Methodological Considerations for Lipid and Polar Component Analyses in Human Skin Stratum Corneum.

Collection of skin very top layer, called stratum corneum, by tape stripping and the analysis of stratum corneum components by mass spectrometry provides multiple advantages for clinical studies that aim to understand the origins of allergic skin diseases and food allergy. However, such a methodology has multiple challenges on the way of complex stratum corneum analysis when molecules of different polarity are needed to be analyzed from minimal amount of skin tape strips. This review provides an overview of current knowledge about lipid and polar molecules in the skin, discusses challenging aspects of sample processing when dealing with skin tape strips, and provides some guidance towards approaches that generate complex, quantitative, normalized to total sample protein data that fit best the purpose of analysis of stratum corneum components for the purpose of clinical trials.

Omega 3 fatty acids stimulate thermogenesis during torpor in the Arctic Ground Squirrel.

Omega 3 polyunsaturated fatty acids (PUFAs) influence metabolism and thermogenesis in non-hibernators. How omega 3 PUFAs influence Arctic Ground Squirrels (AGS) during hibernation is unknown. Prior to hibernation we fed AGS chow composed of an omega 6:3 ratio approximately 1:1 (high in omega 3 PUFA, termed Balanced Diet), or an omega 6:3 ratio of 5:1 (Standard Rodent Chow), and measured the influence of diet on core body temperature (Tb), brown adipose tissue (BAT) mass, fatty acid profiles of BAT, white adipose tissue (WAT) and plasma as well as hypothalamic endocannabinoid and endocannabinoid-like bioactive fatty acid amides during hibernation. Results show feeding a diet high in omega 3 PUFAs, with a more balanced omega 6:3 ratio, increases AGS Tb in torpor. We found the diet-induced increase in Tb during torpor is most easily explained by an increase in the mass of BAT deposits of Balanced Diet AGS. The increase in BAT mass is associated with elevated levels of metabolites DHA and EPA in tissue and plasma suggesting that these omega 3 PUFAs may play a role in thermogenesis during torpor. While we did not observe diet-induced change in endocannabinoids, we do report altered hypothalamic levels of some endocannabinoids, and endocannabinoid-like compounds, during hibernation.

Lysophosphatidic acid is detectable in human bronchoalveolar lavage fluids at baseline and increased after segmental allergen challenge.

BACKGROUND: Lysophosphatidic acid (LPA) is a biologically active lysophospholipid and a component of normal plasma. LPA binds to receptors expressed on circulating and structural lung cells and affects cell growth and activation. Whether LPA is present in the lung has not been previously reported. OBJECTIVE: To develop an assay to measure LPA in bronchoalveolar lavage (BAL) fluids, and to study the association between LPA and allergic airway inflammation. METHODS: Seventeen allergic subjects underwent bronchoscopy and segmental allergen challenge, followed 18 h later by BAL. Supernatants were analysed for LPA content using liquid chromatography and mass spectroscopy. Expression of LPA receptors on primary bronchial epithelial cells was analysed by immunolabelling, and the effects of LPA on epithelial cell barrier function was investigated by measuring transepithelial resistance. RESULTS: LPA was detectable in BAL from control lung segments, and significantly increased 18 h after allergen challenge. Polyunsaturated species of LPA were especially increased following segmental allergen challenge. LPA levels did not strongly correlate with the number or percentages of eosinophils, neutrophils of lymphocytes, whereas MIP-3alpha (CCL20) levels correlated significantly with the allergen-driven influx of lymphocytes. The levels of LPA from control sites correlated inversely with BAL protein content, suggesting that LPA promoted epithelial barrier integrity at baseline. Experiments using primary human bronchial epithelial cells confirmed that LPA tightened the epithelial cell barrier. CONCLUSION: Lysophosphatidic acid is detectable in human BAL fluids at baseline and its expression increases during allergic inflammation. LPA does not appear to be a dominant chemoattractant for eosinophils or lymphocytes during allergic airway inflammation. In the absence of ongoing inflammation, LPA may promote epithelial barrier integrity.

Modification of biological activity of lipopolysaccharide in the complex with chitosan.

