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Biomedical subjects

M G Baines

Publications and source records attributed to M G Baines.

At least 55 records · Page 3Linked to original sources
PubMed1985

A rapid and efficient microtechnique for the analysis of functional transferrin receptors on tumor cells.

A simple and efficient method for the analysis of the affinity and number of functional transferrin receptors (TFR) on human tumor cells is described. The technique is designed to utilize microtitration equipment; and is suitable for easy comparison of up to 8 different cell preparations per assay. Using this technique, 5 established cell lines were evaluated for functional TFR expression. The control erythroleukemic cell line K562 possessed 3.28 X 10(5) functional TFR per cell (+/- 3.69 X 10(4), SEM) Kd = 9.0 X 10(-9) X M-1. Trypsin and heat-pretreated cells were compared to control erythroleukemic K562 cells from the same culture to determine both the effects of receptor removal and cell viability on the assay. Trypsin and heat pretreatment of these K562 cells severely decreased receptor function as indicated by Scatchard analysis as well as by time course and cold competition analysis respectively. Whereas the affinity of trypsin-treated receptors on cells was similar to control values, heat-killed cells displayed an altered cellular affinity for 125I-transferrin underscoring the importance of utilizing cells of high viability in receptor assays.

Cell Line↗
PubMed1985

Studies on the mechanism of specificity of human natural killer cells for tumor cells: correlation between target cell transferrin receptor expression and competitive activity.

Previous studies to determine the nature of the specificity of natural killer (NK) cells for leukemic cells indicated that functional transferrin (Tf) receptors may be one of the determinants recognized by NK cells. To further investigate these observations, the relationship between cellular Tf receptor expression and ability to compete with a control K562 cell preparation in a standard chromium release assay was studied. K562 cells were selected at different phases of growth by removing cells from tissue culture at 1, 3, and 5 days postfeeding. Under these conditions, K562 cells, respectively, displayed relatively high, medium, and low numbers of Tf receptors and corresponding competitive activity against a control K562 cell preparation. K562 cells were modified by either trypsin, heat, or sodium butyrate (differentiation inducer) pretreatment. An NK-resistant clone was also studied. There was a good correlation between Tf receptor expression and cold competitive activity of the above K562 cell preparations (r = 0.82, P less than 0.01). The different tumor target cell lines, K562, Molt-4, Raji, HL-60, and MeWo, which would be expected to express different ranges of specificity, did not show a significant correlation between Tf receptor expression and their cold competitive activities against Cr-51-labeled K562 cells. Rabbit reticulocytes which express high numbers of Tf receptors were tested for their ability to compete with K562 cells for NK cells. These cells were able to compete with K562 cells while mature rabbit red blood cells which do not express Tf receptors did not compete well. These findings support the contention that the Tf receptor may be involved in NK cell recognition of some tumor cells.

Binding, Competitive↗
PubMed1983

Involvement of transferrin and transferrin receptors in human natural killer effector:target interaction.

In this current study one of the determinants of natural killer cell specificity in immunosurveillance against cancer, may be the recognition of transferrin receptors on neoplastic cells by the NK effectors. Human transferrin, when saturated with iron (FeTf), was found to inhibit human natural killer (NK) activity against K562 tumor cells, if included in assay mixtures at physiologically relevant levels. Whereas both FeTf and iron-free transferrin (ApoTf) inhibited initial conjugate formation at the level of the target cell, only FeTf inhibited NK cytolytic activity, as judged by release of chromium from the targets. This suggested a functional role for FeTf on either NK or tumor cells. When either targets or effector lymphocytes were pre-incubated with FeTf, inhibition of killing was strongest when the targets were first exposed to FeTf. The evidence suggested that NK-associated transferrin mediated the interaction with target cells through free target-associated transferrin receptors. The finding that rabbit anti-human transferrin antibody (RaHTf) inhibited killing, when reacted with effector lymphocytes but not with target cells, supported this hypothesis.

Humans↗
PubMed1982

Studies of human natural killer cells. I. In vivo parameters affecting normal cytotoxic function.

The results of natural killer cell (NK) studies on 539 normal healthy donors tested from once to 213 times over a seven-year time span have been presented. NK activity did not vary with donor blood group, Rh type or (in a small sample) HLA type. There was a slight but significant increase in NK activity from birth to adulthood, and between males an females. The male/female difference was present at birth and persisted through adulthood. The relative NK activity of individual donors tested repeatedly over many years was remarkably consistent in spite of variability in the absolute cytotoxicity observed. The expression of NK data in terms of relative NK was found to be superior to other methods, and the values obtained were found to be independent of the NK-sensitive target cell used. Although age and sex differences in NK activity are slight, their existence should be considered when studies of NK activity in patients are analysed.

