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Biomedical subjects

C G Brooks

Publications and source records attributed to C G Brooks.

At least 55 records · Page 3Linked to original sources
PubMed1981

Detection of either rapidly cytolytic macrophages or NK cells in "activated" peritoneal exudates depends on the method of analysis and the target cell type.

The nature of the cytotoxic cells present in the peritoneal cavity of rats treated with Bacille Calmette-Guérin (BCG) or Corynebacterium parvum was investigated using a 6 hr chromium release assay and a quantitative method of analysis based on consideration of target-cell killing as an enzyme-substrate reaction. When the results of cell-fractionation experiments were evaluated in terms of recovery of total lytic units and when appropriate target cells (such as sarcoma Mc7) were used, the simultaneous presence of both cytotoxic macrophages and NK cells in peritoneal exudates could be readily demonstrated. With certain other target cells different results were obtained. Thus, with normal thymocytes, normal hepatocytes, or myeloma P3NSI as targets, NK cells were preferentially detected, whereas with leukaemias L5178Y, P815, and EL4 as targets, cytotoxic macrophages were preferentially detected. These findings resolve the previously conflicting reports concerning the nature of cytotoxic cells in activated peritoneal exudates.

Animals↗
PubMed1980

Quantitative studies of natural immunity to solid tumours in rats. Persistence of natural immunity throughout reproductive life, and absence of suppressor cells in infant rats.

Consideration of cell-mediated cytotoxicity as an enzyme-substrate reaction provided a theoretical and practical basis for the quantification of natural immunity to solid tumours. The natural cytotoxic activity of spleen cells from normal rats towards cultured target cells from solid tumours, measured in a 6 h chromium release assay, developed between 2 and 5 weeks after birth and, in contrast to previous reports of natural cytotoxicity against lymphoid tumour cells, showed no sign of decline up till at least 22 months of age. Both virgin and breeder (multiparous) females showed equally good maintenance of natural immunity, and the apparent specificity of this natural immunity on a panel of four target cells did not change between 8 weeks and 18 months of age. The low level of natural immunity in infant spleen, and also in adult lymph nodes and thymus, was not caused by suppressor cells but rather by an absence of appropriate effector cells.

Aging↗
PubMed1980

Quantitative studies of natural immunity to solid tumours in rats. The nature of the killer cell depends on the type of assay.

Cell fractionation techniques were used to identify the cells in rat spleen responsible for natural killing of a syngeneic sarcoma cell in short-term (6 h and 18 h) and long-term (72 h) cytotoxicity assays. Cytotoxicity was quantified precisely using a method previously derived from consideration of natural cytotoxicity as an enzyme-substrate reaction, and by analysing results in terms of lytic units. Killing in all three assays displayed 'single-hit' kinetics implying that a single effector cell was sufficient to lyse a single target cell. The fractionation studies, using glass adherence, carbonyl iron, nylon wool, EA and EAC monolayers and congenitally athymic rats, revealed two populations of cytotoxic cells. In the 6 h assay most of the activity was due to cells with similar characteristics to the NK cells previously defined using leukaemic targets, but in the 18 h and 72 h assays macrophages played an important rôle. The activity exerted by the macrophages was cell lysis and not cytostasis. No evidence that the macrophages acted by releasing factors which stimulated NK cells could be found.

Animals↗
PubMed1979

High nonspecific reactivity of normal lymphocytes against mycoplasma-infected target cells in cytotoxicity assays.

Several rat tumor cell cultures were deliberately infected with three species of mycoplasma commonly found as contaminants of cell lines grown in vitro, and the effect of mycoplasma infection on the results of cytotoxicity assays was examined. Lymph node cells and spleen cells from normal animals showed an apparently high spontaneous cytotoxic activity against tumor cells infected with either M. arginini or M. hyorhinis, but the reactivity against cells infected with M. orale was not significantly higher than that against uninfected cells. The high reactivity towards tumor cells infected with M. arginini and M. hyorhinis bore a close resemblence to natural cell-mediated immunity in that spleen cells were much more reactive than lymph node cells, spleen cells from nude mice were as effective as spleen cells from normal mice, and the reaction crossed both strain and species barriers. However, closer examination revealed that the cytotoxic effects were directly caused by depletion of arginine or other essential nutrients from the medium. These findings imply that a cautious approach should be taken when interpreting certain aspects of spontaneous cell-mediated cytotoxicity, and that the greatest care be taken to ensure that the cells used as targets in any cytotoxicity test are mycoplasma-free.

