XI. Tagung der Gesellschaft fur Immunologie . 191

Workshop Nr. 2: Clinical Relevance of New Immunological Methods

Departments of Microbiology, Pathology and Medicine, State University of New York at Buffalo, USA

2. 1 A micromethod for the detection of circulating immune complexes using Raji cells

B. ALBINI, E. OSSI, E. PENNER and G. A. ANDRES

FITC- and radio-labeled antisera have been used for the detection of immune complexes (IC) reacted with Raji cells (RC) (THEOPHILOPOULOS et aI., J. Exp. Med. 40: 1230, 1974). The established methods for membrane immunofluorescence with RC are time consuming and do not permit quantification. Methods employing radiolabeled antisera are very sensitive but render high background values; the data, expressed as aggregated IgG equivalant, make the erroneous impression of accurate quantitative findings; finally, presence of Iymphocytotoxic antibodies in test sera may lead to false positive results. In order to minimize these disadvan­tages, an attempt has been made to establish a micromethod based on the membrane immunofluorescence technique described by SCHAUENSTEIN et al. (J. Immunol. Methods. 10: 143,1976). Serum dilutions at 20 loti volume are reacted with an equal volume of a suspension of 107 RC/ml in microhemagglutination trays. After washing, 20 loti of the appropriate FITC- or rhodamine-conjugated antiserum is added. Binding of immune complexes or aggregated immunoglobulins to the Fc receptors of RC is detected in control preparations with decom­plemented sera (EDTA). The results are expressed as titers of Ie. This technique has been used to detect IC formed with IgG, IgM, and IgA antibodies. In vitro formed IC have been used to standardize the technique.

Kinderklinik der Universitat Munchen and Institut fur Virologie und Immunbiologie der Universitat Wurzburg, Federal Republic of Germany

2.2 Impaired lymphocyte function in subacute sclerosing panencephalitis (SSPE) by using buffy coat cells instead of purified peripheral blood lymphocytes

R. F. EIFE and H. W. KRETH

In general in vitro lymphocyte functions in children with SSPE have been found to be normal. Skin tests with common antigens measuring delayed type hypersensitivity reactions, however, are negative in a considerable number of patients with SSPE, implying some kind of primary or secondary immune disturbance.

We have studied several in vitro reactions of SSPE lymphocytes. In the present study we compared the blast cell transformation and the Iymphotoxin secretion by stimulated peripheral

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blood lymphocytes in (A) leukocyte cultures (washed buffy coat cells) and (B) lymphocyte cultures (purified mononuclear cells - about 80% lymphocytes and 20% monocytes). In leukocyte cultures the blast cell transformation induced by mitogens (PHA, ConA, PWM) was somewhat impaired, however, the Iymphotoxin secretion was strikingly reduced; in addition, blast cell transformation could not be induced by specific antigens. In lymphocyte cultures the mitogen induced blast cell transformation was normal, but in contrast to the leukocyte cultures the Iymphotoxin secretion as well as the antigen induced blast transforma­tion were stikingly increased. In mixed lymphocyte/leukocyte cultures the buffy coat cells dominated. When the same experiments were carried out with normal human lymphocytes, a similar but less significant change in reactivity was observed.

From these observations it might be speculated that the defective response of lymphocytes in vitro using buffy coat cells might be due to suppressor cells which are lost by further purification of mononuclear cells and it might be suggested that studies of the cellular immunocompetence of patients with SSPE are more representative when buffy coat cells instead of purified lymphocytes are used.

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 37, B7).

Department of Clinical Immunopathology of the University Childrens' Hospital Hamburg, Federal Republic of Germany

2.3 Quantitation of chord blood-IgM and -IgA samples by fluoroimmunometric technique

K. FISCHER and A. POSCHMANN

Elevated IgM levels of chord bloods (above 0.2-0.3 gil) are suggestive for prenatal infections. Simultaneously elevated IgA levels (above 0.03 gil) can indicate contamination of the chord blood sample by maternal serum. Today, screening of chord bloods for elevated IgM levels is mostly performed by radial immunodiffusion technique (RID). However, results are firstly yielded after 24 hours. Therefore, we compared a new and rapid (1-2 hours) fluoroim­munometric technique (using the FlAX test kits of International Diagnostic Technology, Boehringer, Ingelheim) and RID (using Tri-Partigen plates of Behring, Hoechst, Marburg) for quantitating IgM and IgA levels in chord blood sera (n = 104). Applying flAX, the mean IgM level was 0.150 gil. The correlation coefficient between RID and FlAX was 0.9611. IgA levels in chord sera are often not detectable. However, elevated IgA levels (above 0.03 gil) are accurately determined. Validation and problems of quantitating low level Ig values are discussed, using fluoroimmunometric technique.