Age Factors↗
PubMed1982

Studies of human natural killer cells. III. Neutropenia associated with unusual characteristics of antibody-dependent and natural killer cell-mediated cytotoxicity.

A 52-year-old Caucasian man with chronic neutropenia and recurrent infections was found to have an increased proportion of peripheral T lymphocytes having Fc receptors for IgG (T gamma ). Although levels of antibody-dependent cell-mediated cytotoxicity (ADCC) and "natural" killing (NK) by unfractionated lymphocytes were similar to those of a control donor, the frequency of KN cells was markedly increased. Removal of E rosette-forming cells eliminated both NK and ADCC by the patient's peripheral blood, in marked contrast to the enhanced cytotoxicity seen with control lymphocytes. Both normal and patient ADCC and NK functions were removed by depletion of Fc receptor-bearing cells. These depletion experiments proved that all of the patient's killer cells were E rosette-forming T gamma cells, in contrast to the heterogeneous pattern of null gamma and T gamma killer cells seen in the blood of normal donors. The homogeneity of the T gamma proliferation suggested that ADCC and NK were mediated by the same cell type, albeit acting by different mechanisms. The addition of the patient's serum and lymphocytes to chromium-labelled normal granulocytes caused a low but significant level of cytotoxicity, indicating that the patient's neutropenia may have been caused by a similar mechanism in vivo. There was no evidence of complement-dependent serum antibody-mediated neutrophil lysis, but one serum sample taken over the course of the patient's disease agglutinated granulocytes from four of five donors tested.

Agglutination↗
PubMed1982

Impairment of the thymus-dependent humoral immune response by syngeneic or allogeneic pregnancy.

Pregnancy results in a diminished capacity of the gravid mouse to respond to thymus-dependent antigens. The IgM response to thymus dependent and independent antigens is relatively unaltered whereas the IgG response to thymus-dependent antigens is markedly suppressed in both syngeneic and allogeneic pregnancy. The impairment of the humoral immune response is not due to loss of antibody-forming precursor lymphocytes (AFPL) from the spleen since their number remains unaltered. During pregnancy the spleen becomes enlarged and the precursor frequency is diluted somewhat but the AFPLs retain their normal activity in vitro. This implies an alteration in their amplification by accessory cells (T cells or macrophages) is responsible for the impaired IgG responses.

Animals↗
PubMed1981

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. IX. The quantitation of natural killer cell activity.

On analysis of in vitro assays of human natural killer (NK) cell function the inadequacy of commonly used methods of expressing lytic activity was apparent. A comparison was made of the data obtained using modifications of two equations-the simple exponential fit and the von Krogh equations. Both of these equations were found to satisfy the following essential criteria for use in these assays. First, the majority of the results obtained in the chromium-release assay could be used in data reduction; second, the resultant "dose-response" curve was reduced to linearity; and third, a single numerical expression was obtained which was directly proportional to the cytotoxic activity. Of the two methods the more conventional exponential fit was found to be the simpler to use. The closeness of fit of the experimentally derived data to the ideal curves did not support the possibility that normal lymphocyte preparations contain suppressor cells capable of inhibiting NK activity. Data have also been presented showing that NK-sensitive targets could be categorized with respect to their susceptibility by comparing the slopes of the target cell survival curves obtained using the exponential fit equation. These observations are relevant to the accurate assessment of NK activity in patient populations and to the determination of the effects of disease and its treatment on this activity.

Animals↗
PubMed1981

Quantitation of a whole blood assay for human natural killer cell activity.

A method is described for the measurement of human natural killer cell activity using heparinized whole blood in a 51chromium release assay. Fractionation-reconstitution experiments showed that the cytotoxic activity was abolished by removal of the Fc receptor bearing lymphocytes, but not by the elimination of monocytes and granulocytes. Autologous or pooled plasma was not found to possess inherent cytolytic activity. By analogy to an enzyme kinetic reaction, the results were expressed as kinetic lytic units (KLU) which were defined as the maximum number of target cells that could be lysed per unit time per milliliter of whole blood. The buffy coat cytotoxicity (BCC) assay is quick, easy to perform, and suitable for screening and monitoring of natural cytotoxicity. Since this methodology preserves the actual concentration of natural killer cells, it may represent a truer reflection of in vivo events.