Animals↗
PubMed1979

Spontaneous development of cytotoxic activity in cultured lymphnode cells from tumour-bearing rats.

Incubation in vitro of lymphnode cells (LNC) from rats bearing a transplanted syngeneic methylcholanthrene-induced sarcoma (Mc7) resulted in the generation of a potent cytotoxic activity. Four to seven days' culture was required for development of cytotoxic activity, which was shown to be mediated by a heat-stable soluble factor. The cytotoxicity was not detectable in a 3 h or 15 h 51Cr-release assay, but was demonstrated in a 48 h microcytotoxicity assay, where post-labeling with isotopically labelled cell precursors was used to quantitate cell survival. The cytotoxicity of the cultured tumour-bearer LNC and their supernatant factor was shown to be cross-reactive for tumour cell lines other than sarcoma Mc7, and was also expressed against adult or embryonic fibroblasts.

Animals↗
PubMed1978

Studies on the microcytotoxicity test. III. Comparison of [75Se]selenomethionine with [3H]proline, Na2, 51CrO4 and [125I]iododeoxyuridine for pre-labelling target cells in long-term cytotoxicity tests.

Four intracellular radioisotope labels, [3H]proline, Na2 51CrO4, [75Se]selenomethionine and [125I]iododeoxyuridine, were evaluated for use in a pre-labelling long-term microcytotoxicity assay for cell-mediated immunity. Adherent rat tumour cells established in tissue culture were used as targets and the basic variables studied were labelling efficiency, toxicity and spontaneous release rates. [125I]Iododeoxyuridine was found unsuitable on account of its high toxicity and correspondingly high spontaneous release rate, and Na2 51CrO4 for its toxicity and low labelling efficiency. Of the two other radiolabels, [75Se]selenomethionine had the advantage over [3H]proline of higher labelling efficiency (especially in Ham's F10 medium), lower toxicity, and being a gamma-emitter. Furthermore, released 75Se was shown to be non-reutilisable and its retention by target cells provided an accurate measure of cell survival in an alloimmune system. Methods of calculating the results of pre-labelling cytotoxicity tests based on the total radioactivity in target cells at the beginning of the assay were found to be invalid.

Animals↗
PubMed1978

Studies on the microcytotoxicity test. II. The uptake of amino acids ([3H]leucine or [75Se]methionine) but not nucleosides ([3H]thymidine or [125I]IUdR) or 51CrO24-provides a direct and quantitative measure of target cell survival in the presence of lymphoid cells.

We have studied the suitability of various commonly used radioactive materials for the direct post-labeling of adherent rat target cells in long-term cytotoxicity tests. The use of nucleosides at high concentration avoids the necessity of adding fluorodeoxyuridine to enhance nucleoside uptake by target cells, and reduces the degree of non-specific inhibition of nucleoside uptake caused by products released from effector lymphoid cells. However, when [125I]iododeoxyuridine was used for labelling, such inhibition was not completely avoided even at very high nucleoside concentration, necessitating the washing of target cells prior to labelling. Similarly, without prewashing, the uptake of 51CrO24-ions frequently failed to correlate well with the numbers of surviving target cells as assessed by cell counting. On the other hand, radiolabelled amino acids, when present at semi-saturating concentrations, were taken up quantitatively by target cells under all conditions tested. Furthermore, in comparison to [125I]iododeoxyuridine, radioactive amino acids showed little if any toxicity to target cells. The use of the gamma-emitting amino acid analogue, [75Se]selenomethionine, is particularly recommended.

Animals↗
PubMed1976

Studies on the microcytotoxicity test. I. Evidence that the effects of normal lymphoid cells on tumour cell growth in microtest plates may be caused by non-immunological modifications of the culture medium.

