Macrophage Electrophoretic Mobility Research Articles

A human lymphokine released by mononuclear blood cells upon contact with CEA

As revealed by the macrophage electrophoretic mobility (MEM) technique mononuclear blood cells from certain cancer patients respond to carcinoembryonic antigen (CEA). This phenomenon appeared to be due to a specific lymphokine release. In this study, the lymphokine activity of supernatant pools was stepwise enriched by gel filtration on Sephadex. The mediator activity was recovered within a molecular mass region <47 kDA. The lymphokine was highly enriched during further gel filtration steps and showed a single activity peak in the molecular mass region of 23.5 kDa. Gel filtrations of appropriate control supernatants resulted in biologically inactive fractions. The lymphokine was heat-labile at 56°C, showed a clear-cut, dose-dependent effect on macrophages, and could be blocked by fucose. Preparative gel electrophoresis of radiolabeled and unlabeled lymphokine resulted in two corresponding peaks of biological activity and radioactivity.

A reappraisal of the macrophage electrophoretic mobility (MEM) test for the measurement of lymphokine activity

Using a cell electrophoretic apparatus, which was sensitive in detecting small changes in electrophoretic mobility (EPM), the macrophage electrophoretic mobility (MEM) test was investigated as a routine method for detecting lymphokine activity. Electrophoretic analysis of guinea-pig macrophages revealed 2 main subpopulations, one with an EPM of 0.90 μm cm s −1 V −1 (fast) and the other, an EPM of 0.83 μm cm s −1 V −1 (slow). From 23 experiments the fast and slow populations were found to consist of 90% and 10% cells, respectively. When macrophages were incubated with standard guinea-pig lymphokine preparations there was a significant decrease in the fast population with a corresponding increase in the slow population. This lymphokine induced ‘slowing’ of the macrophages was shown to be very reproducible. Since only 50% of macrophages of high EPM were observed to respond to lymphokine activity, it is not surprising that the MEM test has failed in the past when investigators have accepted as significant a 10–15% reduction in EPM, estimated from measurements made on only 10 macrophages. Parallel bioassays indicated that there were appreciable potency differences for macrophage slowing factor (MSF) and macrophage migration inhibition factor (MIF) activities in the lymphokine preparations used which suggest that these activities may be due to different molecular entities.

Lymphocyte Reactivity in Normal and Malignant Cell Proliferation Against a Phylogenetically Conserved Antigen in Fetal Extracts&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

Lymphocyte reactivity to 3-M KCl extracts from fetuses of different species of origin was shown by the macrophage electrophoretic mobility (MEMB) and/or the leukocyte migration inhibition tests in tumor-bearing mice and humans. In chemical carcinogenesis, reactivity was detectable before tumors developed. Six weeks after sc injection of 1,000 micrograms benzo[a]pyrene [(BP) CAS: 50-32-8] into XVII/Bln mice, the MEMB test became positive. The latent period was 15 weeks after 1.0 micrograms BP, indicating a dose-response relationship of the phenomenon. Painting of mouse skin with 7,12-dimethylbenz[a]anthracene [(DMBA) CAS: 57-97-6], croton oil (CAS: 8001-28-3), or benzene (CAS: 71-43-2) had the same sensitizing effect as BP. In contrast to the strong carcinogens BP and DMBA that caused lymphocyte reactivity to persist until tumors developed, benzene and the promoter croton oil induced only a transient effect. Termination of treatment abolished reactivity within 12 weeks. Lymphocyte reactivity to fetal extract in normal cell proliferation was evident from the fact that two-thirds-hepatectomized rats and BCG-treated mice became MEMB-positive. In hepatectomized rats the effect was reversible according to the completion of liver regeneration. Lymphocytes from tumor-bearing mice reacted with mouse and human fetal extract as well as with extracts from different developmental stages of frogs and fish. Fetal extracts were assumed to contain a phylogenetically conserved antigen.

Human lymphocyte response to CEA: Titration of different CEA samples and neutralization experiments with monoclonal anti-CEA antibodies

By means of the macrophage electrophoretic mobility technique a striking digestive system cancer-associated human lymphocyte response to CEA has been found during a large-scale study including tests in 499 individuals. The question to be answered by this study was whether this response is really CEA-specific. Titration experiments with 3 different CEA preparations in lymphocytes from 5 colorectal cancer patients showed that the threshold dose of CEA necessary to induce lymphocyte responses amounts to 50–100 ng CEA per ml and 106 lymphocytes, regardless of the CEA origin and its state of purity. The CEA specificity of the responses was proved by neutralization experiments with 3 CEA-specific monoclonal antibodies. When allowed to react with CEA before lymphocyte incubation, the MABs prevented CEA from inducing lymphocyte responses. Appropriate murine control myeloma protein did not influence these responses. The reactivity of these lymphocyte samples to a teratocarcinoma extract could not be prevented by treating this material with CEA-specific MABs before incubation. Preliminary attempts to enrich the lymphokine(s) released after CEA stimulation resulted in recovery of the activity within 2 arbitrarily cut Sephadex G-100 fractions comprising the mol. wt range of 3000–47000.

