Probiotic interventions promote metabolic health in high fat-fed hamsters in association with gut microbiota and endocannabinoidome alterations.

Probiotics represent a promising tool to improve metabolic health, including lipid profiles and cholesterol levels. Modulation of the gut microbiome and the endocannabinoidome - two interrelated systems involved in several metabolic processes influenced by probiotics - has been proposed as a potential mechanism of action. This study establishes the impact of probiotics on metabolic health, gut microbiota composition and endocannabinoidome mediators in an animal model of hypercholesterolaemia. Syrian hamsters were fed either a low-fat low-cholesterol or high-fat high-cholesterol (HFHC) diet to induce hypercholesterolaemia and gavaged for 6 weeks with either Lactobacillus acidophilus CL1285, Lactiplantibacillus plantarum CHOL-200 or a combination of the two. Globally, probiotic interventions ameliorated, at least partially, lipid metabolism in HFHC-fed hamsters. The interventions, especially those including L. acidophilus, modified the gut microbiota composition of the small intestine and caecum in ways suggesting reversal of HFHC-induced dysbiosis. Several associations were observed between changes in gut microbiota composition and endocannabinoidome mediators following probiotic interventions and both systems were also associated with improved metabolic health parameters. For instance, potential connexions between the Eubacteriaceae and Deferribacteraceae families, levels of 2­palmitoylglycerol, 2­oleoylglycerol, 2­linoleoylglycerol or 2­eicosapentaenoylglycerol and improved lipid profiles were found. Altogether, our results suggest a potential crosstalk between gut microbiota and the endocannabinoidome in driving metabolic benefits associated with probiotics, especially those including L. acidophilus, in an animal model of hypercholesterolaemia.

[Extracellular molecules controlling the contraction of airway smooth muscle and their potential contribution to bronchial hyperresponsiveness]. / La contractilité du muscle lisse des voies respiratoires est régulée par de nombreuses molécules extracellulaires qui pourraient contribuer à l'hyperréactivité bronchique.

INTRODUCTION: A significant portion of symptoms in some lung diseases results from an excessive constriction of airways due to the contraction of smooth muscle and bronchial hyperresponsiveness. A better understanding of the extracellular molecules that control smooth muscle contractility is necessary to identify the underlying causes of the problem. STATE OF KNOWLEDGE: Almost a hundred molecules, some of which newly identified, influence the contractility of airway smooth muscle. While some molecules activate the contraction, others activate the relaxation, thus acting directly as bronchoconstrictors and bronchodilators, respectively. Other molecules do not affect contraction directly but rather influence it indirectly by modifying the effect of bronchoconstrictors and bronchodilators. These are called bronchomodulators. Some of these bronchomodulators increase the contractile effect of bronchoconstrictors and could thus contribute to bronchial hyperresponsiveness. PROSPECTS: Considering the high number of molecules potentially involved, as well as the level of functional overlap between some of them, identifying the extracellular molecules responsible for excessive airway constriction in a patient is a major contemporary challenge.

Effects of pyrrophenone, an inhibitor of group IVA phospholipase A2, on eicosanoid and PAF biosynthesis in human neutrophils.

BACKGROUND AND PURPOSE: The biosynthesis of leukotrienes (LT) and platelet-activating factor (PAF) involves the release of their respective precursors, arachidonic acid (AA) and lyso-PAF by the group IVA PLA2 (cPLA2alpha). This paper aims at characterizing the inhibitory properties of the cPLA2alpha inhibitor pyrrophenone on eicosanoids and PAF in human neutrophils (PMN). EXPERIMENTAL APPROACH: Freshly isolated human PMN were activated with physiological and pharmacological agents (fMLP, PAF, exogenous AA, A23187 and thapsigargin) in presence and absence of the cPLA2alpha inhibitor pyrrophenone and biosynthesis of LT, PAF, and PGE2 was measured. KEY RESULTS: Pyrrophenone potently inhibited LT, PGE2 and PAF biosynthesis in PMN with IC50s in the range of 1-20 nM. These inhibitory effects of pyrrophenone were specific (the consequence of substrate deprivation), as shown by the reversal of inhibition by exogenous AA and lyso-PAF. Comparative assessment of pyrrophenone, methyl-arachidonoyl-fluoro-phosphonate (MAFP) and arachidonoyl-trifluoromethylketone (AACOCF3) demonstrated that pyrrophenone was more specific and 100-fold more potent than MAFP and AACOCF3 for the inhibition of LT biosynthesis in A23187-activated PMN. The inhibitory effect of pyrrophenone on LT biosynthesis was reversible as LT biosynthesis was recovered when pyrrophenone-treated PMN were washed with autologous plasma. No alteration of phospholipase D (PLD) activity in fMLP-activated PMN was observed with up to 10 microM pyrrophenone, suggesting that the cPLA2alpha inhibitor does not directly inhibit PLD. CONCLUSIONS AND IMPLICATIONS: Pyrrophenone is a more potent and specific cPLA2alpha inhibitor than MAFP and AACOCF3 and represents an excellent pharmacological tool to investigate the biosynthesis and the biological roles of eicosanoids and PAF.

