Significance of lymphocyte fluorescence polarization changes after phytohemagglutinin stimulation in cancer and noncancer conditions.

The double-zone fluorescein fluorescence polarization (FFP) "cancer" test was used to study 540 blood lymphocyte samples from 341 donors, of whom 158 had confirmed cancer: The other donors were noncancer patients, pregnant women, and normal individuals. The FFP response in cancer patients was the reverse of that in normal individuals, but an abnormal response was also obtained in some noncancer conditions, including the chronic inflammatory disorders--rheumatoid arthritis, cholecystitis, and diverticulitis--and in early pregnancy or pregnancy-induced hypertension. Thus the cancer discriminatory value of the test is limited. Examination of its biologic basis suggests that the positive FFP response in cancer and other conditions is due to altered immune status of blood lymphocytes, with associated change in cytoplasmic fluidity affecting the polarization of fluorescence. Incubation of normal blood lymphocytes with cyclic GMP induced an abnormal, cancer-like FFP response.

Lymphocyte fluorescence polarization changes after phytohemagglutinin stimulation in the diagnosis of colorectal carcinoma.

A lymphocyte fluorescence polarization test that measures reactivity to phytohemagglutinin (PHA) has permitted discrimination of a majority of patients with colorectal carcinoma from noncancer individuals. The test involves separation of two lymphocyte fractions from 20 ml venous blood on a modified leukocyte separation gradient, incubation with PHA for 45 minutes, addition of fluorescein diacetate, and analysis of change in fluorescence polarization. Of 19 colorectal patients tested preoperatively, 13 had a positive stimulation index, 3 a zero index, and 3 a negative index. Of 7 patients with other malignant neoplasms, a positive value was obtained in 6 and a negative value in 1. Of 14 patients with other diseases, a negative value was obtained in 9, zero in 3, and positive in 2. Of 31 normal donors a negative value was obtained in 27, zero in 2, and weak positive in 2. This rapid test promises to develop into a useful cancer-diagnostic method, and elucidation of its biological basis should identify a new cancer marker.

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.

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.

Position-dependent cell survival and the microcytotoxicity assay.

For microcytotoxicity, when numbers of effector or target cells are limited, the method of Takasugi and Klein (1970) is at present the most convenient way of assessing cytotoxicity. In this procedure, target cell survival is affected by minor variations in culture conditions or washing vigour and the variations may be greater in some areas of the plate than in others, notably at the sides and especially the corners. A randomized block design for allotment of treatments to the plate is less affected by these position effects than the conventional row by row design. Statistical significance of acquired data may then be assessed by two-way analysis of variance, which is more sensitive than Student's t test in these circumstances. The use of the randomized procedure for the microcytotoxicity assay is strongly recommended.

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.

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.

Criteria of cell killing in vitro.

The attachment of cells to plastic tissue culture plates is a widely used criterion of cell viability in microcytotoxicity assays. When cell suspensions of primary human colon carcinoma and melanoma cells which were of low initial viability (assessed by trypan blue exclusion) were allowed to adhere to tissue culture plates, many of the adhering cells did not satisfy other criteria of cell viability. They did not stain with fluorescein diacetate but did stain with propidium iodide and fluorescein-labelled antinuclear antibodies. Using complement-mediated cytotoxicity, relatively weak activity was inconsistently demonstrated against these cells. On the other hand, strong activity was always demonstrated against highly viable cells from one colon carcinoma and two melanoma cell lines. Immunologically mediated damage to these cells was demonstrated readily by loss of the ability to stain with fluorescein diacetate, but not by cell detachment.

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.

Fluorescent probe assay of early mixed lymphocyte reaction to predict allograft survival in mice.

A rapid fluorescent probe assay of mixed lymphocyte reactivity has permitted prediction of allograft survival in mice. The assay is based on detection of early membrane events in stimulated cells by decreased fluorescence intensity of cell-bound N-phenyl-l-napthylamine (NPN) 30 min after allogenic cell interaction. The NPN-mixed-lymphocyte reaction (NPN-MLR) detected antigenic differences coded for by the whole H-2 complex or by the I region but not by the M locus. The probe assay correlated better with graft survival than did conventional 3H-thymidine assay, which also detects differences at the M locus that are less relevant to allograft rejection. Investigation of the cell types needed to obtain the response detected by the NPN-MLR assay revealed a requirement for mature T cells and plastic-surface-adherent monocytes and macrophages in the responding cell population. The monocytes and macrophages had an essential role in the generation of soluble factors that mediated the NPN-detected response. The NPN-MLR assay offers a reliable, rapid test of recipient-donor compatibility for allograft survival, and a system for studying early events in allogeneic cell interaction.

Serological diagnosis of amoebiasis by immunofluorescence.

