Zeitschrift fur Immunitatsforschung, experimentelle und klinische Immunologie最新文献

Rabbits hyperimmunized with Micrococcus lysodeikticus produce, in addition to anti-cell wall antibodies, considerable amounts of anti-immunoglobulins or rheumatoid factors which can interfere with immune reactions. In particular, in immunodiffusion, they can reveal non precipitating systems for which the immunodiffusion typing sera are usually not tested. In our hands, rabbit allotypes d12 and A8 give rise to visible immunodiffusion reactions in presence of rheumatoid factors. Inhibition of this reaction can be used to type for these markers in sera which do not contain the rheumatoid factors.

Staphylococcus epidermidis peptidoglycans solubilized by sonication or lysozyme digestion, and synthetic peptidoglycan analogs such as HSA-carboxymethyl-Gly-L-Ala-L-Ala-D-Ala-D-Ala (HSA-pentapeptide) or L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala (pentapeptide) have been labeled with 125I and tested for their applicability in the radioactive antigen binding assay. Use of radioiodinated Staph. epidermidis peptidoglycans was found to be considerably impeded by the presence of at least 2 different antigenic sites on such molecules, the pentapeptide and the glycan determinant. Application of labeled HSA-pentapeptide was limited by the necessity to use PEG for precipitation of Ag-Ab-complexes and by short linear potions of binding curves. However, the synthetic pentapeptide hapten, radioiodinated by the active ester method of BOLTON and HUNTER, proved to be a most useful regent for the selective measurement of pentapeptide antibody. Inhibition studies indicated that the immunological specificity of the labeled hapten was retained. Pentapeptide binding curves were linear from 15-500 g/ml of antibody. Generally, there was good agreement between pentapeptide antibody concentrations measured by radioimmunoassay and quantitative precipitation.

This report describes the interaction of peptidoglycan (Streptococcus group A, Staphylococcus epidermidis and Micrococcus lysodeikticus) with 2 serum mediator systems, namely with the anti-IgG system and with complement. The observation that the majority of rabbits hyperimmunized with A-variant streptococcal vaccine produced anti-group carbohydrate antisera containing anti-IgGs and antibodies directed to peptidoglycan suggested that the production of these 2 latter antibodies was related. This view was supported by the finding of a monoclonal 7S anti-IgG with antibody specificity for the pentapeptide of peptidoglycan as evidenced by inhibition of the coprecipitation of 7S anti-IgG with antigen-antibody complexes by the pentapeptide. Inhibition of the anti-idiotype reaction by the pentapeptide provided further evidence for the antibody specificity of 7S anti-IgG for peptidoglycan. When added to normal human sera all peptidoglycan preparations inhibited the hemolytic activity of the sera. Consumption of C3 in C2 deficient serum and consumption of C2 in normal serum indicated the activation of both known complement pathways. Activation of the classical pathway of complement was more efficient since 50 mug of peptidoglycan consumed approximately 70% of C2 per ml normal serum whereas more than 2 mg of the same preparations was required to inactivate 17-24% of C3 in C2 deficient sera. Each of the different peptidoglycan preparations consumed similar amounts of complement in all 20 sera tested. This finding suggested that activation of the classical complement pathway by peptidoglycan was not mediated by anti-peptidoglycan antibodies present in only 20-40% of normal human sera.

A recently described latex agglutination test was used to determine peptidoglycan antibody titers in sera from healthy human subjects and in sera from patients with a history of streptococcal infections or rheumatoid arthritis. Using latex particles coated with group A streptococcal peptidoglycan 32.8% of the sera from 961 healthy donors reacted positively with titers ranging from 1 : 5 to 1 : 320. Peptidoglycan antibodies were more frequently present in the younger population (45.1% in the 20-29 years old) and considerably decreased in advanced age (15.7% in the 70 years or older). Sera from 82 patients with elevated ASO-titers showed detectable peptidoglycan antibody levels in 40.2%; a statistically significant correlation between ASO and peptidoglycan antibody titers could not be substantiated. Sera from 25 patients with rheumatoid arthritis gave a high incidence (56-92%) of positive results. However there was evidence that this may be due to the action of rheumatoid factor present in such sera.

