ß-glucans and cholesterol (Review).

Hypercholesterolemia is one of primary risk factors of cardiovascular disease, together with metabolic syndrome, hypertension and diabetes. Although progress has been made, the search for novel methods of preventing and treating dyslipidemia is ongoing and current therapies for cardiovascular disease induce various side effects. ß­glucans are linear unbranched polysaccharides found in various natural sources, such as mushrooms. Due to their structure they are able to interact with innate immunity receptors, however they also act as dietary fibers in the digestive tract. As there are two forms of ß­glucans, insoluble and soluble forms, they are able to interact with lipids and biliary salts in the bowel and consequently reduce cholesterol levels. Therefore, they may be developed as a suitable therapeutic option to treat patients with dyslipidemia, as they are natural molecules that do not induce any significant side effects. The current review discusses the evidence supporting the effects of ß­glucans on cholesterol levels.

Role for phospholipid acyl chains and cholesterol in pulmonary infections and inflammation.

Bacterial and viral respiratory tract infections result in millions of deaths worldwide and are currently the leading cause of death from infection. Acute inflammation is an essential element of host defense against infection, but can be damaging to the host when left unchecked. Effective host defense requires multiple lipid mediators, which collectively have proinflammatory and/or proresolving effects on the lung. During pulmonary infections, phospholipid acyl chains and cholesterol can be chemically and enzymatically oxidized, as well as truncated and modified, producing complex mixtures of bioactive lipids. We review recent evidence that phospholipids and cholesterol and their derivatives regulate pulmonary innate and adaptive immunity during infection. We first highlight data that oxidized phospholipids generated in the lung during infection stimulate pattern recognition receptors, such as TLRs and scavenger receptors, thereby amplifying the pulmonary inflammatory response. Next, we discuss evidence that oxidation of endogenous pools of cholesterol during pulmonary infections produces oxysterols that also modify the function of both innate and adaptive immune cells. Last, we conclude with data that n-3 polyunsaturated fatty acids, both in the form of phospholipid acyl chains and through enzymatic processing into endogenous proresolving lipid mediators, aid in the resolution of lung inflammation through distinct mechanisms. Unraveling the complex mechanisms of induction and function of distinct classes of bioactive lipids, both native and modified, may hold promise for developing new therapeutic strategies for improving pulmonary outcomes in response to infection.

Comparison of a combined nontreponemal (VDRL) and treponemal immunoblot to traditional nontreponemal and treponemal assays.

BACKGROUND: Serology is the mainstay for the diagnosis and management of patients with syphilis. Newer technologies such as immunoblotting are now available for the diagnosis of syphilis. METHODS: A commercial IgM/IgG immunoblot assay that detects both nontreponemal (VDRL-Venereal Disease Research Laboratory) and treponemal antibodies was compared with standard nontreponemal and treponemal assays. The immunoblot and T. pallidum particle agglutination assay (TP-PA) were performed on 198 samples. Ninety-seven samples were Rapid plasma reagin (RPR)-positive and one hundred one were RPR-negative. Positive RPR samples were titered by VDRL. RESULTS: The agreement, sensitivity, and specificity of the IgM/IgG VDRL results of the immunoblot compared to RPR were 74.2% (95% CI: 67.2-80.2), 77.3% (95% CI: 70.2-83.4), and 71.3% (95% CI: 64.4-77.1), respectively. The agreement, sensitivity, and specificity of the IgM/IgG treponemal immunoblot compared to TP-PA were 100% for all parameters, if the ten equivocal results were not used in the calculation. CONCLUSION: The treponemal portion of the ViraBlot IgM/IgG immunoblot compared well with the treponemal confirmation assay and could be a useful supplemental method to fluorescent treponemal antibody or TP-PA for the confirmation of syphilis. The addition of the detection of nontreponemal antibodies to the immunoblot assay, however, may not be of added benefit to the overall assay, due to decreased sensitivity and specificity compared to standard assays.

Inflammasome-dependent and -independent IL-18 production mediates immunity to the ISCOMATRIX adjuvant.

Adjuvants are an essential component of modern vaccines and used for their ability to elicit immunity to coadministered Ags. Many adjuvants in clinical development are particulates, but how they drive innate and adaptive immune responses remains poorly understood. Studies have shown that a number of vaccine adjuvants activate inflammasome pathways in isolated APCs. However, the contribution of inflammasome activation to vaccine-mediated immunity in vivo remains controversial. In this study, we evaluated immune cell responses to the ISCOMATRIX adjuvant (IMX) in mice. Like other particulate vaccine adjuvants, IMX potently activated the NALP-3-ASC-Caspase-1 inflammasome in APCs, leading to IL-1ß and IL-18 production. The IL-18R pathway, but not IL-1R, was required for early innate and subsequent cellular immune responses to a model IMX vaccine. APCs directly exposed to IMX underwent an endosome-mediated cell-death response, which we propose initiates inflammatory events locally at the injection site. Importantly, both inflammasome-related and -unrelated pathways contributed to IL-18 dependence in vivo following IMX administration. TNF-α provided a physiological priming signal for inflammasome-dependent IL-18 production by APCs, which correlated with reduced vaccine-mediated immune cell responses in TNF-α- or TNFR-deficient mice. Taken together, our findings highlight an important disconnect between the mechanisms of vaccine adjuvant action in vitro versus in vivo.

