Rapid Lipid Modification of Endothelial Cell Membranes in Cardiac Ischemia/Reperfusion Injury: a Novel Therapeutic Strategy to Reduce Infarct Size.

PURPOSE: Plasma membranes constitute a gathering point for lipids and signaling proteins. Lipids are known to regulate the location and activity of signaling proteins under physiological and pathophysiological conditions. Membrane lipid therapies (MLTs) that gradually modify lipid content of plasma membranes have been developed to treat chronic disease; however, no MLTs have been developed to treat acute conditions such as reperfusion injury following myocardial infarction (MI) and percutaneous coronary intervention (PCI). A fusogenic nanoliposome (FNL) that rapidly incorporates exogenous unsaturated lipids into endothelial cell (EC) membranes was developed to attenuate reperfusion-induced protein signaling. We hypothesized that administration of intracoronary (IC) FNL-MLT interferes with EC membrane protein signaling, leading to reduced microvascular dysfunction and infarct size (IS). METHODS: Using a myocardial ischemia/reperfusion swine model, the efficacy of FNL-MLT in reducing IS following a 60-min coronary artery occlusion was tested. Animals were randomized to receive IC Ringer's lactate solution with or without 10 mg/mL/min of FNLs for 10 min prior to reperfusion (n = 6 per group). RESULTS: The IC FNL-MLT reduced IS (25.45 ± 16.4% vs. 49.7 ± 14.1%, P < 0.02) and enhanced regional myocardial blood flow (RMBF) in the ischemic zone at 15 min of reperfusion (2.13 ± 1.48 mL/min/g vs. 0.70 ± 0.43 mL/min/g, P < 0.001). The total cumulative plasma levels of the cardiac injury biomarker cardiac troponin I (cTnI) were trending downward but were not significant (999.3 ± 38.7 ng/mL vs. 1456.5 ± 64.8 ng/mL, P = 0.1867). However, plasma levels of heart-specific fatty acid binding protein (hFABP), another injury biomarker, were reduced at 2 h of reperfusion (70.3 ± 38.0 ng/mL vs. 137.3 ± 58.2 ng/mL, P = 0.0115).  CONCLUSION: The IC FNL-MLT reduced IS compared to vehicle in this swine model. The FNL-MLT maybe a promising adjuvant to PCI in the treatment of acute MI.

Soluble Epoxide Hydrolase Regulation of Lipid Mediators Limits Pain.

The role of lipids in pain signaling is well established and built on decades of knowledge about the pain and inflammation produced by prostaglandin and leukotriene metabolites of cyclooxygenase and lipoxygenase metabolism, respectively. The analgesic properties of other lipid metabolites are more recently coming to light. Lipid metabolites have been observed to act directly at ion channels and G protein-coupled receptors on nociceptive neurons as well as act indirectly at cellular membranes. Cytochrome P450 metabolism of specifically long-chain fatty acids forms epoxide metabolites, the epoxy-fatty acids (EpFA). The biological role of these metabolites has been found to mediate analgesia in several types of pain pathology. EpFA act through a variety of direct and indirect mechanisms to limit pain and inflammation including nuclear receptor agonism, limiting endoplasmic reticulum stress and blocking mitochondrial dysfunction. Small molecule inhibitors of the soluble epoxide hydrolase can stabilize the EpFA in vivo, and this approach has demonstrated relief in preclinical modeled pain pathology. Moreover, the ability to block neuroinflammation extends the potential benefit of targeting soluble epoxide hydrolase to maintain EpFA for neuroprotection in neurodegenerative disease.

Ordered Lipid Domains Assemble via Concerted Recruitment of Constituents from Both Membrane Leaflets.

Lipid rafts serve as anchoring platforms for membrane proteins. Thus far they escaped direct observation by light microscopy due to their small size. Here we used differently colored dyes as reporters for the registration of both ordered and disordered lipids from the two leaves of a freestanding bilayer. Photoswitchable lipids dissolved or reformed the domains. Measurements of domain mobility indicated the presence of 120 nm wide ordered and 40 nm wide disordered domains. These sizes are in line with the predicted roles of line tension and membrane undulation as driving forces for alignment.

Milk Fat Globule Membrane Supplementation in Formula-fed Rat Pups Improves Reflex Development and May Alter Brain Lipid Composition.

