How T lymphocytes recognize lipid antigens

Recognition of lipid antigens by T lymphocytes is well established. Lipids are recognized by T cells when presented in association with CD1 antigen-presenting molecules. Both microbial and self lipids stimulate specific T lymphocytes, thus participating in immune reactions during infections and autoimmune diseases. The immune system uses a variety of strategies to solubilise lipid antigens, to facilitate their internalization, processing, and loading on CD1 molecules. Recent studies in the field of lipid antigen presentation have revealed new mechanisms which allow the immune system to sense lipids as stimulatory antigens.     

Synthetic branched-chain analogues of distearoylphosphatidylcholine: structure-activity relationship in inhibiting and activating protein kinase C

A series of distearoylphosphatidylcholine (DSPC) analogues having various branched alkyl chains were synthesized and tested for their abilities to regulate protein kinase C (PKC). The greatest improvement (about 3-fold) in the PKC inhibitory activity over that seen for the parental lipid (i.e., DSPC) was accomplished by substitution of 8-methylstearate at sn-2 and 16-methylstearate at both sn-1 and sn-2 positions of glycerol; substitutions at both sn-1 and sn-2 with 8-methylstearate, on the other hand, caused a decrease (about 4-fold) in its inhibitory activity. Introduction of butyl, phenyl, or keto functions to various positions in the fatty alkyl chain substituted at both sn-1- and sn-2 positions imparted upon the DSPC analogues an ability to potently stimulate PKC to an extent comparable to those attainable by diacylglycerol or phorbol ester; the analogues having substitution only at the sn-2 position, in comparison, had no or reduced stimulatory activity. The butyl, phenyl, and keto analogues of DSPC, as with DSPC itself and its methyl analogues, inhibited PKC at high concentrations. Kinetic analysis indicated that the methyl DSPC analogues inhibited the enzyme competitively with respect to phosphatidylserine (PS; a phospholipid cofactor) and Ca2+. The butyl analogues activated the enzyme without affecting its affinity for PS or Ca2+, indicating a mechanism different from that seen for diacylglycerol or phorbol ester. The inhibitory activity of the methyl DSPC analogues and the stimulatory activity of the butyl DSPC analogues were reduced when PKC was activated by phorbol ester. Both classes of the analogues were unable to compete for the binding of [3H]phorbol dibutyrate to PKC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipopeptide structure determines TLR2 dependent cell activation level

Bacterial lipoproteins/peptides are composed of di-O-acylated-S-(2,3-dihydroxypropyl)-cysteinyl residues N-terminally coupled to distinct polypeptides, which can be N-acylated with a third fatty acid. Using a synthetic lipopeptide library we characterized the contribution of the lipid portion to the TLR2 dependent pattern recognition. We found that the two ester bound fatty acid length threshold is beyond eight C atoms because almost no response was elicited by cellular challenge with analogues carrying shorter acyl chains in HEK293 cells expressing recombinant human TLR2. In contrast, the amide bound fatty acid is of lesser importance. While two ester-bound palmitic acids mediate a high stimulatory activity of the respective analogue, a lipopeptide carrying one amide-bound and another ester-bound palmitic acid molecule was inactive. In addition, species specific LP recognition through murine and human TLR2 depended on the length of the two ester bound fatty acid chains. In conclusion, our results indicate the responsibility of both ester bound acyl chains but not of the amide bound fatty acid molecule for the TLR dependent cellular recognition of canonical triacylated LP, as well as a requirement for a minimal acyl chain length. Thus they might support the explanation of specific immuno-stimulatory potentials of different microorganisms and provide a basis for rational design of TLR2 specific adjuvants mediating immune activation to distinct levels.     

Synthesis of lipid A analogs: achievements and perspective

Data on the synthesis of analogues of lipid A, a biologically active fragment of gram-negative bacteria's lipopolysaccharides, are summarized. Main types of the compounds obtained are systematized, and problems of the synthesis of various parts of the molecule considered. The results of studying biological activity of lipid A analogues are discussed, which led to some conclusions on the structure-function relation. Perspectives of further studies are briefly outlived.     

Effect of the carcinogenic metabolites of aromatic amino acids on oxidative processes in lipids in vitro and in vivo

A comparative study of the effect produced by endogenous carcinogens (p-hydroxyphenyllactic and 5-methoxyindole-3-acetic acids) and their non-cancerogenic analogues on lipid peroxidation in vitro and in vivo was performed. It has been found that cancerogenic tyrosine and serotonin metabolites, unlike their non-cancerogenic analogues, increase lipid peroxidation in vitro. In vivo, cancerogenic tyrosine metabolite--p-hydroxyphenyllactic acid--is capable both of increasing and decreasing antioxidative lipid activity in the animal liver.     

