactivity of host effectors associated with the manifestation of concomitant tumor immunity.

In this study, we have measured the specific tumoricidal activity of tumor-infiltrating lymphocytes that prevent the growth of secondary tumors in animals harboring progressing primary tumors (concomitant immunity). Since no tumor grows at the challenge site when concomitant immunity is established, tumor cells were inoculated into a preimplanted gelatin sponge whose subsequent solubilization in collagenase permitted the retrieval of leukocytes after tumor challenge. Primary progressing EMT6 tumors were established in normal BALB/c mice and 10 days later they were challenged with a secondary tumor inoculum introduced through a preimplanted gelatin sponge. At 3, 7, and 10 days after the administration of the tumor inoculum challenge, a monodispersed suspension of infiltrating leukocytes was recovered by collagenase digestion of the sponge matrix and tested for cytotoxicity toward EMT6 tumor targets. Cytotoxic T-lymphocytes with tumoricidal activity accumulated at the site of the secondary tumor challenge by 3 days. This antitumor activity was maximal 7 days following challenge and decayed thereafter. Splenic lymphocytes from these animals showed little cytotoxicity. In animals harboring a primary tumor, lymphocytes found in sponges that were not inoculated with tumor cells were not cytotoxic. We interpret these data to indicate that cytotoxic lymphocytes migrate to, and accumulate at the site of the tumor but not at other sites and that peripheral sources of lymphocytes in tumor-bearing animals such as the spleen may not be the best source of effector cells for evaluating the host's immune response to its tumor. The approach described here may also be useful in studying the mechanisms for host control of metastatic disease.

Animals↗
PubMed1988

Characterization of granulocytes and mast cells in cultures of mouse bone marrow stimulated with interleukin-3.

Bone marrow cells in liquid culture with interleukin 3 produce a population of non-adherent granulocytes and mast cells. Flow cytometry was used to identify granulocytes and mast cells on the basis of the physical properties of perpendicular light scatter (PLS) and coulter volume (CV) as well as the expression IgE and CR3 receptors. Multicolor analysis indicated there were subpopulations of Thy1.2 positive cells which transiently appeared in these cultures and also expressed IgE receptors, CR3 receptors or neither of these receptors. The data suggested a differentiation scheme in which Thy1.2 positive precursor cells give rise to granulocytes and mast cells. Further evidence for this differentiation scheme was provided from CV vs. PLS distributions which showed increases in CV and PLS as Thy1.2 positive cells differentiated into mast cells and decreases in CV and PLS as Thy1.2 positive cells differentiated into granulocytes.

Animals↗
PubMed1988

Flow cytometric analysis of I-J expression on murine bone marrow-derived macrophages.

Attempts to analyze bone marrow-derived macrophages (BMDM) by flow cytometry have been prohibited because of their high autofluorescence. Using an autofluorescence reduction method of Steinkamp and Stewart to reduce the autofluorescence of BMDM, we were able to examine several macrophage populations for their expression of I-A, I-J, and Mac-1 cell surface determinants. Bone marrow cells examined immediately after removal from the femur contain 50-60% Mac 1-positive cells (mainly granulocytes). During the next few days granulocytes and nonmacrophage precursor cells die, and the number of Mac 1-positive cells decrease. Once the bone marrow cells have been maintained in L cell conditioned medium (LCM) for 2 to 3 days, the number of cells expressing Mac 1 increases rapidly from 20% to 98% during the next 3 to 4 days. Bone marrow cells grown in LCM do not express I-J until these cells have been in culture 3 to 4 days, and the number of cells expressing I-J (up to 90% positive) parallels the increase in macrophages. Bone marrow cells maintained in LCM did not express detectable I-A during the 14 days these cells were examined. Two other macrophage populations often used in a variety of immunological studies were analyzed by flow cytometry. We found that the majority (up to 80%) of peritoneal cells expressed I-A, and only 20% of peritoneal cells had I-J cell surface determinants. On the other hand, peritoneal exudate cells collected 4 days after thioglycolate medium treatment were predominantly I-J positive (up to 70%), and only about 30% of these cells expressed I-A cell surface antigens. The binding of anti-I-J IgM antibody to BMDM was not to Fc receptors because pretreating these cells with up to 25 micrograms of an IgG2a myeloma protein did not block anti-I-J antibody binding. The addition of 25-200 micrograms of monoclonal anti-Fc receptor antibody was also ineffective in blocking the binding of a monoclonal anti-I-Jk antibody to BMDM. Pretreatment of BMDM with the IgM fraction of several control IgM antibody preparations did not block the specific binding of fluoresceinated anti-I-J IgM antibody. BMDM provide a pure population of macrophages that express a significant level of cell surface I-J antigens. Bone marrow cells grown in LCM are essentially devoid of other contaminating cells, and the increase in the number of I-J-positive cells parallels the increase in macrophages in these cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

