Biomedical subjects
Publications and source records attributed to H Ambrosius.
In order to improve our diagnostic possibilities and the decisive therapeutic consequences resulting from this we began also to include the determination of the alpha-foetoprotein into our diagnostic spectre in tumours of the testicles. Our recent results and the problems appearing are described.
An apparatus derived from data in the literature about different sample harvesters and constructed in our workshop is described. The advantages for the analysis of lymphocyte transformation test of lymph-node cells of guinea pigs is demonstrated. We could show the reproducibility of results after stimulation of cells by mitogens and antigens (bovine gamma globulin, human serum albumin). The optimal experimental conditions for the culture of lymph-node cells of guinea pigs are described.
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Molecular size and polypeptide chain composition of cell membrane immunoglobulin (mIg) on lymphocytes of carp were studied using lactopreoxidase-catalysed surface radioiodination and SDS-polyacrylamide gel electrophoresis. Carp lymphocytes prepared from pronephros, blood and thymus carry mIgM in relatively high quantity. That means about 5-10% of the radiolabelled macromolecular cell surface material precipitates as IgM. Cell surface IgM on carp lymphocytes is present as monomeric IgM (m.w. 220000-260000) and HL subunit (m.w. 110000). There are differences among molecular weights of mIg monomers of pronephric lymphocytes (m.w. 220000) and thymocytes (m.w. 260000), whereas blood lymphocytes show both components. Following reduction and alkylation H and L chains were observed. Additional thymocytic mIg possesses two unidentified components with m.w. 35000-40000 and 110000.
The effectivity of treatment with 10 mg 6-mercaptopurine/kg/day--beginning on the day of antigen injection and continuing for seven days--on the synthesis of complement binding antibodies is very different. The formation of gamma-2-antibodies against BGG was nearly complete suppressed, an influence on the anti-DNP-gamma-2-antibody formation was not detectable and the antibody formation against HSA was enhanced. The results suggest that the kind of antigen is very important for the immunosuppressive activity of 6-mercaptopurine in guinea pigs.
Bovine gammaglobulin-L-asparaginase conjugates were prepared with an enzymic activity of about 30 IU/mg. Glutardialdehyde was used as bifunctional reagent for the cross-linking in an optimal concentration of 0.16 mg/10 mg protein. The both components were found in the conjugates in a ratio of 1:1.5. The recovery was about 25% of the initial protein amounts. The antigenic specificity of the globulin component decreased up to 60%, the enzymic activity also up to 60% by the conjugation.
Following an intradermal immunization of guinea pigs with dinitrophenylated bovine gamma globulin (DNP23-BGG), human serum albumin (HSA) and trinitrophenylated sheep red blood cells (TNP-SRBC) emulsified in complete or in incomplete Freund's adjuvans the effectivity of 6-mercaptopurine (6-MP) on the formation of specific antibodies was investigated. By application of 10 mg 6-MP/kf/day--in guinea pigs a significant depression of antibody formation against BGG could be demonstrated if treatment is started on the day of antigen injection and continued for seven days. The production of anti-SRBC agglutinating antibodies was only suppressed if the antigen was injected together with complete Freund's adjuvans. The anti-DNP and anti-HSA antibody formation was not influenced by this kind of immunosuppressive treatment.
The macrophage disappearance reaction (MDR) is a suitable test for detection of cell mediated immunity against bovine gamma globulin (BGG) and human serum albumin (HSA) in guinea pigs. The MDR is a technical simple, good manipulable, and quantifiable test. The optimal test conditions for the antigens BGC and HSA are the following: Peritoneal exudat cells (PEC) were stimulated with paraffin oil. On the 5th day after receiving oil the animals were injected with 80 microgram BGG or 30 microgram HSA i.p. 5 hours later the PEC were harvested and counted. With the MDR it is possible to detect differences with respect to degree of cell-mediated immunity. Supernatants of sensitized lymphocytes produces the MDR too.
Rabbits treated daily and for seven consecutive days with 6-mercaptopurine bovine gamma globulin conjugates (MPI--n-BGG; 'I'--characterizes the king of chemical binding and 'n' the number of coupled MP-residues per one mole BGG) show an altered immunological reactivity. A following intradermal immunization with BGG alum adsorbate results in a suppressed anti BGG antibody production on the third day after antigen application (antibody titer 1:320, antibody titer of control animals--pretreated with BGG and uncoupled 6-MP equals 1:5120). Already three days later the antibody titers of the test groups show a significant increase and are two dilution stages higher than the titers of the controls. A suppressive effect on the third day is induced by MPI--13-BGG, MPI--26-BGG, MPI--36-BGG and MPI--46-BGG; the later adjuvant effect can only be seen in the MPI--26-BGG, MPI--36-BGG and MPI--46-BGG but not in the MPI--13-BGG pretreated animal group. While the short time suppression was antigen specific--the humoral immune response against a second unrelated antigen was not reduced--the adjuvans effect was not antigen specific. A pretreatment with the substances mentioned above results in an increased anti BGG and anti HSA serum antibody level. Comparing investigations on the unspecific immunosuppression in rabbits by 6-mercaptopurine shows that application of 10 mg 6-MP/kg/day for seven days at first leads to a suppression but later on to an enhancement of antibody production. Application of 10 mg 6-MP/kg/day for 10 days results in a long lasting suppression without enhancement effect. As a reason for these differences the different catabolism of immunosuppressive agent and antigen is discussed. For the phenomena following antigen specific immunosuppression, similar mechanisms can be responsible.