Immunocycte stimulation in vitro by nontoxic bacterial lipopolysaccharide derivatives.

Intact lipopolysaccharides (LPS), considered nonspecific enhancers of B cell responses, as well as nontoxic derivatives from Serratia marcescens LPS, were studied with regard to their ability to stimulate in vitro immune responses to a T-dependent antigen, sheep erythrocytes. Intact LPS, at a dose of 10 to 50 microgram, consistently enhanced the in vitro anti-SRBC immune response by normal splenocytes. The LPS also increased the background PFC response to SRBC in nonimmunized cultures. A chemically detoxified preparation derived from LPS (Mex B) had no stimulatory activity in vitro. A completely nontoxic, relatively small m.w., polysaccharide-rich preparation (PS), free of detectable lipid and protein, was stimulatory in vitro and at a dose of 10 microgram resulted in a 40 to 70% enhancement of the anti-SRBC response. The PS also stimulated an enhanced background response to SRBC as well as several other RBC species in nonimmunized cultures. PS had no mitogenic effect in vitro since addition of this bacterial derivative failed to stimulate thymidine incorporation into mouse splenocytes, as occurred with the intact LPS. The use of nontoxic preparations from gram-negative bacterial LPS for dissecting the stimulatory vs antigenic properties of bacterial products provides a model system for determining the role of a mitogenic stimulus in B cell activation.     

STIMULATION OF BRAIN ACETYL-CoA HYDROLASE BY ATP AND ATP ANALOGUES

The effects of ATP and ATP analogues on the brain acetyl-CoA hydrolase (EC 3.1.2.1) were studied. The enzyme was stimulated reversibly by ATP-Mg²⁺ the presence of Mg²⁺ being absolutely required for the stimulation. The stimulatory effect of ATP was highly specific since adenine nucleotides other than ATP had no stimulatory effects and nucleoside triphosphates other than ATP stimulated the enzyme much less than ATP in the following order: ATP > ITP CTP, UTP GTP. A phosphate modified analogue of ATP, AMP-PNP had a similar stimulatory effect to that of ATP. Other ATP analogues such as AMP-PCP and AMPCPP showed less stimulatory effect than ATP. The order of the stimulatory effects of these ATP analogues was: ATP > AMP-PNP > AMP-PCP > AMPCPP. The concentrations needed for half-maximal stimulation of ATP, AMP-PNP and AMP-PCP were approx 0.11 mm , 0.22 mm, and 0.22 mm , respectively. Double reciprocal plots demonstrated that ATP as well as AMP-PNP produced a significant decrease in the apparent K_m, value for acetyl-CoA and an increase in V_max indicating that these nucleotides increased the affinity for acetyl-CoA through binding at a site other than the catalytic site. The data described above suggest that the rate of hydrolysis of acetyl-CoA may be regulated by the concentration of ATP in the micro-environment of the enzyme.     

Conjugates of synthetic lymphocyte-activating lipopeptides with segments from HIV proteins induce protein-specific antibody formation.

Lipopeptide analogues of bacterial lipoprotein activate macrophages and B lymphocytes. The products formed by coupling these lipopeptides to low molecular mass antigens can be used to induce antigen-specific antibodies in mice. In the present work, it is shown that HIV-1 gp160-derived synthetic oligopeptides coupled to the synthetic lipodipeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-s eryl- serine (P3CSS) induce peptide-specific antibodies in mice without adding further adjuvants. Depending on the peptides applied, the conjugates exhibited different lymphocyte stimulatory activity, immunoglobulin isotype patterns, and boost reactions; lipopeptide conjugates inducing a pronounced secondary immune response are considered to possess both B- and T-cell epitopes. Antibodies induced by the lipopeptide-HIV-1-peptide conjugates were also reactive against the recombinant gp160 of HIV-1.     

Synthesis of lipid A monosaccharide analogues containing acidic amino acid: exploring the structural basis for the endotoxic and antagonistic activities

For elucidation of the structural and conformational requirements on the endotoxic and antagonistic activity of lipid A derivatives, we designed and synthesized lipid A analogues containing acidic amino acid residues in place of the non-reducing end phosphorylated glucosamine. Definite switching of the endotoxic or antagonistic activity was observed depending on the difference of the acidic groups (phosphoric acid or carboxylic acid) in the lipid A analogues.     