Human milk contains nutritional, immunoprotective and developmental components that support optimal infant growth and development. The milk fat globule membrane (MFGM) is one unique component, comprised of a tri-layer of polar lipids, glycolipids, and proteins, that may be important for brain development. MFGM is not present in most infant formulas. We tested the effects of bovine MFGM supplementation on reflex development and on brain lipid and metabolite composition in rats using the "pup in a cup" model. From postnatal d5 to d18, rats received either formula supplemented with MFGM or a standard formula without MFGM; a group of mother-reared animals was used as reference/control condition. Body and brain weights did not differ between groups. MFGM supplementation reduced the gap in maturation age between mother-reared and standard formula-fed groups for the ear and eyelid twitch, negative geotaxis and cliff avoidance reflexes. Statistically significant differences in brain phospholipid and metabolite composition were found at d13 and/or d18 between mother-reared and standard formula-fed groups, including a higher phosphatidylcholine:phosphatidylethanolamine ratio, and higher phosphatidylserine, glycerol-3 phosphate, and glutamine in mother-reared compared to formula-fed pups. Adding MFGM to formula narrowed these differences. Our study demonstrates that addition of bovine MFGM to formula promotes reflex development and alters brain phospholipid and metabolite composition. Changes in brain lipid metabolism and their potential functional implications for neurodevelopment need to be further investigated in future studies.

Lipophilic fractions from the marine sponge Halichondria sitiens decrease secretion of pro-inflammatory cytokines by dendritic cells and decrease their ability to induce a Th1 type response by allogeneic CD4+ T cells.

CONTEXT: Halichondria (Halichondriidae) marine sponges contain components possessing various biological activities, but immunomodulation is not among the ones reported. OBJECTIVE: This study evaluated the immunomodulatory effects of fractions/compounds from Halichondria sitiens Schmidt. MATERIALS AND METHODS: Crude dichloromethane/methanol extracts of H. sitiens were subjected to various chromatographic techniques to obtain fractions/compounds with immunomodulatory activity, using bioassay-guided isolation. The effects of the fractions/compounds were determined by measuring secretion of cytokines and expression of surface molecules by dendritic cells (DCs) and their ability to stimulate and modify cytokine secretion by allogeneic CD4+ T cells. The bioactive fractions were chemically analyzed to identify the immunomodulatory constituents by 1D, 2D NMR, and HRMS data. RESULTS: Several lipophilic fractions from H. sitiens at 10 µg/mL decreased secretion of the pro-inflammatory cytokines IL-12p40 and IL-6 by the DCs, with maximum inhibition being 64% and 25%, respectively. In addition, fractions B3b3F and B3b3J decreased the ability of DCs to induce T cell secretion of IFN-γ. Fraction B3b3 induced morphological changes in DCs, characterized by extreme elongation of dendrites and cell clustering. Chemical screening revealed the presence of glycerides and some minor unknown constituents in the biologically active fractions. One new glyceride, 2,3-dihydroxypropyl 2-methylhexadecanoate (1), was isolated from one fraction and two known compounds, 3-[(1-methoxyhexadecyl)oxy]propane-1,2-diol (2) and monoheptadecanoin (3), were identified in another, but none of them had immunomodulatory activity. DISCUSSION AND CONCLUSIONS: These results demonstrate that several lipophilic fractions from H. sitiens have anti-inflammatory effects on DCs and decrease their ability to induce a Th1 type immune response.

Milk Fat Globule Membrane Supplementation in Formula Modulates the Neonatal Gut Microbiome and Normalizes Intestinal Development.

Breast milk has many beneficial properties and unusual characteristics including a unique fat component, termed milk fat globule membrane (MFGM). While breast milk yields important developmental benefits, there are situations where it is unavailable resulting in a need for formula feeding. Most formulas do not contain MFGM, but derive their lipids from vegetable sources, which differ greatly in size and composition. Here we tested the effects of MFGM supplementation on intestinal development and the microbiome as well as its potential to protect against Clostridium difficile induced colitis. The pup-in-a-cup model was used to deliver either control or MFGM supplemented formula to rats from 5 to 15 days of age; with mother's milk (MM) reared animals used as controls. While CTL formula yielded significant deficits in intestinal development as compared to MM littermates, addition of MFGM to formula restored intestinal growth, Paneth and goblet cell numbers, and tight junction protein patterns to that of MM pups. Moreover, the gut microbiota of MFGM and MM pups displayed greater similarities than CTL, and proved protective against C. difficile toxin induced inflammation. Our study thus demonstrates that addition of MFGM to formula promotes development of the intestinal epithelium and microbiome and protects against inflammation.