The effect of low-dose irradiation on unstimulated and PHA-stimulated human lymphocyte subsets.

A culture system was used to evaluate the radiosensitivity of CD4+ and CD8+ T cells, Leu 19+ cells, and B cells obtained from normal adult males. Unstimulated CD8+ lymphocytes (D0 = 55 cGy) were twice as radiosensitive as CD4+ cells (D0 = 115 cGy). B cells had an intermediate radiosensitivity (D0 = 100 cGy). Leu 19+ cells were much more radioresistant and expressed a D0 of 550 cGy. When lymphoid cells were irradiated 1 or 4 days before phytohemagglutinin (PHA) stimulation, they were more radiosensitive than if they were first stimulated with PHA and then irradiated. When lymphoid cells were irradiated 1 h after PHA stimulation each lymphocyte subset was characterized by an increase in the D0 to 150 cGy for B cells to 290 cGy for CD4+ cells, and to 240 cGy for CD8+ cells. In contrast, Leu 19+ cells exhibited a decrease in their D0 to 290 cGy after they were stimulated by PHA.

Adult↗
PubMed1987

A modular detector for flow cytometric multicolor fluorescence measurements.

A modular detector for measuring multicolor fluorescence from cells illuminated by single or multiple lasers has been developed for flow cytometers. Motion picture projector, camera, and CCTV/video lenses were evaluated for use in the detector by comparing their physical characteristics, image quality, and light collection efficiencies. A 25-mm focal length F/0.95 CCTV lens was selected, based on our criteria and test results. The detector was constructed out of square aluminum extrusion channels. A CCTV lens mounted on the outside of the first channel collected light emitted from cells and collimated it towards filters and secondary CCTV lenses located in each channel. The secondary lenses functioned as relay optics for directing and focusing light onto pinhole spatial filters for measurement by photomultipliers. The detector design allowed any number of channels to be connected together and the versatility for making simultaneous or sequential measurements. Measurements on lymphocytes labeled with four monoclonal antibodies conjugated to fluorescent dyes and measurements on viable tumor cells stained for DNA content and with three fluorescent-labeled antibodies were used to demonstrate the detector's capabilities.

Animals↗
PubMed1987

Regulation of myelopoiesis.

Myeloid cells are produced by the bone marrow from stem cells and progenitor cells. This review summarizes the current understanding of how this process is regulated. Regulation of progenitor cell proliferation and differentiation occurs in microenvironments present within the bone marrow as well as on the systemic level by the release of regulators into the circulation. The regulators of central importance to myelopoiesis are growth factors necessary for the proliferation and differentiation of progenitors. These growth factors have recently been characterized and studies indicate that there is a hierarchy of factors acting upon successive differentiation stages of progenitors. Endotoxin appears to be a major modulator of myeloid growth factor production. Other inhibitors of myelopoiesis are also discussed. Regulation of myelopoiesis therefore involves a balance between growth factor production and inhibition by other factors.

Animals↗
PubMed1987

Analysis of heavy metal immunotoxicity by multiparameter flow cytometry: correlation of flow cytometry and immune function data in B6CF1 mice.