Uptake and intracellular transport pathways of fluorescent lipid analogues inAmoeba proteus (Rhizopoda: Amoebida)

Summary The uptake and intracellular transport of 5 different lipid analogues derived from phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, ceramide, and cholesterol have been studied in livingAmoeba proteus using fluorescence microscopy. Phosphatidylcholine and sphingomyelin are predominantly transported from the external environment to the cell interior in a manner consistent with induced macropinocytosis. On the other hand, phosphatidylethanolamine, ceramide, and cholesterol mainly enter the cellular matrix by carrier-mediated, ATP-dependent transmembrane transport. In general, all lipid analogues are first imported to a large pool of endosomal or lysosomal vacuoles, and then partitioned to numerous tiny cisternal elements; only sphingomyelin remains in the lysosomes and is not exported to other membrane compartments. The ultrastructural localization of ceramide indicates that the cisternal elements result from the decay of the Golgi apparatus into single cisternae during lipid accumulation. As a whole, the transport pathway of lipid analogues inA. proteus from the cell surface to different cell organelles shows many similarities to respective processes in a variety of metazoan cells.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Thymidylate synthetase-deficient mouse FM3A mammary carcinoma cell line as a tool for studying the thymidine salvage pathway and the incorporation of thymidine analogues into host cell DNA.

A thymidylate (dTMP) synthetase-deficient murine mammary carcinoma cell line (FM3A/TS-), auxotrophic for thymidine (dThd), proved extremely useful for studying the dependence of cell growth on the exogenous supply of dThd, the relation between cell growth and DNA synthesis, and the ability of a series of 25 5-substituted 2'-deoxyuridines (dUrd) to substitute for dThd in sustaining cell growth. FM3A/TS-cells did not proliferate unless dThd was supplied to the cell culture medium. The 5-halogenated dUrd derivatives 5-chloro-dUrd, 5-bromo-dUrd and 5-iodo-d Urd also sustained FM3A/TS- cell growth. The extents of incorporation of [methyl-3H]dThd and 5-iodo-[6-3H]dUrd into DNA were closely correlated with their stimulatory effects on FM3A/TS- cell growth. This suggests that the stimulatory effects of the dUrd analogues on the growth rate of FM3A/TS- cells may be considered as evidence for their incorporation into host cell DNA. Based on this premise it is postulated that, in addition to 5-chloro-dUrd, 5-bromo-dUrd, 5-iodo-dUrd and dThd itself, the following dThd analogues are also incorporated into FM3A/TS- cell DNA (in order of the extent to which they are incorporated): 5-hydroxy-dUrd greater than 5-propynyloxy-dUrd greater than 5-ethyl-dUrd greater than 5-ethynyl-dUrd approximately 5-vinyl-dUrd. Thus, the dTMP synthetase-deficient FM3A/TS- cell line represents a unique system to dissociate the de novo and salvage pathways of dTMP biosynthesis and to distinguish those dUrd analogues that are incorporated into DNA from those that are not.     

2-(2-Bromophenyl)-formononetin and 2-heptyl-formononetin are PPARγ partial agonists and reduce lipid accumulation in 3T3-L1 adipocytes

Isoflavones are bioactive compounds that have been shown to decrease lipid accumulation in vitro. However, the knowledge of the isoflavone formononetin is limited. The aim of the study was to assess the effects of formononetin and its two synthetic analogues, 2-(2-bromophenyl)-formononetin and 2-heptyl-formononetin, on lipid accumulation in 3T3-L1 adipocytes and investigate possible mechanisms. Formononetin and the two analogues were added day 0–8 or day 8–10 of the differentiation period, and lipid accumulation, glycerol release and gene expression were measured. Additionally, competitive peroxisome proliferator-activated receptor (PPAR)-γ binding assay, PPARγ transactivation assay and Western blot for phosphorylated AMP-activated protein kinase (AMPK) were performed. Chronic treatment (day 0–8) with formononetin increased lipid accumulation, whereas the two analogues decreased lipid accumulation partly due to decreased differentiation. The two analogues, but not formononetin, also decreased lipid content in mature adipocytes. 2-Heptyl-formononetin increased glycerol release and lipolytic gene expression and decreased lipogenic gene expression. Formononetin did not bind to or activate PPARγ whereas both analogues bound to the receptor and behaved as PPARγ partial agonists in the transactivation assay. Neither of the compounds affected phosphorylation of AMPK. In conclusion, the analogues of formononetin decreased lipid accumulation possibly in part by acting as PPARγ partial agonists.     