Eotaxin promotes eosinophil transmigration via the activation of the plasminogen-plasmin system.

The effect of eotaxin, a potent eosinophil chemotactic factor, on eosinophil transmigration through a reconstituted basal membrane (Matrigel) was evaluated. Eotaxin induced significant eosinophil transmigration in the presence of 10% fetal bovine serum (FBS) and interleukin-5. Its effect was optimal at 0.01 microM, and it plateaued at 18 h. Eotaxin's effect was greater with eosinophils from asthmatic subjects (61.1 +/- 3.4%) than with eosinophils from normal subjects (38.7 +/- 4.2%) (P < 0.001). Inhibition of metalloproteinases decreased eotaxin-induced transmigration by < or = 10.4%, whereas inhibition of the plasminogen-plasmin system decreased eotaxin's effect by < or = 44.4% (P = 0.0002). Moreover, eotaxin-induced transmigration was largely diminished in medium with low concentrations of serum [0.5% FBS: 6.1 +/- 2.4%; 10% FBS: 40.2 +/- 5.8% (P = 0.0001)] but returned to its initial level with the addition of plasminogen (2 U/mL) to 0.5% FBS (43.1 +/- 6.5%). These data show that eotaxin is an efficient promoter of eosinophil transmigration in vitro, that it is more potent with cells from asthmatics than with normal cells, and that its effect depends predominantly on the activation of the plasminogen-plasmin system.

Androgen formation and metabolism in the pulmonary epithelial cell line A549: expression of 17beta-hydroxysteroid dehydrogenase type 5 and 3alpha-hydroxysteroid dehydrogenase type 3.

Surfactant synthesis within developing fetal lung type II cells is affected by testosterone and 5alpha-dihydrotestosterone (5alpha-DHT). The pulmonary epithelial cell line A549, isolated from a human lung carcinoma, like normal lung type II cell, produces disaturated phosphatidylcholines and has been widely used for studying the regulation of surfactant production. Androgen receptor has been detected in A549 cells; however, the capacity of these cells for androgen synthesis and metabolism has not been investigated at molecular level. This study was undertaken to identify the steroidogenic enzymes involved in the formation and metabolism of androgens from adrenal C19 steroid precursors in A549 cells. When cultured in the presence of normal FCS, A549 intact cells converted DHEA to androstenediol, androstenedione principally to testosterone, and 5alpha-DHT to 5alpha-androstane 3alpha,17beta-diol. High levels of 17beta-hydroxysteroid dehydrogenase (HSD) and 3alpha-HSD activities were detected in both cytosol and microsomes isolated from homogenates. Analysis of A549 RNA indicated the presence of 17beta-HSD type 4 and type 5, and of 3alpha-HSD type 3 messenger RNAs. Very low levels of 3beta-HSD type 1 and 5alpha-reductase type 1 messenger RNAs and activities were detected. With regard to active androgen formation, there was little or no capacity for the conversion of DHEA to 5alpha-DHT. In contrast, androstenedione was rapidly transformed to testosterone. The pattern of steroid metabolism was not affected by the use of charcoal-stripped FCS or by the synthetic glucocorticoid dexamethasone. Together, our findings show that A549 cells express a pattern of steroid metabolism in which 17beta-HSD type 5 and 3alpha-HSD type 3 are the predominant enzymes. The level of androgens is regulated at the level of catalysis in intact cells such that the intracellular level of testosterone is stabilized, whereas 5alpha-DHT is rapidly inactivated by reduction to 3alpha,17beta-diol. This pattern of androgen metabolism has implications for the relative importance of testosterone and 5alpha-DHT in normal lung development and surfactant production.

Mini mutagenicity test: a miniaturized version of the Ames test used in a prescreening assay for point mutagenesis assessment.