Local Burn Injury Promotes Defects in the Epidermal Lipid and Antimicrobial Peptide Barriers in Human Autograft Skin and Burn Margin: Implications for Burn Wound Healing and Graft Survival.

Burn injury increases the risk of morbidity and mortality by promoting severe hemodynamic shock and risk for local or systemic infection. Graft failure due to poor wound healing or infection remains a significant problem for burn subjects. The mechanisms by which local burn injury compromises the epithelial antimicrobial barrier function in the burn margin, containing the elements necessary for healing of the burn site, and in distal unburned skin, which serves as potential donor tissue, are largely unknown. The objective of this study was to establish defects in epidermal barrier function in human donor skin and burn margin, to identify potential mechanisms that may lead to graft failure and/or impaired burn wound healing. In this study, we established that epidermal lipids and respective lipid synthesis enzymes were significantly reduced in both donor skin and burn margin. We further identified diverse changes in the gene expression and protein production of several candidate skin antimicrobial peptides (AMPs) in both donor skin and burn margin. These results also parallel changes in cutaneous AMP activity against common burn wound pathogens, aberrant production of epidermal proteases known to regulate barrier permeability and AMP activity, and greater production of proinflammatory cytokines known to be induced by AMPs. These findings suggest that impaired epidermal lipid and AMP regulation could contribute to graft failure and infectious complications in subjects with burn or other traumatic injury.

Archaeosomes display immunoadjuvant potential for a vaccine against Chagas disease.

Archaeosomes (ARC), vesicles made from lipids extracted from Archaea, display strong adjuvant properties. In this study, we evaluated the ability of the highly stable ARC formulated from total polar lipids of a new Halorubrum tebenquichense strain found in Argentinean Patagonia, to act as adjuvant for soluble parasite antigens in developing prophylactic vaccine against the intracellular protozoan T. cruzi, the etiologic agent of Chagas disease. We demonstrated for the first time that C3H/HeN mice subcutaneously immunized with trypanosomal antigens entrapped in these ARC (ARC-TcAg) rapidly developed higher levels of circulating T. cruzi antibodies than those measured in the sera from animals receiving the antigen alone. Enhanced humoral responses elicited by ARC-TcAg presented a dominant IgG2a antibody isotype, usually associated with Th1-type immunity and resistance against T. cruzi. More importantly, ARC-TcAg-vaccinated mice displayed reduced parasitemia during early infection and were protected against an otherwise lethal challenge with the virulent Tulahuén strain of the parasite. Our findings suggest that, as an adjuvant, H. tebenquichense-derived ARC may hold great potential to develop a safe and helpful vaccine against this relevant human pathogen.

Lipid complexes with cationic peptides and OAKs; their role in antimicrobial action and in the delivery of antimicrobial agents.

Antimicrobial agents are toxic to bacteria by a variety of mechanisms. One mechanism that is very dependent on the lipid composition of the bacterial membrane is the clustering of anionic lipid by cationic antimicrobial agents. Certain species of oligo-acyl-lysine (OAK) antimicrobial agents are particularly effective in clustering anionic lipids in mixtures mimicking the composition of bacterial membranes. The clustering of anionic lipids by certain cationic antimicrobial agents contributes to the anti-bacterial action of these agents. Bacterial membrane lipids are a determining factor, resulting in some species of bacteria being more susceptible than others. In addition, lipids can be used to increase the effectiveness of antimicrobial agents when administered in vivo. Therefore, we review some of the structures in which lipid mixtures can assemble, to more effectively be utilized as antimicrobial delivery systems. We describe in more detail the complexes formed between mixtures of lipids mimicking bacterial membranes and an OAK and their usefulness in synergizing with antibiotics to overcome bacterial multidrug resistance.

Oral administration of circulating precursors for membrane phosphatides can promote the synthesis of new brain synapses.

