Shozo Kotani

Structural Requirements of Lipid a for Endotoxicity and Other Biological Activities

For the past ten years, several groups were engaged in synthetic studies of lipid A, namely the lipid portion of bacterial lipopolysaccharides (LPS) that has been assumed to be the bioactive center of LPS, but has not been unanimously approved. Among them, Shiba, Kusumoto, and colleagues, Osaka, Japan have synthesized most energetically and successfully a variety of counterparts of lipid As, biosynthetic lipid A precursors, and their analogs. The endotoxic and related bioactivities of these synthetic compounds were studied by Japanese and German groups, including ours. In 1985, one of the compounds, having an acylation and phosphorylation pattern in beta(1-6)-D-glucosamine disaccharide which was proposed for Escherichia coli F515 lipid A was found to be exhibit full endotoxic and related bioactivities identical to those of the bacterial product. The study was extended by synthesis and examination of bioactivities of variously acylated D-glucosamine di- and monosaccharide phosphates, which correspond to structural components of lipid As, and their analogs or derivatives. Thus, structural requirements have been fairly well elucidated. In this article, first we will review the progress of synthetic and biological studies, with particular emphasis on chemical structure--bioactivities relationships of lipid As, and then we will discuss possible usefulness of some less or nontoxic lipid A-related synthetic compounds in clinical and preventive medicine.

Susceptibility of Rats, Hamsters, and Mice to Carious Infection by Streptococcus mutans Serotype c and d Organisms

Susceptibility of rats, hamsters, and mice to carious infection by S. mutans serotypes c and d was compared. S. mutans serotype c induced a similar level of carious lesions at experimental periods of 68, 82, and 98 d in rats, hamsters, and mice, respectively. On the other hand, S. mutans serotype d developed a high level of caries at those experimental periods in rats and hamsters, whereas in mice it showed weak caries activity.

Minimum structural requirements for encephalitogen and for adjuvant in the induction of experimental allergic encephalomyelitis

Abstract Mycobacteria in the adjuvant used for induction of experimental autoimmune encephalomyelitis (EAE) in guinea pigs can be replaced by synthetic N -acetylmuramyl- l -alanyl- d -isoglutamine. A combination of synthetic encephalitogenic peptides and muramyl dipeptides induces EAE effectively at a dose on the microgram level. In this system, the synthetic heptapeptide, H-Trp-Gly-Ala-Glu-Gly-Gln-Arg-OH, with a sequence identical to those of residues 116 to 122 of the basic protein of human myelin, was the shortest peptide causing EAE. These compounds form a simple system which should be useful in studies on the mechanism of the cell-mediated autoimmune reaction.

Dental caries induction in experimental animals by clinical strains of Streptococcus mutans isolated from Japanese children.

Oral implantation and the cariogenic activity of clinical strains of Streptococcus mutans which had been isolated from Japanese children and labeled with streptomycin‐resistance were examined in specific pathogen‐free Sprague‐Dawley rats. All the seven strains tested were easily implanted and persisted during the experimental period. Extensive carious lesions were produced in rats inoculated with clinical strains of S. mutans belonging to serotypes c, d, e, and f, and maintained on caries‐inducing diet #2000. Noninfected rats did not develop dental caries when fed diet #2000. Type d S. mutans preferentially induced smooth surface caries in the rats. Strains of other serotypes primarily developed caries of pit and fissure origin. Caries also developed in rats inoculated with reference S. mutans strains BH‐TR and FAIR (type b) that had been maintained in the laboratories for many years. However, the cariogenicity of the laboratory strains was found to have decreased markedly. All three S. sanguis strains could be implanted, but only one strain induced definite fissure caries. Two S. salivarius strains could not be implanted well in the rats and therefore they were not cariogenic. Four different species of lactobacilli also failed to induce dental caries in rats subjected to similar caries test regimen on diet #2000. S. mutans strain MT6R (type c) also induce caries in golden hamsters and ICR mice, but of variable degrees.

Correlation of stereochemically specific structure in muramyl dipeptide between macrophage activation and adjuvant activity.

Summary N-acetylmuramyl- l -alanyl- d -isoglutamine, a synthetic substance of a minimal structure required for the adjuvant activity of bacterial cell walls was found to activate macrophages in mice, whereas its diastereomer, N-acetylmuramyl- l -alanyl- l -isoglutamine, which is inactive as adjuvant did not activate them.

Demonstration of serotype d and g specificities of Streptococcus mutans by immunodiffusion

Abstract Although types d and g strains of Streptococcus mutans have been shown to be genetically homogeneous, a serological distinction between these two serotypes was demonstrated in immunodiffusion tests. Antiserum against Strep. mutans strain B13 (serotype d ) strongly cross-reacted with the autoclaved antigen obtained from type g strain OMZ65 and vice versa. Other strains of types d and g showed similar patterns in immunodiffusion tests. When the antiserum against type d or g strain was adsorbed with cross-reacting serotypes a , d or g cells, the adsorbed antiserum gave a specific precipitin band against the homologous antigen. Strep. mutans strain 6715 which had been designated type d was reclassified as type g because of the development of a single precipitin band between 6715 antigen and anti-OMZ65 (serotype g ) serum, adsorbed with types d and a cells. Furthermore, the four AHT substrains in our collection which had been classified as serotype a were found to be type g , on the basis of immunodiffusion pattern using type g and a specific antisera.