Bacterial Vaccines↗
PubMed1981

The use of modified Cunningham chambers for the enumeration of H rosettes, E rosettes, and lymphocyte-tumour cell conjugates.

In this paper, we have described the use of a slide-coverslip Cunningham chamber which is ideal for counting delicate rosettes and lymphocyte-tumour cell conjugates. The ease of construction, facility of use, and economy of cost in comparison with hemocytometers make the use of these chambers the method of choice for rosette counts in general. In contrast to standard double-slide Cunningham chambers, the design of these rosette chambers is such that they can be used with ordinary light microscope objectives (including oil immersion), they can be more easily loaded without trapping air bubbles, and they can be sealed without tilting the slide. As an example of one potential application of these chambers to tumour immunology, data have been presented showing the high proportion of lymphocyte-tumour cell conjugates which can be formed by human peripheral blood cells. The ability of any particular tumour cell to form conjugates did not necessarily reflect the susceptibility of that cell type to natural killer (NK) cell-mediated lysis, nor was there any evidence that E rosette-forming cells preferentially formed conjugate in comparison to non E rosette-forming cells.

Killer Cells, Natural↗
PubMed1980

Human lymphocyte/human erythrocyte rosettes. I. Blood "H" rosettes are high-affinity E-rosetting-forming T lymphocytes occurring in high frequency.

Human lymphocytes will rosette with human red blood cells under certain conditions (H rosettes). The work presented in this paper documents the following: (1) a reproducible short-term assay for the detection of H rosettes in proportions similar to those reported recently in the literature (25-30%); (2) technical improvements on the basic assay system demonstrating that, in fact, 40-65% of human lymphocytes form H rosettes; (3) the fact that, in normal donors, auto and allo H rosettes are formed by identical lymphocyte subpopulations; (4) that H rosettes are formed independently of the ABH, Rh and Thomsen-Freidenreich (TF) red-cell antigens on the indicator red cells; and (5) that H rosette-forming cells consist almost entirely of T lymphocytes having high-affinity receptors for sheep erythrocytes. The large difference in size between sheep and human RBC made it possible to do double rosette assays in which high- and low-affinity E-rosette-forming T cells could be visualized on the same slide. As with T lymphocytes in general, a large proportion of HRFC was found to have Fc-IgM receptors while a smaller proportion has Fc-IgG receptors. Studies done on a small group of leukemic patients supported the T-cell nature of the H rosette-forming cell.

Animals↗
PubMed1978

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. IV. The suppressive effect of normal pregnancy.

The effects of pregnancy and other donor characteristics on spontaneous lymphocyte-mediated cytotoxicity (SLMC) were studied. Sex, age, and stage of the menstrual cycle had no significant effect on the SLMC of the control donors, the SLMC was significantly lower in pregnant donors in the second or third trimester. However, this lowered level of activity was still within the 95 per cent confidience limits of the normal population and parity had no cumulative effect. At delivery, fetal cord blood lymphocytes possessed substantial cytotoxic activity, further evidence that the fetus is immunologically competent at birth. The relevance of these data to the immunologic coexistence of the mother and fetus is discussed.

Adult↗
PubMed1977

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. II. Is the complement receptor necessarily present on the killer cells?

The effect of various lymphocyte depletion techniques on SLMC effector cell activity against the cell line K562 could be attributed to the effects that these procedures had on the proportions of Fc/C3 receptor-bearing cells in the preparations. depletion of cytotoxic activity by removal of Fc receptor-bearing cells could be augmented by the presence of complement on the 7S-EA indicator cells used for rosette depletion. Two methods of assessing the presence of surface receptors on cytotoxic cells are presented: (1) linear regression analysis of spontaneous cytotoxicity plotted as a function of the proportions of receptor-bearing cells remaining after various depletion techniques; and (2) the use of a mathmatical formula to estimate the proportion of cytotoxic cells with any particular receptor, based on the effects of receptor-bearing cell depletion. Linear regression analysis of cytotoxicity vs the proportions of SRBC, Fc or C3 receptor-bearing cells demonstrated that all cytotoxic cells had the Fc receptor, but that cytotoxicity could still occur in the absence of cells with the C3 receptor. This indicated that the C3 receptor is not necessary for the demonstration of SLMC activity. The mathematical formula to predict cytotoxicity after depletion of C3 receptor-bearing lymphocytes showed, however, that a significant proportion of the Fc receptor-positive killer cells were also C3 receptor-positive. No direct evidence was found to support a role for T cells in SLMC, although E rosette depletion resulted in lower than expected cytotoxicity in view of the number of Fc receptor-bearing cells remaining in the preparation.

