Search PubMed⌕ Search

Biomedical subjects

C C Stewart

Publications and source records attributed to C C Stewart.

At least 73 records · Page 4Linked to original sources
PubMed1992

Fluorescent in situ hybridization en suspension (FISHES) using digoxigenin-labeled probes and flow cytometry.

Fluorescent in situ hybridization has become a major technique for visualizing genetic material in fixed cells. Currently, many systems utilize the hybridization of labeled molecular probes to cells that are attached to slides. We have developed a technique that allows for in situ hybridization to be performed using cells in suspension. By using digoxigenin-labeled DNA probes and a fluoresceinated antibody directed against the digoxigenin, we can measure the resulting signal on a flow cytometer and the cells can be attached to microscope slides for visual analysis.

Animals↗
PubMed1991

Identification and comparison of CD34-positive cells and their subpopulations from normal peripheral blood and bone marrow using multicolor flow cytometry.

Four-color flow cytometry was used with a cocktail of antibodies to identify and isolate CD34+ hematopoietic progenitors from normal human peripheral blood (PB) and bone marrow (BM). Mature cells that did not contain colony forming cells were resolved from immature cells using antibodies for T lymphocytes (CD3), B lymphocytes (CD20), monocytes (CD14), and granulocytes (CD11b). Immature cells were subdivided based on the expression of antigens found on hematopoietic progenitors (CD34, HLA-DR, CD33, CD19, CD45, CD71, CD10, and CD7). CD34+ cells were present in the circulation in about one-tenth the concentration of BM (0.2% v 1.8%) and had a different spectrum of antigen expression. A higher proportion of PB-CD34+ cells expressed the CD33 myeloid antigen (84% v 43%) and expressed higher levels of the pan leukocyte antigen CD45 than BM-CD34+ cells. Only a small fraction of PB-CD34+ cells expressed CD71 (transferrin receptors) (17%) while 94% of BM-CD34+ expressed CD71+. The proportion of PB-CD34+ cells expressing the B-cell antigens CD19 (10%) and CD10 (3%) was not significantly different from BM-CD34+ cells (14% and 17%, respectively). Few CD34+ cells in BM (2.7%) or PB (7%) expressed the T-cell antigen CD7. CD34+ cells were found to be predominantly HLA-DR+, with a wide range of intensity. These studies show that CD34+ cells and their subsets can be identified in normal PB and that the relative frequency of these cells and their subpopulations differs in PB versus BM.

Adult↗
PubMed1991

Expression of the c-fgr and hck protein-tyrosine kinases in acute myeloid leukemic blasts is associated with early commitment and differentiation events in the monocytic and granulocytic lineages.

Two members of the src proto-oncogene family of intracellular tyrosine kinases, c-fgr and hck, are selectively expressed in differentiated myeloid cells. To study the expression of these genes in acute myeloid leukemia (AML) and to determine the specific myeloid lineages and stages of myeloid differentiation at which the expression of these genes is acquired, we used a series of 79 cases of de novo AML as a differentiation model. The levels of c-fgr, hck, and c-fms (encoding the colony-stimulating factor-1 receptor) mRNA transcripts were correlated with the presence of specific cell surface antigens and the morphologic and cytochemical features in these AML blasts. Relatively undifferentiated leukemic myeloblasts with an HLA-DR, CD34, CD33, CD13+/- cell surface immunophenotype (French-American-British [FAB] M1 or M2) were characterized by a lack of c-fms and c-fgr expression, while low levels of c-fms and c-fgr could be detected in undifferentiated myeloblasts (FAB M1 or M2), which also expressed CD14 at low antigen density. The hck transcripts were either undetectable in these cells or were expressed at low levels. In contrast, only hck mRNA transcripts could be identified in blasts with progranulocytic morphology (FAB M3), while c-fms, c-fgr, and hck were all expressed at high levels in blasts with differentiated myelomonocytic or monocytic features (FAB M4 and M5). No c-fms, c-fgr, or hck transcripts were evident in leukemic cells of the erythroid lineage (FAB M6). When undifferentiated leukemic myeloblasts (HLA-DR, CD34, and CD33) were induced to differentiate in vitro to cells with monocytic characteristics, the expression of c-fms, c-fgr, and the CD14 cell surface antigen were induced to high levels, accompanied by the acquisition of hck and CD13 expression. In contrast, when HLA-DR, CD34, and CD33 blasts were induced to differentiate in vitro to cells with granulocytic characteristics, only hck and CD13 expression were induced. Our data suggest that the acquisition of c-fgr and/or hck expression is associated with early commitment and differentiation events in distinct myeloid lineages. Assessment of the expression of these kinases may provide a molecular tool to assign lineage in AML in conjunction with morphology, cytochemistry, and cell surface antigen expression.

