Oil supplementation with a special combination of n-3 and n-6 long-chain polyunsaturated fatty acids does not protect for exercise induced asthma: a double-blind placebo-controlled trial.

BACKGROUND: Many patients suffering from exercise-induced asthma (EIA) have normal lung function at rest and show symptoms and a decline in FEV1 when they do sports or during exercise-challenge. It has been described that long-chain polyunsaturated fatty acids (LCPUFA) could exert a protective effect on EIA. METHODS: In this study the protective effect of supplementation with a special combination of n-3 and n-6 LCPUFA (sc-LCPUFA) (total 1.19 g/ day) were investigated in an EIA cold air provocation model. PRIMARY OUTCOME MEASURE: Decrease in FEV1 after exercise challenge and secondary outcome measure: anti-inflammatory effects monitored by exhaled NO (eNO) before and after sc-LCPUFA supplementation versus placebo. RESULTS: Ninety-nine patients with exercise-induced symptoms aged 10 to 45 were screened by a standardized exercise challenge in a cold air chamber at 4 °C. Seventy-three patients fulfilled the inclusion criteria of a FEV1 decrease > 15% and were treated double-blind placebo-controlled for 4 weeks either with sc-LCPUFA or placebo. Thirty-two patients in each group completed the study. Mean FEV1 decrease after cold air exercise challenge and eNO were unchanged after 4 weeks sc-LCPUFA supplementation. CONCLUSION: Supplementation with sc-LCPUFA at a dose of 1.19 g/d did not have any broncho-protective and anti-inflammatory effects on EIA. TRIAL REGISTRATION: Clinical trial registration number: NCT02410096. Registered 7 February 2015 at Clinicaltrial.gov.

A combination of LCPUFA ameliorates airway inflammation in asthmatic mice by promoting pro-resolving effects and reducing adverse effects of EPA.

Lipid mediators derived from omega (n)-3 and n-6 long-chain polyunsaturated fatty acids (LCPUFA) play key roles in bronchoconstriction, airway inflammation, and resolution processes in asthma. This study compared the effects of dietary supplementation with either a combination of LCPUFAs or eicosapentaenoic acid (EPA) alone to investigate whether the combination has superior beneficial effects on the outcome of asthmatic mice. Mice were sensitized with house dust mite (HDM) extract, and subsequently supplemented with either a combination of LCPUFAs or EPA alone in a recall asthma model. After the final HDM and LCPUFA administration, airway hyperresponsiveness (AHR), bronchoalveolar lavages, and lung histochemistry were examined. Lipid mediator profiles were determined by liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). The LCPUFA combination reduced AHR, eosinophilic inflammation, and inflammatory cytokines (IL-5, IFN-γ, and IL-6) in asthmatic mice, whereas EPA enhanced inflammation. The combination of LCPUFAs was more potent in downregulating EPA-derived LTB5 and LTC5 and in supporting DHA-derived RvD1 and RvD4 (2.22-fold and 2.58-fold higher levels) than EPA alone. Ex vivo experiments showed that LTB5 contributes to granulocytes' migration and M1-polarization in monocytes. Consequently, the LCPUFA combination ameliorated airway inflammation by inhibiting adverse effects of EPA and promoting pro-resolving effects supporting the lipid mediator-dependent resolution program.

A combination of LCPUFAs regulates the expression of miRNA-146a-5p in a murine asthma model and human alveolar cells.

