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Biomedical subjects

D L McFarlane

Publications and source records attributed to D L McFarlane.

6 recordsLinked to original sources
PubMed1990

A flow cytometric assay for target binding by NKH1A+ cells using a single laser system.

A modified method of analysis by flow cytometry has been adapted to the NK/target cell system to monitor conjugate formation within a mixed population. The two-parameter assay is based on the use of two colour fluorescent markers in which an indirect FITC-conjugated NKH1A antibody (green fluorescence) is used to detect NK-specific effector cells and an intracellular strain, hydroethidine (red fluorescence) is used to detect target cells. The two cell populations and their conjugates are discernible by the presence of both green and red fluorescence, using a single laser beam tuned to 488 nm. In comparison, a manual count conjugate assay was applied using a hemacytometer and fluorescence microscope. Of the two methods, the automated technique using flow cytometry provides a lower but more precise representation of conjugate formation, due to the avoidance of technical/observer error that is common to manual count assays. The cytometric method has proven to be reproducible and superior in consistency to the manual count assay and can be adapted to almost any effector/target system under investigation. In comparison with previous methods using dual laser, the single laser system provides advantages of cost and time efficiency due to setup simplicity, as well as improved availability.

Antibodies, Monoclonal↗
PubMed1988

Flow cytometric analysis of the phenotypic changes in tumour cell lines following TPA induction.

Single-cell analysis by flow cytometry has enabled us to analyze the effects of a phorbol ester and known tumour promoter, TPA, on the phenotypes of four tumour lines. TPA is capable of triggering a variety of cellular alterations that can affect gene expression and the biochemical balance of intracellular events. We have investigated the effect of TPA on such properties as rate of proliferation, differentiation, expression of cell surface molecules, and susceptibility to natural killer (NK) cell-mediated cytolysis. Four human leukemia and lymphoma cell lines; K562, MOLT 4, Raji, and HL60, were studied in their response to TPA treatment. Based on measurements of the defined cellular properties, we have characterized the pleiotropic responses of each tumour cell line to the phorbol ester in relation to intensity and time of onset of each response. The effects of TPA are highly varied, ranging in time of onset from minutes to days, and in intensity from strong to weak within the four cell lines studied. However, within all the processes that are affected, the activation of protein kinase C appears to be a common initiating event of phorbol ester induction.

Antigens, Neoplasm↗
PubMed1988

Transferrin receptor and 4F2 expression by NK-sensitive and NK-resistant tumour cell lines.

Upon stimulation with a phorbol ester and known tumour promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), the human erythroleukemia K562 cell line, a standard target for human natural killer (NK) cells, shows a significant reduction in expression of transferrin receptors (TfR) and becomes resistant to NK-mediated cytolysis. Prompted by the initial finding that expression of TfRs by K562 cells correlates with target sensitivity to NK cytotoxicity, we have investigated the role of two proposed NK-target structures, the TfR and 4F2 molecule in three other tumour cell lines which vary in their susceptibility to NK activity. We report that similar to K562 cells, MOLT 4, Raji and HL60 cells demonstrate phorbol-ester-induced down-regulation of TfR expression. The expression of TfRs alone does not determine target sensitivity. However, TfRs are probably located close to the NK-target structure in NK-sensitive cell lines. Based on the properties of phorbol ester induction, it is possible that the NK-target molecule is down-regulated in response to phorbol ester induction in a similar, if not identical manner to that of the TfR; thus, rendering NK-sensitive cells resistant to NK killing, after TPA exposure. Conversely, the target molecule is probably upregulated in MOLT 4 cells after TPA treatment as indicated by the acquired sensitivity of these cells to NK-mediated cytolysis. The expression of 4F2 molecules was not influenced by TPA treatment and does not correlate with NK sensitivity.

Antigens, Surface↗
PubMed1978

The multicellular spheroid as a model tumor allograft. II. Characterization of spheroid-infiltrating cytotoxic cells.

Alloimmune lymphoid cells infiltrating multicellular spheroids of EMT6 mammary sarcoma cells (a solid tumor allograft model) have been characterized according to their morphological and functional properties. Both lymphocytes and macrophages were found within spheroids at the time of peak tumor cell damage. Cytotoxic cells specific for allograft antigens were also present. Using a short-term 51-Cr release assay, the cells responsible for cytotoxicity were characterized as a nonadherent, nonphagocytic T cell population. Velocity sedimentation cell separation further demonstrated that these cytotoxic cells had the physical properties of small lymphocytes. Some evidence for selective spheroid infiltration by specifically alloimmune cells was also obtained. The possible relationship of this cellular infiltrate to graft damage is discussed.

Animals↗
PubMed1978

Reversible changes in cytolytic T lymphocyte function induced by hyperthermia.

The ability of cytolytic T lymphocytes (CTL) generated in vitro in mixed leucocyte cultures (MLC) to "repair" functional damage induced by moderate hyperthermia has been investigated. Repair of CTL function was assessed in a split dose system in which MLC cells were exposed to a total of 20 min at 43 degrees C delivered either as a single treatment or as two 10 min fractions separated by an interval at 37 degrees C. Significant repair was observed within 15 min at 37 degrees C in the split-dose protocol and maximum repair (corresponding to a 30-100-fold increase in lytic activity) was seen after 1.5-2 h. Repair was both temperature and energy dependent and was apparently related to a reversible lesion in the cytolytic mechanism itself. The effect of a number of metabolic inhibitors (and other agents) on repair was determined, but the precise nature of the heat-induced lesion could not be identified.

Animals↗