Immunostimulating agents: what next? A review of their present and potential medical applications.

Many chemical entities, either from natural sources or prepared by synthesis, are known to exert stimulating activities on various functions of the immune system, such as antibody production, resistance to infections, rejection of malignant cells, etc. In this review, the origin, chemical structures and main activities of several immunostimulants are described, with special emphasis on their present or potential medical usefulness. An attempt is made to envisage the future of this type of pharmacological agents, excluding however from the presentation the endogenous modulators of the immune system (cytokines), the production and activities of which are influenced by the immunostimulants themselves.

Characterization and localization of the rat, mouse and human testicular phosphatidylethanolamine binding protein.

A cytosolic 23kDa protein was initially purified from bovine brain and shown to bind phosphatidylethanolamine. Later, it was also characterized in rat and human brain, and it is now known to be widespread, having been found in numerous tissues in several species. Here, we report the high level of mRNA and phosphatidyl ethanolamine binding protein expression in rat testis and to a lesser extent in mouse testis. In human testis, although it was not detectable by Northern blot analysis, the mRNA was shown to be present when PCR amplification was performed. Immunohistochemical experiments revealed that the testicular phosphatidylethanolamine binding protein (tPBP) is principally expressed in the elongated spermatids of both rat and mouse testis. This finding, and the association of tPBP with cellular membranes, suggest its possible implication in membrane remodelling during spermatid maturation.

In situ localization of NF-H neurofilament subunit mRNAs in rat brain.

The expression of NF-H neurofilament subunit mRNAs was investigated in the rat brain at different ontogenic stages. The levels of NF-H mRNAs vary 15-fold among brain regions with the highest level in the brainstem. In situ localization studies revealed that the NF-H mRNAs are mainly concentrated in the brainstem motoneuron nuclei. By increasing the sensitivity of the hybridization method, NF-H mRNAs could also be localized in neurons present in the cortex, thalamus and hippocampus areas. Minor amounts of NF-H mRNAs were already detected at 17-day embryonic stages.

Structural variability of the neutral carbohydrate moiety of cow colostrum kappa-casein as a function of time after parturition. Identification of a tetrasaccharide with blood group I specificity.

New neutral oligosaccharides from cow colostrum kappa-casein were identified and characterized by 500-MHz 1H-NMR spectroscopy. Their structures are Gal beta(1----3)GalNAc-ol, Gal beta(1----3)[GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Fuc alpha(1----3)[Gal beta(1----4)]GlcNAc beta(1----6)]GalNAc-ol. The tetrasaccharide and the cow colostrum kappa-caseinoglycopeptide which contains this oligosaccharide inhibit the hemagglutination of blood group I human erythrocytes. In cow mature milk only the disaccharide is characterized. The variability of these neutral oligosaccharides in cow kappa-casein as a function of time after calving is studied.

Casein, a prohormone with an immunomodulating role for the newborn?

Maternal colostrum and milk, the earliest food of the newborn, should not only be considered as supplying nutrients, but also as agents providing protection against aggressions from the new environment. Indeed by enzymatic digestion of the main milk proteins, the caseins, biologically active peptides are released; they may be implicated in the stimulation of the newborn's immune system. From this point of view a 'strategic active zone' has been characterized in beta-casein. A possible role of casein as a 'prohormone' for the newborn is suggested.

Immunostimulating hexapeptide from human casein: amino acid sequence, synthesis and biological properties.

A hexapeptide obtained from human casein by enzymatic digestion has been purified, sequenced and synthesized; its structure is: Val-Glu-Pro-Ile-Pro-Tyr. In vitro this hexapeptide stimulates the phagocytosis of opsonized sheep red blood cells by murine peritoneal macrophages. Administered intravenously to adult mice, it enhances the resistance to infection with Klebsiella pneumoniae.

Localisation of the prosthetic sugar groups of bovine colostrum kappa-casein.