Structural specificity of synthetic peptide adjuvant for induction of experimental allergic encephalomyelitis

Abstract The peptide N -acetylmuramyl- l -alanyl- d -isoglutamine (MDP), which has adjuvant activities, and 17 of its derivatives and analogs were synthesized and assayed to elucidate the structure necessary for adjuvant activity in induction of experimental allergic encephalomyelitis (EAE) in guinea pigs. The results revealed the importance of the d configuration and the α-carboxamide group of the isoglutaminyl residue of MDP for adjuvant activity. Replacement of the l -alanyl residue of MDP by d -alanine, but not by l -serine or glycine, resulted in a marked decrease in the activity. The β-methyl glycoside of MDP was found to be more active than the α-methyl derivative. 6- O -Stearoyl- N -acetylmuramyl- l -alanyl- d -isoglutamme showed activity.

Macrophage-stimulating effect of a synthetic muramyl dipeptide and its adjuvant-active and -inactive analogs for the production of T-cell activating monokines.

Abstract A synthetic muramyl dipeptide (MDP), which has the minimal adjuvant-active structure that can substitute for Mycobacterium in complete Freunds adjuvant, could stimulate peritoneal macrophages of the guinea pig to produce T-cell activating soluble factors. The culture supernatant of MDP-stimulated macrophages could help the proliferative response of the T-lymphocyte-enriched fraction of lymph nodes to PHA. It also helped the antigenic activation of immune T lymphocytes deprived of macrophages to produce macrophage migration inhibitory factor (MIF). Gel filtration of the culture supernatant showed that both of the helping activities were found mainly in the fraction emerging at the 53,000- to 85,000-MW range. A series of MDP analogs which are known to be adjuvant active or inactive was tested for their ability to stimulate macrophages to produce these factors. The adjuvant activity of these synthetic compounds was found to be parallel to the ability to stimulate the production of these T-cell activating monokines. Since both of these activities are highly dependent on the precise stereochemical structures of the MDP analogs, the parallelism of these activities would suggest the possibility that the immunopotentiating effect of the MDP and its analogs is related at least partially to their activity to stimulate macrophages to produce T-cell activating monokines.

Activation of the human complement cascade by bacterial cell walls, peptidoglycans, water-soluble peptidoglycan components, and synthetic muramylpeptides--studies on active components and structural requirements.

Cell walls isolated from 29 strains of 24 gram‐positive bacterial species, whose peptidoglycans belong to the group A type of Schleifer and Kandlers classification, with one exception (Arthrobacter sp.), were shown to activate the complement cascade in pooled fresh human serum mainly through the alternative pathway and partly through the classical one. The complement‐activating effect of cell walls (5 species) possessing group B type peptidoglycan, except those of Corynebacterium insidiosum, was weaker than that of the walls with group A type peptidoglycan. Preparations of peptidoglycan isolated from cell walls of Staphylococcus aureus, Streptococcus pyogenes, and Lactobacillus plantarum also activated the alternative pathway of the complement cascade, but less effectively than the respective parent cell walls. A water‐soluble “polymer” of peptidoglycan subunits (SEPS), which was prepared from Staphylococcus epidermidis peptidoglycans by treatment with a cross‐bridge degrading endopeptidase, retained most of the complement‐activating ability of the parent cell walls. A peptidoglycan “monomer,” SEPS‐M, which was obtained by hydrolysis of the glycan chain of SEPS with endo‐N‐acetylmuramidase to disaccharide units did not activate complement. In conformity with this finding, neither synthetic N‐acetylmuramyl‐l‐alanyl‐d‐isoglutamine (MDP) nor MDP‐l‐Lys‐d‐Ala activated the complement cascade. Among several lipophilic derivatives of MDP, 6‐O‐(3‐hydroxy‐3‐docosylhexacosanoyl)‐MDP‐l‐Lys‐d‐Ala (BH48‐MDP‐l‐Lys‐d‐Ala) and 6‐O‐(2‐tetradecylhexadecanoyl)‐MDP (B30‐MDP) were shown to activate complement through the alternative as well as the classical pathway and exclusively through the classical pathway, respectively. The finding that a d‐isoasparagine analog of B30‐MDP caused the same effect as the parent molecule strongly suggests that the activation of complement by B30‐MDP is different from that caused by cell wall peptidoglycans and a water‐soluble “polymer” of peptidoglycan subunits.

Polyarthritis induced in the rat with cell walls from several bacteria and two Streptomyces species.

Summary Various cell wall preparations from 9 different gram-positive strains were tested for AA induction in the Lewis rat by the lymph node injection method. A striking arthritogenic capability of the cell walls of M. bovis, BCG, and C. diphtheriae was noted to be essentially equivalent to that of M. tuberculosis. The cell walls from Streptomyces fradiae and lavendulae, L. plantarum, and S. aureus were less potent, and several other bacterial strains were not arthritogenic at all. However, when high molecular weight water soluble fractions were isolated from some of these strains, they too were often found to be arthritogenic, when administered in water-in-oil emulsions. We thank Barbara Vande Sande, Robin Yeaton, and Shigeki Nagao for skilled technical assistance.