Zeitschrift fur Immunitatsforschung, experimentelle und klinische Immunologie最新文献

A rabbit antiserum of restricted specificity elicited by the sequential polypeptide, (L-Ala-D-Glu-L-Lys-D-Ala-Gly)n, was purified by column chromatography using Sepharose 4B to which the inhibitor, Gly-L-Ala-D-Glu-Gly, was covalently attached. Approximately 95% of the antibody activity was retained on the immunoabsorbant. After elution, the purified antibody was shown by electrofocusing in a sucrose gradient to be of restricted heterogeneity. The entire antibody fraction was contained within the pH range of 6.9-8.9. The normal immunoglobulin pool from this rabbit, on the other hand, was found by electrofocusing to be distributed throughout the pH range studied (3.6-9.2).

1. The cell walls from some 20 species of gram-positive bacteria, with only few exceptions, were found to be definitely adjuvant-active in both stimulation of increased serum antibody levels and induction of delayed-type hypersensitivity to ovalbumin when administered to guinea pigs in the form of a water-in-oil emulsion. 2. By the use of various cell wall lytic enzymes, the immunoadjuvant principles were solubilized with the full retention of adjuvant activities observed with the cell walls of S. aureus, Str. pyogenes, Str. mutans, L. plantarum, C. diphtheriae, Myc. smegmatis and A. viscosus. N-acetylmuramyl peptide monomers (either L-Lys or meso-Dap type) were shown to be the unit chemical structure responsible for the manifestation of adjuvant activities to stimulate both antibody-mediated and cell-mediated immune responses. 3. Several N-Acetylmuramyl peptides were prepared by condensation of benzyl N-acetyl-4,6-O-benzylidene-alpha-muramide with each peptide benzyl ester by means of dicyclohexylcarbodiimide-N-hydroxysuccinimide method and removal of the protecting groups by hydrogenolysis. N-acetylmuramyl-L-alanyl-D-isoglutamine was identified as the minimum structural entity essential for the immunoadjuvant activities characteristic of bacterial cell walls. Neither synthetic N-acetylmuramyl-L-alanine nor L-alanyl-D-isoglutaminyl-L-lysyl-D-alanin was found to be active.

An immunoperoxidase technique developed for the detection and measurement of anti-peptidoglycan antibodies is described. The technique is based on the Mancini single radial immunodiffusion test with the addition of a reaction step with peroxidase-labelled anti-immunoglobulins. The dark-brown colour produced by the enzyme and a H2O2-diaminobenzidine solution increased the sensitivity considerably. The test was found to be highly specific and valuable both for the screening of total amounts of anti-peptidoglycan antibodies in sera as well as in studies of antibody specificity.

The peptidoglycan is a heteropolymer composed of polysaccharide chains which are cross-linked through short peptides. The polysaccharide moiety (glycan) is made up of beta-1,4 glycosidically linked N-acetylglucosamine and N-acylmuramic acid residues. The carboxyl group of muramic acid is usually substituted by a peptide which consists of alternating L- and D-amino acids. These peptide subunits are cross-linked between the diamino acid in position 3 and the C-terminal D-alanine in position 4 of an adjacent peptide subunit either in a direct way or via an interpeptide bridge (Group A). In some coryneform bacteria the cross-linkage extends from the alpha-carboxyl group of D-glutamic acid in position 2 to D-alanine of a neighbouring peptide subunit (Group B). In the latter case a diamino acid is always found in the interpeptide bridge. A chemical modification of the peptidoglycan may occur in some bacteria due to growth in a quite unbalanced medium. The influence of glycine-rich and glycine-deficient growth medium on the chemical structure of the peptidoglycan of S. aureus will be discussed. Inhibiting concentrations of penicillin, glycine or D-amino acids can also modify the peptidoglycan. Further modification can occur through different extraction procedures which are used to obtain a clean peptidoglycan free of non-peptidoglycan cell wall material. Little is known about the molecular basis of the biological activity. The chemical composition is at least important for the antigenic determinants. The lysozyme susceptibility and the size of the preparation may be other crucial points for the biological activity of the peptidoglycan.

The chemical structure of the adjuvant active fraction of mycobacterial cell walls has been investigated. It had been shown previously that soluble peptidoglycan fragments obtained from cell walls of Mycobacteria by lysozyme digestion or by other treatments act as adjuvants for increasing both humoral and cellular immunity. We then found that even the monomer subunit of the peptidoglycan of Mycobacteria (i.e. a disaccharide-tetrapeptide) is adjuvant active; then, similar compounds from other strains of bacteria were tested; the monomeric subunits of meso-diaminopimelic acid as well as L-lysine containing peptidoglycans were found to be adjuvant active. The smallest active compound studied so far is N-acetyl-muramyl-L-alanyl-D-isoglutamine synthesized for us by SINAY et al. (1975).