Bone marrow and spleen cells obtained from female B6C3F1 mice given a single i.p. exposure to cadmium acetate (0.9 mg/kg), lead acetate (12 mg/kg), or sodium acetate (12 mg/kg), were studied using flow cytometry, immunologic, and hematologic assays. Significant changes were detected in subpopulations of bone marrow cells using multiparameter flow cytometry within 1 day following treatment with cadmium or lead. Bone marrow cells obtained from B6C3F1 mice 5 days after treatment with cadmium or lead were found to have a decreased number of cells expressing Mac-1, 55-7.2, 14.8, and Lyt-1 antigens, suggesting a shift to immature cell types. An increase in the number of progenitor cells (CFU-C) obtained from the bone marrow of mice treated with heavy metals was also noted 5 days after exposure to cadmium or lead. A time-dependent suppression of the in vitro primary humoral immune response of spleen cells to SRBCs, TNP-Ficoll and TNP-LPS was produced by cadmium or lead treatment. Suppression of the mitogenic response of spleen cells to Con A, PHA, and LPS was also found to be time-dependent. Spleen cell surface marker expression (Mac-1, Lyt-1, Lyt-2 and 14.8) was altered in response to cadmium or lead treatments, but these changes did not appear to correlate with the humoral immunity or mitogen-induced proliferation data. These studies demonstrate that changes in cell surface markers on discrete subpopulations of lymphoid cells present in the spleens of heavy metal exposed mice may not correlate with alterations in the functional activity of these cells. However, changes in murine bone marrow surface markers in response to cadmium or lead treatment predicts a shift to immature cell types, which appeared to correlate with the increase in CFU-C activity.

Acetates↗
PubMed1987

Differential expression and regulation of the c-src and c-fgr protooncogenes in myelomonocytic cells.

To study the expression of src-related protooncogenes during the development of myeloid cells and the regulation of these genes by the colony-stimulating factors that control myelopoiesis, normal monocytic cells at distinct stages of differentiation were derived from murine bone marrow with the monocytic lineage colony-stimulating factor CSF-1. Protooncogene expression was also examined in uncultured human myeloid leukemia cells. While c-src transcripts were detected in myeloid leukemia cells representative of all stages of differentiation, the highly related gene c-fgr was expressed at high levels only at later developmental stages, both in normal cells committed to the monocytic lineage and in leukemic cells with a differentiated myelomonocytic phenotype. When bone marrow-derived monocytic cells were synchronized and stimulated to proliferate with CSF-1, c-fgr transcripts (but not transcripts from the highly related genes c-src or c-yes) were induced 8 hr after the addition of CSF-1 and decreased to low levels by 20 hr as the monocytic cells entered S phase. The selective induction of c-fgr mRNA by CSF-1 suggests that this tyrosine kinase may have a unique function in normal monocytic cells, distinct from other src-related tyrosine kinases.

Animals↗
PubMed1986

Dual-laser, differential fluorescence correction method for reducing cellular background autofluorescence.

A method has been developed for reducing the intrinsic autofluorescence background component in cells labeled with fluorescent antibodies, thus permitting low levels of antibody-binding on highly autofluorescent cells to be quantified. The method is based on the broad autofluorescent excitation spectra compared to the well-defined spectra of the fluorescent label. Two laser wavelengths were used, one optimally to excite the fluorescent label plus autofluorescence and the second to excite only the autofluorescence. Two fluorescence measurements were made in the same wavelength region and the signals were subtracted on a cell-by-cell basis using a difference amplifier to zero the autofluorescence and amplify the signal from the fluorescent label. Test results on unlabeled autofluorescent macrophages showed that the autofluorescence component was reduced by balancing the signal inputs to the difference amplifier. When labeled macrophages were analyzed, the autofluorescence was reduced and the fluorescent-labeled antibody-binding component was amplified. The method was also able to resolve labeled lymphocytes from unlabeled autofluorescent macrophages.

Cell Line↗
PubMed1986

A model for the regulation of myelopoiesis by specific factors.

Hematopoietic cells originate from stem cells that generate progenitor cells programmed to differentiate along specific cell lineages. Recently, a number of factors have been identified that are involved in regulating the proliferation of myeloid lineage cells. We propose a model in which a hierarchy of specific factors acts sequentially during defined stages of maturation to regulate myelopoiesis. The role of these factors as competence or progression factors in causing cells to enter and traverse the cell cycle is discussed. Experimental evidence supports the model.

