Jean-François Petit

Fate of the synthetic immunoadjuvant, muramyl dipeptide (14C-labelled) in the mouse.

Synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide or MDP) represents the smallest unit that can substitute for whole Mycobacteria in Freunds complete adjuvant. In this paper the fate of 14C-labelled (on the muramyl moiety) MDP is reported. Following intravenous or subcutaneous injection into mice, more than 50% of 14C-MDP was recovered in the urine after 30 min and more than 90% after 2 h. The labelled compound was found unchanged in the urine, as shown by detailed analyses. However, MDP was sequestered for a longer time at the site of injection when administered as a water-in-oil emulsion. Considering the relatively rapid elimination observed, it is suggested that the biological effects of MDP and related compounds, when administered in an aqueous medium, may be due to their activity at minute concentrations and/or an immediate action at the cellular level.

Preparation and characterisation of liposomes containing mannosylated phospholipids capable of targetting drugs to macrophages

We have prepared liposomes from mannosylated phosphatidylmyo-inositol, derived from mycobacteria, and cholesterol. The size of the particles so formed could be controlled by membrane filtration. The vesicles encapsulated a significant amount of aqueous phase (about 8 microliter per mg phospholipid). Markers of the liposomal membrane and aqueous phase rapidly associated with mouse peritoneal macrophages and, more slowly, with rat alveolar macrophages. The uptake was saturable at high liposome concentrations, although phagocytosis of latex particles of the same mean diameter was not saturable at these concentrations. An excess of unlabelled liposomes composed of phosphatidylcholine and phosphatidylserine, which were also taken up readily by macrophages, did not inhibit the uptake of mannosylated liposomes. The uptake of fluorescent mannosylated bovine serum albumin was inhibited by these liposomes, suggesting a specific interaction with the macrophage mannose-fucose receptor. We conclude that this type of liposome would be useful for the delivery of immunomodulators to reticuloendothelial cells.

Occurrence of N-glycolylmuramic acid in bacterial cell walls: a preliminary survey

The N-acyl substituent of muramic acid in the cell walls of some species of Actinomycetales and of the related family of Corynebacteriaceae has been studied. The N-glycolyl derivative, recently identified in Mycobacterium smegmatis, has been found in the three other species of Mycobacterium studied: M. kansasii, M. tuberculosis BCG, and M. phlei. The classical N-acetyl derivative has been found in Streptomyces albus and Corynebacterium fermentans.

Protection by glutathione against the antiproliferative effects of nitric oxide: Dependence on kinetics of no release☆

Pretreatment by L-buthionine sulfoximine (BSO), which inactivates gamma-glutamylcysteine synthetase and, therefore, inhibits glutathione (GSH) synthesis, greatly increased the sensitivity of tumor cells to the antiproliferative effects of several NO-donating compounds. The sensitization that resulted from depletion of cellular GSH pools was observed in tumor cells exhibiting different degrees of resistance to NO. In contrast, GSH depletion of tumor target cells did not affect their sensitivity to the cytostatic activity of activated macrophages and other NO-producing cells (EMT6 cells treated by interferon gamma and LPS). The kinetics for NO generation is a parameter that may differentiate NO-producing cells and short-lived NO donors. To study the relationship between the magnitude of NO fluxes and the increased toxicity on BSO-pretreated cells, two NO-releasing zwitterions derived from polyamines (NONOates) with different half-lives were selected. NO fluxes as a function of time were simulated, according to the donor half-life and initial concentration, and antiproliferative effects on control and BSO-treated cells were compared. GSH depletion increased the sensitivity of tumor cells in the case of the less stable NO donor only. We, thus, propose that intracellular GSH is specifically protective against high fluxes of NO.

Low response of BALB/c macrophages to priming and activating signals.

