Masachika Tsujimoto

Polyclonal B Cell Activation by Cell Wall Preparations of Gram‐Positive Bacteria

The effects of polyclonal B cell activation (PBA) of cell walls and their cell wall fractions obtained from several kinds of gram‐positive bacteria were studied using the anti‐sheep red blood cell (SRBC) or anti‐trinitrophenylated (TNP) SRBC plaque forming cell (PFC) responses of cultured spleen cells from Balb/c, athymic nu/nu, their littermates (nu/+), C3H/He (LPS‐responder), C3H/HeJ (LPS‐non‐responder), (CBA/N × Balb/c) F1 male with an X‐linked defect in B cell function and the F1 female mice. The cell walls of Staphylococcus epidermidis (ATCC 155), Lactobacillus plantarum (ATCC 8014), Micrococcus lysodeikticus (NCTC 2665), Mycobacterium rhodochrous (ATCC 184), Streptomyces gardneri (ATCC 23911) and Nocardia corynebacteriodes (ATCC 14898) had the ability to induce polyclonal B cell responses in the spleen cells of Balb/c, nu/nu, nu/+, C3H/He and C3H/HeJ mice. The cell wall fractions prepared by enzymatic digestion from the cell walls of S. epidermidis, S. gardneri or N. corynebacteriodes were also capable of inducing polyclonal B cell responses. The responses of spleen cells from (CBA/N × Balb/c) F1 male mice to these active preparations, except the cell walls of M. rhodochrous, were much lower than those of the F1 female mice. These findings indicate that the majority of the cell wall preparations lacks PBA ability for spleen cells with the CBA/N defect, except for the cell walls of M. rhodochrous which possess this ability. The PBA‐ability of synthetic peptidoglycan, muramyl dipeptide (N‐acetylmuramyl‐L‐alanyl‐D‐isoglutamine, MDP), was also examined, and a similar activity was observed in MDP.

Enhancement of humoral immune responses against viral vaccines by a non-pyrogenic 6-O-acyl-muramyldipeptide and synthetic low toxicity analogues of lipid A

6-O-Acyl derivatives of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and synthetic, low toxicity lipid-A analogues were examined for their ability to enhance the potency of current viral vaccines. 6-O-(2-Tetradecylhexadecanoyl)-MDP (B30-MDP) in non-irritative vehicles such as physiological saline, phosphate-buffered saline (PBS), squalene-PBS emulsion, Intralipid or liposomes, significantly stimulated the primary and secondary antibody production of guinea-pigs against influenza split or subunit vaccine and inactivated the hepatitis B virus surface (HBs) antigen. Mice seemed less responsive to the adjuvanticity of B30-MDP than guinea-pigs. Two low toxicity lipid A analogues, acylated beta(1-6)-D-glucosamine disaccharide bisphosphates (which do not have amide-bound or ester-bound 3-acyloxyacyl groups unlike fully toxic Escherichia coli-type lipid A), caused significantly enhanced antibody responses, primary or secondary, when administered to mice by incorporation into liposomes with inactivated HBs antigen.

Possible existence of a novel amphipathic immunostimulator in the phenol-water extracts of mycobacteriaceae

The extracts having diverse immunostimulating activities were obtained as a water‐phase fraction from four bacterial species representing the 4 genera (Mycobacterium, Nocardia, Gordona, and Rhodococcus) of Mycobacteriaceae by the phenol‐water method, which is commonly used for extraction of endotoxic lipopolysaccharides (LPS) from gram‐negative bacteria and amphipathic substances from gram‐positives. These fractions, especially those of G. aurantiaca and R. terrae, showed strong stimulatory effects on murine splenocytes, macrophages of mice and guinea pigs, the immunoadjuvant activities in guinea pigs and mice, and the distinct activities inducing a tumor necrosis factor and interferons α/β and γ in primed mice. The fractions from G. aurantiaca and R. terrae exhibited potent pyrogenicity and the ability to activate the clotting enzyme cascade of the horseshoe crab (Tachypleus tridentatus). Some of these biological activities were not very different from the potency of the reference endotoxic LPS derived from Escherichia coli or Fusobacterium nucleatum. But the test fractions neither showed the activity to prepare rabbit skin to the local Shwartzman reaction, nor reacted with anti‐lipid A conventional and monoclonal antibodies. Furthermore, unlike LPS, these fractions stimulated the splenocytes of C3H/HeJ mice (LPS‐Nonresponder). Although the fractions showing the above biological activities have not yet been adequately purified, they contained polysaccharides, whose main constituent sugar is mannose with a smaller amount of arabinose, fatty acids consisting primarily of palmitic, stearic, and tuberculostearic acids, and small amounts of peptides and amino sugars. Since components characteristic of known immunomodulators of bacterial origin, namely endotoxins (lipid As), cell wall peptidoglycans, lipoteichoic acids, cord factors (trehalose dimycolates), or deoxyribonucleic acids, were practically not detected in these fractions, the agent responsible for the above bioactivities is considered to be a novel substance different from the known, bacterial immunomodulators.

Chemical composition and immunobiological activities of sodium dodecyl sulphate extracts from the cell envelopes of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Fusobacterium nucleatum.