The maximum porosity of Bacillus subtilis and Bacillus licheniformis cell walls was estimated by two independent and relatively simple methods. Peptidoglycan was isolated from Bacillus subtilis cell wall preparations and used as an insoluble support for exclusion chromatography of dextrans of known average molecular size. In an alternative approach the leakage of radioactively labelled proteins from Bacillus licheniformis cells incubated in butanol-saline mixtures was measured and their size estimated by exclusion chromatography on Sephadex G-100. Under these conditions the permeability barrier of the cytoplasmic membrane was destroyed with preservation of the structural integrity of the outer cell wall. The apparent exclusion threshold of the cell wall of either organism as determined by these means corresponded to molecules with a diffusional radius of not more than 2.5 nm.

The tetrasaccharide-heptapeptide (TH), when injected with mineral oil, exerted a strong adjuvant effect. It stimulated B and T cells, but did not increase the phagocytic activity of the reticulo-endothelial system. While BCG exerted significant preventive effect on the growth of sarcoma 180, leucosarcomatosis an EHRLICH ascitic tumor, TH, at the doses used, was devoid of such activity.

Studies on the immunology of peptidoglycan received impetus from the initial observation that the rabbit Group A-variant streptococcal antisera were a rich source of antibodies to peptidoglycan. Indeed, quantitative precipitin studies revealed concentrations as high as 7-10 mg/ml of antiserum. The amount of antibody after Group A-variant streptococcal immunization is much greater than the amount in the sera following immunization of rabbits with the Group A or C streptococci. Furthermore, earlier studies had shown that the purified peptidoglycan obtained as a residue following extraction of streptococcal cell walls with hot formamide was a poor antigen. Both the hexosamine polymer and the peptide moiety are antigenic. Use of the solid phase synthesized pentapeptide L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala and related similar peptides facilitated the determination of the fine structure of the immunodominant site of pentapeptide. The evidence points to the C-terminal D-Ala-D-Ala as the immunodominant determinant. Because of the similarity of the peptidoglycans of a number of different bacteria, it would be anticipated that they would cross-react immunologically, and this has been shown to be the case. The biological and medical significance of antibodies to peptidoglycan has yet to be determined. Certainly the exposure to this ubiquitous substance which occurs in all the indigenous bacteria of the respiratory and the gastrointestinal tract must mean that from an early age and through advancing years there is a constant stimulation of the immune response to peptidoglycan. Because the immunochemistry of these substances is now firmly established, there is a scientific basis for proceeding with the medical and biological implications of peptidoglycan immunity.

In previous investigations we could show that incubation of cross-linked dextran (Sephadex) with normal human and normal guinea pig serum results in the binding of C3 to Sephadex. This binding was found to be due to activation of C3 via an alternate pathway. In this paper data are presented which show that components bound to Sephadex can be recovered after enzymatic digestion of serum-reacted Sephadex beads. The digests were characterized with immuno-electrophoresis and double immunodiffusion techniques. It could be shown that the main component present in the digest was converted C3. Apart from C3 under our test conditions only minute amounts of C3A but no other serum proteins were detectable. The observation that almost exclusively C3 is bound to Sephadex is further supported by the finding that immunization of rabbits with serum-reacted Sephadex beads results in the exclusive formation of anti C3 antibodies. Implications from these findings and possible applications are discussed.

BCG cell walls contain approximately 30% free lipids like other mycobacterial cell walls. The insoluble skeleton of the cell wall is made up of two covalently linked polymers, a peptidoglycan and an arabinogalactan mycolate, with which are associated non peptidoglycan amino acids and a glucan. We present data on two structural features: 1. The "non peptidoglycan" amino acids; they form two kinds of compounds: peptide chains which can be solubilized by proteolytic enzymes and a trypsin-chymotrypsin insensitive poly-alpha-L-glutamic acid. 2. Presence of meso-DAP-meso-DAP1) interpeptide linkages in the peptidoglycan: this new type represents at least 50% of the interpeptide linkages of the cell wall of the BCG strain.

Peripheral blood T and B lymphocytes were determined in normal humans at different ages. Spontaneous rosette formation with sheep red blood cells (SRBC) was used as a marker for T cells. B cells were detected by immunofluorescent staining of membrane-bound immunoglobulins. Blood samples from old individuals contained significantly lower T lymphocyte numbers than those from children. This diminution of circulating T cells caused a reduction of the total lymphocyte count in the elderly persons. No significant differences were between the T cell values of young and old adults. Whereas the percentages of B cells indicated an increase of this lymphocyte population in old humans, the absolute numbers of B lymphocytes remained almost unchanged during aging.