Trehalose dimycolate (TDM), a mycobacterial glycolipid, is a powerful macrophage‐priming agent. However, its efficiency seems limited in the case of BALB/c mice. Peritoneal macrophages harvested from TDM‐treated BALB/c mice did not control BCG growth in vitro as efficiently as similar macrophages from two other mouse strains, (B6 × D2)F1 and C57BL/6, which are respectively Bcg r and Bcg s. BALB/c macrophages elicited by TDM also exhibited a low capacity to produce hydrogen peroxide and, after activation by lipopolysaccharide (LPS), weak cytostatic activity against P815 mastocytoma cells. Finally, alkaline phosphodiesterase, a marker of resident and inflammatory macrophages, was still expressed at a high level in macrophages of BALB/c mice treated with TDM. Low responsiveness of BALB/c macrophages to stimuli was not observed with TDM only; activation for tumor cytotoxicity of thioglycolateelicited macrophages from BALB/c mice required also higher doses of interferon‐γ, and LPS. l‐Arginine–dependent production of nitric oxide was inducible in macrophages from BALB/c mice, but the conditions required for its induction were more stringent. Thus, the reduced antiproliferative effects of BALB/c macrophages may be due to uncomplete induction of NO synthase after suboptimal stimulation.

Importance, localization and functional properties of the cell-associated form of plasminogen activator in mouse peritoneal macrophages

In inflammatory macrophages, plasminogen activator exists in two active forms, a soluble form released into the extracellular medium and a cell-associated form. This communication describes some properties of the cellular form of plasminogen activator, in intact macrophages and in cell lysates. Cellular plasminogen activator is a membrane protein, associated with the outer face of the plasma membrane; in intact macrophages, it participates in the activation of exogenous plasminogen and, thus, has to be considered as an ectoenzyme. A plasminogen activator activity can be detected in cell lysates (macrophage monolayers lysed in 0.1% Triton X-100) only when plasmin production is followed by the use of small synthetic substrates because a soluble inhibitor, released during extraction, blocks plasmin fibrinolytic activity. In these lysates, plasminogen activator molecules exist as high molecular weight unstable complexes exhibiting a high affinity for plasminogen.

Nonspecific immunoprevention of l1210 leukemia by cord factor (6-6' dimycolate of trehalose) administered in a metabolizable oil.

SummaryThe present report confirms that L1210 leukemia can be prevented by administration — in saline — of “interphase material” (IPM) which is a composite preparation extracted from nonpathogenic mycobacteria, such as M. smegmatis. IPM was more active than the different BCG cell wall preparations tested under the same conditions (i.e., in saline). The activity of the cord factor, which could be present in IPM, was evaluated in comparison. Since this chemically well-defined fraction (6–6′ dimycolate of trehalose) is insoluble in water, it was injected in an emulsion of mineral or vegetable oil. Cord factor administered alone in Bayol F showed a marked antitumor activity against this systemic, syngeneic murine leukemia. Moreover, this protection could also be demonstrated in an emulsion of saline and a metabolizable oil.

MDP and LPS act synergistically to induce arginine-dependent cytostatic activity in rat alveolar macrophages.

Rat alveolar macrophages can be activated in vitro for cytostatic activity against tumor cells by MDP and LPS acting in synergy. MDP can be substituted for by analogs active as adjuvants. Macrophage activation correlates with an increased production of nitrite and citrulline. NG-monomethyl-L-arginine, a specific inhibitor of the L-arginine metabolism having nitrite and citrulline as end products, abolishes the cytostatic activity. We therefore conclude that, in this model, the main effector mechanism of the cytostatic activity is mediated by molecules derived from L-arginine through the newly described NOo-generating pathway.

Photoaffinity labelling of macrophages and B-lymphocytes using 125I-labelled aryl-azide derivatives of muramyldipeptide

Two 125I-labelled aryl-azide derivatives of MDP have been synthesized. Photoaffinity labelling experiments demonstrated cell surface binding sites for muramylpeptides on activated B-lymphocytes and intracellular muramylpeptides binding protein(s) of 40-45 kD in rat alveolar macrophages.

Structural study of the poly-l-Glutamic acid of the cell wall of Mycobacterium tuberculosis var hominis, strain Brevannes.

Summary Trypsin-chymotrypsin-treated delipidated cell walls from M.tuberculosis Brevannes contain a poly-L-glutamic acid which can be extracted from the cell walls after partial acid hydrolysis as a molecule of about 35,000 daltons. Elementary analysis of this polymer suggests that it is at least partly amidated. The partial amidation and the α-linkage could be established up to the 14th residue by automatic Edman degradation.