Antigen-specific enhancement of natural human IgG antibodies to phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol-4-phosphate, cholesterol, and lipid A by a liposomal vaccine containing lipid A.

Natural IgG antibodies (NA) to lipids are ubiquitously distributed in sera of healthy humans and are believed to serve beneficial functions. Although NA to lipids generally exhibit germ line or near germ line binding specificities, the antibodies commonly increase transiently in the acute phases of most, if not all, infectious diseases and may serve as a first line of defense. In order to determine whether similar anti-lipid antibodies can be induced by a vaccine in humans, we examined stored sera obtained from volunteers who had previously received a candidate vaccine to Plasmodium falciparum. The vaccine had consisted of liposomes that contained both the recombinant protein antigen and also contained monophosphoryl lipid A (MPLA) as an adjuvant. All of the pre-immune sera contained NA to one or more of the liposomal lipids in the vaccine: dimyristol phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), cholesterol, and MPLA. After initial immunization, followed by a boost, increased levels of IgG antibodies to all of the liposomal lipids, especially DMPG and MPLA, were observed by ELISA. Antibodies to phosphatidylinositol-4-phosphate (PIP) above the normal pre-immune NA to PIP were also observed. Although PIP was not present in the immunizing liposomes, based on the adsorption of anti-PIP antibodies by DMPG the anti-PIP antibodies were thought to represent cross-reacting anti-DMPG antibodies. The immune response was apparently antigen-specific in that NA to unrelated lipids, other than PIP, that were not present in the liposomes, galactosyl ceramide and ganglioside GM1, were not increased by the immunization. We conclude that antibodies to DMPC, DMPG, PIP, cholesterol, and MPLA can be induced in humans by immunization with liposomes containing MPLA.

The role of adjuvants in vaccines for seasonal and pandemic influenza.

Current seasonal influenza vaccines aim to induce high-titred virus-neutralizing antibody to the viral hemagglutinin (HA), which is the best form of protection against infection, but these vaccines can be poorly efficacious in the elderly and other target groups that rely on them most. Furthermore, little cross-protection is provided against significantly drifted strains and even less against different subtypes of virus with pandemic potential. Adjuvants could theoretically have two different roles in improving control of influenza through vaccination. Firstly, a role in enhancing the antibody response in situations where the split virus preparation is poorly immunogenic or if there is an imperative to provide "dose sparing" in the context mass vaccination with a virus to which the population is immunologically naïve. Secondly, adjuvants could be used to allow induction of additional arms of the immune response that are not stimulated by current split virus vaccines. Briefly reviewed here are our efforts to investigate the role of adjuvants in both these contexts.

Oral amphipathic peptides as therapeutic agents.

Cholesterol can promote inflammation by its ability to stimulate the production of reactive oxygen species that result in the formation of pro-inflammatory oxidised phospholipids. High-density lipoproteins (HDLs) are part of the innate immune response and can be either pro- or anti-inflammatory independently of plasma HDL-cholesterol levels. During systemic inflammation as occurs with atherosclerosis, Apolipoprotein A-I can be altered, reducing its ability to promote reverse cholesterol transport and HDL can become pro-inflammatory. Amphipathic peptides with either a class A amphipathic helix (D-4F) or a class G* amphipathic helix (D-[113-122]apoJ), or even those that are too small to form a helix (KRES and FREL) have some similar characteristics. Their interaction with lipids leads to a reduction in lipoprotein-lipid hydroperoxides that releases HDL-associated antioxidant enzymes, such as paraoxonase, therefore providing antiatherosclerosis and anti-inflammatory activity. In addition, the peptide D-4F stimulates the formation and cycling of pre-beta HDL. These amphipathic peptides appear to have therapeutic potential as oral agents.

Design, characterization and preclinical efficacy of a cationic lipid adjuvant for influenza split vaccine.

We prepared a series of cationic lipid vesicles comprising a cationic cholesterol derivative, DC-Chol with or without a neutral phospholipid, DOPC or DOPE. The vesicles were tested for their ability to bind and adjuvant split inactivated influenza vaccines. We found that DC-Chol-containing liposomes are capable to strongly bind influenza vaccine antigens upon simple mixing with the vaccine. The resulting formulations induced robust anti-influenza immune responses both after s.c. and i.n. administration in BALB/c mice while neutral Cholesterol/DOPC liposomes displayed virtually no stable antigen binding and no adjuvant effect. The parenteral adjuvant effect of DC-Chol on trivalent split influenza vaccines was then confirmed in outbred mice and monkeys. Among the most potent formulations tested, a simple mixture of the vaccine with a microfluidized dispersion of DC-Chol in an aqueous buffer is being considered for further development to produce an improved influenza vaccine.

