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J M Rolland

Publications and source records attributed to J M Rolland.

At least 73 records · Page 4Linked to original sources
PubMed1981

Lymphocyte stimulation by concanavalin A studied by the fluorescent probe acridine orange.

Viable mouse thymocytes or spleen leucocytes stained with acridine orange (AO) were divided into one part used for stimulation, and the other part for control. Analysis of cellular green-fluorescence emission enabled physicochemical changes in lymphocytes to be detected after 30 min stimulation with the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM). No change in fluorescence was observed with the nonmitogenic reagent wheat germ lectin (WGL) or with allogeneic cell stimulation (MLR). When green fluorescence intensity of individual cells was monitored by microfluorimetry, 30 min stimulation with Con A induced an increase, whereas PWM induced a decrease. When analysed by fluorescence spectrophotometry, Con A induced a 2 nm blue shift in emission maximum and a decrease in polarization values.

Acridine Orange↗
PubMed1981

Early changes in concanavalin A-stimulated lymphocytes detected by the fluorescent probe N-phenyl-1-naphthylamine.

The hydrophobic fluorescent cell-membrane probe N-phenyl-1-naphthylamine (NPN) is a useful investigative tool for studies of early lymphocyte activation. NPN-labelled mouse thymus cells incubated with 5 micrograms/ml concanavalin A (Con A) for 30 min at 37 degrees C gave a reproducible increase in mean cell-fluorescence intensity measured by microfluorimetry on 100 single cells. The dose-response curve was similar to that obtained by 3H-thymidine assay. Increased fluorescence was not observed in the presence of 10 mM alpha-methyl mannoside, 5 mM sodium azide, 10(-5) M cytochalasin B, or Ca2+-free culture medium. However, incubation with 10(-5) M colchicine did not alter the probe response. Fluorescence change was also shown by spleen cells from a normal mouse but not from an athymic mouse, indicating T cell dependence of the response. Comparison with other lectins showed that increased fluorescence followed incubation with phytohaemagglutinin, and the non-mitogenic wheat germ lectin, but there was no change with succinyl-Con A, and decreased fluorescence with pokeweek mitogen. Use of fluorescent-labelled lectins showed that the NPN fluorescence change did not correlate with surface receptor patching and capping. Increased phospholipid-fatty acid turnover and subsequent increased membrane fluidity with alteration of molecular polarity are suggested as likely explanations of increased NPN fluorescence.

1-Naphthylamine↗
PubMed1980

Fluorescent probes to detect lymphocyte activation.

Fluorescent probes can monitor events in lymphocytes stimulated by mitogens and antigens. Early activation is associated with conformational changes in membrane macromolecules, and has been studied by measurement of fluorescence intensity or polarization of the membrane-localizing probes ANS, NPN, DPH and TMRITC. Subsequent changes in cytoplasmic macromolecules have been detected by altered fluorescence polarization of intracellular fluorescein. Altered metabolic activity in the activated lymphocyte is also revealed by fluorescent probes: the increased red fluorescence of lysosomes seen by AO staining, is attributable to altered lysosome membrane permeability. AO fluorescence has also detected early changes in the nuclear nucleoprotein complex. The later synthesis of new DNA is readily demonstrated by increased staining with the nuclear probes AO, ethidium bromide, propidium iodide, mithramycin and the Hoechst dyes. Adaptation of fluorescent probe analyses to the now rapidly developing flow microfluorimeters is providing rapid and sensitive assays of lymphocyte stimulation. Such methods will permit routine detection of lymphocyte response to particular antigens or mitogens, as well as identification of antigenic substances by their stimulation of known reactive lymphocytes. Last but not least, fluorescent probes are providing new understanding of the cellular events and regulatory mechanisms associated with lymphocyte activation.

Acridine Orange↗
PubMed1979

Detection of early lymphocyte activation by the fluorescent cell membrane probe N-phenyl-1-naphthylamine.

N-phenyl-1-naphthylamine (NPN) becomes fluorescent after binding to hydrophobic regions of cell membranes. Rat and mouse lymphoid cell suspensions stained with NPN showed changes in fluorescence emission 30 min after stimulation with mitogen or antigen, detected by microfluorimetry. Incubation of NPN-labelled mouse and rat thymocytes with phytohaemagglutinin or concanavalin A (Con A) caused an increase in mean cell fluorescence intensity. The response to Con A was inhibited by sodium azide and alpha-methyl mannoside. Stimulation of spleen cells from mice by allogeneic cells, or from tumour-bearing rats by tumour antigen consistently resulted in decreased fluorescence. The 'mixed lymphocyte response' detected only certain genetic differences between mouse strains and was proportional to the ratio of stimulator to responder cell number. The NPN staining procedure offers a simple and rapid assay of immunoreactivity and a means of studying early subcellular changes following lymphocyte activation.

1-Naphthylamine↗
PubMed1979

Rhodamine as a fluorescent probe of lymphocyte activation.

Fresh rat and mouse lymphoid cells have been labelled by stable linkage with tetramethylrhodamine isothiocyanate (TMRITC). A change in intensity, either an increase or decrease of the fluorescent emission of the cells, detected by microfluorimetry, was induced by mitogen stimulation or the mixed lymphocyte reaction. The change in fluorescence was observed within 3 h of mitogen stimulation and within 0.5 h in the mixed lymphocyte test. These early cellular responses were detectable consistently whether the labelling was done before or after mitogen stimulation; post-labelling only was studied in the mixed lymphocyte reaction. The method should provide a time-saving practical procedure for early detection of the lymphoid cell responses and would readily lend itself to flow cytofluorimetry for possible routine diagnostic use.

Animals↗
PubMed1976

Acridine orange fluorescence cytochemistry for detecting lymphocyte immunoreactivity.

