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Biomedical subjects

H R MacDonald

Publications and source records attributed to H R MacDonald.

At least 325 records · Page 18Linked to original sources
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↗
PubMed1977

Energy metabolism and T-cell-mediated cytolysis. I. Synergism between inhibitors of respiration and glycolysis.

The energy requirements for T-cell-mediated cytolysis have been investigated. Cytolytic thymus-derived lymphocytes (CTL) were generated in vitro in mixed leukocyte cultures and assayed for cytotoxicity on 51Cr-labeled mastocytoma target cells. Cytolysis was only slightly reduced in the absence of exogenous glucose (less than 5 micrometer) or under conditions of extreme hypoxia (less than 0.2 micrometer oxygen). Furthermore, neither the glucose analogues 2-deoxy-D-glucose and 5-thio-D-glucose nor the respiratory antagonists sodium azide and 2,4-dinitrophenol were very effective inhibitors of cytolysis when used individually. However, these glucose analogues were highly effective in inhibiting cytolysis in the absence of oxygen, and the respiratory antagonists inhibited cytolysis to a much greater extent in the absence of glucose. In addition, synergistic effects were observed when the glycolytic and respiratory inhibitors were combined. Taken together, these results indicate that T-cell-mediated cytolysis is an energy-dependent process which can be supported by either oxidative or glycolytic energy pathways.

Animals↗
PubMed1977

Energy metabolism and T-cell-mediated cytolysis. II. Selective inhibition of cytolysis by 2-deoxy-D-glucose.

The effect of the hexose analogue 2-deoxy-D-glucose (2-DG) on T-cell-mediated cytolysis has been investigated. 2-DG inhibited cytolysis in glucose-free medium but not in medium containing equimolar concentrations of glucose. This inhibition was reversible and quantitatively competitive with glucose. Among other natural sugars examined, only mannose competed effectively with 2-DG and reversed the inhibition of cytolysis, whereas sodium pyruvate, fructose, galactose, fucose, and glucosamine were without effect. Mannose and glucose were equally effective in competing with 2-DG on a molar basis. When other glucose analogues such as 5-thio-D-glucose (5-SH-G) and 3-O-methylglucose were investigated under the same conditions, no inhibition of cytolysis was observed however, 5-SH-G (but not 3-O-methylglucose) was able to reverse the inhibitory effect of 2-DG in a competitive fashion. Taken together with the data presented in the accompanying paper, these findings provide strong evidence that 2-DG inhibits T-cell-mediated cytolysis by a mechanism that is unrelated to energy production. The possibility that inhibition is related to interference with membrane glycoprotein synthesis is discussed.

Animals↗
PubMed1977

Inhibition of clone formation as an assay for T cell-mediated cytotoxicity: short-term kinetics and comparison with 51Cr release.

The short-term kinetics of T cell-mediated cytotoxicity was investigated using a cloning inhibition assay. Murine cytotoxic thymus-derived lymphocytes (CTL) generated in vitro in mixed leukocyte cultures (MLC), were incubated for various periods of time at 37 degrees C with allogeneic mastocytoma target cells. The mixtures were then plated in soft agar, and mastocytoma clone formation was assessed after 5-7 days incubation. Using this technique, it was demonstrated that events leading to the loss of cloning ability could be detected after 1-3 min incubation at 37 degrees C, and after 20-30 min, 95% of the clone forming cells had been inactivated. When these results were compared directly with those obtained using the conventional 51Cr-release assay, it was found that the events leading to loss of cloning ability occurred more rapidly than indicated by the isotope assay. However, a modification of the 51Cr-release assay involving EDTA addition, gave comparable results to the cloning inhibition assay. These results raise the possibility that the events leading to 51Cr-release of tumor target cells may be related in time to those leading to the loss of cloning ability.

Animals↗
PubMed1977

Multicellular spheroids: a new model target for in vitro studies of immunity to solid tumor allografts.

Multicellular spheroids of EMT6 mammary sarcoma cells of BALB/c origin were incubated with normal spleen cells or alloimmune spleen cells generated in vitro in mixed leukocyte cultures (MLC). After 24 hours, spheroids were trypsinized and assayed for surviving tumor cells by use of a cloning technique. Under these conditions a 60-80% reduction in clone-forming tumor cells was observed after incubation of spheroids with immune lymphocytes as compared to normal lymphocyte controls. This cytotoxic effect occurred in situ, and alloimmune cells sensitized against unrelated antigens were much less cytotoxic than were specifically sensitized cells. In parallel autoradiographic studies, some immune lymphoid cells that had been labeled with tritiated thymidine during the proliferative phase of the MLC could be demonstrated within spheroids after 24 hours. These results suggested that multicellular spheroids will be a useful in vitro model for more detailed analysis of the factors controlling infiltration in in situ destruction of solid tumor grafts.