The study of glycopeptides obtained after enzymic digestions of bovine colostrum kappa-caseinoglycopeptide obtained 15 minutes after calving, demonstrated the presence of two prosthetic sugar groups linked to threonine residues, instead of only one and up to 10 in the case of bovine (normal) and human caseinoglycopeptides, respectively. The secondary structure of bovine kappa-caseinoglycopeptide was reinvestigated.

Characterization of alpha 1-microglobulin in human colostrum and milk.

The mean concentration of alpha 1-microglobulin in human colostrum and milk, estimated by electroimmunoassay, was found to be about 0.4-0.6 mg/l and 0.1-0.2 mg/l, respectively. Both liquids contained alpha 1-microglobulin in mono- and dimeric forms, while the presence of higher polymeric forms, as characterized in plasma, could not be demonstrated.

Adjuvant and immunostimulating activities (in the absence of freund's incomplete adjuvant) of chemically modified low molecular weight mycobacterial peptidoglycans.

Relatively low molecular weight peptidoglycan fragments extracted from two strains of Mycobacterium tuberculosis var. hominis were chemically coupled with lauric acid. The fatty acid conjugates were compared with the native substances with respect to some immunopotentiating activities. In vitro, the mitogenic effect on murine spleen lymphocytes was significantly enhanced following conjugation. One of the lauric acid conjugates stimulated, upon intravenous administration in mice, the formation of antibody-producing cells in the spleen, while the native substance was devoid of such activity. In adjuvanticity tests performed in the guinea pig in the absence of mineral oil, the fatty acid conjugates generally exerted a higher adjuvant effect on antibody production or on delayed type hypersensitivity reactions than did the native preparations.

Adjuvant activities of chemically modified water-soluble substances from Mycobacterium tuberculosis.

Water-soluble peptidoglycan fragments extracted from the cells of two strains of Mycobacterium tuberculosis var. hominis were chemically conjugated with lauric or with palmitic acid. The coupling reaction was confirmed by physicochemical procedures. The native and the fatty acid-conjugated substances were studied for their adjuvant activity in the induction of delayed type hypersensitivity (DTH) reactions in guinea-pigs and on the production of circulating antibodies in the rabbit. Palmitic acid conjugation of one of the substances increased its adjuvanticity on DTH in the presence of mineral oil; lauric or palmitic acid conjugation rendered the substances adjuvant-active on DTH in the absence of mineral oil. Lauric acid, but not palmitic acid conjugation conferred on both substances an adjuvant activity on antibody production in the absence of mineral oil. Furthermore, lauric acid conjugation of one of the substances led to the appearance of an in vitro mitogen-like activity for murine spleen lymphocytes. In conclusion, fatty acid conjugation exerted a significant modifying effect on the immuno-potentiating activities of these peptidoglycan fragments, and such a chemical procedure may lead to the development of substances exerting a full adjuvant activity without the need of injecting them in an oily vehicle.

Low molecular weight water-soluble peptidoglycans as adjuvants and immunostimulants.

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.

The sugar part of kappa-caseins from cow milk and colostrum and its microheterogeneity.

Cow kappa-casein contains only three different sugars (Gal, GalNAc, NeuNAc). However detailed analyses achieved mainly by gas liquid chromatography suggested a microheterogeneity at the sugar level. After alkaline borohydride treatment, filtration on Bio-Gel P4 and paper chromatography, different carbohydrate parts were obtained. The two main compounds had the following molar compositions: GalNAc (1), Gal(1) and NeuNAc (1) and GalNAc (1), Gal(1) and NeuNAc (2). From these data and our previous sequence studies, some formulae of the polysaccharide part were proposed. One of them was closely related to the sugar sequence of a glycopeptide with MN activity which was in agreement with our observation concerning a cross antigenic reactivity between the N blood group substances and the caseinoglycopeptides. All the polysaccharide parts isolated from colostrum caseinoglycopeptide were much more complex than those obtained from the normal glycopeptide, confirming an evolution of the sugar part as a function of time after parturition.