In the complex with chitosan, lipopolysaccharide partially lost its ability to induce lymphokines tumor necrosis factor and interleukin-8, but retained immunostimulating properties and increased phagocytic function of macrophages by improving digestion of bacteria.

[Effect of the cannabinoid receptor ligand WIN-55,212-2 on the TNF-alpha production by human mononuclear cells]. / Vliianie liganda kannabinoidnykh retseptorov WIN-55,212-2 na vyrabotku TNF-al'fa mononuklearnymi kletkami cheloveka.

Synthetic cannabinoid WIN-55,212-2 produces a dose-dependent decrease on the TNF-alpha production by LPS-stimulated human mononuclear cells in vitro. The most pronounced inhibition of the TNF-alpha synthesis was observed for a drug concentration of 10 microM.

Cannabinoid receptor-inactive N-acylethanolamines and other fatty acid amides: metabolism and function.

Although it is now generally accepted that long-chain N-acylethanolamines and their precursors, N-acylethanolamine phospholipids, exist as trace constituents in virtually all vertebrate cells and tissues, their possible biological functions are just emerging. While anandamide (N-arachidonoylethanolamine) has received much attention due to its ability to bind to and activate cannabinoid receptors, the saturated and monounsaturated N-acylethanolamines, which usually represent the vast majority, are cannabinoid receptor-inactive but appear to interact with endocannabinoids and to have other signaling functions as well. Also, primary fatty acid amides, including the amide of oleic acid, which acts as a sleep-inducing agent, do not interact with cannabinoid receptors but are catabolically related to endocannabinoids. Here we review published information on the occurrence, metabolism, and possible signaling functions of the cannabinoid receptor-inactive N-acylethanolamines and primary fatty acid amides.

Cannabinoid-receptor-independent cell signalling by N-acylethanolamines.

Anandamide and other polyunsaturated N-acylethanolamines (NAEs) exert biological activity by binding to cannabinoid receptors. These receptors are linked to G(i/o) proteins and their activation leads to extracellular-signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAP kinase) activation, inhibition of cAMP-dependent signalling and complex changes in the expression of various genes. Saturated and monounsaturated NAEs cannot bind to cannabinoid receptors and may thus mediate cell signalling through other targets. Here we report that both saturated/monounsaturated NAEs and anandamide (20:4(n-6) NAE) stimulate cannabinoid-receptor-independent ERK phosphorylation and activator protein-1 (AP-1)-dependent transcriptional activity in mouse epidermal JB6 cells. Using a clone of JB6 P(+) cells with an AP-1 collagen-luciferase reporter construct, we found that 16:0, 18:1(n-9), 18:1(n-7), 18:2(n-6) and 20:4(n-6) NAEs stimulated AP-1-dependent transcriptional activity up to 2-fold, with maximal stimulation at approx. 10-15 microM. Higher NAE concentrations had toxic effects mediated by alterations in mitochondrial energy metabolism. The AP-1 stimulation appeared to be mediated by ERK but not JNK or p38 signalling pathways, because all NAEs stimulated ERK1/ERK2 phosphorylation without having any effect on JNK or p38 kinases. Also, overexpression of dominant negative ERK1/ERK2 kinases completely abolished NAE-induced AP-1 activation. In contrast with 18:1(n-9) NAE and anandamide, the cannabinoid receptor agonist WIN 55,212-2 did not stimulate AP-1 activity and inhibited ERK phosphorylation. The NAE-mediated effects were not attenuated by pertussis toxin and appeared to be NAE-specific, as a close structural analogue, oleyl alcohol, failed to induce ERK phosphorylation. The data support our hypothesis that the major saturated and monounsaturated NAEs are signalling molecules acting through intracellular targets without participation of cannabinoid receptors.

Activation of PAF receptors results in enhanced synthesis of 2-arachidonoylglycerol (2-AG) in immune cells.

