The binding of IgG1 containing immune complexes to the FcR of allogenically activated T cells induces changes in the membrane potential and the cell surface charge.

The effect on membrane potential and cell surface charge of binding immune complexes containing IgG1 and IgG2a monoclonal antibodies to Fc receptors was studied in resting and allogenically activated murine T cells. IgG1 complexed by antigen or heat aggregation induced electrophysiological changes on activated T cells. A biphasic alteration of membrane potential was detected by measurement of the intra- and extracellular distribution of the fluorescent dye, DiOC6. A short-lived hyperpolarization, detectable for 4-6 min after adding the respective ligand, was followed by a longer lasting depolarization. The cell surface charge, measured by cell electrophoresis, was also changed. This alteration was detected 2-4 hr after addition of immune complexes and disappeared by the 8th hr of incubation. Monoclonal antibody 2.4.G2, reactive with mouse FcR, induced a similar membrane potential response on activated T cells, but did not affect the cell surface charge. Monomeric IgGs and complexes of IgG2a did not modify these parameters. FcR ligands had no effect on the studied characteristics of resting T cells.

A comparison of the kinetics of the macrophage electrophoretic mobility (MEM) and the tanned sheep erythrocyte electrophoretic mobility (TEEM) tests.

In this study the macrophage electrophoretic mobility (MEM) test was modified by using tanned sheep erythrocytes in place of guinea pig peritoneal macrophages as the indicator cells of lymphocyte sensitization to antigens. This modification is named the tanned sheep erythrocyte electrophoretic mobility (TEEM) test, and a comparison of the kinetics of the two systems allowed the following conclusions to be made: 1) Treatment of freshly drawn sheep red blood cells with a concentration of 1/40,000 tannic acid produced optimum results in the TEEM test. 2) Lymphocyte-antigen and lymphocyte-number response curves show similarity in the two test systems. 3) A plateau response with slowing factor is achieved at a lower dilution in the TEEM test than in the MEM test. 4) Whilst similarity in the first stage reaction was found in the two systems, in the second stage of the test (at 37 degrees C) tanned sheep red cells gave a plateau response after 45 min instead of the 60 min found in the MEM test. 5) The two slowing factors showed similar gel filtration patterns with molecular weights between 13,000 and 15,000 daltons, and had equivalent activity in both test systems. 6) The disadvantages of the guinea pig macrophage as an indicator cell are discussed. 7) The TEEM test seems simpler to perform than the MEM test and may be widely applicable in clinical immunology for the estimation of lymphocyte sensitization.

[The macrophage electrophoretic mobility LAD test - a diagnostic method for multiple sclerosis.]. / Der Makrophagen-Elektrophorese-Mobilitäts-LAD-Test als diagnostisches Verfahren für die multiple Sklerose.

With the MEM test (Field) one can establish a cellular immune reaction because the sensitized lymphocytes release the macrophage slowing factor (MSF) upon interaction with the appropriate antigen. A macrophage migration inhibition was detected in some neurological diseases with destruction of the parenchyma. The modification MEM-LAD (linoleic acid depression) test made further differentiation possible in the 146 neurological patients and normals. The reduction of macrophage mobility inhibition was 94.7+/-4.7% in multiple sclerosis (MS) cases as compared with that of normals of 55.1+/-3.7% and of other neurological diseases of 47.8+/-7.1%. There were no significant differences due to the course and duration of the disease or to immunosuppressive therapy. The pathogenically important results in relatives of MS patients with values between the MS and normal group (78.5+/-0.7%) in mothers suggested a familial (genetic) disposition. The same value was found in a monozygotic twin of an MS patient. The results in the children studied showed that besides the endogenic metabolic component the aetiopathogenically important exogenic factors can operate early in life. In correlation with the principle of the MEM-LAD test the suppressive action of linoleic acid can result in a further therapeutic concept.