BACKGROUND: LCPUFAs are suggestive of having beneficial effects on inflammatory diseases such as asthma. However, little is known about the modulative capacity of omega-(n)-3 and n-6 LCPUFAs within the epigenetic regulation of inflammatory processes. OBJECTIVE: The aim of this study was to investigate whether a specific combined LCPUFA supplementation restores disease-dysregulated miRNA-profiles in asthmatic mice. In addition, we determined the effect of the LCPUFA supplementation on the interaction of the most regulated miRNA expression and oxygenase activity in vitro. METHODS: Sequencing of miRNA was performed by NGS from lung tissue of asthmatic and control mice with normal diet, as well as of LCPUFA supplemented asthmatic mice. Network analysis and evaluation of the biological targets of the miRNAs were performed by DIANA- miRPath v.3 webserver software, TargetScanMouse 7.2, and tool String v.10, respectively. Expression of hsa-miRNA-146a-5p and activity of COX-2 and 5-LO in LCPUFA-treated A549 cells were assessed by qPCR and flow cytometry, respectively. RESULTS: In total, 62 miRNAs were dysregulated significantly in murine allergic asthma. The LCPUFA combination restored 21 of these dysregulated miRNAs, of which eight (mmu-miR-146a-5p, -30a-3p, -139-5p, -669p-5p, -145a-5p, -669a-5p, -342-3p and -15b-5p) were even normalized compared to the control levels. Interestingly, six of the eight rescued miRNAs are functionally implicated in TGF-ß signaling, ECM-receptor interaction and fatty acid biosynthesis. Furthermore, in vitro experiments demonstrated that upregulation of hsa-miRNA-146a-5p is accompanied by a reduction of COX-2 and 5-LO activity. Moreover, transfection experiments revealed that LCPUFAs inhibit 5-LO activity in the presence and absence of anti-miR-146a-5p. CONCLUSION: Our results demonstrate the modulative capacity of LCPUFAs on dysregulated miRNA expression in asthma. In addition, we pointed out the high regulative potential of LCPUFAs on 5-LO regulation and provided evidence that miR-146a partly controls the regulation of 5-LO.

A specific combined long-chain polyunsaturated fatty acid supplementation reverses fatty acid profile alterations in a mouse model of chronic asthma.

BACKGROUND: The immune-modulating potential of long-chain polyunsaturated fatty acids (LCPUFAs) based on their conversion into lipid mediators in inflammatory situations has been proven by several studies. Respecting the immune-modulative role of lipid mediators in bronchoconstriction, airway inflammation and resolution of inflammatory processes, LCPUFAs play an important role in asthma. To design a disease-specific and most beneficial LCPUFA supplementation strategy, it is essential to understand how asthma alters LCPUFA profiles. Therefore, this study characterizes the alterations of LCPUFA profiles induced by allergic asthma. In addition, this study explores whether a simple eicosapentaenoic acid (EPA) alone or a specific combined LCPUFA supplementation could restore imbalanced LCPUFA profiles. METHODS: Mice were sensitized with a daily dose of 40 µg house dust mite (HDM)-extract in a recall model and fed with either normal diet, EPA or a specific combined (sc)-LCPUFA supplementation containing EPA, docosahexaenoic acid (DHA), γ -linolenic acid (GLA) and stearidonic acid (SDA) for 24 days. After recall with HDM, mice were sacrificed and blood and lung tissue were collected. Fatty acid profiles were determined in plasma, blood cells and lung cells of asthmatic mice by capillary gas-chromatography. RESULTS: In lung cells of asthmatic mice, arachidonic acid (AA, p < 0.001) and DHA (p < 0.01) were increased while dihomo-γ-linolenic acid (DGLA, p < 0.05) was decreased. EPA supplementation increased only EPA (p < 0.001) and docosapentaenoic acid (DPA, p < 0.001), but neither DGLA nor DHA in lung cells of asthmatic mice. In contrast, a specific combined dietary supplementation containing n-3 and n-6 LCPUFAs could decrease AA (p < 0.001), increase EPA (p < 0.001), DPA (p < 0.001) and DHA (p < 0.01) and could reverse the lack of DGLA (p < 0.05). CONCLUSIONS: In summary, allergic asthma alters LCPUFA profiles in blood and lung tissue. In contrast to the EPA supplementation, the distinct combination of n-3 and n-6 LCPUFAs restored the LCPUFA profiles in lung tissue of asthmatic mice completely. Subsequently, sc-LCPUFA supplementation is likely to be highly supportive in limiting and resolving the inflammatory process in asthma.

Direct spray drying and microencapsulation of probiotic Lactobacillus reuteri from slurry fermentation with whey.