The chemical composition and immunobiological activities in vivo and in vitro of sodium dodecyl sulphate extracts (SDS-SE) derived from periodontopathic bacteria (three strains of Actinobacillus actinomycetemcomitans, two strains of Bacteroides gingivalis, and one strain of Fusobacterium nucleatum) were investigated. The main components of SDS-SE were protein and lipid, with negligible amounts of peptidoglycan and lipopolysaccharide. Immunopotentiating activity was detected in both delayed-type hypersensitivity and antibody formation against the elicitation of a protein antigen with the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and B. gingivalis 381 and 1021. On the other hand the SDS-SE of A. actinomycetemcomitans ATCC 29522 enhanced only the induction of a delayed-type hypersensitivity response. All the SDS-SE preparations had mitogenic activity to splenocytes from BALB/c nu/nu, C3H/HeN and C3H/HeJ mice. Migration-stimulating activity for human peripheral blood monocytes was detected especially in the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and Y4. All of the SDS-SE samples inhibited [3H]thymidine uptake in human gingival fibroblasts and caused degradation of the cells. The results suggest that the cell membrane components extractable with sodium dodecyl sulphate from periodontopathic bacteria are involved in the pathogenesis of periodontal disease.

Higher Immunoadjuvant Activities of N‐Acetyl‐β‐D‐glucosaminyl‐(1–4)‐N‐acetylmuramyl‐L‐alanyl‐D‐isoglutamine in Comparison with N‐Acetylmuramyl‐L‐alanyl‐D‐isoglutamine

In the previous paper (3) the present authors (S. Ku, K.Y., and T.S.) reported the first successful synthesis of N-acetyl-ƒÀ-D-glucosaminyl(1-4)-N-acetylmuramyl L-alanyl-D-isoglutamine (GMDP) via the following steps : 1) synthesis of a ƒÀ (1-4 ) linked glucosamine disaccharide, 2) selective transformation of the glucosamine residue at the reducing terminal into muramic acid, 3) coupling of the muramic acid moiety of the disaccharide with the dipeptide, and 4) the final deprotection . Since the immunoadjuvant activities of the synthetic disaccharide dipeptide (GMDP) , which corresponds to the larger building block of bacterial cell wall peptidoglycans than N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), have never been examined before, the abilities of GMDP to stimulate the cellular and humoral immune responses against a protein antigen have been quantitatively compared with those of MDP under the standard assay conditions (1, 2) . Groups of five female, random-bred albino guinea pigs, weighing about 250 g , were injected into the left hind foot pad with three-fold graded doses of either GMDP or MDP with one mg (per animal) of crystalline ovalbumin (Grade V, Sigma ) emulsified in 0.2 ml aliquots of a water-in-oil mixture consisting of 1/75 M phosphate buffered saline (pH 7.0), Arlacel A and Drakeol 6VR in a ratio of 5 : 1 : 4, by volume . Two and three weeks after the immunizing injection, the guinea pigs were examined concerning the induction of delayed-type hypersensitivity by intracorneal injection of an ovalbumin solution. One week after the second corneal test, the animals were tested for delayed skin reaction with the antigen, and then bled for determination of circulating anti-ovalbumin antibody by the quantitative precipitin reaction after a further 3-4 days . The results summarized in Figs. 1 and 2 revealed that GMDP was significantly more potent than MDP in induction of delayed-type hypersensitivity and in stimula tion of circulating antibody levels. The minimum effective dose of GMDP for induc ing definitely positive corneal and delayed skin reactions was found to be around 1.7 ƒÊg (2.5 nmole, equivalent to 1.23 ƒÊg of MDP on a molar basis) while the cor responding effective dose of MDP was around 3.7 to 11.1 ƒÊg (7.5 to 22.5 nmole) . Determination of circulating anti-ovalbumin antibody titers also indicated that

Immunomodulating and Related Biological Activities of Bacterial Cell Walls and Their Components, Enzymatically Prepared or Synthesized

In 1959, we isolated the cell walls of tubercle bacilli (BCG) which have long been known to have a strong immunoadjuvant activity, especially in the stimulation of cell-mediated immune responses, and elucidated the chemical and immunological properties of this cell wall fraction (32, 31). The most remarkable finding on the chemical composition of this subcellular fraction was that various lipids, especially ethanol-ether insoluble but chloroform-soluble lipids and bound lipids, which characterized tubercle bacilli were almost exclusively localized in their cell walls. It was also demonstrated that the cell walls were the most active fraction of subcellular components isolated from sonicated BCG cells, in inducing tuberculin hypersensitivity in guinea pigs and that the cell walls were involved in the development of an acquired cell-mediated resistance of mice to tuberculous infection.

Muramyl Dipeptides: Prospect for Cancer Treatments and Immunostimulation

The immunopharmacological activities of bacterial cell walls and muramyl peptides were collected in table form with a comprehensive literature. The past and present studies emphasizing the host-defense enhancing activities of muramyl peptides for antitumor immunotherapy were surveyed along three possible approaches: 1) the nonspecific enhancement of natural defense ability of host against tumor cells themselves; 2) the enhancement of nonspecific resistance of host to microbial infections which are frequently encountered and difficult to treat in the advanced stage of tumor patients; and 3) the stimulation of immunity against tumor-specific or tumor-associated immunogens. Finally, the prospects of successful antitumor immunotherapy with muramyl peptides and their derivatives was discussed.