Reactivity in Neoplasia, Preneoplasia, and Pregnancy of Lymphocytes Against Fetal Extracts: Cross-Reactions Between Man and Mouse&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;

KCl extracts (3 M) from human fetuses were tested by macrophage electrophoretic mobility (MEMB), leukocyte migration inhibition, and/or leukocyte adherence inhibition techniques against lymphocytes-leukocytes from tumor-bearing humans and control persons. A positive MEMB test was found in 209 of 284 (73%) patients with various types of clinically manifest tumors. Only 15 of 134 (11%) controls gave a positive reaction by the MEMB test. Leukocyte migration was significantly inhibited in 48 of 64 (75%) tumor patients, and leukocyte adherence was significantly inhibited in 40 of 50 (80%) tumor patients versus 7 of 50 (14%) and 10 of 54 (19%) in the corresponding controls. Lymphocyte reactivity to fetal extracts is also detectable in inbred XVII/Bln, CBA/Bln, and C3H/Bln mice bearing spontaneous or transplanted tumors of different etiology. Human and mouse fetal extracts gave cross-reactions. Lymphocytes from tumor-bearing mice were reactive against fetal extracts from the cow, pig, sheep, guinea pig, cat, and chicken. Mice bearing benign skin warts induced by 7,12-dimethylbenz[a]anthracene (DMBA) as well as DMBA-treated mice without visible skin lesions gave a positive MEMB test. Likewise, pregnancy is associated with lymphocyte reactivity to fetal extracts.

Sensitiziation of human lymphocytes to carcinoembryonic antigen (CEA): neutralization experiments with anti-CEA sera

By means of the macrophage electrophoretic mobility technique we could show lymphocytes of patients suffering from cancers of the digestive system to be sensitized to carcinoembryonic antigen (CEA). These findings conflict with the common view that CEA is not immunogenic in humans. The aim of the present study was to look as to whether conventional anti-CEA sera can neutralize the activity of a CEA preparation which is responsible for the human lymphocyte response. When 60 ng CEA were preincubated with highly diluted anti-CEA serum and the resulting immune complexes were thereafter co-precipitated by protein A-sepharose, positive lymphocyte responses could no longer be obtained. This effect was observed with 3 anti-CEA sera in 3 cancer patients (colon cancer, stomach cancer, teratocarcinoma), who's lymphocytes responded to CEA by lymphokine release. Normal serum had no neutralizing effect. The anti-CEA sera did not influence the activity of another tumour-relevant extract (teratocarcinoma-derived), to which cancer patients' lymphocytes reacted regardless of the tumour site. The lymphocytes from an oesophagus carcinoma patient, though reacting to the teratocarcinoma preparation, did not respond to CEA, thus, logically, all other tests with normal serum and anti-CEA sera were negative, too. The results show that the digest system cancer-associated lymphocyte reactivity to CEA can be abrogated by conventional anti-CEA sera, which finding indicates that there exist closely CEA-associated "tumour-specific" antigenicities.

Macrophage migration stimulation factor.

Macrophage migration stimulation factor.

A common tumour-specific antigen: III. Preparation of small fragments incorporating the cell-sensitizing determinant.

Intact cells of various human tumours and tumour cell lines, and acid extracts of various human tumours and normal tissues, each of which react with the lymphocytes of cancer patients as detected by the macrophage electrophoretic mobility (MEM) test, have been subjected to proteolysis. Activity was destroyed by some enzymes, and the apparent molecular size of the active material was reduced by others. An active low-mol.-wt fragment has been partially purified from papain digests of several tumours. Peptides with normal tissue and tumour-characteristic activities have been separated chromatographically from tryptic digests of tumour extracts.

Cancer diagnostic test of Field and Caspary: Part II. A review and interpretation of the collected data

Tables are presented showing data collected to date on the clinical trials of the macrophage electrophoretic mobility test for cancer of Field and Caspary. Seven variations on the original technique are summarized. The original antigen used in these tests was the basic protein of CNS myelin. Four additional proteins have been shown to function in these tests in place of the basic protein. There appears not to be a strong immunological crossreaction between these proteins. This paper discusses a possible solution to the problem of why non-crossreactive proteins appear to crossreact when testing cancer patients with the macrophage electrophoretic mobility related tests. All lymphocytes in saline suspension without added protein will aggregate. This is prevented in normals by the addition of some proteins to the culture, but is not so prevented in cancer and other diseases. Aggregation results in lymphokine release that makes the macrophage electrophoretic mobility test and variants positive. Aggregation occurs in the first incubation step of these tests and this non-immunological aggregation is the underlying phenomenon detected rather than specific antigen recognition as has been heretofore presumed.