Immunocompetent cells obtained from NIP-RGG immunized mice were fractionated on bead columns coated with antigen or anti-immunoglobulin serum. The separated cell fractions were examined for their capacity to be stimulated by the antigen in short term culture, to produce antigen specific antibodies in the plaque assay and to bind radioactive labeled antigen. Cells which produce hapten specific antibodies or bind radioactive labeled hapten are removed from the cell population passed through a hapten-carrier complex coated column. Cells stimulated by the antigen to an increased DNA-synthesis are also retained by columns coated with the hapten-carrier-complex or the carrier alone; the fractionation seems to be carrier specific. The fractionation of cells is blocked by free antigen in the columnar fluid. However, the fractionation patterns of cells passed through anti-Ig-serum coated columns are different when antibody producing cells and cells stimulated by the antigen are compared. Whereas antibody producing cells and antigen binding cells are almost completely retained by anti-Ig-serum coated columns the cells which are stimulated by the hapten carrier complex are not removed from the passed cells. Studies to characterize the fractionated cell populations according to their sensitivity to anti-theta-serum, to the presence of Ig-receptors and to the phytohemagglutinin stimulation indicate that the antibody producing cells and the antigen binding cells have to be attributed to B-cells whereas the question whether the antigen stimulated cells are T- or B-cells cannot be definitely answered.

To get more information about the mechanism of stimulation of the immune response during immunosuppression under certain conditions, stimulating factor (SF) was collected 48 hours after treatment of NMRI-mice with cyclophosphamide. SF was characterized by different steps of separation using centrifugation, dialysis and salt-fractionation. For demonstration of the SF animals were sensitized with SRBC and given different substrates separately. The stimulation of the antibody-production was demonstrated on the cellular level using the plaque-technique. SF was detected by comparing the counts of plaque-forming cells in the spleen of treated mice compared with controls merely sensitized or given the same substrate of normal animals. In this way it was shown that the SF is a soluble factor in the serum with a molecular weight of more than 20,000, but that it is no immunoglobulin.

Peritoneal exudate cells from sensitized guinea pigs were incubated in culture with staphylococcal cell walls. Filtered supernatant fluids from the cultures were chemotactic for horse leukocytes. Maximum chemotactic activity was observed in supernatant fluids of cultures incubated 1.5 hours. Activity disappeared after 4.5 and 18 hours additional incubation.

An active C3 cleaving enzyme is generated when properdin factor B (glycine-rich beta-glycoprotein, GBG) is activated (GBG is cleaved) by factor D in the presence of C3b and Mg++. Factor D can be replaced by trypsin in this reaction but even then C3b and Mg++ are required. In the absence of C3b and/or Mg++ trypsin does not generate C3 cleaving activity from GBG although it cleaves GBG and releases its GGG fragment (B) under these conditions as well. Addition of C3b to GGG immediately after its release does not yield a C3 cleaving enzyme. These findings indicate that C3b, GBG and Mg++ interact, and that only in association with C3b can GBG be cleaved in a way that its enzyme activity, residing in a C3b, GGG complex, is expressed. The complex is labile (half-life at 20 degrees C: 9 minutes); Mg++ does not affect its stability nor is it essential for the activity. It was possible to sequentially fix on agarose the essential components of the C3 cleaving enzyme and thus to elucidate the single steps and the order of its formation. C3 was activated and the resulting C3b fragment fixed on agarose by incubating C3 with trypsin covalently bound to the agarose. The agarose-C3b intermediate was capable of binding GBG provided Mg++ was present. GBG could then be cleaved by factor D or trypsin added in solution; the solid-phase-fixed complex obtained had C3 cleaving activity, in the presence as well as absence of Mg++. Omission of any of these steps or components, or changes in the sequence did not give rise to an active enzyme. Mixtures of C3b, GBG and Mg++ have weak C3 cleaving activity by themselves. C3 cleavage in such incubation mixtures proceeds slowly for hours and is not accompanied by cleavage of GBG. There is thus complete analogy between the CVF-dependent and C3b-dependent C3 cleaving systems. C3b and CVF act in the same way, they form a reversible, weakly active C3 cleaving complex with GBG and Mg++, the activity of which is markedly enhanced but becomes subject to decay when GBG is cleaved by trypsin-like enzymes while bound to CVF or C3b.

The lipopolysaccharides from P. aeruginosa, S. minnesota and mucopeptide from Streptococcus group A injected intravenously into rats induce a dose-dependent changes of temperature. Simultaneously, a profound disturbance of sleep occurs. The administration of salicylate, which markedly suppressed the fever does not influence the mucopeptide-caused sleep disturbance. The most prominent change in the sleep pattern is a marked decrease of the total time of paradoxical sleep. The measurement of turnover rates of 5-hydroxytryptamine (5-HT) and noradrenaline (NA) in hypothalamus and midbrain, areas involved in temperature and sleep control, after injection of streptococcal mucopeptide demonstrated a significant increase of 5-HT turnover in both areas during fever and paradoxical sleep deprivation. Small electrolytic lesions of the dorsal raphe nuclei which are the largest collection of neural cells containing 5-HT completely eliminated the pyrogenic potency of mucopeptide. The findings suggest that some bacterial products might increase the body temperature through the interference with activity of 5-HT-containing neurons of the raphe complex.