Shiro Oka

Parallel antitumor, granuloma-forming and tumor-necrosis-factor-priming activities of mycoloyl glycolipids fromNocardia rubra that differ in carbohydrate moiety: Structure—activity relationships

SummaryMultiple intravenous injections (30 µg, ten times) in ICR mice of trehalose dimycolate and glucose monomycolate fromNocardia rubra, containing C36–48 mycolic acids, showed a prominent antitumor effect on a subcutaneously implanted sarcoma-180, an allogeneic sarcoma of mice with a significant granuloma formation in lungs, spleen and liver. On the other hand, mycoloyl glycolipids other than glucose monomycolate and trehalose dimycolate, such as mannose or fructose mycolate, showed no significant activity for tumor regression or granuloma formation in mice.Trehalose dimycolate and glucose monomycolate fromN. rubra, and glucose monomycolate with C56–60 mycolic acids fromRhodococcus terrae also showed a distinctive priming activity for tumor necrosis factor (TNF), when lipopolysaccharide fromEscherichia coli was administered as an eliciting agent. The TNF activity in the sera of mice was abrogated almost completely by anti-(murine TNFα) antibody with protein-A—agarose. Again in contrast, mannose and fructose mycolate fromN. rubra and glucose monomycolate with C30–34 mycolic acids fromRhodococcus equi did not show such activities in mice.Meth-A, a syngeneic fibrosarcoma of BALB/c mice, was less sensitive to administration of glycolipids than sarcoma-180. These results indicated that the existence of a glucose or trehalose molecule was necessary for the expression of immunomodifying activities among various mycoloyl glycolipids differing in carbohydrate structure. However, since the administration of lipopolysaccharide was essentially required as an eliciting agent for the induction of TNF, while no eliciting agent was required for the antitumor activities, TNF does not seem to contribute directly to the antitumor activities of mycoloyl glycolipids in our systems. There was, however, a parallel structure-activity relationship among granuloma-forming, antitumor and TNF-priming activities, indicating that the structures of both the carbohydrate moiety and the mycoloyl residues influenced an initial step, such as macrophage activation, commonly and profoundly.

Production and Partial Characterization of Antibody to Cord Factor (Trehalose 6,6′-Dimycolate) in Mice

Antibody production against the trehalose 6,6′‐dimycolate (TDM, cord factor) of Rhodococcus ruber, a non‐pathogenic species of the Actinomycetales group, was investigated in mice by repeated intraperitoneal injection of TDM in water‐in‐oil‐in‐water micelles without carrier protein. The antigenic TDM was isolated and purified chromatographically from the chloroform‐methanol extractable lipids of R. ruber. The hydrophobic moiety of this TDM was composed of two molecules of monoenoic or dienoic α‐mycolic acids with a carbon chain length ranging from C44 to C48 centering at C46. To detect the antibody, an enzyme‐linked immunosorbent assay (ELISA) system was employed using plastic plates coated with TDM. The antibody reacted against the TDM of R. ruber. The antibody was reactive in similar fashion against glycosyl monomycolates differing in the carbohydrate moiety, such as that of glucose mycolate (GM) and mannose mycolate (MM), obtained from R. ruber. Moreover, the antibody reacted against mycolic acid methyl ester itself when it was used as the antigen in ELISA, and trehalose did not absorb the antibody to TDM or inhibit the reaction. These results indicate that the epitope of TDM recognized by the antibody is mycolic acid, an extremely hydrophobic part of the molecule. Next, we prepared monoclonal anti‐TDM antibody (moAb) in mice myeloma cells to examine its biological activities and the role of humoral immunity in mycobacterial infection. MoAb reacted against the TDM, glycosyl mycolate, and mycolic acid methyl ester in ELISA in the same manner as our polyclonal antibody did. The administration of moAb suppressed granuloma formation in the lungs, spleen, and liver induced by TDM and inhibited the production of interleukin‐1 (IL‐1) and chemotactic factor, which is reported to precede granuloma formation.

Stimulatory effect of gangliosides on phagocytosis, phagosome–lysosome fusion, and intracellular signal transduction system by human polymorphonuclear leukocytes