The rosette forming cell (RFC) response of different lymphoid organs (spleen, lymph node, peripheral blood) of guinea pigs was examined by immunocytoadherence. The method has been adapted to work with the soluble antigen BGG. The number of antigen binding cells following immunization with BGG in CFA reached the highest level on day 6 in Ln. popliteus and on the 10th day in spleen and peripheral blood. It is shown that the highest level of antigen binding cells appears generally before the maximum of circulating antibodies. After i.p.-immunization to alum-adsorbed BGG we only detected a small number of RFCs. After pretreatment with 6-mercaptopurineI-25-BGG (MP-BGG) and a control substance (toluylI-25-BGG) over 8 days and in increasing doses we obtained an antigenspecific suppression of BGG binding cells following MP-BGG treatment. In contrast to these findings the antibody titres were only slightly decreased in the beginning of the immune response.
The affinities of anti-DNP-antibodies (IgM) of carp, immunized with different doses of dinitrophenylated human serum albumin in complete Freund's adjuvant, were determined by a modified Farr-technique using the hapten epsilon-DNP-L-Lysine. Very low affinity antibodies were formed in the early immune response (Ko = 0.2 - 1.0-10(5) M-1). A progressive increase in IgM antibody affinity occurred during the immune reaction. After immunization with low doses of antigen (0.01 and 0.1 mg) the affinity increased up to four weeks (Ko = 3.2 and 9.1-10(5) M-1) and decreased in the subsequent three weeks (K = 0.8 and 1.2-10(5) M-1). Following immunization with a higher dose of antigen (1 and 10 mg), the affinity increased up to seven weeks (Ko = 4.9 and 11.9-10(5) M-1). The highest antibody concentration in all groups were observed at the maximum of affinity.
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The most important mechanisms for the specific depression of immune reactions--immuno-tolerance, enhancement, transfer of antibodies, drug induced tolerance, immunological suicide, application of antibody-toxin-complexes--are discussed with regard to their possible application in the clinical practice. A tentative hypothesis for induction of antigen specific suppression is proposed, basing on the use of antigen-immunosuppressive agent-conjugates (AIC). Antigen binding lymphocytes are supposed to bind the AIC and to pick them up through endocytosis. After breakdown of the AIC in the lymphoid cells the free immunosuppressive agent can become effective causing damage to the specific cell clones.
The maintenance of specific immunogenicity of carrier proteins is a necessary condition for the successful use of antigen-immunosuppressive agent-conjugates (AIC) for an antigen specific suppression of the immune response. The experimental results indicate that, in spite of the binding of 6-mercaptopurine (6-MP) and toluyl (T) to bovine gamma globulin (BGG) and human serum albumin (HSA), the carrier specific immunogenicity is not significantly altered. The intradermal application of 6-MP-BGG and T-BGG emulsified with complete Freund's adjuvant in guinea pigs results, in all cases, in a well detectable anti BGG hemagglutination and precipitation titer. This kind of immunization leads also to a formation of anti 6-mercaptopurine and anti new antigen determinants (NAD's) antibodies.
6-mercaptopurine (MP) conjugates of bovine gamma globulin (BGG) and human serum albumin (HSA) with increasing numbers of MP-groups per protein molecule were prepared and some physicochemical and antigenic properties studied. The electrophoretic mobility increased proportionally to the degree of substitution. In the same manner the formation of high molecular weight aggregates increased. A loss of BGG and HSA specific determinants was observed too. The physicochemical and antigenic properties of these antigen-suppressive agent-conjugates with low and medium coupling degree (to MP26-BGG and MP20-HSA) were similar to the unmodified antigens. The affinity of rabbit anti-DNP-antibodies decreased from K0 = 1,4 - 10(6) M-1 of the unmodified antibodies to 6,5 - 10(5), 3,6 - 10(5) and 3,4 - 10(5) M-1 of the antibodies conjugated with 15, 21 and 29 MP-groups per antibody molecule. The precipitation capacity of the MP-conjugated antibodies decreased to 89, 85 and 61% for the same coupling ratios. These results suggest that the upper limit of the coupling ratio of the antibodies is 20 to 1.
Bovine gamma globulin (BGG) antigens were modified by the binding of 6-mercaptopurine and toluyl residues, and their influence on the humoral immune response in guinea pigs was investigated. The antigen-immunosuppressive agent-conjugates (AIC) were different, depending on the method used for their preparation and the number of coupled residues per one molecule of BGG. Conjugates denoted as MPI-n-BGG were prepared by special chemical binding of corresponding thioisocyanates. MPII-n-BGG were synthetized by acetylation, and MPIII-n-BGG conjugates, by reductive alkylation. Pretreatment of guinea pigs with MPIII-19-BGG, MPII-16-BGG resulted in a stimulatory effect on the subsequent humoral immune response induced by BGG application. A significant suppressive influence was detectable if the animals had been pretreated with MPII-6-BGG and MPI-26-BGG. MPI-13-BGG and MPI-36-BGG had no effect on the later induced anti-BGG antibody formation. The immune response against a second antigen (human serum albumin) was not influenced by this kind of pretreatment of the animals. Therefore it seems justified to conclude that both stimulatory and suppressive effects seen here were antigen specific and that both the method for chemical modification and the number of coupled 6-MP residues are very important for their effectivity.