[Demonstration of a factor in cerebrospinal fluid with inhibitory activity for electrophoretic cell mobility in multiple sclerosis (author's transl)]. / Nachweis eines Faktors mit elektrophoretischer Zellmobilitätshemmung im Liquor cerebrospinalis bei Multipler Sklerose

Inhibition of electrophoretic cell migration using cerebrospinal fluid (CSF) directly was investigated by the modified MEM (macrophage electrophoretic mobility) and TEEM (tanned sheep erythrocyte electrophoretic mobility) tests, respectively. An inhibitory activity of macrophage slowing factor (MSF)--one of in vivo lymphokines--in CSF was established in cases of multiple sclerosis (17.5 +/- 3.8%), and neurolues. The value of this MSF assay turned out to be significantly different from the remaining inflammatory ailments of the nervous system (10.1 +/- 6.8%). Results of other neurological diseases were found to be very much lower (5.1 +/- 4.2%). It seems important, for immunopathogenesis and the diagnosis of neuroimmunological diseases with enhanced cellular immunoreaction, to evaluate MSF activity in CSF. To characterize the active factor in CSF (and serum) these fluids were fractionated by gel filtration chromatography as well as supernatants from lymphocyte-antigen incubation in MS patients. The main activity for inhibition of electrophoretic cell mobility was eluated in the same fraction in these fluids. It could be shown that units have a molecular weight of about 15000 Daltons; this value for MSF lies below those for other inhibitory lymphokines.

Anomalous lymphocyte-antigen reaction in relatives of multiple sclerosis patients. A study of a possible genetic factor in the disease.

A combined familial study of multiple sclerosis (MS) in England and in the Rostock area of the GDR using the macrophage electrophoretic mobility (MEM)-LAD test embracing 132 relatives has revealed a closely similar pattern of distribution of "anomalous" LAD (Linoleic Acid Depression) values in relatives (77% type of reaction) to that originally reported in the British study. The anomaly in predominantly associated with females--all mothers of MS patients being affected, whilst daughters and sisters are also represented. In addition unusual full MS type of reaction (90% reduction) has been found in some children related to patients. There is clearly a genetic element in the development of MS probably mainfested in the inborn mishandling of unsaturated fatty acids suggested by Thompson; no recognizable pattern of inheritance is noticeable even within the combined material. There is evidence that the metabolic anomaly alone does not inevitably lead to MS, and the full abnormality may be present at an early age. A survey about the examinations and a selection of characteristic family trees of MS are given, illustrating the manner in which the 77% type anomaly is distributed with occasional omission of a generation.

Aspects of cellular immunity in multiple sclerosis. Antigen-reactivity of lymphocytes and lymphokine activity.

Some new results of cell-mediated immunity in multiple slcerosis (MS) are presented, based on the determination of charge-changing lymphokines as products of antigen sensitive lymphocytes (C PAL), obtained by several forms of the electrophoretic mobility (EM) method. Lymphocytes from MS react to myelin basic protein (BP), the reactivity in other neurological diseases depending on the degree of destruction of nervous parenchyma. Applying a membrane-associated antigen from normal brain (NTA), positive reactivity of MS lymphocytes was obtained. Besides the usual determination of lymphokines in vitro the sensitive EM test allows the demonstration of lymphokine activities in vivo; i.e. in body fluids such as cerebrospinal fluid (CSF) and serum. In CSF of MS a high lymphokine activity was found. The differentiation of lymphokines and comparison between in vitro and in vivo activity were carried out. Moreover, a characteristic lymphokine pattern for MS with high activities in all regions of molecular weight, especially in the CSF, could be detected. On the basis of these findings, important also from the pathogenetic point of view, a diagnostic scheme for MS is suggested, consisting of a program of determination of the immuno-reactive CSF syndrome and some special procedures, including examination of lymphocyte reactivity.

The use of protein beads as immunoadsorbent for the column fractionation of lymphocytes.

Beads of calf serum proteins (CSB) with antigen-antibody complexes or antigen by activation with glutaraldehyde were used as immunoadsorbents for the fractionation of mouse lymphocytes. T-lymphocytes could be separated by this method with high purity. The precursors of antibody-forming cells were completely eliminated from spleen and bone marrow cell suspensions. The unspecific binding of lymphocytes to CBS or Sepharose was similar and not selective for T or B lymphocytes.

Determination of cell mediated immunity against tumour associated antigens in patients of ENT.

In an unconventional assay system (MEM Test) Caspary & Field claimed in 1971 to have detected lymphocyte sensitization to a common tumour antigen in all patients with cancer. There was no evidence of histogenetic specificity to the reaction and their conclusions are in direct contradiction to those of all workers who have studied the cytotoxic activity of lymphocytes on tumour cells. To improve the specificity of the test system, another type of tumour antigen was used in MEM Test incubation. Tumour-associated antigens were prepared according to the hypertonic salt extraction method introduced by Reisfeld, Leonard, Meltzer et al. As shown by the results, information could be gained concerning the existence of a malignant tumour and its location.