Animals↗
PubMed1986

Flow cytometric characterization of alveolar macrophages.

Phenotypes of lung free cells (FC) harvested from Fischer-344 rats by episodic lavage were characterized by flow cytometry. Parameters evaluated included electronic volume (EV), axial light loss (ALL), 90 degrees light scatter (LS), blue autofluorescence (BA), and green-yellow autofluorescence (G-YA). Three phenotypic populations, FC-A, FC-B, and FC-C were identified by their differing LS characteristics. FC-C represented 90% of the cells and were exclusively alveolar macrophages. Two subpopulations in FC-C, FC-CI and FC-CII, were further distinguished by their unique ALL features. The morphologic appearances of these subpopulations by light microscopy clearly differed in sorted preparations. Based on their patterns of autofluorescence, these FC-CI and FC-CII phenotypes were found to be composed of eight subpopulations. In FC populations harvested during further lavage episodes of the same lungs, the relative contributions of FC-CI to the FC-C subpopulation decreased as FC-CII correspondingly increased. This study demonstrates 1) that subpopulations of lavaged AM can be categorized according to their optical phenotypes by flow cytometry and 2) that the relative frequency of retrieval of some phenotypes depends on how exhaustively the lungs are lavaged. With regard to the latter, bronchoalveolar lavage does not randomly sample the underlying AM population in the alveolar compartment.

Animals↗
PubMed1986

Comparison of five short-term assays that measure nonspecific cytotoxicity mediated to tumor cells by activated macrophages.

Five different short term assays (less than 48 h) used to measure macrophage-mediated, nonspecific cytotoxicity were compared under similar conditions in the same laboratory using the same reagents. The purpose was to determine the extent to which results were comparable. Three of the assays were dependent on the release of a radioisotope to measure cytotoxicity, one was dependent on cell counting, and the last was dependent on flow cytometric quantification of remaining viable tumor target cells after they had been exposed to macrophages. The variables examined were the following: three different populations of macrophages; four different kinds of target cells; two types of radioisotopes; and two different agents that trigger the expression of cytolytic activity by primed macrophages. Recombinant gamma interferon was used as the priming agent in all the experiments. There was unexpectedly good agreement between the results of the various assays. No differences were found among the different macrophage populations, the isotopes or the triggering agents. Perhaps the most important finding was that differences in target cell susceptibility to killing by activated macrophages, which were apparent in assays of less than 24 h duration, disappeared when the same kinds of targets were compared in assays of greater than 40 h duration. The results of this study are an important first step toward standardizing the way in which macrophage-mediated, nonspecific cytotoxicity is measured in short-term assays, laboratory to laboratory.

Cell Line↗
PubMed1986

Flow cytometry Coulter volume analysis of lead- and cadmium-induced cellular alterations in bone marrow obtained from young adult and aged Balb/c mice.

Flow cytometry Coulter volume analysis was used to examine the effects of an acute exposure to cadmium or lead on subpopulations of Balb/c bone marrow cells. A significant shift in the volume of Balb/c bone marrow cells was detected in response to a single i.p. injection of cadmium acetate (Cd) or lead acetate (Pb) compared to sodium acetate (Na)-treated mice. An increase in the relative number or size of myeloid/monocytic cells was noted in the bone marrow of cadmium or lead-treated mice. This effect was more pronounced in aged Balb/c mice than in young adults. These studies suggest the flow cytometry Coulter volume analysis may be a useful and sensitive technique for the assessment of cellular changes that occur in the bone marrow in response to xenobiotic exposure.

Aging↗
PubMed1986

Translocation of particles to the tracheobronchial lymph nodes after lung deposition: kinetics and particle-cell relationships.