Klinisches Labor der Chirurgischen Universitatsklinik Heidelberg, Federal Republic of Ger­many

2. 4 Rapid detection of IgM antibodies to rubella virus and cytomegalovirus by elisa-technique

H. P. GEISEN, H. W. DOERR, E. ADOLF and G. ENDERS

The most important tool to diagnose infectious diseases during pregnancy are serologic test techniques. The demonstration of specific IgM in single serum specimens enables the approx-

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imative determination of the onset on infection. The IgM determination is performed by developed test techniques. Serum IgM is isolated by specific immunosorption to silanized controlled porous glass (CPG), to which heavy chain specific anti-IgM antibodies have been fixed covalently. The isolated serum IgM antibodies are subsequently identified by perox­idaselabelled cytomegalovirus (CMV) and rubellavirus antigens. Many of serum specimens originating from adult and newborn patients were investigated. The results are in a good agreement to control tests carried out by the indirect immunofluorescence (CMV) and by the hemagglutination inhibition test after separation of IgM (rubella) either on a sucrose density gradient or by polystyrol particles sensibilized with specific IgM antibodies. The interference of RF factor is significantly reduced in the CPG test system. IgM antinuclear antibodies are excluded easily by the use of a negative control antigen (CMV). The CPG charges once used can be simply regenerated by washing them in 1 M propionic acid and equilibrating them in neutral buffer solution.

1. Med. Klinik, Univ. d. SaarI., Homburg, Federal Republic of Germany

2. 5 An enzyme-immunoassay for quantitation of human Ig

H. J. HAMMER, G. KUMEL, M. HOFFMANN, P. G. SCHEURLEN and H. MAUCH

For a study concerning the secretion of IgG from human lymphocytes in micro-tissue culture, we developed two solid-phase immunoassays for the determination of low amounts of human IgG (h-IgG). A monospecific anti-h-IgG immunoglobulin fraction was prepared from rabbit hyperimmune-IgG by affinity chromatography and bound to polystyrene by an optimimized adsorption procedure.

1. A competitive assay:

In competition with the h-IgG to be analysed (test-IgG), peroxidase-labelled standard h­IgG was incubated with the solid-phase-bound anti-h-IgG. The amount of bound labelled IgG is determined by the enzyme reaction. It quantitates the ratio of standard IgG to test-IgG and the concentration of the latter can thus be determined.

2. A sandwich assay:

The test h-IgG is bound to the solid-phase-bound anti-h-IgG as the antigen, in a second reaction step it is quantitated by enzyme-labelled anti-h-IgG antibody.

The sensitivity of both assays is sufficient to determine h-IgG in nanogram amounts in 200 III samples. The competitive assay has the advantage of taking only 2';' hours for its performance. Both assays are characterized by an intra- and interassay variance of about 2-6%.

Medizinische Univ. Poliklinik Munster, Federal Republic of Germany

2.6 Laser-nephelometry, a quantitative method to determine rheumatoid factors in paraproteinemias

H. W. INTORP and H. LEYSSENS

In previous studies laser-nephelometry was found to be a sensitive method to quantitatively determine immunoglobulins and other serumfactors in plasma as well as other body fluids. To

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find out whether antibodies to immunoglobulins are present in sera of patients with para­proteinemias a newly developed technique was applied which permits to detect rheumatoid factors by laser-nephelometry.

Sera from 175 patients with monoclonal gammopathies of the IgG, IgM and IgA class were incubated with aggregated human IgG. The resulting antigen-antibody-complexes that are formed were detected by measuring the light scattering of a helium-neon laserbeam. Of the 175 paraproteinemic sera 60 contained rheumatoid factors, indicating that antibodies are present in more than 30% monoclonal gammopathies. In contrast to the latex agglutination and Waaler­Rose test this technique permits the detection of antiglobulin factors of the IgM and IgG class. The percentage of positive results is significantly higher than with the other methods used.

The detection of the IgG-rheumatoid factor correlates to the clinical symptoms of arthritis not rarely associated with paraproteinemias.