A positive serological diagnosis of amoebiasis could be made by immunofluorescence in 66 of 78 established cases, taking a serum titre of 16 or higher as diagnostic: at this level there were no false positives among 94 control sera. The test is simple and may be carried out on amoebic smears stored for several months in 2-octanol. The serological activity is largely confined to the IgG immunoglobulin fraction and is specific for Entamoeba histolytica; cross reactions were not detected with other protozoa. Gel diffusion serological analysis permitted a positive diagnosis of amoebiasis in 60 of the 78 cases, and, combining this with the immunofluorescence test, raised the diagnostic score to 71 cases.

Immunofluorescent staining of rat gastric parietal cells by human antibody unrelated to pernicious anaemia.

Immunofluorescence tests on 94 human sera reacting with rat gastric parietal cells revealed that 41 (44%) of the sera contained antibody to a rat parietal cell antigen that was distinct from the pernicious anaemia autoantigen. Ten of the sera contained antibodies to both parietal cell antigens. The remaining 53 (56%) sera contained only parietal cell antibodies of the pernicious anaemia type. We recommend that mouse gastric mucosa, which does not react with the heterologous rat parietal cell antibody, replace rat gastric mucosa for immunofluorescence diagnostic tests.

Immunohistological patterns of carcinoembryonic antigen in colorectal carcinoma. Correlation with staging and blood levels.

Forty-four primary adenocarcinomas of the large bowel and 2 liver metastases were stained for carcinoembryonic antigen (CEA) in tissue sections by indirect immunofluorescence. All tumours were positive and showed either one or more of 3 different patterns--luminal; linear at surface of the tumour cells; cytoplasmic. In most cases (83%), two or all 3 patterns were seen in the same or in different parts of a tumour. The immunohistological staining was concordant with preoperative blood levels of CEA in 31 cases (67%) in that 26 tumours showed strong immunofluorescence associated with blood CEA above 2.5 micrograms/l, and 5 showed weak staining and blood CEA values less than 2.5 micrograms/l. However, in 7 strong staining was associated with low blood CEA, and in 8 weak staining was associated with high blood levels. The dissociation between histological and blood CEA findings in 1/3 of the cases, together with the marked variation within the same tumour and differences between one of the primaries and its liver recurrence, suggest that CEA immunohistology is of no better prognostic value than blood CEA levels. There was no association between CEA immunohistology and tumour staging or differentiation. However, blood CEA levels were significantly higher in tumours with extensive local or distant spread (stage D) and in poorly differentiated tumours.

Mucinous colorectal carcinoma: immunopathology and prognosis.

A total of 519 colorectal carcinomas were examined for the presence or absence of mucinous differentiation by means of microscopical morphometry. Of these, 28% had objectively measurable amounts of mucinous tumour epithelium. Tumours with > 50% mucinous areas (14%) had significantly poorer prognosis than non-mucinous in stages A and C, while mucinous differentiation did not correlate with prognosis in stages B and D. Lymph nodes regional to mucinous tumours had significantly less paracortical response, and those with < 50% mucinous differentiation, significantly less perivascular lymphocyte cuffing at the tumour margins. These lymph node and stromal compartments are putative T-lymphocyte areas, and hence our findings suggest that mucinous tumours are either less stimulatory or perhaps inhibitory of cell-mediated immunity.

Persistence of organ- and iso-antigens in vitro in a long-term culture cell line of colonic carcinoma.

Cells of colonic carcinoma line HT-29, cultured over more than 170 generations and extensively used in immunological studies of patients with colorectal tumours, still express several immunologically valuable characteristics presumably present since its inception. The cell line can induce a poorly differentiated adenocarcinoma in the nude mouse, it has human antigenic characteristics, it expresses blood group A antigen, and produces a colon-specific mucin and CEA, though not colon cancer-specific mucin (CCM). It remains useful as a target for in vitro testing of anti-tumour immunoreactivity in colorectal cancer patients.

Regional lymph node and stromal immunomorphology in colorectal carcinoma and relation to tumour spread.

A quantitative morphometric study of lymphocyte patterns in the stroma and regional lymph nodes was made in 509 cases of colorectal adenocarcinoma and 17 non-invasive adenomas. An increase in the perivascular lymphocytes, and in the size of lymph nodes, germinal centres and paracortical areas was most obvious in 'localized' invasive tumours and significantly more in Dukes' stage B than in stage C. Stage C1, defined as cases where the primary tumours were confined to the wall but with lymph node spread, showed hardly any perivascular lymphocyte aggregates, although in the regional lymph nodes there was relative paracortical, i.e. T-lymphocyte, hyperplasia. It is concluded that the presence of cuffs of perivascular lymphocytes at the tumour edge, and the increased size of tumour-free regional lymph nodes together with the relative and absolute abundance of germinal centres (B-lymphocyte) and paracortical areas (T-lymphocyte), are stage dependent. This is most obvious in stage B. These morphological expressions of host immunoreactivity may well reflect favourable anti-tumour mechanisms.