Cell Differentiation↗
PubMed1991

Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations.

Flow cytometric determination of viable versus nonviable cells in fixed samples can be accomplished by utilizing the irreversible binding of photoactivated ethidium monoazide (EMA). EMA is a positively charged molecule which is excluded by cells with intact membranes (viable cells), included by cells with damaged membranes, and can be photochemically crosslinked to nucleic acids using visible light. EMA fluorescence can be excited using a standard argon laser operating at 488 nm and is able to be distinguished from fluorescein and phycoerythrin. Fixation is important when analyzing cells from a potentially infectious origin. EMA is photochemically crosslinked and therefore unable to leak out of cells when removed from the extracellular media, unlike propidium iodide (PI) or other viability stains, which were heretofore commonly used. We demonstrate the usefulness of EMA in combination with fluoresceinated and phycoerythrin labeled monoclonal antibodies in immunophenotyping. The photoaffinity labeling technique allows for a quick and efficient means of identifying nonviable cells which cannot be distinguished on the basis of light-scattering properties.

Affinity Labels↗
PubMed1991

Sorting minor subpopulations of cells: use of fluorescence as the triggering signal.

Flow cytometric cell sorting is commonly used to obtain purified subpopulations of cells for use in in vitro and in vivo assays. This can be time-consuming if the subpopulations of interest represent very low percentages of the cell suspension under study. Often the desired subpopulations are identified by two-color immunofluorescence staining. Generally, cell sorting is performed with a flow cytometer configured to trigger on light scatter signals, then sort windows are set based upon the signals from both fluorescent markers. We demonstrate that triggering the cytometer with the fluorescence signal from antibody staining common to both of the desired subpopulations, then sorting the subpopulations based upon staining of a second marker, substantially increases the speed of cell sorting vis-à-vis traditional methods. This is because undesired events are not analysed, allowing an increase in the throughput rate. While desired subpopulations of cells can be obtained by this method, undesired (i.e., nonstaining) cell "contaminants" increase and may require a second sort. The combined time for the initial enrichment sort and a second sort can be less than sorting once using standard methodology. Alternatively, the degree of contamination may be controlled by adjusting the concentration of the cell suspension and by the sample flow rate.

Animals↗
PubMed1991

Gelatin sponge model of effector recruitment: tumoricidal activity of adherent and non-adherent lymphokine-activated killer cells after culture in interleukin-2.

This study examined the specific tumoricidal activity of lymphokine-activated killer (LAK) cells derived from tumor-infiltrating lymphocytes that prevent the growth of secondary tumors in animals harboring progressing primary tumors. A pre-implanted gelatin sponge was employed to capture infiltrating host effectors during the expression of concomitant tumor immunity. Additionally, this study compared the cytolytic activity of these sponge-derived cells with those of counterpart splenic lymphocytes. The cells from both sources were cultured for 4 days in IL-2 to generate LAK cells which were further expanded in IL-2-containing medium for up to 11 days. The cytotoxic activities of these cells were measured in a Chromium-51 release assay. The data revealed that the culture of splenic, or sponge-derived lymphocytes results in the emergence of non-adherent and adherent cell populations with LAK activity. The 4-day sponge-derived LAK cells (adherent and non-adherent) exhibited significant cytolysis of EMT6 cells while the spleen-derived counterparts showed minimal cytotoxicity toward these targets. Some NK activity in LAK cells derived from both sources was evident by their lysis of YAC-1 cells. LAK cells from both sources were incapable of lysing histo-compatible EL-4 (H-2b) tumor cells. The lysis of the EMT6 cells by the sponge-derived LAK cells was maintained over an 11-day period of culture in IL-2. Conversely, the spleen-derived LAK cells were unable to significantly lyse EMT6 cells during this period of in vitro culture. These results show the superior specific tumoricidal activity of LAK cells derived from lymphocytes mediating tumor rejection in vivo (sponge-derived) over that of counterpart splenic lymphocytes.