AIMS: Formulations of dietary probiotics have to be robust against process conditions and have to maintain a sufficient survival rate during gastric transit. To increase efficiency of the encapsulation process and the viability of applied bacteria, this study aimed at developing spray drying and encapsulation of Lactobacillus reuteri with whey directly from slurry fermentation. METHODS AND RESULTS: Lactobacillus reuteri was cultivated in watery 20% (w/v) whey solution with or without 0·5% (w/v) yeast extract supplementation in a submerged slurry fermentation. Growth enhancement with supplement was observed. Whey slurry containing c. 10(9)  CFU g(-1) bacteria was directly spray-dried. Cell counts in achieved products decreased by 2 log cycles after drying and 1 log cycle during 4 weeks of storage. Encapsulated bacteria were distinctively released in intestinal milieu. Survival rate of encapsulated bacteria was 32% higher compared with nonencapsulated ones exposed to artificial digestive juice. CONCLUSIONS: Probiotic L. reuteri proliferate in slurry fermentation with yeast-supplemented whey and enable a direct spray drying in whey. The resulting microcapsules remain stable during storage and reveal adequate survival in simulated gastric juices and a distinct release in intestinal juices. SIGNIFICANCE AND IMPACT OF THE STUDY: Exploiting whey as a bacterial substrate and encapsulation matrix within a coupled fermentation and spray-drying process offers an efficient option for industrial production of vital probiotics.

Effect of n-3 polyunsaturated fatty acids in asthma after low-dose allergen challenge.

BACKGROUND: We investigated the anti-inflammatory potential of n-3 polyunsaturated fatty acids (PUFA) on specific bronchial inflammation. Allergic asthmatics were challenged using a low-dose allergen provocation model. METHODS: Our parallel double-blinded study randomly assigned 23 house dust mite-allergic asthmatics (aged 22-29 years; 13 females, 10 males) to dietary supplementation with either an n-3 PUFA-enriched fat blend (0.69 g/day) or placebo for 5 weeks. After 3 weeks, the patients were challenged daily with low doses of mite allergen for 2 weeks. Primary outcome parameters were effects on lung function (forced expiratory volume in 1 s, FEV(1)) and exhaled nitric oxide (eNO) as a marker of bronchial inflammation. RESULTS: Even before the bronchial challenge, eNO was significantly lower in the n-3 PUFA group (p=0.014). Levels of eNO increased during allergen exposure in both groups, but differences in means were significantly lower in the n-3 PUFA group (p=0.022). During the low-dose allergen challenge, there were no differences between the groups with regard to symptoms, FEV(1) or the allergen dose required to induce deterioration of lung function (PD(20)). Numbers of sputum eosinophils did not differ significantly, while serum eosinophils (10.1+/-0.1.84 vs. 5.79+/-0.69%) as well as changes in eosinophilic cationic protein (20.5+/-9.93 vs. -1.68+/-4.36 ng/ml) and in vitro cysteinyl leukotriene release (2,889+/-872 vs. 1,120+/-173 ng/ml) were significantly lower in the n-3 PUFA group (p<0.05 each). CONCLUSION: Our results provide evidence that dietary supplementation with n-3 PUFA is able to reduce bronchial inflammation even after low-dose allergen challenge.

[Kinetics of Pseudomonas aeruginosa virulence gene expression during chronic lung infection in the murine model]. / Cinétique d'expression des gènes de virulence de Pseudomonas aeruginosa au cours d'une infection pulmonaire chronique dans le modèle murin.

UNLABELLED: Pseudomonas aeruginosa is a Gram-negative bacillus frequently encountered in human diseases. P. aeruginosa produces a large number of secreted and cell associated virulence factors. Their production is coordinated by various systems of gene regulation. The correlation and sequential intervention of regulation systems during a pulmonary infection have not been determined yet. OBJECTIVE: The aim of this study was to analyze the expression of three P. aeruginosa virulence genes (exoS, lasI, and algD) during the first seven days of chronic lung infection. To do so, mice were infected intratracheally with agarose beads containing P. aeruginosa. RESULTS: The results were a progressive decrease of exoS transcription and an increase of algD, and lasI transcription during infection. This dynamic evolution was consistent with the clinical observation, which demonstrated a progressive loss of type III secretion system function and an increase in the mucoid phenotype development in P. aeruginosa strains from cystic fibrosis patients. CONCLUSION: The development of a P. aeruginosa pulmonary chronic infection associates a decrease of gene expression related to a type III secretion system and an increase of alginate production.