Acridine orange staining reveals changes within 3 hours of in vitro stimulation of normal rat lymphocytes with mitogens, and of immune rat lymphocytes with the sensitizing antigen. An increased number of red fluorescent cytoplasmic organelles, presumably lysosomes are seen by fluorescence microscopy. Fluorimetry of the supernatants from stained cell suspensions suggests an overall decreased cell uptake of the dye. The microscopy and fluorimetry detected early events in the reaction of lymphocytes from tumour-bearing rats with the target tumour cells. It would appear that the changes in intracellular behaviour of the dye and in overall cell uptake after immune stimulation are a reflection of dissociated variations in internal and external cell membrane permeability, and may provide simple general means for recognizing cellular immune reactions.

Acridines↗
PubMed1976

Lymphoid cell fractionation by aggregated immunoglobulin-agarose columns.

Fractionation by columns of aggregated rat immunoglobulin (Agg Ig)-agarose was investigated as a method of separating different populations of lymphoid cells. With rat spleen cells, Agg Ig columns retained phagocytes, IgM- and IgG-antibody-forming-cells, cells mediating antibody- or PHA-induced lysis of chicken erythrocytes, and specifically immune splenocytes lytic to chicken erythrocytes without exogenous antibody. Agg Ig columns did not selectively remove 'B lymphocytes' (surface-Ig-bearing lymphocytes with or without EAC' receptors), or T lymphocytes capable of PHA-induced proliferation or graft-versus-host reactivity. With mouse spleen cells, Agg Ig columns retained alloimmune cytotoxic T cells.

Animals↗
PubMed1975

Analysis of inhibition of lymphocyte cytotoxicity in human colon carcinoma.

Serum inhibition of autochthonous lymphocyte cytotoxicity for tumour cells has been studied in 112 cases of colonic carcinoma. Addition of patient's serum to the lymphocyte tumour cell reaction mixture resulted in decreased cytotoxic reactivity of lymphocytes from 8 of 39 cytotoxic positive cases. It was also shown that sera could inhibit if separately preincubated with the lymphocytes (4 cases) or the target cells (2 cases). A tumour antigen preparation inhibited only when incubated with the lymphocytes. Inhibition by serum or antigen appeared to be specific for colon carcinoma. Four cases were specially studied to determine the mode of lymphocyte killing of tumour cells: in 3 it was mediated largely if not entirely by T lymphocytes, and in the fourth by both T and non-T cells. The findings support the view that T lymphocytes lose their anti-tumour reactivity in vivo in the presence of circulating antigen or antigen-antibody complexes such as would occur with progressive tumour growth.

Adult↗
PubMed1973

Lymphocyte anergy in patients with carcinoma.

All of 10 patients with colonic carcinoma and 5 with malignant melanoma of skin showed no sign of immunoreactivity against the cultured tumour cells by the lymphocyte populations residing within the tumours. More than half of these patients did show cytotoxic reactivity by their blood lymphocytes. Possible cytotoxic reactivity by the regional lymph node lymphocytes was also investigated in 57 tumour cases (44 colonic, 13 melanoma, and including 12 of the 15 examined for intrinsic lymphocyte activity). One third of the cases showed positive blood lymphocyte immunoreactivity, but in only 4 tumours (3 colonic) did the node lymphocytes show any cytotoxicity against the tumour cells. This state of anergy of intrinsic and regional lymphocytes is presumably acquired during the development of the cancer and would permit local tumour spread and metastasis to lymph nodes. Its cause has not been identified but appears to be lymphocyte inhibition rather than selective change in lymphocyte population. In particular, no special pattern can be seen in the relative proportions of T and B cells in patients' blood, lymph node or intrinsic carcinoma lymphocytes.

B-Lymphocytes↗
PubMed1973

Immune response to a syngeneic rat tumour: development of regional node lymphocyte anergy.

The development of cellular immunity to a syngeneic squamous cell carcinoma in Wistar rats was studied by in vitro microcytotoxicity assay. Reactivity of lymphocytes from lymph nodes, spleen and blood was tested throughout the period of tumour growth. Maximum lymphocyte cytotoxicity against the tumour was observed at 2 weeks in regional lymph nodes, 4 weeks in intermediate nodes, spleen and blood, and 6 weeks in distant nodes; the intensity of these cytotoxic responses subsequently declined. In the regional nodes, lymphocytes became totally unresponsive despite the maintenance of significant cytotoxicity in intermediate nodes, spleen and blood. Local anergy may account for tumour spread to the regional node in an otherwise immunocompetent host. This anergy may be due to high local concentration of tumour antigen or antigen-antibody complexes, but it was not associated with selective changes in T and B cell proportions.

Animals↗
PubMed1973

Immune response to a syngeneic rat tumour: evolution of serum cytotoxicity and blockade.

The development of serum factors by Wistar rats during the growth of a syngeneic squamous cell carcinoma has been investigated to clarify the nature of the local lymphocyte anergy reported previously in this system. Sera from tumour bearing animals were tested for cytotoxicity against tumour cells by in vitro microassay, and their ability to inhibit cell mediated cytotoxicity was also studied. Serum cytotoxicity was first detected after 2 weeks of tumour growth, reaching a peak at 4 weeks and then declining. Inhibitory activity was found only in the sera of animals with advanced tumours. Anti-tumour antibody either in the sera or bound to tumour cells was not detected by immunofluorescence techniques. No evidence of general immunological debilitation was found, the animals showing normal immune responses to sheep erythrocytes and killed Brucella abortus organisms throughout tumour growth. Serum inhibitory factors may be responsible for the decline in anti-tumour immunoreactivity and the local lymphocyte anergy observed in tumour bearing hosts.

Animals↗