Animals↗
PubMed1977

Effect of hyperthermia on the functional activity of cytotoxic T-lymphocytes.

The effect of hyperthermia on the functional activity of murine cytotoxic thymus-derived lymphocytes (CTL's) has been investigated. CTL's were generated in vitro in mixed leukocyte cultures (MLC's) and assayed for cytotoxicity on 51Cr-labeled allogeneic P-815 mastocytoma cells. Exposure of MLC cells to temperatures of 43 degrees C or above rapidly inactivated CTL function. The degree of inactivation depended on temperature and time of exposure to heat. CTL function was much more sensitive to hyperthermia than was the reproductive capacity of tumor target cells (assessed by a cloning assay), and the heat sensitivity of CTL's appeared to depend on their stage of differentiation. CTL function dramatically increased when MLC cells were incubated at 37 degrees C after exposure to hyperthermia or when a total exposure to heat was split into two equal fractions separated by an interval at 37 degrees C. These latter findings were operationally analogous to the repair of potentially lethal and sublethal radiation damage, respectively, and suggested that further studies are required to assess the complex interrelationships between hyperthermia, cell-mediated immunity, and tumor cell survival.

Animals↗
PubMed1976

Increased cytolytic T lymphocyte activity induced by 2-mercaptoethanol in mixed leukocyte cultures: kinetics and possible mechanisms of action.

The 20- to 50-fold increase in cytolytic T lymphocyte (CTL) activity caused by the addition of 50 muM 2-mercaptoethanol (2-ME) at the onset of a one-way murine mixed leukocyte culture (MLC) between C57BL/6 and DBA/2 splenic lymphocytes appears to be unrelated to early events in the culture: if 2-ME was present for the first 24 hr of culture only, there was no increase on day 4, but if addition of 2-ME was delayed until the last 24 hr of culture, the CTL activity was almost as high as that of cultures that were exposed to 2-ME for the entire 4-day culture period. The increase of CTL activity caused by delayed addition of 2-ME ("2-ME rescue") was used to investigate the mechanism by which the thiol induces differentiation of CTL from precursor cells. 2-ME rescue was mimicked by two other thiols, dithiothreitol and cysteamine phosphate, but at higher concentrations. Because the latter compound has no free sulhydryl group until it diffuses into cells and is enzymatically dephosphorylated, we conclude that thiols may increase the differentiation of CTL from precursor cells by an intracellular process involving free sulphydryl groups rather than by interaction with membrane sulfhydryls or destruction of inhibitor cells or their products. Cell separation experiments indicated that 2-ME rescue was independent of the presence of B lymphocytes and of adherent cells (macrophages) and was restricted to a subpopulation of T lymphocytes that developed into large lymphoid precursor cells during the first 3 days in culture even without 2-ME. The development of this subpopulation required DNA synthesis between 24 nad 72 hr after the onset of MLC. When 2-ME was added to day-3 MLC, CTL activity increased slightly as early as 4 hr later, but the major increase occurred during the second half of the 24 hr "rescue"period. Because this increase was inhibited by cytosine arabinoside (ARA-C), it seems likely that DNA synthesis is associated with and may be required for the differentiation of large precursor lymphoid cells into CTL after the addition of 2-ME.

Animals↗
PubMed1976

Characterization of effector lymphocytes associated with immunity to murine sarcoma virus (MSV) induced tumors. I. Physical properties of cytolytic T lymphocytes generated in vitro and of their immediate progenitors.

Cytolytic T lymphocytes (CTL) were generated in secondary mixed leukocyte-tumor cell cultures (MLTC) with syngeneic RB1-5 tumor cells as stimulating cells and with responding spleen cells from regressor mice that had rejected a murine sarcoma virus (MSV)-induced tumor. CTL precursor cells were found to be exclusively of thymic origin and non-T cells were apparently not required for CTL generation. When the size variations of CTL from syngeneic MLTC were analyzed by velocity sedimentation it appeared that a transition from small precursor cells to larger effector cells occurred during the first 5 days in culture; this change in cell size was then followed by a shift toward small-sized cells. Furthermore, the CTL generated in syngeneic MLTC in the MSV tumor immune system were compared with those CTL obtained in allogeneic mixed leukocyte cultures (MLC) and were shown to exhibit fundamental similarities.

