Haruhiko Takada

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.

Structural Requirements of Lipid a for Endotoxicity and Other Biological Activities—An Overview

Lipopolysaccharide (LPS), a major constituent of outer membranes of gram-negative bacteria, exhibits a wide variety of bioactivities (Table 1) (61). In 1954 Westphal and Luderitz proposed that the lipid moiety of LPS is responsible for most of endotoxicities, and designated it as lipid A (Fig 1) (60). However, this extremely important discovery was neither adequately nor unanimously accepted by endotoxin investigators at that time. One of the main reasons for this controversy is that the high hydrophobicity of the lipid A molecule made its manipulation as a test material for bioactivities difficult. Another reason is concerned with inherent microheterogeneity in fine structures even among preparations derived from the same bacterial

Structural requirements of endotoxic lipopolysaccharides and bacterial cell walls in induction of interleukin-1

A variety of compounds, synthetic, semisynthetic or bacterial, which corresponds to structural components of endotoxic lipopolysaccharides (LPS) and bacterial cell wall peptidoglycans were studied for their activity to enhance interleukin-1 (IL-1) production of murine peritoneal macrophages and the ability to activate the complement cascade in fresh adult human serum. Not only bacterial LPS and cell walls or peptidoglycans but also their structural components with appropriate size and structure induced IL-1 production by macrophages and activated the human complement cascade, which may lead to the IL-1 production by monocytes/macrophages.

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.

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.

Enhancement of Dengue Virus Infection in Cultured Mouse Macrophages by Lipophilic Derivatives of Muramyl Peptides

Dengue virus multiplication in cultures of a murine myelomonocytic cell line (WEHI‐3) as well as mouse peritoneal macrophages was enhanced by treatment of the cells with lipophilic derivatives of muramyl peptides for 2 or 3 days before virus inoculation, but not for 2 hr before virus inoculation or during the adsorption period. The infection‐enhancing activity of the materials was dependent on their chemical structure, correlating with their immunoadjuvanticity. The infection enhancement in WEHI‐3 cells was due primarily to an increase in the number of virus‐infected cells which was accompanied by an increased cellular capacity to bind latex particles to their cell surfaces.

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.

Mitogenic Activity of Cytoplasmic Membranes Isolated from L-Forms of Staphylococcus aureus

Cytoplasmic membranes of L‐forms of Staphylococcus aureus exerted a strong mitogenic effect on splenocytes of athymic nude mice as well as normal mice, while a cytoplasmic fraction of the same bacteria did not show definite mitogenicity. The mitogenic principle(s) of the membrane fraction was resistant to treatment with trypsin and was heat stable (at 100 C for 10 min). The active principle(s) in the insoluble residue of the membrane fraction digested with trypsin was not extracted with cold acetone, but could be solubilized by extraction with a cold chloroform‐methanol mixture (2:1, v/v). The mitogenic principle(s) in the extract was fractionated by silicic acid column chromatography. Among five fractions separated by chromatography, fractions eluted with chloroform‐methanol mixtures (1:1 and 1:20, v/v) were found to be strongly mitogenic.

Mitogenic Effect of Cytoplasmic Membranes and a Cytoplasmic Fraction of Staphylococcus aureus L-Forms on Human Peripheral Blood Lymphocytes

The cytoplasmic membranes and a cytoplasmic fraction of Staphylococcus aureus L‐forms increased the incorporation of [3H] thymidine by human lymphocytes in the presence of fetal bovine serum. Both fractions stimulated cord blood lymphocytes as well as adult peripheral lymphocytes, suggesting the possibility that the observed effect was not due to an antigen‐specific reaction, but to an immunologically nonspecific action.