Lymph node cells from normal Wistar rats were found to affect the growth/survival of syngeneic chemically-induced solid tumour cells in the microcytotoxicity test. Whether inhibition or enhancement of tumour cell numbers occurred depended on the particular tumour cell type and, in some cases, on the particular in vitro subline used. Fractionation of LNC on nylon wool columns revealed that the two effects were mediated by distinct subpopulations of lymphoid cells: column-retained cells showed predominantly an inhibitory effect and column-eluted cells predominantly an enhancing effect. When column-retained and column-eluted cells were cultured under the conditions of the microcytotoxicity test but in the absence of tumour cell growth. The inhibitory activity was maximal within one hour of lymphocyte culture, while the enhancing effect developed slowly during incubation. Furthermore, the tumour cells themselves were found to produce growth-enhancing activity. It is proposed that interaction between these various supernatant activities accounts at least in part for the non-specific effects of normal lymphoid cells in the microcytotoxicity test.

Animals↗
PubMed1975

Neonatally induced transplantation tolerance: in vitro evidence supporting a clonal inactivation mechanism.

Mixed lymphocyte culture (MLC) tests on the cells of mice made fully tolerant to allogeneic tissues in neonatal life showed an absence of specific antigen-reactive cells. Similary, no cells cytotoxic in a chromium release test could be detected. The absence of reactivity in the tolerant populations could not be accounted for by either suppressor cell activity or serum blocking factors. By contrast, mice in which partial tolerance had been deliberately or inadvertently induced often possessed detectable numbers of MLC-reactive cells and, after grafting, their sera inhibited the MLC reactivity of normal cells. These results are discussed in terms of possible mechanisms of self tolerance.

Animals↗
PubMed1975

The effects of cell density, incubation temperature, syngeneic serum, and syngeneic red blood cells on mouse lymphocyte responses in vitro.

Various parameters affecting the quantitative assessment of mouse mixed lymphocyte response (MLR) in microplates were investigated. In agreement with the findings of others cell density was found to be particularly critical, maximal proliferative efficiency occurring at 3 X 10(6) viable cells/cm2. Slight reduction in the degree of viable cell--cell interaction caused by either reduction in cell density or insertion of irradiated cells into the cultures greatly reduced the efficiency of the proliferation. An incubation temperature of 33-34 degrees C was found to have distinct advantages over 37 degrees C, there being improved cell survival and reproducibility at this lower temperature. Transformation of mouse lymphocytes was strongly inhibited by low concentrations of normal syngeneic serum, the degree of inhibition depending on the concentration of foetal calf serum (FCS) in culture and on the method of preparation of serum. Inhibition of mouse cell responses was also caused by syngeneic red blood cells (RBC), in contrast to the pronounced enhancement of guinea pig lymphocyte responses to phytohaemagglutinin (PHA) in the presence of syngeneic RBC.

Animals↗
PubMed1975

Specific unresponsiveness to skin allografts in mice. IV. Immunological reactivity of mice treated with liver extracts, Bordetella pertussis, and antilymphocyte serum.

The strain-specific unresponsiveness to H-2 incompatible skin allografts induced by treatment of adult mice with single inoculations of donor strain liver extract and Bordetella pertussis vaccine, as well as three doses of antilymphocyte serum, has been investigated by several in vivo and in vitro methods, with a view to elucidating it mechanism. Lymphoid cells from mice with long surviving skin grafts were found to be reactive in graft-versus-host assays (as measured by splenomegaly or popliteal lymph node enlargement), and mixed lymphocyte culture tests gave positive results. Attempts to cause lethal runting of F1 hybrid mice injected at birth with spleen cells from unresponsive mice gave variable results. However, the injection of F1 hybrid cells into the footpads of unresponsive animals failed to elicit a significant host-versus-graft response. Although lymphoid cells from unresponsive animals did not include detectable numbers of cytotoxic cells, such cells could be generated by previous in vitro mixed lymphocyte culture stimulation or, to some degree, by the injection of the animals with F1 hybrid cells. Attempts to prevent mixed lymphocyte culture stimulation or cytotoxicity with serum from unresponsive mice failed at the serum concentrations used. The data indicate that long-term unresponsiveness in this system is maintained by the production in the hosts of factors that interfere with the cell-mediated response.

Animals↗