The endocannabinoid signaling system is believed to play a down-regulatory role in the control of cell functions. However, little is known about the factors activating endocannabinoid synthesis and which of two known endocannabinoids, 2-arachidonoylglycerol (2-AG) or N-arachidonoylethanolamine (20:4n-6 NAE, anandamide), is of physiological importance. We approached these questions by studying a possible link between cell activation with 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor, PAF) and the generation of 2-AG and anandamide in human platelets and mouse P388D1 macrophages. Human platelets responded to stimulation with the production of various 1- and 2-monoacylglycerols, including 2-AG, whereas stimulation of P388D1 macrophages induced the rapid and selective generation of 2-AG, which was immediately released into the medium. The effect of PAF was receptor mediated, as PAF receptor antagonist BN52021 blocked the effect. The treatment did not change the content of anandamide in either macrophages or platelet-rich plasma. The inhibitors of PI- and PC-specific phospholipases C (U73122 and D609) as well as PI3-kinase inhibitor (wortmannin) attenuated PAF-induced 2-AG production in macrophages. These data suggest a direct role for the endocannabinoid system in controlling immune cell activation status and indicate that 2-AG rather than anandamide is the endocannabinoid rapidly produced in response to proinflammatory stimulation of immune cells.

Immunomodulatory Properties of Cucumariosides from the Edible Far-Eastern Holothurian Cucumaria japonica.

Holothurian triterpene glycosides (cucumariosides) are known to possess multiple biological activities. Here we show that cucumariosides from the Far Eastern edible holothurian (sea cucumber), Cucumaria japonica, and their semisynthetic derivatives possess potent immunomodulatory properties. Intraperitoneal injection of cucumariosides (0.2-20 ng per mouse) induced macrophage lysosomal activity in a dose-dependent manner (up to 250% of control). The stimulatory effect was related to the chemical structure of cucumariosides and was especially influenced by the number and position of sulfate groups in the carbohydrate moiety of the molecules. In vitro, an inhibitory rather than a stimulatory effect of cucumariosides on phagocytosis and release of tumor necrosis factor-alpha (TNF-alpha) was found. Virtually all cucumariosides inhibited latex bead phagocytosis by human peripheral blood granulocytes in a dose-dependent manner. Also, the lipopolysaccharide-induced TNF-alpha production by immune cells in human blood diluted with RPMI-1640 was decreased, without a clear relation to the structure and dose of the compounds. The data are discussed in terms of possible mechanisms underlying immunomodulatory properties of triterpene glycosides from C. japonica.

Cannabinoid receptors and the regulation of immune response.

Cannabinoid research underwent a tremendous increase during the last 10 years. This progress was made possible by the discovery of cannabinoid receptors and the endogenous ligands for these receptors. Cannabinoid research is developing in two major directions: neurobehavioral properties of cannabinoids and the impact of cannabinoids on the immune system. Recent studies characterized the cannabinoid-induced response as a very complex process because of the involvement of multiple signalling pathways linked to cannabinoid receptors or effects elicited by cannabinoids without receptor participation. The objective of this review is to present this complexity as it applies to immune response. The functional properties of cannabinoid receptors, signalling pathways linked to cannabinoid receptors and the modulation of immune response by cannabinoid receptor ligands are discussed. Special attention is given to 'endocannabinoids' as immunomodulatory molecules.

Stress-induced generation of N-acylethanolamines in mouse epidermal JB6 P+ cells.

It has long been known that N-acylethanolamine phospholipids [N-acylphosphatidylethanolamine (N-acyl PE)] and N-acylethanolamines (NAEs) accumulate in mammalian tissues undergoing degenerative membrane changes associated with necrosis. Here we studied the effects of stress factors (UVB irradiation and serum deprivation) on the endogenous levels of N-acyl PE and NAE in mouse epidermal JB6 P(+) cells. We found that 16:0, 18:0, 18:1,n-9 and 18:1,n-7 are the predominant amide-linked fatty acids in both N-acyl PE and NAE in these cells. UVB irradiation and serum deprivation resulted in significantly increased levels of N-acyl PE and NAE, especially 18:1, n-9 N-acyl PE and NAE. UVB challenge increased the cellular content of anandamide (20:4,n-6 NAE), but this increase was the lowest of all NAEs measured. Serum deprivation resulted in a decreased cellular anandamide level, as well as a decrease in 20:4,n-6 N-acyl PE. Interestingly, the replacement of serum-free medium with medium containing 5% (v/v) fetal calf serum after 36 h of serum deprivation restored N-acyl PE and NAE levels almost completely within 4-8 h. These data suggest the involvement of N-acyl PE and NAE in cellular responses to stress.

