Skin cancer--childhood protection.
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Adult↗
Biomedical subjects
R C Nairn
Publications and source records attributed to R C Nairn.
Analysis of early lymphocyte activation events by fluorescence polarization flow cytometry.
After short-term (up to 4 h) stimulation with mitogen or antigen, lymphocytes were incubated with fluorescein diacetate and the polarization of fluorescence from intracellular fluorescein was measured on a specially adapted FACS II. This flow cytofluorimetric method to assay early changes in activated lymphocytes gave a reproducible response to the mitogens phytohaemagglutinin (PHA), concanavalin-A and the monoclonal antibody OKT3, recognized at 1 h by decreased polarization. A response by immune spleen cells to the antigen dinitrophenyl-ovalbumin was revealed at 4 h. The calcium ionophore A23187 induced an increase in polarization after only 10 min. The PHA polarization response was shown to be dependent on PHA binding, PHA dose, T cells, calcium ions and an intact cytoskeleton. The cellular events monitored by the polarization change are presumably altered fluidity of the probe's microenvironment due to conformational change in macromolecules to which the probe has bound or to dissociation of the probe into the aqueous phase. The fluorescein fluorescence polarization assay is a reliable and sensitive monitor of early lymphocyte activation events and, coupled with the use of a flow cytometer, permits study of particular subpopulations of responding cells.
Acetylgalactosamine↗
Flow cytofluorimetry of fluorescein fluorescence polarization to assay lymphocyte activation.
Change in fluorescence polarization of intracellular fluorescein measured with a specially adapted flow cytometer reliably reflected subtle biophysical changes in cells, such as those accompanying increased temperature or osmolality of the suspending medium. This system was developed to monitor changes in lymphocytes one hour after stimulation with the mitogen phytohaemagglutinin, and provided a sensitive and rapid assay of lymphocyte activation.
Animals↗
Fluorescence polarization assay by flow cytometry.
Fluorescence polarization measurement on cell suspensions provides a highly sensitive means for detecting subtle changes in the cells, such as occur early after lymphocyte activation or on malignant transformation. We review here the principles of fluorescence polarization, its measurement by a commercially available flow cytometer and application of such assays especially in cellular immunology.
Animals↗
Changes in lymphocyte status and responsiveness in pregnancy detected by a fluorescent cell probe.
Lymphocyte reactivity assessed by a fluorescent lipophilic probe test of responsiveness to concanavalin A (con A) was shown to differ from normal in early pregnancy. The difference was most marked in multiparas. Abnormal reactivity was detected in the earliest pregnancy examined (5 weeks' gestation) and up to about the 20th week; after 20 weeks, reactivity was normal in all of the multiparas and most of the nulliparas studied. However, in pregnancy induced hypertension (PIH), a disorder of late pregnancy, the same responsiveness as in early pregnancy was found. When unstimulated lymphocytes were examined, abnormal reactivity associated with increased fluorescence was observed in early pregnancy and in PIH, compared with normal late pregnancy, reflecting alteration in lymphocyte membrane phospholipids. It is postulated that pregnancy is associated with sequential change in immunity, disturbance of which may result in immunologically-determined obstetric morbidity.
Adult↗
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↗
Immunoreactivity by intrinsic lymphoid cells in colorectal carcinoma.
Mononuclear leucocytes were separated by Hypaque--Ficoll from 60 unselected primary colorectal carcinomas, and then fractionated by rosetting with sheep erythrocytes, either alone (E) or coated with antibody and complement (EAC). The E-rosetting cells, putative T lymphocytes, were cytotoxic in vitro to autologous tumour cells in 18 of the 60 cases, whilst the EAC-rosetting cells were unreactive. This intrinsic T-lymphocyte anti-tumour immunoreactivity was significantly associated with the presence of "cuffs" of small dark lymphocytes at the mesocolic or pararectal edge of the primary tumours, but there was no correlation with antitumour cytotoxic lymphocytes in the patient's blood at the time of operation.
Adenocarcinoma↗
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↗
Ulcerative colitis. Animal model: immunoreactive inflammation in fetal colon implants in syngeneic adult rats.
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Animals↗
Experimental model of ulcerative colitis.
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Animals↗
Sequential antitumour immunoreactivity and carcinoembryonic antigen levels as a guide to prognosis in colorectal carcinoma.