A contribution to immunological tumour diagnostics in urology.

Fifty-four patients with malignant and other diseases of the urinary bladder and the prostate were examined for lymphocyte sensitization by means of electrophoretic mobility test (EM test). In this antigens from brain tissue (EF) and from malignomas of kidney, urinary bladder and prostate were used. Positive HEP values were obtained in 40 patients out of 42 with malignant tumours and in 5 patients out of 12 with other kidney diseases. With the corresponding tumour-associated antigens (TAA) in the EM-test, the histological tumour diagnosis could be confirmed in every case. Positive findings after using EF as well as the corresponding TAA are with great probability an indication of a malignant disease.

[Method of electrophoretic mobility of macrophages and its use in clinical immunology]. / Metod élektroforeticheskoi podvizhnosti makrofagov i ego primenenie v klinicheskoi immunologii.

A test of measurement of the electrophoretic mobility of macrophages (EMM) is used for detection of the product secreted by the sensitized lymphocytes after the contact with the antigen. Thus, by reduction of the macrophage mobility it is possible to assess the sensitization level of the lymphocyte population under study. This offers a possibility of using this test for the diagnosis of some infectious diseases, for provisional diagnosis of malignant growth, and also of destructive affections of the nervous system. The EMM test finds wide application in the determination of compatibility of donor's and the recipient's tissues before the transplantation, and also to assess the efficacy of immunodepressive therapy.

[Methods of isolating immune cells--critical assessment, artifacts]. / Methoden zur Isolierung von Zellen des Immunsystems--kritische Einschätzung, Artefakte. Mit 1 Abbildung.

This paper describes common used methods of immune cell separation such as separation by density-gradient, cell electrophoresis, cell adherence, affinity chromatography, isolation of cell populations by antisera and complement, by cell sorting, by monolayer and lectins. The authors give a critical evaluation in respect of yield and purity in relation to the extent of the experimental procedure and regard eventual artefacts.

Electrophoretic mobility test for lymphocyte sensitization using tanned sheep erythrocytes.

The M.E.M.-test was modified by using tanned sheep erythrocytes in place of guinea pig peritoneal macrophages as indicator calls. This modification is named the tanned sheep erythrocyte electrophoretic mobility (T.E.E.M.)-test. The present study was undertake to compare the kinetics of the two indicator systems. The T.E.E.M.-test appears to be simpler to perform than the M.E.M.-test and may be widely applicable in clinical immunology for the estimation of lymphocyte sensitization.

[Aspects of immunologic diagnosis of brain tumors. Results with tumor-associated membrane antigens and the electrophoretic mobility test (author's transl)]. / Aspekte der immunologischen Diagnostik von Hirntumoren. Ergebnisse mit tumorassoziierten Membranantigenen und dem Elektrophorese-Mobilitäts-Test.

In cell-mediated immune processes the products of antigen-sensitive lymphocytes are detectable as charge-changing lymphokines by means of the cell electrophoretic mobility method (EM test). the improvement of the method concerns the measuring procedures (indicator particle and automation) and the application of specific antigens. The employment of special membrane antigens (3 M KCl extracts) from tissue of normal brain and brain tumors is decisive for further differentiating results. The application of tumor-associated membrane antigens (TAA) from various kinds of brain tumors shows some diagnostically important reaction patterns. Using a common tumor antigen, the TAA-X3 mixture, a (1) characteristic result of general significance for neoplasms can be obtained; in applying the different TAA of brain tumors the findings in EM test point out an (2) organospecific reactivity and/or a (3) histogenetic reaction pattern. The distinct neuroepithelial and mesodermal brain tumors produce predominantly an organospecific reaction and only partly a histogenetical profile. For this type of reactivity to the TAA in brain tumors, a series of factors depending on neuroimmunological specialities are important.

Flow cytometric estimation of transmembrane potential of macrophages--a comparison with microelectrode measurements.