Div. of Clinical Immunology, Dept. of Medicine, Medizinische Hochschule, Hannover, Federal Republic of Germany

2. 7 A Microtiter® conglutinin-binding RJA for detection of circulating immune complexes

W. LIMAN, M. FRICKE and H. DEICHER

A solid phase radioimmuno assay for the detection and quantitation of circulating immune complexes (dC) has been developed which employs purified bovine conglutinin. 300-400 samples can be tested at once using a Microtiter® system with special polystyrol tubes (Immunolon®), a multichannel pipetter, and an automatic washer.

After an incubation with 5% BSA to saturate the unspecific binding capacity of the tubes, sera to be tested are allowed to bind to the solid phase conglutinin. Bound dC's are detected using a radiolabelled anti-IgG antibody.

The conglutinin binding test (KgBT) preferentially detects large complexes, is minimally influenced by monomeric IgG, and can be inhibited by calcium chelation or decomplementa­tion. The lower limits of sensitivity has been found at 5-10 ug/ml aggregated human IgG. To test the clinical relevance of the KgBT, groups of patients with RA, SLE, and myeloma have been screened for dC's using a Clq binding test, a Raji cell test, and the KgBT.

(Supported by the Deutsche Forschungsgemeinschaft, SFB-G3)

Dermatology Dept. Ludwig-Maximilians-University Miinchen, Federal Republic of Ger­many, Scripps Clinic Res. Fdtn. La Jolla, USA

2. 8 In vitro histamine release as possible indicator of contrast media hypersensitivity

J. RING, R. SIMON, C. ARROYAVE

Radiographic contrast media (RCM) have been shown to release histamine from peripheral human leukocytes in vitro (1). This study was designed to compare in vitro histamine release in patients with prior RCM incompatibility and normal controls.

Methods: Peripheral leukocytes of 10 patients with positive history of RCM hypersensitiv-

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ity and 19 normal volunteers were stimulated in vitro with different RCM in various concentrations and the amount of histamine release into the supernatant was measured.

Results: Histamine release induced by low doses (0.02-0.1 M) of RCM was significantly higher in patients than in normals, while there was no difference at high concentrations (0.2-0.3 M). Leukocytes from 4 patients were stimulated preferentially by the dye responsible for the incompatibility. 6 patients showed no such «preference». The increased releasability of the patients' leukocytes could not be transferred by serum.

Conclusions: An increased non-immunological response of basophil leukocytes to RCM stimulation might, in part, account for the clinical hypersensitivity reactions. In vitro his­tamine release might be used as a diagnostic tool for detecting patients with a high risk of developing contrast media reactions.

Reference: 1. J. RING, C. M. ARROYAVE, M. J. FRITZLER, E. M. TAN: In vitro histamine and serotonin release by radiographic contrast media (RCM). Complement-dependent and inde­pendent release reaction and changes in ultrastructure of human blood cells. Clin. expo Immuno!. 32, 105 (1978)

Medical University Clinic Tiibingen, Dept. II, Tiibingen, Federal Republic of Germany

2. 9 Demonstration of a trypsin insensitive subcellular antigen as a marker reacting only with sera from a subgroup of patients with cholestatic liver disease

T. J. SAYERS, K. H. WIEDMANN and P. A. BERG

It was previously shown that antimitochondrial antibodies (AMA) of different specifities can occur in chronic cholestatic liver diseases. Marker antigens may be used to differentiate primary biliary cirrhosis (PBC) from other cholestatic liver diseases (mixed forms). In order to determine whether these antigens are biochemically different purified mitochondrial ATPase fraction (PBC-Ag), submitochondrial particles (SMP) as well as various tissue sections were treated with a variety of enzymes. Proteases, particularly trypsin at a concentration of 50 !-l/ml for 30 min. at 37°C, were found to be the most effective in discriminating between various antigens.

Sera from 30 patients with PBC and 40 pat. with mixed form liver disease diagnosed serologically by the presence of antibodies against both a PBC-specific antigen and another as yet undefined subcellular antigen were tested in the complement fixation (CFT) using SMPs as well as in the immunofluorescence test (IFL).

All sera from patients with PBC became negative in the CFT and IFL after the trypsin treatment. In contrast 15 out of 40 sera from patients with mixed forms still remained strongly positive in both tests. From analysis of the reaction pattern obtained with these various antigen fractions the following conclusions can be made: 1. PBC sera seem to react exclusively with the trypsin sensitive ATPase associated antigen. 2. Mixed form, probably one of two different types of mixed form I (trypsin sensitive group) and mixed form II (trypsin insensitive group) revealed that histological features of chronic active hepatitis were predominantly found in the trypsin insensitive group.