Polyunsaturated fatty acid supplementation stimulates differentiation of oligodendroglia cells.

Dietary polyunsaturated fatty acids (PUFAs) have been postulated as alternative supportive treatment for multiple sclerosis, since they may promote myelin repair. We set out to study the effect of supplementation with n-3 and n-6 PUFAs on OLN-93 oligodendroglia and rat primary oligodendrocyte differentiation in vitro. It appeared that OLN-93 cells actively incorporate and metabolise the supplemented PUFAs in their cell membrane. The effect of PUFAs on OLN-93 differentiation was further assessed by morphological and Western blot evaluation of markers of oligodendroglia differentiation: 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), zonula occludens-1 (ZO-1) and myelin-associated glycoprotein (MAG). Supplementation of the OLN-93 cells with n-3 and n-6 PUFAs increased the degree of differentiation determined by morphological analysis. Moreover, CNP protein expression was significantly increased by gamma-linolenic acid (GLA, 18:3n-6) supplementation. In accordance with the OLN-93 results, studies with rat primary oligodendrocytes, a more advanced model of cell differentiation, showed GLA supplementation to promote oligodendrocyte differentiation. Following GLA supplementation, increased numbers of proteolipid protein (PLP)-positive oligodendrocytes and increased myelin sheet formation was observed during differentiation of primary oligodendrocytes. Moreover, increased CNP, and enhanced PLP and myelin basic protein expression were found after GLA administration. These studies provide support for the dietary supplementation of specific PUFAs to support oligodendrocyte differentiation and function.

Breastfeeding, soluble CD14 concentration in breast milk and risk of atopic dermatitis and asthma in early childhood: birth cohort study.

BACKGROUND: Breast milk contains a variety of bioactive substances, among them, soluble CD14 (sCD14), which plays an important role in innate immunity. OBJECTIVE: We analysed data of a large prospective birth cohort study to examine the determinants of sCD14 in breast milk, and investigated whether breastfeeding practice and sCD14 concentrations in breast milk are determinants of the risk of atopic dermatitis (AD) and asthma in children. METHODS: Eight hundred and three mothers and their newborns were included in this analysis. We measured sCD14 concentrations in breast milk samples collected 6 weeks post-partum. During a 2-year follow-up the cumulative incidences of AD and asthma were recorded. RESULTS: Overall, AD was reported for 20.6% of the 2-year-olds and asthma was reported for 19.6%. We found the lowest incidence of physician-reported AD in children of mothers without a history of atopic diseases if breastfed for 6 to less than 9 months. Furthermore, we found an inverse association between duration of breastfeeding and risk of asthma, which was especially evident in children with mothers without a history of atopic disease (P=0.01). These patterns persisted after control for other factors by multivariate analysis methods. The protective effect of breastfeeding seemed to be synergistic with sCD14 concentrations in breast milk (P for trend 0.0005). CONCLUSIONS: The results of this prospective birth cohort study suggest that a longer duration of breastfeeding does decrease the risk for asthma in early childhood, especially in children of mothers without a history of atopic disease. The beneficial effects of breastfeeding might be further supported by high levels of sCD14 in breast milk.

Lipoproteins from Borrelia burgdorferi applied in liposomes and presented by dendritic cells induce CD8(+) T-lymphocytes in vitro.

Borrelia burgdorferi (Bb) is the tick-borne etiologic agent of Lyme borreliosis, which has many aspects of autoimmune diseases. Bb is unable to recycle synthesized membrane lipids and lipoproteins. Consequently, a large amount of liposome-like vesicle (Bb-blebs) is shed from the outer bacterial membrane. The influence of Bb-blebs on the cellular immune response is not yet known. As a Bb-blebs model, we established standardized Bb-liposomes, produced from freshly extracted lipids and lipoproteins of live Bb. Bb-liposomes were incorporated via nonendocytotic mechanisms by different human cell types, namely dendritic cells (DC), lymphocytes, and fibroblasts, as visualized by immunofluorescence and transmission electron microscopy. Bb-liposomes were localized in the cytosol and in the nucleus of the cells. With this in mind, we generated in vitro Bb-specific T-cells from nonadherant peripheral blood mononuclear cells by use of Bb-liposomes loaded autologous DC. More than 95% of those T-cells were CD8(+) and they killed autologous Bb-liposome-loaded T-cell blasts. These results suggest that Bb-blebs may be responsible for the autoimmune-like appearance of Lyme disease.

