Structural requirements of endotoxic lipopolysaccharides and bacterial cell walls in induction of interleukin-1.

A variety of compounds, synthetic, semisynthetic or bacterial, which corresponds to structural components of endotoxic lipopolysaccharides (LPS) and bacterial cell wall peptidoglycans were studied for their activity to enhance interleukin-1 (IL-1) production of murine peritoneal macrophages and the ability to activate the complement cascade in fresh adult human serum. Not only bacterial LPS and cell walls or peptidoglycans but also their structural components with appropriate size and structure induced IL-1 production by macrophages and activated the human complement cascade, which may lead to the IL-1 production by monocytes/macrophages.

Use of a synthetic peptidoglycan-precursor immunogen and its antibodies as a probe of infectious diseases in man.

Antibodies to a peptide sequence found in peptidoglycan (PG)- precursors (Lys-D-ala-D-Ala) have been found in man and their titers are elevated in several human diseases. Antibodies in rabbits were elicited by a synthetic immunogen containing these PG-precursor sequences, affinity purified with the precursor linked to Sepharose and shown to cross-react with bacterial by-products, such as the high molecular weight soluble peptidoglycan (SPG) from Staphylococcus aureus, which contain these sequences. SPG are secreted by some gram-positive bacteria when grown in the presence of penicillin. Serological studies have indicated that SPG may be the natural immunogen in man. The affinity purified antibodies cross-reactive with Lys-containing SPG plus vancomycin which has a similar specificity have been used to develop an ELISA capable of detecting as little as 50 pg/ml of SPG. Using this ELISA, about half of the human volunteers that took an oral 250 mg dose of penicillin V had detectable levels of SPG in their urine six hours later. There are structural differences among SPG which may be useful in identifying the bacteria responsible for the SPG. The SPG from Bacillus subtilis has diaminopimelic acid (A2pm) instead of lysine. Using the antibody to the synthetic immunogen, the A2pm-containing SPG gave a strong color reaction with the ELISA. However, the sensitivity was about an order of magnitude less than that for the Lys-containing SPG from S. aureus.

Adjuvant activity of 6-O-mycoloyl-N-acetylmuramuyl-L-alanyl-D-isoglutamine.

Immunological properties of synthetic 6-O-mycoloyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (6-O-mycoloyl-N-acetylmuramyldipeptide) and 6-O-mycoloyl-N-acetylmuramic acid were examined in guinea pigs and mice in comparison with those of BCG cell-wall skeleton, N-acetylmuramyl-L-alanyl-D-isoglutamine, and 6-O-stearoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine. 6-O-Mycoloyl-N-acetylmuramyldipeptide showed a potent adjuvant activity for the induction of delayed type hypersensitivy to N-acetyl-L-tyrosine-3-azobenzene-4'-arsonic acid (ABA-N-acetyltyrosine). It was also found that 6-O-mycoloyl-N-acetylmuramyldipeptide treated with oil droplets or suspended in phosphate-buffered saline was as effective as oil-attached BCG cell-wall skeleton for the generation of cell-mediated cytotoxic effector cells to mastocytoma P815-X2 cells in the spleen of C57BL/6J mice in vivo. However, 6-O-mycoloyl-N-acetyl-muramyldipeptide was less active as adjuvant than BCG cell-wall skeleton and N-acetylmuramyldipeptide in enhancing the circulating antibody formation to T-independent antigen, 2,4-dinitrophenyl-lysyl (DNP-Lys)-Ficoll in vivo, and on the generation of helper function of carrier-primed T-cells, and was inactive as a mitogen on normal mouse spleen cells. On the other hand, although 6-O-mycoloyl-N-acetylmuramic acid was shown to be inactive as adjuvant on immune systems described above, it was active as a mitogen on normal mouse spleen cells.

Immunochemical studies with synthetic immunogens chemically related to peptidoglycan.

Rabbits were immunized with synthetic immunogens HSA-(Gly-L-Ala-L-Ala-D-Ala-D-Ala)39 and HSA-(Gly-gamma-D-Glu-L-Ala-D-Ala-D-Ala)40, respectively. Antibodies against HSA-(Gly-L-Ala-L-Ala-D-Ala-D-Ala)39 showed a strong precipitin reaction with the homologous antigen, with HSA-(Gly-gamma-D-Glu-L-Ala-D-Ala-D-Ala)40 and with solubilized peptidoglycan containing peptide subunits with C-terminal D-alanyl-D-alanine. The albumin-peptide conjugates also cross-reacted with rabbit antisera to Streptococcus A-variant, which contain antibodies directed against the peptide moiety of peptidoglycan. The proof for identical determinant groups of peptidoglycan of Streptococcus A-variant and HSA-(Gly-L-Ala-L-Ala-D-Ala-D-Ala)39 was furnished by Ouchterlony gel diffusion studies and by the appropriate inhibition tests of the quantitative precipitin reaction. Immunization of rabbits with HSA-(Gly-gamma-D-Glu-L-Ala-D-Ala-D-Ala)40 yielded antisera which, besides the specificity of antisera to HSA-(Gly-L-Ala-L-Ala-D-Ala-D-Ala)39, showed an additional specificity.