In addition these findings may indicate differences in the aetiology of the various subgroups of chronic cholestatic liver disease.

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Medical University Clinic Tiibingen, Dept. II, Tiibingen, Federal Republic of Germany

2. 9 Differential diagnosis of cholestatic liver disease using a complement fixing PBC - and mixed form (MF) - specific subcellular antigen

E. STECHEMESSER and P. A. BERG

It has previously been shown that antimitochondrial antibodies (AMA) of different specificities may be associated with various types of cholestatic liver disease. Since no reliable procedure of antigen preparation was up to now available we tried to purify the antigen further and establish a standard method of antigen isolation. The primary biliary cirrhosis (PBC) antigen known to be associated with the mitochondrial ATPase complex was prepared from beef heart according to BEECHEY et al. (Biochem. j., 148, 533, 1975). The mixed form antigen (MF) was isolated from rat liver using a modification of the density gradient method of DALLNER (Meth. Enzymol., 31, 191, 1974). This microsomal-rich fraction was free of PBC­antigenic activity.

Fifty six sera from patients with various AMA-positive liver diseases were tested in the complement fixation test against these marker fractions. Positive results were obtained with all sera tested against the mitochondrial ATPase-associated antigen. However, only 22 of the 56 sera also fixed complement with the MF-fraction. These antibodies occured predominantly in patients who had morphologically cholestatic chronic active liver disease with hypergamma­globulinaemia and frequent relapses.

Thus, a certain form of cholestatic CAH may be characterized by the presence of two different types of CF-antibodies reacting simultaneously with the PBC-associated antigen and either an outer mitochondrial membrane or microsomal antigen.

Institute for General and Experimental Pathology, Univ. of Innsbruck, Austria

2. 10 Laser immunofluorescence: Bleaching characteristics of FITC conjugates specifically and non-specifically bound to antigen coated insoluble carriers

K. SCHAUENSTEIN, G. BOCK and G. WICK

A main shortcoming in immunofluorescence is the rapid fading of fluorescence under continuous illumination. As shown in earlier studies, however, this problem may be overcome by pulsed high power excitation since fading turned out to be recoverable under these conditions (1). In the recent past bleaching and recovery of free and antibody bound fluorescein-isothiocyanate (FITC) were systemically investigated using a fluorescence micro­scope equipped with an Argon ion laser and fast, electronically gated shutter and photodetect­ing devices. The results of this work can be summarized as follows: 1) According to the fluorescence intensity curves as monitored on an oscilloscope, fading of FITC seems to be a biphasic process: A very rapid decrease is followed by a moderate, linear phase. 2) Binding of FITC to protein markedly enhances the first rapid phase without changing the characteristical shape of the curve. 3) Pronounced differences of bleaching characteristics, however, are observed with FITC-antibodies specifically or non-specifically bound to antigen-coated

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Sephadex beads. According to these first data, analysis of fluorescence kinetics during the first I-Isec of excitation may be a tool to elucidate the nature of binding of the respective FITC­tagged antibody molecule. 1. K. SCHAUENSTEIN, G. BOCK, and G. WICK, 1978. in: W. KNAPP, K. HOLUBAR, G. WICK

(eds.): Immunofluorescence and Related Staining Techniques. Elsevier/North Holland Biomedical Press, p. 81 Supported by the Legerlotz Foundation, Innsbruck.

2. Medizinische Abteilung, Landeskrankenhaus, Graz, Austria, and Institutes of Medical Sciences, Pacific Medical Center, San Francisco, California, USA