Potential-dependent accumulation of the lipophilic cationic dye 3,3' dihexyloxacarbocyanine (DiOC6(3)) in macrophages has been investigated. Resulting fluorescence of cells was measured by flow cytometry. Alterations of membrane potential of macrophages were induced by ionophore treatment (valinomycin and gramicidin) in a dose-dependent (10(-5) M-10(-7) M) and time-dependent (0 min-45 min) manner. Resulting changes in relative fluorescence intensity were compared with changes of transmembrane potential measured by intracellular recordings obtained by applying glass microelectrodes. The comparative studies offer the possibility to calibrate the flow cytometric estimate of membrane potential of suspended cells. Equilibration of dye partition between cells and surrounding medium is strictly potential-dependent at dye concentrations between 5 X 10(-8) M and 10(-7) M and within an incubation interval from 10 min up to 30 min after addition of dye. Conclusions are drawn concerning the field of application of the optical method. Dynamics of electrical processes following ionophore treatment are discussed in terms of molecular mechanisms of altered ionic transport.

[Heterogenous immune reactivity in brain tumors: results of cellular immunity in relation to brain tissue antigens]. / Heterogene Immunreaktivität bei Hirntumoren: Ergebnisse zur zellulären Immunität gegenüber Hirngewebsantigenen.

In brain tumors and other neurological diseases cell-mediated immune reactions to fetal brain tissue antigens (FBA), normal tissue antigen of adult brain (NTA) and tumor-associated antigens of different brain tumors (TAA) have been analysed. The detection of sensitized lymphocytes using the MEM-(macrophage-electrophoretic-mobility-) test revealed general tumor-related results applying the FBA, in some extent a cross reactivity and partly no kind of reaction. A phase-specific reactivity to normal brain antigens could not be found, only cases of multiple sclerosis produced restrictive results employing the NTA. By testing tumor-associated brain antigens different reaction types were seen: The common TAA caused a tumor-characteristic reaction; the histo-specific TAA predominantly presented a organotypic form of reaction, confined a histo-specific reaction pattern; in some cases there were found inadequate, non-corresponding reactions as well as unreactivity. With regard to different types of reaction the problems of heterogeneity of the brain tumors and the cellular immune response--i.e. a heterogeneity of 1. or 2. order--were discussed including further factors concerning several special conditions in the nervous system.

Flow-cytometric analysis of glucose induced changes of the transmembrane potential and optical density in pancreatic islet cell suspensions.

Glucose-induced insulin release of beta cells of pancreatic islets is associated with islet cell membrane electrical activity. We measured the membrane potentials of neonatal rat islet cell suspensions by means of "optical probes" by flow cytometry. Moreover, we determined the narrow angle light scattering and light absorption in direction of the laser beam, respectively. In both these cases two or three clusters of the whole cell population showing different responses to glucose could be distinguished. Glucose-dependent depolarization as well as loss in optical density of cells of pancreatic islets should be associated with the physiological function of insulin producing beta-cells. It is concluded that changes of the membrane potential and/or optical density of islet cells produced by insulin secretagogues can be analysed by flow cytometry which might provide the basis for sorter experiments to separate specific subpopulations of cells of pancreatic islets.

[Phagocytic reactivity of granulocytes in the peripheral blood of patients with multiple sclerosis and normal probands]. / Phagozytoseaktivität von Granulozyten aus dem peripheren Blut bei Patienten mit Multipler Sklerose und Normalprobanden.

For estimation of phagocytic activity the uptake by granulocytes of heat-inactivated, opsonised yeast particles (Saccharomyces cerevisiae) was measured. Peripheral granulocytes of multiple sclerosis patients revealed an enhanced ability for phagocytosis in comparison to normal healthy controls, if tested with normal serum as opsonic source. An activated state of cells in multiple sclerosis is supposed. The selective influence on granulocyte phagocytosis of treatment by ultraviolet-irradiated blood in multiple sclerosis patients supports the view of altered reactivity of these cells in comparison to normal controls.

[Autoimmunity in chronic inflammatory eye disease (author's transl)]. / Zur Autoimmunität entzündlicher Augenerkrankungen

Preparations of allogenous and xenogenous retinal rod outer segments (ROS) were found to be adequately antigenic for detection of cellular immunity in human chorioretinitis. The electrophoretic mobility test was used for estimation of delayed-type hypersensitivity. No significant results with ROS were found in patients with panophthalmia or in control subjects. It is assumed that the results reported here in chronic ophthalmic inflammation may be produced by or connected with autoimmune processes.

Lymphokines which influence electrophoretic mobility of macrophages.

Supernatants of blood lymphocytes from immunized guinea pigs after incubation with purified protein derivative (PPD) were fractionated on Sephadex G-75. The fractions were tested for activity to change the electrophoretic mobility of macrophages. Dependent on incubation time, three different regions of activity with an average molecular weight of 13 000, 40 000 and more than 100 000 were estimated.