One of the pathways for particle clearance from the lung involves the translocation of particles from the alveoli to the tracheobronchial lymph nodes. Details of the mechanisms involved in this translocation process have not been well delineated. In the present study, we: assessed the kinetics of particle transfer to the tracheobronchial lymph nodes in the rat over a 30-day period following the intrapulmonary deposition of 4 X 10(8), 1.9 micron, fluorescent polystyrene microspheres; utilized multiparameter flow cytometric technology to quantitatively differentiate between "free" and cell-associated particles that accumulated in the lymph nodes; and assessed the role of alveolar macrophages (AM) in carrying particles to the lymph nodes by comparing the distributions of particles in lavaged AM with distributions of particles found in nodal mononuclear phagocytes (NMP). The accumulation of particles, most of which were extracellular, in the nodes was biphasic with the most rapid phase occurring within the first 24 hr. Over the Day 1-30 period, the numbers of particles in the lymph nodes increased linearly to approximately equal to 1.2 X 10(6) microspheres, or approximately equal to 0.3% of the originally instilled lung burden. The percentages of the nodal particles that were associated with NMP over the course of the study were inversely proportional to nodal particulate burdens, even though the percentage of cells with engulfed particles increased; the percentage of NMP asymptotically approached a maximum value over the range of nodal burdens of 6-12 X 10(5) particles. The distributions of the microspheres in the NMP were essentially identical on Days 1, 14, and 30. Major differences in the distributions of particles in lavaged AM and NMP were not consistent with the notion that the latter represented translocated AM.

Animals↗
PubMed1986

Analysis of lymphocyte-target conjugates by flow cytometry. I. Discrimination between killer and non-killer lymphocytes bound to targets and sorting of conjugates containing one or multiple lymphocytes.

The use of flow cytometric analysis and sorting techniques for the enumeration and purification of lymphocyte-target conjugates was investigated. Murine cytotoxic T-lymphocytes (CTL) with killer effector function were identified and quantitated during a 3-hour cell-mediated cytotoxicity reaction using multiparameter analysis. Resolution of conjugates containing single and multiple lymphocytes was achieved by two-color fluorescence, and individual conjugate subpopulations were subsequently sorted for further analysis. To measure total and cytotoxic conjugate frequencies, CTL were labelled with FITC-conjugated Thy 1.2 antibody and dead target cells were stained with propidium iodide (PI). Size difference between the CTL and P815 tumor target cells, as measured by Coulter volume and axial light loss, facilitated detection of conjugates which were identified as both large and Thy 1.2-positive. Conjugates containing dead target cells possessed red fluorescence due to PI uptake. The frequency of conjugates containing cytotoxic activity increased with time during the cytotoxicity period and correlated with frequencies obtained in single-cell assays. Analysis of the distribution of single and multiple lymphocyte-bound conjugates was done by co-centrifugation of Hoechst-stained CTL and FITC-labeled P815 target cells. Analysis by two-color fluorescence effectively resolved conjugate populations containing different numbers of CTL and allowed their purification by cell sorting. The purity of the separate populations was confirmed by fluorescence microscopic inspection. The results of these studies demonstrate that flow cytometry can resolve target-bound and free CTL, measure cytolytic efficiency and specifically sort out cytometrically defined subgroups within the effector cell population.

Animals↗
PubMed1986

Simultaneous measurements of macrophage-induced cytostasis and cytotoxicity of EMT6 cells by flow cytometry.

Cytostasis and cytotoxicity as well as survival and growth of EMT6 cells following exposure to tumoricidal macrophages have been examined using multiparameter flow cytometry. Macrophages were resolved from tumor cells, and the number of surviving EMT6 cells at different interaction times was determined by adding, to each cell suspension, fluorescent latex particles the concentration of which was known and counting them along with the cells. The percentages of EMT6 cells in S phase were determined by analysis of DNA histograms and compared to the percentages as determined by autoradiography. The progression of cells through the cell cycle was also examined during exposure to macrophages by dual parameter analysis of DNA content and bromodeoxyuridine incorporated into DNA prior to exposure to macrophages. The results showed that, by 4 h of interaction with tumoricidal macrophages, EMT6 cells stopped progressing; tumor cell progression was inhibited in all phases of the cell cycle. By 24 h, there was an absolute decrease in survival. By 48 h, however, surviving tumor cells which escaped the tumoricidal activity of macrophages exhibited a population doubling rate similar to control cells.

Animals↗