2. 11 The radioactive antiglobulin test

P. SPATH, P. YAM and 1. D. PETZ

Weak red cell sensitization by hemolysing antibodies may not be detected by the standard antiglobulin test. For the diagnosis of certain immune hemolytic anemias more sensitive methods are needed. Anti-IgG was purified as F (ab')z by pepsin digestion and labelled with 1251 using the chloramine T method. 15 X 109 cells were washed and suspended in 0.1 ml of 6% albumin in saline. After incubation with 0.1 ml of 125 I-anti-IgG F (ab')2 for 30 minutes, the cells were washed and the uptake of radioactivity was counted using a gammacounter. Standard cells were coated with a known concentration of an IgG anti-D preparation in the range of 50-1000 molecules per red cell. The sensitivity for the detection of red cell sensitization was at least 50 molecules per red cell. Because of varying binding ratios between the labelled anti-IgG reagent and different red cell antibodies the determination of the exact number of IgG molecules on the red cells is difficult. However, this very sensitive and reproducible method shows practical advantages in comparison with a complement fixation antibody consumption method with which corresponding results were obtained. Beside the evaluation of antiglobulin test negative autoimmune hemolytic anemias, this method may be applied for the determination of the specificity of weak red cell alloantibodies not detectable by standard blood bank procedures.

Institute of Immunology, Univ. Vienna and Children-Clinic, Garmisch-Partenkirchen, Austria and Federal Republic of Germany

2. 12 Demonstration of antibodies to denatured type I and type II collagen in juvenile rheumatoid arthritis, Still's syndrome and controls by 14C-collagen radioimmunoassay

C. STEFFEN, 1. SANGER and J. MENZEL

From 88 sera of patients with juvenile rheumatoid arthritis 30.6% showed antibodies to denatured type I collagen and 31.8% antibodies to type II collagen, a percentage which corresponds to radioimmunoassay results in adult RA. Rheumatoid factors were demonstrated with Waaler Rose test in 14.7% and with Latextest in 6.8% of investigated patients. Results on

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investigations with type I and II collagen correlated in 73.7%. Whilst type I collagen antibodies appeared equally in active and non-active stages, type II antibodies were twice as frequent in active stages as in non-active stages. 16 sera of children with non-rheumatoid diseases had no collagen antibodies. JRA-sera and controls differed in regard to collagen antibodies with statistical significance.

36 sera of children with Still's syndrome showed in 13.8% antibodies to type I collagen and in 33.3% to type II collagen. Both types of antibodies appeared more frequently in clinically active stages. The sera differed in regard to collagen antibodies from controls with statistical significance.

Med. Poliklinik der Universitat Gie/~en, Federal Republic of Germany

2. 13 The clinical relevance of Ig-classes and complement fixation of thyroid antibodies and immuncomplexes in various thyroid diseases

J. TEUBER, K. HELMKE, B. SCHIESSEL, B. MICHEL and K. FEDERLIN

Thyroid AB are found in varying titers in thyroid diseases and in some cases in persons without thyroid disorders. Up to now the pattern of Ig-classes among the antibodies has found little interest. Therefore we investigated the Ig-classes and complement fixing ability of different thyroid antibodies in patients with thyroiditis, thyrotoxic goitre (M. Basedow) and hyperthyroidism. Simultaneously we determined the circulating immuncomplexes in these sera.

The Ig-classes of thyroid antibodies were detected by indirect immunfluorescencetechnique using specific antisera against IgA, IgG, IgM. The results were compared to HAG-techniques after incubation and absorption with precipitating antibodies against the different Ig-classes. Complement fixing ability was tested in the same kind after decomplementation and following adding of fresh guinea pig complement. Circulating immuncomplexes as well as the specific antibodies involved in these complexes were detected by a laser nephelometric method.

These results were correlated with clinical situation, course of disease and treatment.

Med. Univ. Poliklinik Gie/~en, Federal Republic of Germany

2. 13 Comparative studies of various tests for the detection of thyroid antibodies, their sensitivity and specivity

J. TEUBER, K. HELMKE, M. UMBACH, E. MASER and K. FEDERLIN

Sera of 130 patients with various thyroid diseases and 50 controls were investigated for AB against microsomal and colloidal fractions of the thyroid. For this purpose we used three different commercial testsystems (3 various HAG-test, 1 RIA and 1FT).

The sensitivity of the various methods was estimated according to the AB-titer of HAG and

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binding capacity of serum in RIA. The specifity of the methods was tested by previous incubation and absorption of the sera with different thyroid AG.

In the different HAG-testsystems we found a varying sensitivity using the same sera. While the specifity of these tests in general was shown well comparable, in some instances there were clear cut discrepancies. This effect may be related to differences in antigenicity or concentra­tion of thyroid substrat prepared in different testkits.

These results show that there is a necessity for standardization of the various tests and techniques to get comparable outcome in different laboratories. In this context the clinical relevance of the AB detection depends essentially on the sensitivity and specificity of the methods for their determination.