Although cognitive performance in humans and experimental animals can be improved by administering omega-3 fatty acid docosahexaenoic acid (DHA), the neurochemical mechanisms underlying this effect remain uncertain. In general, nutrients or drugs that modify brain function or behavior do so by affecting synaptic transmission, usually by changing the quantities of particular neurotransmitters present within synaptic clefts or by acting directly on neurotransmitter receptors or signal-transduction molecules. We find that DHA also affects synaptic transmission in mammalian brain. Brain cells of gerbils or rats receiving this fatty acid manifest increased levels of phosphatides and of specific presynaptic or postsynaptic proteins. They also exhibit increased numbers of dendritic spines on postsynaptic neurons. These actions are markedly enhanced in animals that have also received the other two circulating precursors for phosphatidylcholine, uridine (which gives rise to brain uridine diphosphate and cytidine triphosphate) and choline (which gives rise to phosphocholine). The actions of DHA aere reproduced by eicosapentaenoic acid, another omega-3 compound, but not by omega-6 fatty acid arachidonic acid. Administration of circulating phosphatide precursors can also increase neurotransmitter release (acetylcholine, dopamine) and affect animal behavior. Conceivably, this treatment might have use in patients with the synaptic loss that characterizes Alzheimer's disease or other neurodegenerative diseases or occurs after stroke or brain injury.

Effect of lipid-containing, positively charged nanoemulsions on skin hydration, elasticity and erythema--an in vivo study.

Dry skin and other skin disorders such as atopic dermatitis are characterized by impaired stratum corneum (SC) barrier function and by an increase in transepidermal water loss (TEWL) leading to a decrease in skin hydration. The possibility that dermatological and cosmetic products containing SC lipids could play a part in the restoration of disturbed skin barrier function is of great interest in the field of dermatology and cosmetics. The aim of the present study was to evaluate the effect of positively charged oil/water nanoemulsions (PN) containing ceramide 3B and naturally found SC lipids (PNSC) such as ceramide 3, cholesterol, and palmitic acid on skin hydration, elasticity, and erythema. Creams of PNSC were compared to PN creams, to creams with negatively charged o/w nanoemulsion and SC lipids (NNSC) and to Physiogel cream, a SC lipid containing formulation, which is already on the market. The formulations (PN, PNSC, and NNSC) were prepared by high-pressure homogenization. After adding Carbopol 940 as thickener, particle size and stability of the creams were not significantly changed compared to the nanoemulsions. The studies were carried out on three groups, each with 14 healthy female test subjects between 25 and 50 years of age, using Corneometer 825, Cutometer SEM 575 and Mexameter 18 for measurements of skin hydration, elasticity, and erythema of the skin, respectively. The creams were applied regularly and well tolerated throughout the study. All formulations increased skin hydration and elasticity. There was no significant difference between PNSC and Physiogel. However, PNSC was significantly more effective in increasing skin hydration and elasticity than PN and NNSC indicating that phytosphingosine inducing the positive charge, SC lipids and ceramide 3B are crucial for the enhanced effect on skin hydration and viscoelasticity.

Lipid replacement/antioxidant therapy as an adjunct supplement to reduce the adverse effects of cancer therapy and restore mitochondrial function.

The most common complaints of cancer patients undergoing chemo- or radiotherapy are fatigue, nausea, vomiting, malaise, diarrhea and headaches. These adverse effects are thought to be due to damage of normal tissues during the course of therapy. In addition, recent evidence indicates that fatigue is related to reduced mitochondrial function through loss of efficiency in the electron transport chain caused by membrane oxidation, and this occurs during aging, in fatiguing illnesses and in cancer patients during cytotoxic therapy. Lipid Replacement Therapy administered as a nutritional supplement with antioxidants can prevent oxidative membrane damage to normal tissues, restore mitochondrial and other cellular membrane functions and reduce the adverse effects of cancer therapy. Recent clinical trials using patients with chronic fatigue have shown the benefit of Lipid Replacement Therapy plus antioxidants in restoring mitochondrial electron transport function and reducing moderate to severe chronic fatigue by protecting mitochondrial and other cellular membranes from oxidative and other damage. In cancer patients a placebo-controlled, cross-over clinical trial using Lipid Replacement Therapy plus antioxidants demonstrated that the adverse effects of chemotherapy can be reduced in 57-70% of patients. Dietary use of unoxidized membrane lipids plus antioxidants is recommended for patients undergoing cancer therapy to improve quality of life but should not be taken at the same time of day as the therapy.