Macrophage electrophoretic mobility (MEM) test in immunodiagnostics of Papio hamadryas malignant lymphomas

Summary Reactivity of lymphocytes from P. hamadryas monkeys carrying malignant lymphomas directed against soluble antigens from allogeneic lymphomas was investigated using macrophage electrophoretic mobility (MEM) test. Twenty-three monkeys have been examined. 14 of them carried malignant lymphoma including those with initial manifestations of malignant lymphoma, animals with generalized processes and those in the period of spontaneous remission, and 9 control animals (8 healthy baboons and one animal with hepatoma). The macrophage electrophoretic mobility reduction higher than 10% was considered as positive. Positive reaction was observed in 78% of monkeys with malignant lymphoma. The MEM-test was positive already in the initial stage of the disease and remained positive in the period of spontaneous remission.

MACROPHAGE ELECTROPHORETIC MOBILITY TEST AS A SENSITIVE PROBE OF TRANSPLANTATION IMMUNITY IN MICE

Transplantation immunity was examined in mouse H-2- and non-H-2-disparate strain combinations by the macrophage electrophoretic mobility (MEM) assay. Lymph node cells from the recipients rejecting histoincompatible skin grafts were incubated in the presence of solubilized histocompatibility antigens prepared from spleens of the grafts donors. Product(s) of lymphocyte-antigen interaction in appropriate combinations (sensitized lymphocytes with the sensitizing antigen) significantly diminished the electrophoretic mobility of guinea pig macrophages in all of the histoincompatible strain combinations examined. The data indicate that transplantation immunity in mice can be detected by the MEM assay.

On the mechanism of binding of human lymphocyte products to guinea pig macrophages.

When lymphocytes from a majority of patients with cancer are incubated with encephalitogenic factor, a lymphocyte product is released that reduces the anodic electrophoretic mobilities of guinea pig macrophages and fixed, tanned sheep erythrocytes. Although these reactions are not specific for cancer, it is distinctly possible that in patients with cancer, products from stimulated lymphocytes are capable of altering the surfaces of the patients' own macrophages, thereby modifying the course of their disease. In this paper, we attempt to elucidate some mechanisms for the binding of lymphocyte products to macrophages, such as occurs in the macrophage electrophoretic mobility (MEM) test, since this may be of general interest. Binding of lymphocyte product to macrophages has been monitored by measurements of their electrophoretic mobilities and by electron microscopic determination of the density of binding of electron-dense, cationic colloidal iron hydroxide particles to their surfaces. The results show that the lymphocyte products reduce the net surface negativity of the macrophages by (coulombic) binding of this net positively charged material to sialic acids at the macrophage surface. Product-binding can be prevented by prior treatment of the macrophages with neuraminidase. It appears that only a minority of sialic acids are involved in the binding process, which occurs without demonstrable blocking of adjacent sialic acids or redistribution of such sites over the macrophage surface. Parallel experiments with fixed tanned erythrocytes also suggest that binding of lymphocyte product is not solely determined by surface sialic acids, although it cannot occur without them.

Simplified cell microelectrophoretic method applied to the macrophage electrophoretic mobility test for cancer diagnosis.

Simplified agarose-coated capillary microelectrophoresis was adapted to the Macrophage Electrophoretic Mobility (MEM) test for cancer detection. Guinea pig alveolar macrophages gave superior reproducibility to peritoneal macrophages. As good or better reproducibility was obtained with cryopreserved, as with fresh macrophages. The electrophoretic mobilities of patients' lymphocytes themselves, after incubation with Encephalitogenic Factor (EF) showed a significant increase in electrophoretic mobility, while in control lymphocytes a decrease occurred. Thus, for the detection of the results of the interaction of EF on human lymphocytes, a direct lymphocyte electrophoretic mobility test may suffice, and guinea pig macrophages may no longer be required at all.