Antibodies, Neoplasm↗
PubMed1977

Lymphocyte populations in peripheral blood during normal human pregnancy.

The effects of normal pregnancy on peripheral blood lymphocytes were investigated. The frequencies of thymus-derived and bone-marrow-derived lymphocytes in maternal peripheral blood does not vary significantly throughout pregnancy. This observation implies qualitative rather than quantitative alterations in lymphocyte functions to explain the reduced cell-mediated immune responses seen during pregnancy.

B-Lymphocytes↗
PubMed1977

Effects of pregnancy on the maternal lymphoid system in mice.

The effects of pregnancy on the lymphocyte populations in the mouse have been studied. Though cell numbers decreased in the thymus and increased in the lymph nodes draining the parauterine area, no significant changes were seen in the proportions of thymus derived (theta allo-antigen positive) and bone marrow derived (Fc receptor positive) lymphocytes. The humoral response to sheep erythrocytes was suppressed during both syngeneic and allogeneic pregnancies following a decline coincidental with that of thymic mitotic activity. Though the observed immunosuppression could be explained by a selective lymphocyte depletion, it appears more likely to be a nonspecific effect of the steroid milieu of pregnancy.

Animals↗
PubMed1976

Spontaneous human lymphocyte-mediated cytotoxicity againts tumour target cells. I. The effect of malignant disease.

Spontaneous human lymphocyte-mediated cytotoxicity (SLMC) against tumour-cell targets was examined in a series of patients with localized or malignant disease, both treated and untreated, and patients with untreated chronic lymphocytic leukemia (CLL). The level of SLMC was assessed by means of two previously established assay systems; the xenogeneic assay involving the mouse mastocytoma line P815, and the allogeneic assay in which the human chronic myelogenous leukemia-derived line, K562, was used. The assay systems involve the use of Ficoll-Isopaque-separated, iron-plus-magnetism-purified lymphocytes in an overnight 51chromium release assay, and reflect the cytotoxic ability of human non-T, complement receptor-, Fc receptor-positive lymphocytes. In the present paper, lymphocytes from all normal donors tested showed significant activity in the SLMC assay, with some variation from day to day. This variation was markedly reduced when different normal donors were tested on the same day and under identical experimental conditions. In contrast, lymphocytes from many patients with malignant disease had decreased SLM activity, and this decrease was highly significant in patients with treated or untreated metastatic disease, or untreated CLL. This was also the case when the data were expressed relative to the number of cytotoxic cells in the normal control population, or in comparison to the relative SLMC activity of lymphocytes from patients with other conditions. Markedly decreased SLMC was observed in some patients in spite of normal T and B lymphocyte proportions, or the presence of the ability to mount a vigorous delayed hypersensitivity reaction to PPD. A comparison of the xenogeneic and allogeneic assays showed that the same information with respect to whether SLMC was normal or abnormal was obtained with both assays in the majority of cases. The significance of the data is discussed with respect to the possible role of SLMC in vivo and the relevance of SLMC to the assessment of specific cell-mediated cytotoxicity in malignant disease.

Adult↗
PubMed1976

Immunofluorescence using dichlorotriazinylaminofluorescein (DTAF). I. Preparation and fractionation of labelled IgG.

Dichlorotriazinylaminofluorescein (DTAF), the product of the reaction of aminofluorescein with cyanuric chloride, is an effective reagent for conjugating fluorescein to immunnoglobulins. DTAF has absorption and emission properties nearly identical to fluoresceinisothiocyanate (FITC) and DTAF and FITC-labelled antibodies are similar in terms of preparation and specificity of immlnofluorescence. However, DTAF is superior to FITC with regard to cost, purity and stability. Also, DTAF-labelled rabbit IgG conjugates can by quickly and efficientyly fractionated by simple ammonium sulfate precipitation procedures to yield preparations free of both over- and under-conjugated material. In addition, over-conjugated protein can be readily removed from DTAF:IgG conjugates by appropriate adjustment of pH and temperature.

Animals↗