Instiute for General and Experimental Pathology, University of Innsbruck, Austria, and Broegelmann Research Laboratory for Microbiology, University of Bergen, Norway

2.14 Localization of cell markers in human lymphoid tissue

S. THuNoLD"'), R. MATRE and O. T0NDER

The distribution of E, Fcy and C receptors in normal human lymphoid tissue was studied using haemadsorption (HA) to cryostat sections in closed chambers. Indicator cells were sheep erythrocytes (E), E treated with 2-aminoethylisothiouronium bromide-hydrobromide (E-AET), E coated with rabbit IgG antibodies (EA) and E coated with rabbit IgM antibodies and human C3b or C3d (EAC3b, EAC3d).

E receptors were detected mainly in thymus, while E-AET receptors were detected in thymus and in peripheral T-lymphocyte areas. The Fcy receptors were present primarily in interfol­licular areas (B-lymphocytes/macrophages), but also in lymphoid follicles (B-lymphocytes). C3b receptors were found in and between the follicles while the C3d receptors exclusively appeared in follicles. The distribution of the C receptors indicates that follicle cells have receptors for both C3b and C3d, whereas interfollicular cells have only receptors for C3b.

The HA technique distinguishes markers of T- and B-lymphocytes and macrophages in tissue sections, and combines specificity with morphological localization of the reactions. It can be used for the characterization of cell infiltrates in organs in various diseases, and for the immunological classification of malignant lymphomas.

".) Visiting Professor from Department of Pathology, University of Bergen, Norway.

Inst. for Gen. & Exper. Pathology, Univ. of Innsbruck, Austria

2. 15 Demonstration of anti-mucopolysaccharide (MPS) antibodies fol­lowing treatment of patients with glycosaminoglycan-polysulfate

H. WOLF and G. WICK

A new method has been developed for the demonstration of antibodies to heparin or sulfated mucopolysaccharides in general. Heparin, applied as an anticoagulant, has sofar been

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considered to be non-immunogenic in spite of various procedures for immunization and serological analysis. Green et al. (1), however, found that patients treated with heparin occasionally had an increased incidence of thrombocytopenia. We were interested to see whether this phenomenon has an immunological basis. A test system was developed utilizing heparin-coated normal blood group 0 thrombocytes for the detection of suspected antibodies against this complex. After addition of human serum or serum from guinea pigs or rabbits immunized with heparin to a suspension of highly purified heparin- or MPS-coated platelets, agglutination was microscopically determined under phase contrast. Indirect membrane immunofluorescence tests were performed in a similar way. After a first incubation of the platelet suspension with the patients sera and a second one using FITC-conjugates against total human Ig or class specific conjugates (a-y, a-I!, a-a) the specimens were examined using a fluorescence microscope. Positive staining was observed with a-Ig and a-IgG conjugates. These data provide evidence for the presence of antibodies to MPS or to antigenic determinants evolving from the binding of MPS to the thrombocyte surface in the few patients developing thrombocytopenia after treatment with sulfated mucopolysaccharides. 1. D. GREEN, K. HARRIS, N. REYNOLDS, M. ROBERTS and R. PATTERSEN Clin. Med. 91: 167 (1978)

Abt. Immunol. der Universitat Kiel, Federal Republic of Germany

2. 16 New immunopharmacological approaches at modulation of reactivity in variously sensitized individuals

M.-Y. Yu, K. ULRICHS, W. MOLLER-RuCHHOLTZ

So-called immunosuppressive drugs appear to be the more clinically relevant when clearly effective in presensitized organisms. A recent methodological approach to this aim consists of repeated administration of defined combinations of drug plus antigen. T -effector cell reactivity of BALBlc mice was investigated after treating the animals with cyclophosphamide, procar­bazine and vinblastine plus allo-antigen (C3H-lymphocytes). It was found, that each of these drugs induces a characteristic specific reaction pattern, showing either immunosuppression or immunostimulation, depending on (1) the degree of sensitization (not or 3-fold presensitized), (2) the type of drug used, (3) the dose of the drug injected (20% versus 70% LDso i. p.), (4) the time of drug-application in relation to antigenic treatment (up to 6 days before or 6 days after the antigen respectively), (5) the time of testing (5-7,18 and 40 days) after the treatment. These observations indicate that only detailed immunopharmacological studies will allow sufficient knowledge to be obtained for manupulations of an immune response in either direction, both for clinical and experimental purposes.