Comparison between the macrophage electrophoretic mobility (mem) and the fixed tanned erythrocyte electrophoretic mobility (fteem) tests in the detection of cancer

When peripheral lymphocytes from patients with a history of cancer are incubated with encephalitogenic factor (EF), in 90% of cases the resulting products reduce the net surface negativity of guinea-pig macrophages, used as detector cells, as revealed in the macrophage electrophoretic mobility (MEM) test. The MEM test is positive in 36% of people with no history of cancer. Formaldehyde-fixed tanned sheep erythrocytes have been used as detector cells in place of guinea-pig macrophages, in a fixed tanned erythrocyte electrophoretic mobility (FTEEM) test, with lymphocyte products identical to those used in MEM tests. In patients with a history of cancer, positive results were obtained in 28/42 cases with the FTEEM test compared with 32/42 in the MEM test. In people with no history of cancer, negative results were obtained in 16/18 cases with the FTEEM test, compared with 12/18 in the MEM test in the present series, and 51/69 in a more extensive series. These differences are not significant. Cases in which discrepancies are revealed between the two tests are discussed in terms of individual case histories.

Solubilized tumour-associated antigens of methyl-cholanthrene-induced mouse sarcomas. Comparative studies by in vitro sensitization of lymph-node cells, macrophage electrophoretic mobility assay and transplantation tests.

AbstractCell‐mediated immune response to tumour‐associated antigens (taa) extracted from syngeneic methylcholanthrene (mc)‐induced mouse sarcomas with 3 m kci was simultaneously examined by in vitro sensitization of lymph‐node cells (lnc), macrophage electrophoretic mobility (mem) assay and transplantation tests. the solubilized taa elicited in vivo both primary and secondary immune responses as judged from induction of transplantation resistance. the resistance to transplantation of the corresponding sarcoma cells was tumour‐specific and relative in nature, i.e., only partial and weaker than that induced by immunization with irradiated tumour tissue. treatment of mice with solubilized taa prior to immunization with irradiated tumour tissue inhibited the induction of transplantation resistance by irradiated tumour cells. the interaction of the solubilized taa preparations in vitro, either with lnc from animals immunized against the corresponding tumour or with lnc from tumour‐bearing animals, gave rise to a macrophage‐slowing factor detectable by the mem test. taa detected by the mem assay in kci extracts of individual mc‐induced sarcomas were found to be non‐cross‐reacting, individually distinct, like those detected by transplantation tests. individual specificity of taa present in the mc‐induced sarcomas was also demonstrated in vitro by sensitization of normal syngeneic lnc on monolayers of irradiated sarcoma cells followed by detection of the generated cytotoxic lnc in a cytotoxicity assay utilizing the inhibition of [3h]thymidine incorporation. whereas sensitization of lnc on layers of irradiated sarcoma cells gave rise to lymphocytes cytotoxic for cells of the sensitizing tumour, sensitization of lnc with solubilized taa isolated from the same sarcoma has thus far given negative results.

Macrophage electrophoretic mobility and structuredness of cytoplasmic matrix.

Macrophage electrophoretic mobility and structuredness of cytoplasmic matrix.

Failure to obtain positive MEM tests in either cell-mediated immune conditions in the guinea-pig or in human cancer.

The macrophage electrophoretic mobility test described by Caspary and Field (1971) and modified by Pritchard et al. (1973) was investigated in various models of cell-mediated immune conditions in the guinea-pig and in cancer in man. No positive results were obtained in 92 guinea-pig experiments. Only 17 of 154 experiments on 74 patients gave definite positives in experiments with human cancer and a few positive results were obtained with normal healthy subjects.

Failure to Confirm the Macrophage Electrophoretic Mobility Test in Cancer

A series of patients with a variety of histopathologically confirmed cancers have been examined using the MOD-MEM test as described by Pritchard et al. (1973). Despite the closest possible adherence to the experimental protocols recommended by these authors, no positive reactions to the test were observed in this series: neither were we able to demonstrate the release of a “macrophage-slowing factor” by a panel of normal donors when challenged with tubercle PPD. We conclude that the test has no present application to the diagnosis of cancer.

Workshop on Macrophage Electrophoretic Mobility (MEM) and Structuredness of Cytoplasmic Matrix (SDM) Tests

Workshop on Macrophage Electrophoretic Mobility (MEM) and Structuredness of Cytoplasmic Matrix (SDM) Tests

The macrophage electrophoretic mobility test: Results on carcinoma of the colon and rectum.

The macrophage electrophoretic mobility (MEM) test was performed on guinea-pig macrophages treated with the interaction products of encephalitogenic protein and peripheral lymphocytes from 44 patients with colorectal cancer and 33 "healthy" controls. In 54/60 tests involving patients, statistically significant reductions in electrophoretic mobilities were observed, compared with 12/33 in controls. Our overall results on the reductions in macrophage mobilities by lymphocyte products are in accord with the work of some other workers, but not all. In contrast to many other studies, the standard procedures used here to express the results should permit an exchange of data on an international basis and allow a more rapid, general appraisal of the MEM test.