Twenty-five patients with colorectal carcinoma were tested for blood-lymphocyte anti-tumour cytotoxicity and carcinoembryonic antigen (CEA) levels at three-monthly intervals for eighteen months or more after resection, and examined clinically every three to six months. Twelve of the patients were followed for two years and one for four years. The six patients whose tumours recurred showed positive blood lymphocyte antitumour cytotoxicity and elevated plasma CEA levels at some time from six months after operation, usually well before the recurrence was clinically detectable.
Adenocarcinoma↗
Monolayer cultures for immunofluorescent staining from tumor primary explants.
Epithelial cells growing around primary explants of carcinomas in plastic ware are well-suited for modern incident light immunofluorescence microscopy. Epithelial outgrowths in growth culture medium are flushed with phosphate-buffered saline (PBS) and absolute ethanol and snap-frozen in liquid nitrogen-isopentane. The walls of the plastic containers bearing the dried monolayer outgrowths are cut out to form microscopic slides. Immunofluorescence tests are made on circular areas demarcated on the monolayers by using small metal cylinders to mask against a water-repellent plastic spray. More than 20 immunofluorescence tests can be performed on a culture 40 X 50 mm.
Antigens, Neoplasm↗
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↗
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↗
Characterization of serum factors modulating splenic cytotoxicity in a syngeneic rat tumour system.
During the terminal stages of tumour growth (6-8 weeks) in Wistar rats bearing a syngeneic squamous cell carcinoma (Sp1), their sera can block in vitro anti-tumour cytotoxicity by immune splenic T lymphocytes. At an earlier stage of tumour growth (4-6 weeks) the sera do not block this cytotoxicity, but can induce anti-tumour cytotoxicity by non-immune spleen cells in the absence of complement. Sera taken at these 2 stages of tumour growth have been fractionated by ion-exchange chromatography, using DEAE-cellulose. The fractions have been examined by immunoelectrophoresis and tested for anti-tumour reactivity. Blocking activity was found in the Week-8 serum fraction eluted with 0-005M phosphate buffer, pH 7-4, whilst the "cytotoxic" activity of Week-4 serum was eluted with 0-02M phosphate buffer, pH 6-2. It is suggested that different IgG sub-classes are responsible for the 2 activities.
Animals↗
Characterization of cytotoxic spleen cells and effects of serum factors in a syngeneic rat tumour system.
Splenocytes from inbred Wistar rats bearing a syngeneic squamous cell carcinoma (Spl) were fractionated by several techniques to characterize the lymphoid cells cytotoxic to the tumour in vitro. The anti-tumour cytotoxicity is presumably mediated primarily by T lymphocytes because it was greatly reduced by removal of T lymphocytes with heterologous anti-T serum plus complement but not by removal of other cell types. Cytotoxicity could be blocked at the tumour cell but not at the effector cell by sera taken late in tumour growth. Sera taken earlier in tumour growth could induce cytolysis of tumour cells by normal splenocytes but only if the tumour cells were treated with serum and washed before addition of the effector cells. Although splenocytes from normal and tumour-bearing rats were equally effective at lysing antibody-coated target cells it is unlikely that this mechanism is important in vivo as sera from early in tumour growth onwards contained factors (immune complexes?) which inhibited antibody-induced lymphocytolysis.
Animals↗
Correlation of regional lymph node in vitro antitumor immunoreactivity histology with colorectal carcinoma.
Lymph nodes from resected specimens of human colorectal carcinoma were investigated for in vitro lymphocyte cytotoxicity against primary cultures of autologous tumor cells. Regional lymph node lymphocytes were cytotoxic in 32 of 142 cases (23%). Altogether 200 nodes were examined and the cytotoxicity correlated directly with sinus histiocytosis, seen in 43 nodes from 35 cases, and with hyperplasia of B- and T-lymphocyte areas combined, seen in 92 nodes from 65 cases. Lymph nodes with combined B- and T-cell hyperplasia were significantly more common in cases of good tumor differentiation. The findings suggest that sinus histiocytosis and hyperplasia of both major lymphocyte populations are morphological expressions of in vitro antitumor immunoreactivity in the regional lymph node.
B-Lymphocytes↗
Colonic carcinoma: clinicopathological correlation with immunoreactivity.
The relation between tumour spread, histological differentiation, and in-vitro antitumour immunoreactivity was studied in 132 cases of carcinoma of the large bowel. Positive correlations were found between blood lymphocyte antitumour cytotoxicity and both tumour differentiation and absence of recurrence or metastatic spread.
Adenocarcinoma↗