Monophosphoryl lipid A analogues with varying 3-O-substitution: synthesis and potent adjuvant activity

Structurally defined immunostimulatory adjuvants play important roles in the development of new generation vaccines. Here described are the syntheses of three monophosphoryl lipid A analogues (1-3) with different substitution at 3-O-position of the reducing sugar and their potent immunostimulatory adjuvant activity. The syntheses involve the preparation of glycosylation acceptors benzyl 3,4-di-O-benzyl-2-deoxy-2-[(R)-3-tetradecanoyloxytetradecanamido]-beta-D-glucopyranoside (16) and benzyl 3-O-allyl-4-O-benzyl-2-deoxy-2-[(R)-3-tetradecanoyloxytetradecanamido]-beta-D-glucopyranoside (17). The glycosylation reactions between the donor 4,6-di-O-benzylidene-2-deoxy-2-(2',2',2'-trichloroethoxycarbonylamino)-alpha-d-glucopyranosyl trichloroacetimidate (21) and acceptors 16 and 17 provide the desired beta-(1-->6)-linked disaccharides 22 and 23, respectively. Selective reductive ring opening of the 4,6-di-O-benzylidene group, installation of a phosphate group to the 4'-hydroxyl group, and the final global debenzylation produce the designed monophosphoryl lipid A analogues 1-3. All three synthetic analogues induce antigen specific T-cell proliferation and interferon-gamma (IFN-gamma) production in ex vivo experiments with a totally synthetic liposomal vaccine system. The immunostimulatory potency of compound 1-3 is in the same order of magnitude as that of the detoxified natural lipid A product isolated from Salmonella minnesota R595 (R595 lipid A). The substituent at the 3-O-position of the reducing sugar does not have much effect on the adjuvant activity of monophosphoryl lipid A analogues. The preliminary lethal toxicity study indicates that the 3-O-acylated hepta-acyl monophosphoryl lipid A may not be more toxic than its 3-O-deacylated hexa-acyl analogue.     

Characterization of monoclonal lipid A antibodies with synthetic lipid A analogues

Abstract The specificity of monoclonal antibodies directed against the Salmonella minnesota R595 lipid A and the structural requirements of lipid A epitopes were studied with chemically synthesized lipid A analogues by enzyme-linked immunosorbent assay and its inhibition test. The results suggest that lipid A has specific and common epitopes, in which the specificities are derived from the chemical and conformational structures of the backbone and/or acyl groups.     

Maturation of dendritic cells with lipopeptides that represent vaccine candidates for hepatitis C virus

The ability of antigens to elicit immune responses depends upon their initial recognition, uptake, processing and presentation by dendritic cells. This fact has been recognized by many workers and dendritic cells are now regarded as natural 'adjuvants' in the business of vaccine design. One way of persuading dendritic cells to become interested in foreign material is to decorate it with lipid moieties found in bacteria. This approach has been used in the context of synthetic peptide-based immunogens and depending on the nature of the epitopes included, can provide highly immunogenic structures capable of eliciting antibody or cytotoxic T cell responses. In this paper we describe the results of experiments in which the stimulatory effects of peptide-based vaccine candidates on human dendritic cells are examined. Our findings indicate that lipidated structures comprising vaccine target sequences of viral origin coupled to the synthetic lipid groups of bacteria are able to induce the maturation of dendritic cells, as measured by the expression of cell surface MHC class II molecules.     

Adenosine and its analogues, possibly acting at A2 receptors, stimulate mouse sperm fertilizing ability during early stages of capacitation

Earlier studies have provided indirect evidence that the availability of endogenous adenosine can modulate the fertilizing ability of mouse spermatozoa during capacitation. More direct evidence has been sought by evaluating the effect of exogenous adenosine present during the early stages of capacitation. A concentration-dependent stimulation of in-vitro fertilizing ability was observed, with 10 microM- and 100 microM-adenosine significantly increasing the proportion of eggs fertilized compared with drug-free controls. The adenosine-induced stimulation was observed in the presence of 0.01 microM- and 0.1 microM-dipyridamole, an inhibitor of adenosine uptake, suggesting that adenosine is acting at an external site. Comparison of adenosine with its analogues 2'-deoxyadenosine and 2-chloroadenosine indicated that the analogues at 10 microM were able to stimulate fertilization in a manner similar to adenosine. While neither adenosine nor 2'-deoxyadenosine was consistently effective at 1 microM, 2-chloroadenosine significantly stimulated fertilization at both 1 microM and 0.1 microM. In addition, 5'-N-ethylcarboxamidoadenosine (NECA) and (R)-N6-phenylisopropyladenosine (R-PIA), potent analogues in somatic cell systems, proved to be so with mouse sperm suspensions, NECA being stimulatory at greater than or equal to 0.01 microM and R-PIA at greater than or equal to 0.1 microM. Subjective evaluation of motility patterns indicated that more cells exhibited hyperactivated motility in the presence of stimulatory concentrations of adenosine or analogues. Assessment of capacitation state using chlortetracycline fluorescence patterns indicated that incubation in 2'-deoxyadenosine resulted in significantly fewer cells expressing the uncapacitated F pattern and significantly more cells with the capacitated AR (acrosome-reacted) pattern, compared with drug-free counterparts. It is concluded that adenosine promotes capacitation by interacting with externally-directed receptors, possibly on adenylate cyclase to increase the intracellular availability of cyclic adenosine monophosphate (cAMP); cAMP is known to stimulate mouse sperm fertilizing ability. The greater sensitivity to NECA, 2-chloroadenosine and R-PIA, relative to adenosine and 2'-deoxyadenosine, is consistent with interaction at stimulatory A2 adenosine receptors.     