Quercus suber cork extract displays a tensor and smoothing effect on human skin: an in vivo study.

Recently, it has become indispensable for anti-aging active ingredients to provide a visible and immediate smoothing antiwrinkle effect. In Quercus suber, suberin is the most important structural component of cork cell walls. Studies have shown that suberin is made up mostly of hydroxycarboxylic acids and that it is endowed with many special mechanical and chemical properties that evoke a possible smoothing effect on the surface of the skin. Therefore, we were interested in investigating the effect of this cork extract on the skin's surface in a double-blind clinical study. The study was conducted in 15 healthy volunteers, aged 22 to 52 years. The volunteers applied a gel formula with 3% of cork extract, or placebo gel, on each forearm. Skin surface roughness was evaluated visually by pictures and by silicone replicas 1 and 2 h after application, followed by statistical analysis using the matched-pairs McNemar statistical test. McNemar analysis of the pictures revealed that application of cork extract on the skin resulted in a highly significant reduction of roughness 1 h after application. This effect was observed in 73.3% of volunteers. Two hours after cork extract application, a highly significant improvement of skin roughness was found in 78.6% of volunteers. Moreover, silicone replica treatment confirmed significant improvement in average of roughness at 2 h. These results demonstrate that cork extract provides a remarkable and highly significant tensor and smoothing effect on the skin, which could be of great use in anti-aging skin care products.

In vivo activity of released cell wall lipids of Mycobacterium bovis bacillus Calmette-Guérin is due principally to trehalose mycolates.

The hallmark of Mycobacterium-induced pathology is granulomatous inflammation at the site of infection. Mycobacterial lipids are potent immunomodulators that contribute to the granulomatous response and are released in appreciable quantities by intracellular bacilli. Previously we investigated the granulomagenic nature of the peripheral cell wall lipids of Mycobacterium bovis bacillus Calmette-Guérin (BCG) by coating the lipids onto 90-microm diameter microspheres that were mixed into Matrigel matrix with syngeneic bone marrow-derived macrophages and injected i.p. into mice. These studies demonstrated that BCG lipids elicit proinflammatory cytokines and recruit leukocytes. In the current study we determined the lipids responsible for this proinflammatory effect. BCG-derived cell wall lipids were fractionated and purified by liquid chromatography and preparative TLC. The isolated fractions including phosphatidylinositol dimannosides, cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, trehalose monomycolate, trehalose dimycolate, and mycoside B. Trehalose dimycolate, when delivered to bone marrow-derived murine macrophages, induced the greatest secretion of IL-1beta, IL-6, and TNF-alpha in vitro. Trehalose dimycolate similarly induced the greatest secretion of these proinflammatory cytokines in ex vivo matrices over the course of 12 days. Trehalose monomycolate and dimycolate also induced profound neutrophil recruitment in vivo. Experiments with TLR2 or TLR4 gene-deficient mice revealed no defects in responses to trehalose mycolates, although MyD88-deficient mice manifested significantly reduced cell recruitment and cytokine production. These results demonstrate that the trehalose mycolates, particularly trehalose dimycolate, are the most bioactive lipids in the BCG extract, inducing a proinflammatory cascade that influences granuloma formation.

Liposomal delivery of CTL epitopes to dendritic cells.

The induction of strong and long lasting T-cell response, CD4+ or CD8+, is a major requirement in the development of efficient vaccines. An important aspect involves delivery of antigens to dendritic cells (DCs) as antigen presenting cells (APCs) for the induction of potent antigen-specific CD8+ T lymphocyte (CTLs) responses. Protein or peptide-based vaccines become an attractive alternative to the use of live cell vaccines to stimulate CTL responses for the treatment of viral diseases or malignancies. However, vaccination with proteins or synthetic peptides representing discrete CTL epitopes have failed in most instances due to the inability for exogenous antigens to be properly presented to T cells via major histocompatibility complex (MHC) class I molecules. Modern vaccines, based on either synthetic or natural molecules, will be designed in order to target appropriately professional APCs and to co-deliver signals able to facilitate activation of DCs. In this review, we describe the recent findings in the development of lipid-based formulations containing a combination of these attributes able to deliver tumor- or viral-associated antigens to the cytosol of DCs. We present in vitro and pre-clinical studies reporting specific immunity to viral, parasitic infection and tumor growth.