Arlette Adam

Minimal structural requirements for adjuvant activity of bacterial peptidoglycan derivatives

Abstract We have recently shown that the monomeric subunit of mesodiaminopimelic acid containing bacterial peptidoglycans, i. e. the disaccharide tetrapeptide N-acetyl glucosaminyl-N-acetyl muramyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelyl-D-alanine can replace whole killed mycobacteria in Freunds adjuvant. We now report further structural simplifications of the active molecule; natural N-acetyl-muramyl-tripeptides have been found active; the smallest adjuvant molecule found is a synthetic N-acetyl muramyl-dipeptide prepared by Sinay et al. (1).

Total synthesis and adjuvant activity of bacterial peptidoglycan derivatives

Abstract 2-Acetamido-2-deoxy-3-O-(D-2-propionyl-L-alanine)-D-glucopyranose ( 5 ) and 2-acetamido-2-deoxy-3-O-(D-2-propionyl-L-alanyl-D-isoglutamine)-D-glucopyranose ( 10 ) have been synthesized by condensation of benzyl 2-acetamido-4,6-O-benzylidene-3-O-(D-1-carboxyethyl)-2-deoxy-β-D-glucopyranoside ( 1 ) respectively with the L-alanine derivative 2 and the dipeptide 7 , followed by debenzylidenation and hydrogenolysis. Compound 10 is adjuvant active, whereas 5 is inactive, so that 10 is the smallest adjuvant active structure for the time being.

Adjuvant activity of monomeric bacterial cell wall peptidoglycans.

Abstract We have previously shown that lysozyme solubilized cell walls of Mycobacteria or Nocardiae can replace whole mycobacterial cells or Wax D in Freunds complete adjuvant and it was found quite recently that hydrosoluble peptidoglycans, free of neutral sugars, are also adjuvant active. We show now that the simplest fragment tested — the disaccharide tetrapeptide (I) — increases circulating antibodies to ovalbumin and induces a delayed hypersensitivity toward this antigen. Similar compounds obtained from the basal layer of the cell wall of E. coli are also active. Thus the immunoadjuvant activity of soluble cell wall peptidoglycans is a property of the monomeric unit and is not restricted to acid fast bacteria.

Correlation of structure and adjuvant activity of N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), its derivatives and analogues. Anti-adjuvant and competition properties of stereoisomers.

Abstract The synthetic N-acetyl muramyl-dipeptide (MDP) 1 has been shown to be fully adjuvant active in a water-in-oil emulsion; we now report a study on the adjuvant activity of 1O derivatives and analogues of MDP under similar conditions. NaBH 4 reduction of MDP 1 leads to the inactive muramicitol dipeptide 2 ; β-elimination gives the lactyl-dipeptide 3 , which seems to inhibit adjuvant activity. Shortening of the lactyl side chain of MDP gives nor-MDP, 4 , which is less active. Amongst the analogues in which one amino acid of the dipeptide moiety is replaced by another one, the L-Ser analogue 5 is fully active, whereas replacement of L-Ala by Gly or of D-iso-Gln by D-Glu-OH, or D-Glu (OMe) 2 , or D-Glu (OMe)-NH 2 gives less active compounds ( 6 , 8 , 9 , 1O ). The diastereoisomer 7 where L-Ala is replaced by D-Ala, shows an anti-adjuvant activity.

IL-4 inhibits apoptosis and prevents mitochondrial damage without inducing the switch to necrosis observed with caspase inhibitors.

We previously demonstrated that the broad-spectrum caspase inhibitor, zVAD-fmk, totally deviated apoptosis to necrosis in B lymphocytes. We report here that, in contrast with zVAD-fmk, IL-4 protected B cells from spontaneous and from dexamethasone-induced apoptosis and actually maintained cell viability. This was assessed by morphological and biochemical criteria and accompanied by the maintenance of mitochondrial transmembrane potential (ΔΨCm) and elevated glutathione (GSH) levels. Under these conditions, zVAD-fmk also totally inhibited apoptosis in thymocytes, but it partly preserved cell viability with a parallel increase in the percentage of cells exhibiting high ΔΨCm and elevated GSH levels. Nevertheless, non-rescued cells were deviated to necrosis. Therefore, the pathway leading to either apoptosis or necrosis appears to involve common mitochondrial dysfunctions which could not be reversed by caspase inhibition, suggesting that the pharmacological inhibition of cell death should occur at an earlier stage.

Non-specific MIF-like activity induced by the synthetic immunoadjuvant: N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP)

Abstract N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) which represents the active part of mycobacteria for adjuvanticity is able to inhibit the migration of peritoneal exudate cells. This inhibitory action of MDP is not due to cytotoxicity because colchicine which has been shown to inhibit MIF also restores the migratory properties of MDP treated cells, and pretreatment of peritoneal cells with MDP has no influence on their later migration. There could be a relation between structural requirements for adjuvant activity and macrophage migration inhibition because the D-D-stereo-isomer of MDP which is inactive as an adjuvant, has no inhibitory properties.

In vitro immune response to sheep erythrocytes in macrophage depleted cultures. Restoration with interleukine 1 or a monokine from resident macrophages and stimulation by N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP)

The involvement of macrophages in the adjuvanticity of N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) has been examined. The stimulation of the in vitro primary immune response to sheep red blood cells (SRBC) has been studied, because it is known that macrophages cooperate through the mediation of soluble compounds for the induction of the anti-SRBC response. The cultures depleted of macrophages by passing spleen cells on Sephadex G-10 were unable to give any response to SRBC. Their immune responsiveness was fully restored by the addition of either Interleukine 1 (IL 1) obtained from P388D1 cells or a factor able to replace macrophages (FRM) obtained from resident peritoneal macrophages. MDP alone, at any dose, was unable to induce any response in such macrophage depleted cultures, but it was able to enhance the antibody response of these cultures reconstituted with monokines, with the same characteristics in dose effect and timing dependence than in whole spleen cells.

Chemical Structure of Mycobacterial Cell Walls

Many of the immunostimulant effects of living or killed cells of mycobacteria can be obtained with isolated cell walls: protection against tuberculosis [25, 26], stimulation of nonspecific resistance against infections [14, 22], adjuvant activity [4, 11], stimulation of resistance to tumors [12] and tumor suppression [34].

N-acetylmuramyl-l-alanyl-d-isoglutamine stimulates the immune response of B cells : Use of fast protein liquid chromatography to purify a novel monokine involved in this effect

The immune response of murine B cells, which requires the presence of various cytokines, was stimulated by N-acetylmuramyl-L-alanyl-D-isoglutamine. A novel monokine involved in this effect has been partially purified from the conditioned medium of peritoneal macrophages by using affinity chromatography on Blue-Sepharose and high-performance liquid chromatography. Hydrophobic interaction chromatography was highly efficient in removing almost all protein contaminants. The resulting biologically active material exhibited heterogeneity in ion-exchange chromatography, and apparent isoelectric points between 5.2 and 5.5 on chromatofocusing. Its apparent molecular weight, as determined by size-exclusion filtration, was about 35,000. Preliminary results on the kinetics of appearance of intracellular biological activity suggested that the monokine could also be obtained from cytoplasmic extracts.