Dual biological effects of the cytokines interleukin-10 and interferon-γ

It is generally thought that each cytokine exerts either immune stimulatory (inflammatory) or immune inhibitory (antiinflammatory or regulatory) biological activities. However, multiple cytokines can enact both inhibitory and stimulatory effects on the immune system. Two of these cytokines are interleukin (IL)-10 and interferon-gamma (IFNγ). IL-10 has demonstrated antitumor immunity even though it has been known for years as an immunoregulatory protein. Generally perceived as an immune stimulatory cytokine, IFNγ can also induce inhibitory molecule expression including B7-H1 (PD-L1), indoleamine 2,3-dioxygenase (IDO), and arginase on multiple cell populations (dendritic cells, tumor cells, and vascular endothelial cells). In this review, we will summarize current knowledge of the dual roles of both of these cytokines and stress the previously underappreciated stimulatory role of IL-10 and inhibitory role of IFNγ in the context of malignancy. Our progressive understanding of the dual effects of these cytokines is important for dissecting cytokine-associated pathology and provides new avenues for developing effective immune therapy against human diseases, including cancer.     

Effects of polyunsaturated fatty acids and of their oxidation products on cell survival

The stimulatory, cytostatic and cytotoxic effects of polyunsaturated fatty acids, prostaglandins, thromboxanes, hydroperoxy fatty acids, hydroxy fatty acids and leukotrienes on normal and tumor cells are described. Their effects are related to the ability of the cells to undergo lipid peroxidation. The significance of controlled peroxidation of selected polyunsaturated fatty acids in the control of tumor development is examined. It is suggested that selected polyunsaturated fatty acids if used at appropriate concentrations may have a protective role against cancer development by inducing and/or mediating cytotoxic reactions in malignant cells directly or indirectly through the intermediacy of immune cells.     

Biological activities of analogues of lipid A based chemically on the revised structural model. Comparison of mediator-inducing, immunomodulating and endotoxic activities

Lipid A analogues were chemically synthesized based on the model structure recently revised, and biological activities of the analogues were tested. The analogue, (beta-1,6)-linked glucosamine disaccharide carrying ester-bound 3-hydroxytetradecanoic acids at 3 and 3' position of reducing and nonreducing glucosamine in addition to amide-bound 3-hydroxytetradecanoic acids and glycosidic-linked and ester-linked phosphate groups, showed much stronger activities for mediator inducing and immunomodulating as well as endotoxic activities than those exhibited by the previously synthesized analogues based on the old model. Among the activities tested, induction of interferon and tumor necrosis factor as well as mitogenicity, adjuvanticity and pyrogenicity were, however, not expressed so strongly as natural lipid A used as controls. In contrast, the analogue exhibited comparable activities to those of control lipid A in the test of lethal toxicity to mice and gelating activity of Limulus amebocyte lysate. Other synthetic analogues carrying a phosphate group showed comparable, slightly stronger or weaker activities depending on the test, but nonphosphorylated analogue exhibited no apparent or only very weak activities.     

Lipid carrier proteins and ethanol

Ethanol has a pronounced effect on lipid homeostasis. It is our overall hypothesis that certain lipid carrier proteins are targets of acute and chronic ethanol exposure and that perturbation of these proteins induces lipid dysfunction leading to cellular pathophysiology. These proteins include both intracellular proteins and lipoproteins. This paper examines recent data on the interaction of ethanol with these proteins. In addition, new data are presented on the stimulatory effects of ethanol on low-density-lipoprotein (LDL)-mediated cholesterol uptake into fibroblasts and direct perturbation of the LDL apolipoprotein, apolipoprotein B. A cell model is presented that outlines potential mechanisms thought to be involved in ethanol perturbation of cholesterol transport and distribution.