A sensitive endocannabinoid assay. The simultaneous analysis of N-acylethanolamines and 2-monoacylglycerols.

Mammalian cells produce both N-arachidonoylethanolamine (20:4n-6 NAE, anandamide) and 2-arachidonoylglycerol (2-AG), lipid signaling molecules that activate cannabinoid receptors. Because both agonists occur in the presence of receptor-inactive congeners, we have developed a sensitive method for the simultaneous assay of N-acylethanolamines (NAEs) and 2-monoacylglycerols (2-MAG). These lipid classes are isolated from total lipids by solid phase extraction and converted to tert-butyldimethylsilyl (tBDMS) derivatives in the presence of deuterated analogs. The tBDMS derivatives are analyzed by gas chromatography/mass spectrometry using selected ion monitoring programs specific for NAE and 2-MAG. Individual NAEs and 2-MAGs can be quantified in the nanogram and subnanogram range. The NAE and 2-MAG compositions of rat organs and cultured JB6 cells are reported.

Inhibition of sea urchin fertilization by fatty acid ethanolamides and cannabinoids.

The influence of saturated and unsaturated fatty acid ethanolamides as well as delta9-tetrahydrocannabinol (delta9-THC), WIN 55,212-2 and cannabinoid CB1 receptor antagonist SR 141716 on sea urchin fertilization was studied. The ethanolamides of arachidonic, oleic and linoleic acids but not saturated fatty acid (C14-C20) derivatives inhibited fertilization when pre-incubated with sperm cells. Delta9-THC and WIN 55,212-2 also inhibited fertilization, delta9-THC being ten times as potent as WIN 55,212-2. Selective cannabinoid CB1 receptor antagonist SR 141716 also blocked fertilization and did not antagonize the action of delta9-THC. The obtained results indicate that different unsaturated fatty acid ethanolamides may control sea urchin fertilization, and that sea urchin sperm cell cannabinoid receptor may differ from the known cannabinoid receptor subtypes.

Effects of cannabinoid receptor ligands on LPS-induced pulmonary inflammation in mice.

The effects of cannabinoid receptor agonists WIN 55,212-2, delta9-tetrahydrocannabinol (delta9-THC), arachidonoylethanolamide (anandamide) and palmitoylethanolamide on lipopolysaccharide (LPS) -induced bronchopulmonary inflammation in mice were investigated. WIN 55,212-2 and delta9-THC induced a concentration-dependent decrease in TNF-alpha level in the bronchoalveolar lavage fluid (BALF) (maximum inhibition 52.7% and 36.9% for intranasal doses of 750 nmol x kg(-1) and 2.65 mmol x kg(-1), respectively). This effect was accompanied by moderately reduced neutrophil recruitment. Palmitoylethanolamide (750 nmol x kg(-1)) diminished the level of TNF-alpha in BALF by 31.5% but had no effect on neutrophil recruitment. Anandamide (7.5-750 nmol x kg(-1)) did not influence the inflammatory process but TNF-alpha level and neutrophil recruitment were decreased by 28.0% and 62.0%, respectively, with 0.075 nmol x kg(-1). These results demonstrate that the cannabinoid receptor ligands inhibited LPS-induced pulmonary inflammation and suggest that this effect could be at least in part mediated by the cannabinoid CB2 receptor.

Influence of fatty acid ethanolamides and delta9-tetrahydrocannabinol on cytokine and arachidonate release by mononuclear cells.