Animals↗
PubMed1975

Generation of cytotoxic T lymphocytes in vitro. IV. Functional activation of memory cells in the absence of DNA synthesis.

Re-exposure of day 14 mixed leukocyte culture (MLC) cells to the original stimulating alloantigens (secondary response) has previously been shown to result in significant proliferation and in rapid reappearance of high levels of cytolytic T-lymphocyte (CTL) activity within the next 4 days. Moreover, evidence has been presented that CTL precursor cells in day 14 MLC populations, while they derived from cells were large at peak of the primary response (day 4) were themselves small lymphocytes which developed into large CTL after restimulation. In this study, inhibition of DNA synthesis by cytosine arabinoside (ARA-C) was used to investigate whether CTL formation could be dissociated from proliferation during the secondary response. It was found that within the first 24 h after restimulation (a) CTL activity increased 6-to-20-fold, (b) 60-70% of the small T lymphocytes became medium- to large-sized cells, and (c) both events were independent of DNA synthesis. By using two successive cell separations by velocity sedimentation at unit gravity, before and after stimulation of day 14 MLC cells for 24 h in the presence or absence of ARA-C, direct evidence was obtained that small CTL precursor cells developed into large CTL, irrespective of DNA synthesis. The presence of ARA-C for periods longer than 24 h inhibited any further increase in CTL activity, in contrast to a parallel increase in lytic activity and cell number from day 1 to day 4 in control restimulated cultures. Taken together with the finding that 90% of the medium- and large-sized lymphoid cells in control restimulated cultures underwent DNA synthesis within 24 h, these results thus suggest that during a secondary MLC response there is initially a differentiation step leading to the formation of CTL which, although it can be clearly dissociated from DNA synthesis, is under normal conditions followed by proliferation of these effector cells.

Animals↗
PubMed1975

Effect of delayed addition of 2-mercaptoethanol on the generation of mouse cytotoxic T lymphocytes in mixed leukocyte cultures.

The generation of mouse cytolytic T lymphocytes (CTL) in primary or secondary mixed leukocyte cultures (MLC) is greatly enhanced by the addition of 2-mercaptoethanol (2-ME) to the culture medium. This enhancement can be equally demonstrated with either reduced or oxidized 2-ME. Addition of 2-ME to MLC as late as 3 days after the initiation of the culture results in a peak CTL response which is nearly as high as the one in cultures containing 2-ME throughout the 4 day-incubation period. Quantitative analysis of the CTL response in MLC supplemented with 2-ME on day 3 shows a 50-fold increase in lytic activity within 24 h, suggesting that cell differentiation, in addition to proliferation may be affected by this agent.

Animals↗
PubMed1975

Early detection of potentially lethal events in T cell-mediated cytolysis.

The short-term kinetics of the interaction between mouse cytolytic T lymphocytes (CTL) and 51Cr-labeled target cells was investigated. It was found that addition of EDTA to mixtures of CTL and target cells instantaneously blocked de novo lytic interactions, but did not inhibit the release of 51Cr from already damaged target cells. Using this information, a modified cytolytic assay was developed. By applying this assay to highly active CTL populations generated in secondary mixed leukocyte cultures, it was possible to detect appreciable target cell damage as early as 30 seconds after exposure to CTL. Quantitative studies demonstrated linear relationships between cytolysis and time and between rate of cytolysis and cell number under these assay conditions.

Animals↗
PubMed1975

Heterogeneity of in vitro colony- and cluster-forming cells in the mouse marrow: segregation by velocity sedimentation.

C57BL bone marrow cells were separated on the basis of their sedimentation velocity at unit gravity and cell fractions cultured in agar using three types of colony stimulating factor (CSF). Colony-forming cells separated as a single peak (s equal 4.4 mm/hr) in cultures stimulated by mouse lung conditioned medium (CSFMLCM) or endotoxin serum (CSFES). Cluster-forming cells were separable into two peaks and the majority were larger than colony-forming cells (s equal 5.7 mm/hr). Partial segregation of colony-forming cells was observed according to the morphological types of colonies generated, large cells tending to generate macrophage colonies and small cells, granulocytic colonies. Large colony-forming cells were more responsive to stimulation by CSF than small cells. Human urine (CSFHU) appeared unable to proliferation of most small colony-forming cells. Colony-forming cells appear to be a highly heterogeneous population with intrinsic differences in responsiveness to CSF and with differing capacities to generate colonies whose cells differentiate to granulocytes of macrophages.