Animals↗
PubMed1991

Murine eosinophil granulocytes bind the murine macrophage-monocyte specific monoclonal antibody F4/80.

Studies designed to identify a panel of monoclonal antibodies useful for the separation of murine eosinophil myeloid progenitors revealed that F4/80, an antigen heretofore thought to be expressed only by murine monocyte/macrophage lineage cells, was expressed by eosinophil granulocytes. Eosinophils from several strains of mice stained positively with specific antibody for the epitope. A novel pathway for myeloid differentiation is proposed in which neutrophil progenitors exit a common lineage prior to a common progenitor of monocytes and eosinophils. Moreover, the results demonstrate that binding of anti-F4/80 can no longer be viewed as exclusive for mononuclear phagocytes.

Animals↗
PubMed1991

The gene encoding human TFE3, a transcription factor that binds the immunoglobulin heavy-chain enhancer, maps to Xp11.22.

TFE3, a member of the helix-loop-helix family of transcription factors, binds to the microE3 motif of the immunoglobulin heavy-chain enhancer and is expressed in many cell types. We have localized human TFE3 to the proximal short arm of the X chromosome using a somatic cell hybrid panel. A frequent RsaI RFLP detected by the TFE3 cDNA was found and used to confirm this location by linkage analysis in 20 pedigrees. Two-point and multipoint lod scores place TFE3 near markers in Xp11.22 with the most likely order DXS7-DXS255-TFE3-DXS146-DXS14.

Animals↗
PubMed1991

Stochastic versus deterministic in haemopoiesis: what is what?

'Stochastic' models of haemopoiesis are discussed in juxtaposition to 'deterministic' concepts. It is argued that certain characteristic features attributed to stochastic models are conjectures, not supported by the observations quoted to provide an experimental basis: for example, an assumption of extrinsic control and another assumption that such control is inaccessible to external influences up to the very latest stages of cell development. Furthermore, it is argued that quantitative relationships between early progenitors (stem cells) and the number of colonies produced in vitro, cited as evidence supporting the proliferation and differentiation scheme of the stochastic model of random restriction, are also compatible with other models, providing that some sufficiently complex mechanism is involved in the control process. A simple model based on the principle of ordered development (usually denoted 'proliferation tree') is presented as an example and the calculated distributions are shown to have the same form as the experimental ones.

Hematopoiesis↗
PubMed1990

13-cis retinoic acid augments the production of macrophages in mouse bone marrow cultures stimulated with interleukin 3.

Mouse bone marrow cells in liquid culture with interleukin 3 generate nonadherent granulocytes, mast cells, and macrophages. The addition of 13-cis retinoic acid (13cRA) (10(-8)-10(-6) M) enhanced proliferation of the nonadherent cells, and concentrations greater than 5 x 10(-7) M stimulated a sixfold increase in adherent macrophages. Four-color flow cytometry was used to identify the lineages present using the following antibodies: MAC1 (granulocytes and macrophages), F4/80 (macrophages), B54.2 (mast cells), and H12 (anti-Thy1.2 to identify myeloid precursors). This analysis demonstrated a twofold increase in MAC1+ F4/80+ cells, which were sorted and identified morphologically as macrophages. 13cRA also increased by 60%-95% the numbers of colony-forming cells responsive to interleukin 3 (IL-3) and macrophage colony-stimulating factor (M-CSF) but did not significantly change the colony-forming cells responsive to granulocyte-macrophage colony-stimulating factor (GM-CSF). These data suggest that 13cRA increases the production of macrophages by modulating the commitment of IL-3-expanded progenitor cells to the macrophage lineage.

Animals↗
PubMed1989

Regulation of MHC class II gene expression in macrophages by hematopoietic colony-stimulating factors (CSF). Induction by granulocyte/macrophage CSF and inhibition by CSF-1.

CSF-1 and granulocyte/monocyte CSF (GM-CSF) were shown to modulate the levels of Ia gene and protein expression in bone marrow-derived macrophages (BMM). Recombinant GM-CSF induced high levels of Ia expression, similar to the levels induced by INF-gamma, while IL-3 had no effect. In contrast, recombinant CSF-1 not only suppressed the basal levels of Ia gene and protein expression in BMM, but also inhibited the induction of Ia by IFN-gamma and GM-CSF. Basal levels of Ia were not inhibited by recombinant CSF-1 until after 16-24 h of culture, suggesting an indirect mechanism of suppression. IFN-alpha/beta and PGE2 were shown not to be involved in the CSF-1 inhibition of basal levels of Ia expression. However, the CSF-1-mediated suppression of both the basal levels of Ia expression and the induction of Ia in BMM by IFN-gamma and GM-CSF did correlate with the induction of cellular proliferation. These data imply that in addition to regulating hematopoiesis, CSFs may regulate the initiation of the immune response through their effects on Ia expression in macrophages.

