Tatsuji Yasuda

Membrane-damaging action of staphylococcal alpha-toxin on phospholipid-cholesterol liposomes

The mechanism of membrane damage by staphylococcal alpha-toxin was studied using carboxyfluorescein (internal marker)-loaded multilamellar liposomes prepared from various phospholipids and cholesterol. Liposomes composed of phosphatidylcholine or sphingomyelin and cholesterol bound alpha-toxin and released carboxyfluorescein in a dose dependent manner, when they were exposed to alpha-toxin of concentrations higher than 1 or 8 micrograms/ml, respectively. In contrast, the other liposomes composed of phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol or phosphatidylinositol plus cholesterol were not susceptible to the toxin even at high concentrations up to 870 micrograms/ml. The insensitive liposomes containing either phosphatidylserine or phosphatidylglycerol were made sensitive to alpha-toxin by inserting phosphatidylcholine into the liposomal membranes. In addition, phosphorylcholine inhibited the toxin-induced marker release from liposomes. These results indicated that the choline-containing phospholipids are required for the interaction between alpha-toxin and liposomal membranes. Susceptibility of liposomes containing phosphatidylcholine or sphingomyelin increased with the increase in cholesterol contents of the liposomes. Based on these results, we propose that the choline-containing phospholipids are possible membrane components or structures responsible for the toxin-membrane interaction, which leads to damage of membranes. Furthermore, cholesterol may facilitate the interaction between alpha-toxin and membrane as a structural component of the membrane.

A simple method to measure anti-glycolipid antibody by using complement-mediated immune lysis of fluorescent dye-trapped liposomes

A simple, reproducible, and micro quantity method is described to measure the antibody against glycolipid antigens. The multilamellar liposomes containing carboxyfluorescein (CF), which is self-quenched at high concentration, are prepared by vortexing the dried lipid films consisting of egg lecithin, cholesterol, phosphatidic acid and Forssman glycolipid antigen. On addition of anti-glycolipid serum plus active complement, liposome lysis occurs, and trapped CF is released. The dilution of CF in the external volume abolishes the quenching, resulting in a high fluorescence signal. Experimental conditions to measure anti-glycolipid antibody is established in this paper.

Liposome immune lysis assay (LILA). Application of sandwich method to determine a serum protein component with antibody-bearing liposomes

A complement-dependent liposome immune lysis assay (LILA) using carboxyfluorescein (CF)-entrapped liposomes bearing antibody was developed to measure C-reactive protein (CRP) antigen as a model of protein antigens in human sera. Goat anti-CRP antibody was covalently coupled to liposomes, and a specific lysis of the liposomes could be observed when the liposomes were incubated with both rabbit anti-CRP antibody (secondary antibody) and CRP antigen in sera in the presence of guinea pig complement. In this assay system, so-called sandwich assay, CRP (a multivalent antigen) bound to the liposomes bearing anti-CRP antibody and subsequently secondary antibody, which activated complement efficiently. The amount of CF released by a complement-dependent liposome immune lysis was proportional to CRP concentrations. This sandwich assay was simple, fast, highly sensitive, and covered the ranges 10-300 ng of CRP/ml in a homogeneous mode, that is, one where no separation step was employed. The results correlated well with those obtained by single radial immunodiffusion and enzyme immunoassay. This assay system would be applicable to the measurement of other protein antigens.

Humoral immune response to liposomes

Since the introduction of liposomes in 1965, these model membranes have influenced research in every biological discipline that can lay claim to membraneassociated phenomena. The first immunological application (reviewed in Ref. 1) was the elaboration of liposomes that could serve as targets for humoral mediated membrane damage via the antibody-dependent (classical) or antibody-independent (alternative) complement pathways. Unfortunately, attempts to develop analogous liposomes that can function as surrogate substrates for either antibody-dependent or -independent cell mediated lysis have been so far notably less successful. More recently, liposomes have been used to study the induction of afferent humoral and cellular responses. In this article, we shall focus attention on several features of antibody formation elicited by liposomes that have been made immunogenic (sensitized) by a novel class of synthetic lipid antigens: N-(hapten)-substituted derivatives of phosphatidylethanolamine (PE). Our intention is to review certain aspects of liposomal immunogenicity that have been reasonably well established and to point out a few areas that, in our opinion, are controversial and merit additional study.