The bacterial reverse mutagenicity test on Salmonella typhimurium, known as the Ames test, is widely used by regulatory agencies, academic institutions and chemical companies to assess the mutagenic potential of raw compounds. Several attempts have been made to miniaturise the Ames test in order to fit the industrial constraint of screening more products at the low quantities available. The major limitation of these miniaturised versions of the Ames test lies in the impossibility to work with all the six strains used in the regular Ames test, especially with those showing a low spontaneous revertant frequency. We describe here a mini version of the regulatory Ames test protocol that allows a significant reduction of the quantity of test substance needed (300 mg) but remains applicable to all Salmonella strains used in the regulatory protocol. In a preliminary study, 10 in-house chemical compounds have been evaluated in the Mini Mutagenicity Test (MMT) together with some positive control substances. A first set of historical data obtained in 1999 as well as the predictivity and the sensitivity of the MMT are presented and compared to those of the regular Ames test.

Leukotrienes: mediators that have been typecast as villains.

As befalls many mediators that act upon the human stage, leukotrienes have become identified with their most powerful roles as villains of the immune system. They are well known for their leading roles in allergic diseases, including asthma. They also have gained recognition for their dramatic role as promoters of inflammation. As new roles for these lipid messengers are sought, it is becoming apparent that the leukotrienes have been typecast as bad guys of the immune system. As examples, their more recent roles have been in atherosclerosis, pulmonary fibrosis and cancer. However, upon further evaluation, we can begin to see their versatility. Thus, leukotrienes stimulate innate immunity against pathogens. In addition, they promote the expression of mediators, receptors and other molecules that are important for immune defense. In these lesser known roles, they lead the fight against bacterial, fungal and viral infection. This review is intended to shed light on the leukotrienes, where they come from and what we really know about them.

In vivo distribution of free long-chain sphingoid bases in the human stratum corneum by high-performance liquid chromatographic analysis of strippings.

Conditions were established for the in vivo determination of free long-chain bases (phytosphingosine, sphingosine and sphinganine) in human stratum corneum layers by reversed-phase HPLC analysis of adhesive-tape strippings. Long-chain bases were first extracted from individual strippings with pure methanol in an ultrasound bath, then o-phthalaldehyde derivatives were separated on a C18 column with a gradient from methanol-water (80:20, v/v) to pure methanol. By performing a second series of strippings for protein determination it was possible to express the amounts of free long-chain bases per milligram of protein for each individual stratum corneum layer. Co-elution of endogenous phytosphingosine, sphingosine and spinganine was demonstrated in separate experiments by addition of standards to typical strips. From a study of three human volunteers, average concentrations of free long-chain bases in the stratum corneum were found to be 2.8, 1.2 and 0.5 nmol/mg of protein for phytosphingosine, sphingosine and sphinganine, respectively.

5-Oxo-6,8,11,14-eicosatetraenoic acid induces important eosinophil transmigration through basement membrane components: comparison of normal and asthmatic eosinophils.

Basement membrane transmigration is an important step in tissue recruitment of eosinophils into inflamed tissue. Recent reports showed that this phenomenon is modulated by platelet-activating factor (PAF) in combination with cytokines and proteinases. We investigated the in vitro efficacy of 5-oxo-6,8,11, 14-eicosatetraenoic acid (5-oxo-ETE), a metabolite of arachidonic acid and known as a potent eosinophil chemotactic factor, in promoting the transmigration of blood eosinophils from normal and asthmatic subjects through a Matrigel basement membrane. 5-Oxo-ETE proved to be a more potent (> 10-fold) inducer of eosinophil transmigration than PAF, and this effect was similar in cells from normal and asthmatic subjects (82.0 +/- 3.7% and 88.1 +/- 3.7%, respectively). Moreover, 5-oxo-ETE was active in the absence of interleukin (IL)-5, although this cytokine amplified the effect of 5-oxo-ETE from 61.3 +/- 3.3% to 92.8 +/- 1.8% (p = 0.003). The membrane receptor for urokinase plasminogen activator (CD87), a serine protease, was observed on eosinophils, and its expression was increased by IL-5. The inhibition of both metalloproteinases (MMP) and plasmin/plasminogen complex with inhibitor or monoclonal antibodies decreased cell transmigration by about 50%. Combination of an MMP inhibitor with anti-CD87 antibodies had no additive effect. These data show that 5-oxo-ETE is an efficient promoter of eosinophil transmigration in vitro, and is much more potent in this respect than PAF. The data suggest that 5-oxo-ETE could play an important role in eosinophil recruitment in vivo. Moreover, they demonstrate that in addition to MMP, the plasmin/plasminogen system could be involved in eosinophil transmigration.