Gangliosides are known to be differentiation-inducing molecules in mammalian stem cells. We studied the interaction between the molecular structure of glycosphingolipids (GSLs) and their promoting mechanisms of the phagocytic processes in human polymorphonuclear leukocytes (PMN). The effect of various gangliosides from mammalian tissues on adhesion, phagocytosis, phagosome–lysosome (P–L) fusion and superoxide anion production was examined by human PMN using heat-killed cells of Staphylococcus aureus coated with GSLs. Gangliosides GM3, GD1a, GD3 and GT1b showed a marked stimulatory effect on the phagocytosis and P–L fusion in a dose-dependent manner, while ganglioside GM1, asialo GM1 and neutral GSLs did not. The relative phagocytic rate of ganglioside GM3-coated S. aureus was the highest among the tested GSLs. Both P–L fusion rate and phagocytosis of S. aureus were elevated significantly when coated with ganglioside GD1a, GD3 or GT1b, and GT1b gave a five times higher rate than that of the non-coated control. These results suggest that the terminal sialic acid moiety is essential for the enhancement of phagocytosis and that the number of sialic acid molecules in the ganglioside is related to the enhancement of the P–L fusion process. On the other hand, the superoxide anion release from PMN was not affected by ganglioside GM2, GM3, GD1a or GT1b. Furthermore, to clarify the trigger or the signal transduction mechanism of phagocytic processes, we examined the effect of protein kinase inhibitors such as H-7, staurosporine (protein kinase C inhibitor), H-89 (protein kinase A inhibitor), genistein (tyrosine kinase inhibitor), ML-7 (myosin light chain kinase inhibitor), and KN-62 (Ca2+/calmodulin-dependent protein kinase II inhibitor) on ganglioside-induced phagocytosis. H-7, staurosporine and KN-62 inhibited ganglioside-induced phagocytosis in the range of concentration without cell damage, while H-89, genistein and ML-7 did not. Moreover, H-7 and KN-62 inhibited ganglioside-induced P–L fusion. These results suggest that protein kinase C and Ca2+/calmodulin-dependent protein kinase II may be involved in the induction of phagocytosis and P–L fusion stimulated by gangliosides.

Induction of Interferons (IFNs) and Tumor Necrosis Factor (TNF) in Mice by a Novel Glycolipid Trehalose 2, 3, 6'-Trimycolate from Rhodococcus aurantiacus (Gordona aurantiaca)

The immunomodifying activity of a novel mycoloyl glycolipid, trehalose 2,3,6′‐trimycolate (GaGM), from a unique psychrophilic acid‐fast bacterium, Rhodococcus aurantiacus, was examined. ICR mice were primed intravenously (i.v.) or intraperitoneally (i.p.) with liposomes containing GaGM (300 μg/mouse), and were administered LPS dissolved in saline (25 μg/mouse, i.v.) 2 weeks later. Two hours after injection of LPS, interferons (IFNs) and tumor necrosis factor (TNF) were induced significantly in mice sera. The increase in activities of IFNs and TNF was approximately paralleled with granuloma formation in spleen of mice primed with GaGM. However, IFNs and TNF were not induced either in mice primed with GaGM but not elicited with LPS, or in those primed with GaGM and elicited by GaGM. Both activities induced were lower in mice primed with trehalose mono‐ or dimycolate from R. aurantiacus (GaTMM, GaTDM) or TDM from Nocardia rubra than in GaGM‐primed mice. Time course study showed that the maximum activity of each interferon (α, β, or γ) was observed at different stages after LPS administration; IFN‐α, IFN‐β, and IFN‐γ appeared 3, 2, and 6 hours most abundantly after LPS administration, respectively.

Stimulation of phagocytosis and phagosome—lysosome (P-L) fusion of human polymorphonuclear leukocytes by sulfatide (galactosylceramide-3-sulfate)

Recently, extensive attention has been paid to the physiological function of glycosphingolipids (GSLs) of mammalian cell membranes. Among a variety of GSLs, sulfatide (galactosylceramide-3-sulfate) has been proposed to be a specific receptor or binding molecule to microorganisms. However, no report has appeared on the direct stimulation by sulfatide for cellular function differentiation in phagocytic cells. We found that sulfatide showed a marked stimulation for phagocytic processes of human peripheral polymorphonuclear leukocytes (PMN) using heat-killed cells of Staphylococcus aureus coated with isolated lipid. Among mammalian acidic GSLs, sulfatide showed the highest stimulative activity for adhesion, phagocytosis and phagosome-lysosome (P-L) fusion by PMN. On the other hand, neutral GSLs did not stimulate essentially. Relative phagocytic rate of sulfatide-coated staphylococci was six times higher than that of the non-coated control and P-L fusion rate was ten times at maximum, respectively. Although the promotion mechanism of sulfatide for such phagocytosis or P-L fusion is not clear, it was strongly suggested that the existence of negative charges on carbohydrate moiety may be essential for the induction of differentiation of phagocytic cell function via signal transduction systems.

Research articleEnzyme-linked immunosorbent assay (ELISA) using a glycolipid antigen for the serodiagnosis of melioidosis

The serodiagnosis of melioidosis is commonly performed with tests using protein or polysaccharide as antigen. However, due to the low sensitivity, specificity and difficulty in the preparation of the antigens, more simple, precise and reproducible diagnostic tests were required. A purified glycolipid antigen (GL) which is a specific lipid component of Burkholderia pseudomallei has been used in an ELISA. With this antigen, specific immunoglobulin G (IgG) was detected in 49 out of 50 melioidosis sera. IgG was also detected in 2 out of 185 (Japanese) and 16 out of 181 (Vietnamese) control sera. Thus, the sensitivity was 98.0%, and specificity was 98.9% and 91.1% in the Japanese and Vietnamese sera, respectively. When the ELISA and indirect haemagglutination (IHA) tests were combined, a sensitivity of 100% and specificity of 97.8% were achieved. The advantages of the glycolipid antigen are ease of preparation, stability, high sensitivity and specificity.