Regulation by glycophorin of complement activation via the alternative pathway

Abstract The effect of glycophorin on complement activation via the alternative pathway was examined by incorporating it into the liposome membrane with trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine (TNP-Cap-DPPE). Liposomes having incorporated TNP-Cap-DPPE onto the membrane activate the alternative complement pathway of guinea pig as reported previously, and the additional insertion of glycophorin was found to reduce their activating capacity on the alternative complement pathway. This inhibitory effect was cancelled by pretreatment of the glycophorin-containing liposomes with neuraminidase indicating that the sialic acid in glycophorin is playing a role in the regulation of alternative complement pathway-activation on the biological membrane.

Experimental allergic encephalomyelitis induced by basic protein with synthetic adjuvant in comparison with Freund's complete adjuvant. Role of antibodies in correlation with the clinicopathological features.

Experimental allergic encephalomyelitis (EAE) was induced in guinea pigs with bovine myelin basic protein (BP) with adjuvant of either synthetic muramyl dipeptide (Mdp) or Mycobacterium tuberculosis (Tbc). The following results were obtained: (1) The body temperature of the animals was studied serially after sensitization and its elevation was shown to be an early sign of EAE. (2) Several animals developed the clinical and histological signs of hyperacute EAE. (3) An optimal combined dosage of BP and adjuvant was found for induction of clinical EAE and for the the production of complement fixing (CF) antibodies. (4) Little passive hemagglutinating (PH) antibody was produced by single immunization. These results displayed no essential difference in EAE induced by either adjuvant. (5) Detectable PH antibodies developed later in addition to CF antibodies in a few animals immunized with Tbc adjuvant. These animals were skin-tested to BP, and had recovered from body weight loss or limb weakness. The results suggest that humoral antibodies play a role in modifying the disease process, even if they are not essential in production of EAE.

Immunoassay Using Fluorescent Dye-Trapped Liposomes Liposome Immune Lysis Assay (LILA)

The liposome immune lysis assay (LILA) system was developed by S. C. Kinsky and coworkers in 1968 (Haxby, 1968). They used glucose as an internal liposome marker, and measured the glucose release in an enzymatic reaction. They also introduced umbelliferone phosphate and alkaline phosphatase as the first fluorescent marker system (Six, 1974). The enzyme reaction resulted in both the generation and the amplification of fluorescence. In 1977, Smolarsky et al. developed a novel LILA system using a complex of a fluorescent molecule (1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS)) and a quencher (dipyridinium-p-xylene) as a marker trapped in liposomes. The complex, when entrapped in liposomes, shows little fluorescence. Upon lysis of the liposomes, dilution of the quencher in the external volume leads to a high fluorescence signal. One of the problems of the use of ANTS as a fluorophore is, however, that its excitation and emission wavelengths coincide with those of a fluorescent component existing in complement sources, and precise measurements may not be possible because of an increase in the background fluorescence.

Binding of C3 molecules to membranes via the SH-residue generated by the cleavage of a thioester bond can initiate complement activation.

Dithiopyridine (DTP)-dipalmitoylphosphatidylethanolamine (DTP-DPPE) was incorporated into liposome membranes to prepare DTP-liposomes. The DTP-liposomes could be lysed by reaction with the alternative complement pathway of any kind of serum tested. Activation of the alternative complement pathway has been shown to be mediated by the binding of C3 molecules to DTP on the liposomes via the SH-residue generated by the cleavage of thioester bond in the alpha-chain of the molecules.