Animals↗
PubMed1989

Resolving leukocytes using axial light loss.

Axial light loss (ALL) is the measurement of the total light lost from the laser beam at 0 degrees when a particle passes through the beam. Used in combination with the monoclonal antibody CD45, ALL can effectively resolve lymphocytes, monocytes, granulocytes, and dead cells in viable or fixed preparations of human peripheral blood. A bivariate display of ALL vs. CD45 clearly resolves all granulocytes from lymphocytes; although degranulated granulocytes cannot be resolved with forward-angle and right-angle light scatter, they are clearly resolved in right-angle scatter vs. CD45. A blood differential can be performed, with a single laser flow cytometer and three colors of fluorescence, when ALL is combined with fluoresceinated CD45 to resolve leukocytes, phycoerythrin-labeled NKH1 to resolve natural killer cells, and biotinylated CD3 in combination with DuoCHROME, the phycoerythrin/Texas red conjugate fluorochrome from Becton Dickinson, to resolve T-cells. B-cells are the only cells negative for both phycoerythrin and Texas red. When PE CD4 is included, the CD3+ CD4+ T-cell subset is resolved from the CD3+ CD4- subset comprising mainly the CD3+ CD8+ T-cell subset. The addition of propidium iodide is unnecessary since ALL clearly resolves dead cells in a viable preparation of human peripheral blood. Furthermore, since ALL resolves these cells even after fixation in paraformaldehyde, all samples can be fixed prior to analysis, thereby minimizing the potential biohazard risk.

Antibodies, Monoclonal↗
PubMed1989

Quantitation of the host cell infiltration kinetics of the nonimmunogenic colon 26 tumor by multiparameter flow cytometry.

In this study, we examined the kinetics of host cell infiltration into the nonimmunogenic Colon 26 tumor. We found that 1 x 10(4) cells were required to produce tumors in 100% of mice. The vivo doubling time was 42.5 h, and a barely palpable tumor contained 8 x 10(6) cells. No evidence of concomitant immunity was found. The number of host cells infiltrating the in vivo tumors increased at the same rate as the number of tumor cells, but averaged only 22% of total cells. Cycling T lymphocytes were present in the host cell infiltrate of this tumor. In addition, approximately 50% of in vivo Colon 26 cells were Thy-1.2 positive. The observed characteristic of low immunogenicity makes it a useful murine model for studying human malignant tumors.

Animals↗
PubMed1989

High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules.

We are developing a laser based technique for the rapid sequencing of large fragments (approximately 40 kb) of DNA based upon the detection of single, fluorescently tagged nucleotides cleaved from a single DNA fragment. We have demonstrated significant progress on several of the important steps of this technique. The projected rate of sequencing is several hundred bases per second which is orders of magnitude faster than existing methods. Once developed, this technology could be utilized by investigators for rapid sequencing of genetic material from virtually any source.

Base Sequence↗
PubMed1989

General principles of multiparameter flow cytometric analysis: applications of flow cytometry in the diagnostic pathology laboratory.

Recent developments in the design of flow cytometric instrumentation and advances in computerized methods of data analysis have produced flow cytometers that are compatible with the clinical laboratory setting. Because flow cytometry is impacting on virtually every area of diagnostic pathology (particularly the subspecialties of hematology, immunology, infectious disease, transplantation monitoring, neoplasia, and genetics), it is essential that pathologists develop a general understanding of flow cytometric techniques. In addition to new developments in instrumentation, simplification of laser design and advances in fluorochrome chemistry have provided several new sophisticated tools to make multiple correlative biological measurements on normal and neoplastic cells in the clinical setting. Flow cytometric analysis thus allows the pathologist to more effectively diagnose, predict prognosis, and monitor disease progression. This report reviews the basic components of flow cytometric instrumentation, the physical and biological parameters that may be assessed using flow cytometric techniques, and the multiparametric data analysis systems available for the clinical laboratory.

Antibodies, Monoclonal↗