Animals↗
PubMed1975

Growth of mouse megakaryocyte colonies in vitro.

Mouse bone marrow and spleen cells formed pure or mixed colonies of up to 80 megakaryocytes in agar cultures after stimulation by medium conditioned by activated mouse lymphoid cells. Megakaryocytes were identified on the basis of their morphology, polyploid mitoses and DNA content, and high cytoplasmic content of acetylcholinesterase. Megakaryocyte colony-forming cells were relatively small with a peak sedimentation velocity of 4.2 mm/hr. Spleen, lymph node, and thymus cells produced the factor stimulating megakaryocyte proliferation after culture in medium containing 2-mercaptoethanol, with or without added mitogens or allogeneic spleen cells. Peak activity in conditioning medium was associated with the small lymphocyte fractions in mouse spleen.

Animals↗
PubMed1975

A comparison of the effector cells involved in cell-mediated lympholysis and antibody-dependent cell-mediated cytotoxicity in man.

Some physical and mechanistic properties of cytotoxic lymphocytes and antibody-dependent killer cells were compared. Cytotoxic lymphocytes were measured by the cell-mediated lympholysis (CML) technique after sensitization of human peripheral blood leukocytes (PBL) in unidirectional mixed leukocyte culture (MLC). Killer cells were measured before and after MLC in an antibody-dependent cell-mediated cytotoxicity (ADCMC) assay using 51-Cr-labeled mouse mastocytoma cells as targets. Quantitative analysis of the cytotoxic activity of populations separated by velocity sedimentation at 1 g indicated that cytotoxic lymphocytes in MLC were a relatively heterogeneous population of rapidly sedimenting cells that corresponded morphologically to lymphoblasts. Further studies in which PBL were separated before MLC suggested that such cytotoxic lymphocytes were derived from small lymphocyte progenitors. Analysis of ADCMC in separated populations showed that most killer cells in PBL had the sedimentation properties of small lymphocytes. After stimulation of PBL in MLC, increased ADCMC activity was detected, and the increased activity was associated with a population of rapidly sedimenting cells, Comparison of the sensitivity of cytotoxic lymphocytes and killer cells to inhibition by EDTA, cytochalasin B, and dimethyl sulfoxide showed no significant differences between the two types of effector cells.

Cell Separation↗
PubMed1975

Antibody-dependent cell-mediated cytotoxicity: heterogeneity of effector cells in human peripheral blood.

We have compared antibody-dependent cell-mediated cytotoxicity (ADCMC) of human peripheral blood leukocytes (PBL) in three model systems. target cells were 51Cr-labeled mouse mastocytoma cells, chicken erythrocytes (CRBC), and human erythrocytes (HRBC) coated with appropriate heterologous or isologous antisera. Effector cells were characterized on the basis of their adherence, phagocytosis, radiosensitivity, and sedimentation velocity(s) at 1 g. In predominantly mononuclear (Ficoll-Isopaque-purified) PBL preparations (MPBL) HRBC were lysed by an adherent, phagocytic population of cells that was markedly radio-resistant. Sedimentation velocity analysis further established that these effector cells were restricted to rapidly sedimenting fractions (s greater than 4.5 mm/hr). On the other hand, mastocytoma cells were lysed by a population of MPBL that was nonadherent, nonphagocytic, and relatively radiosensitive. These cells mainly restricted to slowly sedimenting fractions (s greater than 4.5 mm/hr) following 1 g velocity sedimentation. CRBC appeared to be susceptible to lysis by both types of mononuclear effector cell. In some experiments, enriched populations of polymorphonuclear leukocytes (PMN) were isolated. These cells were found to lyse both HRBC and CRBC very efficiently, whereas mastocytoma cells were lysed very little if at all by the same effector populations. Taken together, these results suggest that antibody-coated mastocytoma cells are lysed uniquely by effector cells in human peripheral blood with the physical properties of lymphocytes, whereas antibody-coated HRBC are lysed by both monocytes and PMN, but not by lymphocytes. Antibody-coated CRBC would appear to be lysed by all of the three effector cell types tested.

Animals↗