The effects of arachidonic acid ethanolamide (anandamide), palmitoylethanolamide and delta9-tetrahydrocannabinol on the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-4, interleukin-6, interleukin-8, interleukin-10, interferon-gamma, p55 and p75 TNF-alpha soluble receptors by stimulated human peripheral blood mononuclear cells as well as [3H]arachidonic acid release by non-stimulated and N-formyl-Met-Leu-Phe (fMLP)-stimulated human monocytes were investigated. Anandamide was shown to diminish interleukin-6 and interleukin-8 production at low nanomolar concentrations (3-30 nM) but inhibited the production of TNF-alpha, interferon-gamma, interleukin-4 and p75 TNF-alpha soluble receptors at higher concentrations (0.3-3 microM). Palmitoylethanolamide inhibited interleukin-4, interleukin-6, interleukin-8 synthesis and the production of p75 TNF-alpha soluble receptors at concentrations similar to those of anandamide but failed to influence TNF-alpha and interferon-gamma production. The effect of both compounds on interleukin-6 and interleukin-8 production disappeared with an increase in the concentration used. Neither anandamide nor palmitoylethanolamide influenced interleukin-10 synthesis. delta9-Tetrahydrocannabinol exerted a biphasic action on pro-inflammatory cytokine production. TNF-alpha, interleukin-6 and interleukin-8 synthesis was maximally inhibited by 3 nM delta9-tetrahydrocannabinol but stimulated by 3 microM delta9-tetrahydrocannabinol, as was interleukin-8 and interferon-gamma synthesis. The level of interleukin-4, interleukin-10 and p75 TNF-alpha soluble receptors was diminished by 3 microM delta9-tetrahydrocannabinol. [3H]Arachidonate release was stimulated only by high delta9-tetrahydrocannabinol and anandamide concentrations (30 microM). These results suggest that the inhibitory properties of anandamide, palmitoylethanolamide and delta9-tetrahydrocannabinol are determined by the activation of the peripheral-type cannabinoid receptors, and that various endogenous fatty acid ethanolamides may participate in the regulation of the immune response.

Phosphodiesterase 4 inhibitors and db-cAMP inhibit TNF-alpha release from human mononuclear cells. Effects of cAMP and cGMP-dependent protein kinase inhibitors.

We investigated the effects of specific inhibitors of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) on the inhibitory activity of phosphodiesterase (PDE) type 4 inhibitors and of the cell permeable analogue of cAMP, db-cAMP on LPS-induced TNF-alpha release from human mononuclear cells. Incubation from 30 min of mononuclear cells with dbcAMP (10(-5) to 10(-3) M), rolipram (10(-9) M to 10(-5) M) or Ro 20-1724 (10(-9) M to 10(-5) M) significantly inhibited LPS-induced TNF-alpha release. When mononuclear cells were preincubated for 30 min with the selective PKA inhibitor, H89 (10(-4) M), but not with the selective PKG inhibitor, Rp-8-pCPT-cGMPs (10(-4) M), a significant reduction of the inhibitory effect of db-cAMP was noted. Thirty min incubation of mononuclear cells with Rp-8-pCPT-cGMPs induced a significant reduction of the inhibitory activities of both rolipram and Ro 20-1724 (10(-9) to 10(-5) M) on LPS-induced TNF-alpha release, whereas H89 elicited a moderate, but significant inhibition. The present data indicate that db-cAMP inhibits TNF-alpha release from human mononuclear cells through a PKA-dependent mechanism. In contrast, PDE 4 inhibitors elicit their in vitro anti-inflammatory activities via a PKG-dependent rather than PKA-dependent activation.

Sea urchins--a new model for PAF research in embryology.

This study reports on the effect of PAF on sperm motility, fertilization and early embryo development in the sea urchin Strongylocentrotus intermedius. PAF proved to be the amplifier of sperm motility and fertilizing capability at 10(-8)-10(-9) M and was toxic at higher concentrations. BN 52201 did not counteract the PAF action. The advantages of using sea urchins for PAF research in embryology are discussed.

[Interaction of 1-O-alkyl-2-methoxy-sn-glycero-3-phosphocholine and platelet activating factor with blood and tumor cells]. / Vzaimodeistvie 1-O-alkil-2-metoksi-sn-glitsero-3-fosfokholina i faktora aktivatsii trombotsitov s kletkami krovi i opukholevymi kletkami.

The effects of the platelet-activating factor (PAF) and the methoxy-PAF analog, 1-O-alkyl-2-methoxy-sn-glycero-3-phosphocholine, on platelet activation, oxidative burst in neutrophils and proliferation of K-562 and P815 cells have been studied. It has been found that the biological activity of methoxy-PAF for human platelets and neutrophils is commensurate with that of PAF. However, the cytotoxic properties of methoxy-PAF are much stronger than the cytotoxicity of PAF for tumour cells. It is concluded that the biological activity of methoxy-PAF is, to a great extent, due to its close structural resemblance to PAF as well as to the inability of cells to perform rapid metabolism of methoxy-PAF.