Antibodies to cholesterol: biological implications of antibodies to lipids.

Injection of silicone gel or silicone oil intraperitoneally into BALB/c mice induced the formation of antibodies that reacted by ELISA with highly purified crystalline cholesterol and, to a much lesser extent, antibodies that reacted with a phospholipid (dimyristoyl phosphatidylglycerol). Although IgM and IgG antibodies to cholesterol were detected, the titers of IgG antibodies were low when compared with IgM. The titers of IgM antibodies to cholesterol in certain sera exhibited activities that reached baseline values at dilutions as high as 1:5000, thus making them equivalent to titers that have been previously published for ascites fluid containing murine monoclonal antibodies to cholesterol. The antibodies to cholesterol induced by silicone compounds are indistinguishable in their binding to crystalline cholesterol from naturally-occurring antibodies to cholesterol in normal human serum. They are also indistinguishable from antibodies induced by a proposed vaccine to cholesterol that is currently in late preclinical development for prevention of hypercholesterolemia in humans. The anti-cholesterol vaccine, which consists of liposomes heavily laden with cholesterol as an antigen and lipid A as an adjuvant, induces antibodies that react with low density lipoproteins (LDL) and opsonize them for removal by liver macrophages. It appears that silicone gel or silicone oil causes recruitment and adsorption of cholesterol at the injection site, and also serves as an adjuvant that may have immunostimulant properties similar to lipid A for inducing antibodies to lipids. Antibodies to lipids such as cholesterol or phospholipids are not harmful to intact cell membranes because of steric hindrance from surrounding lipids and larger macromolecules that block binding of the antibodies.

Inhibition by cholesterylphosphorylserine of T-cell-mediated immune responses in mice.

The synthetic analogue of phosphatidylserine, cholesterylphosphorylserine (CPHS) inhibits T-cell-mediated immune responses in mice. Tested in cultured mouse spleen cells, CPHS inhibits concanavalin A-induced activation of DNA synthesis (IC50, 3.5 microM). Injected i.p. during the efferent phase, CPHS (25-100 mg/kg) inhibits the manifestations of delayed-type of hypersensitivity. The compound (25 mg/kg i.p., daily) reduces the acute graft-versus-host reaction when given for 5 days to donor mice before the isolation of spleen cells used for the inoculum. These data suggest that the addition of a phosphorylserine group to a steroid ring may produce immunoregulatory compounds.

Obesity: does it modulate infectious disease and immunity?

Obesity, high-fat diets, or excess lipids interact with infectious agents and immunocompetent cells in the following ways: Some infections and autoimmune diseases are enhanced in inbred mice. In man, surgical wound infections are increased; the risk of tubercular death is decreased; no data exist on the interaction of lipids and autoimmune disease. Certain fatty acids and cholesterol are potent modulators of T lymphocyte and phagocyte functions in laboratory animals and in leukocyte cultures. However, in humans, the modulation of immune function by dietary lipids is still uncertain. Precisely how lipids interact with the immune system opens an important and exciting area for future research.

Immunochemical properties of phosphatidyl cholesterol and its homologue.

1,2-Dipalmitoyl-rac-glycerol-3-phosphoryl-3'-cholesterol (PCH) and 1,2-dipalmitoyl-rac-glycerol-3-phosphoryl-20'-(3-hydroxy norpregn-5-ene) (PET) were combined with poly-L-lysine to form a PCH-poly-L-lysine complex and a PET-poly-L-lysine complex, respectively. These complexes were subcutaneously injected into rabbit foot pad with Freund's complete adjuvant. PCH antiserum showed specificites against the phosphatidyl group, the cholesterol moiety and the side chain of cholesterol. PET antiserum contained the specific antibodies against the phosphatidyl group, the cholesterol moiety and the OH-group at the C3 position of the cholesterol molecule.

Liposomes in immunology: impairment of the adjuvant effect of liposomes by incorporation of the adjuvant lysolecithin and the role of macrophages.

The immune response against HSA (human serum albumin) was studied in rabbits after intravenous injection of various HSA preparations. When HSA was injected one day after, together with or coupled to lysolecithin, a late response was found in twelve out of thirteen rabbits, whereas a minority of the rabbits responded when lysolecithin was omitted. These results confirm the adjuvant activity of lysolecithin. A rapid response starting on day 6 was found in rabbits injected with HSA entrapped in liposomes which had been composed of lecithin, phosphatidic acid and cholesterol (PPC liposomes). The response against liposome entrapped HSA was delayed for about one day when the phospholipid adjuvant lysolecithin was incorporated in the liposomes (LPPC liposomes). Results lend support to the hypothesis that the adjuvant activity of lysolecithin and its opposite inhibition of the adjuvant activity of liposomes are mediated by the same mechanism, i.e. inhibition of enzymatic digestion in lysosomes of macrophages.