The lipid component of lipoproteins from Borrelia burgdorferi: structural analysis, antigenicity, and presentation via human dendritic cells.

The spirochaetal bacteria Borrelia burgdorferi (Bb) is the tick-borne causative agent of lyme disease. The major membrane immunogens of Bb are outer surface proteins. The lipid component of these lipoproteins is relevant for the immunogenicity of Bb-lipoproteins. To characterize the antigenic properties, the native lipid component of lipoproteins was isolated and the detailed molecular structure was analyzed. The molecular structure of the lipoprotein-lipid component turned out to be S(propane-2',-3'diol)-3-thio-2-aminopropanic acid (S-glyceryl-cysteine) with one ester-linked fatty acid, one acetyl group, and one N-terminal amide-bound fatty acid. Fatty acid analysis of the lipid component indicated a heterogeneous composition comprising C16:0, C18:0, C18:1, C18:2, and C 20:0. The antigenicity was tested with in vitro bioassays using human blood-derived dendritic cells (DCs) as antigen-presenting cells and autologous Bb-specific T-cells. We found that human DCs present the lipid component of Bb-lipoproteins via MHC class II inducing an antigen-specific T-cell immune response in vitro.

Venodilatory effects of nicorandil in healthy volunteers.

The effects of a single dose of nicorandil (40 mg orally) on human dorsal hand veins in vivo were assessed and compared with the effect of nitroglycerin (0.8 mg sublingually). The hand veins had been preconstricted with norepinephrine to approximately one-third of their initial size. We demonstrated that a venous relaxation occurred after administration of nicorandil and nitroglycerin. In the doses studied, the effect was more pronounced and lasted longer after nicorandil. The pharmacodynamic effects of nicorandil in humans may be explained in part by this venous relaxation.

Oral administration of carvedilol and prazosin inhibits the prostaglandin F2 alpha- and noradrenaline-induced contraction of human hand veins in vivo.

Carvedilol is a beta-blocker with additional vasodilating activity. This study was performed in order to determine whether the vasodilator action of orally administered carvedilol in man is based upon an alpha-adrenoceptor antagonism exclusively or if evidence for an additional mechanism could be confirmed. The influence of carvedilol (50 mg p.o.) and prazosin (2 mg p.o.) upon the vasoconstrictor effect of noradrenaline and prostaglandin F2 alpha, infused into superficial hand veins, was established in 8 healthy male volunteers. Increasing dosages of the vasoconstrictors below their threshold of systemic activity were employed in order to obtain dose-response curves of the hand veins congested at a venous occlusion pressure of 40 mmHg. These dose-response curves were repeated 1 and 3.5 h after oral administration of either carvedilol, prazosin, or placebo. The ex vivo, in vitro alpha 1-receptor occupancy in plasma was measured before and after each vasoconstrictor dose-response curve, using an alpha 1-radioreceptor binding assay. Washout periods of 48 h were kept between study days, investigating the influence of one orally administered drug upon one of the local vasoconstrictor dose-response curves at a time. In the alpha 1-radioreceptor assay, plasma concentrations from 0.9- to 1.7-fold the equilibrium dissociation constant (Ki) of carvedilol could be evaluated 1 as well as 3.5 h after medication, corresponding with a receptor occupancy of 44%-63%. After prazosin, 9-13 times the Ki values were determined, which amounts to an alpha 1-adrenoceptor occupation of about 90%-93%.(ABSTRACT TRUNCATED AT 250 WORDS)

Constriction of human dorsal hand veins in vivo with several vasoconstrictors and the influence of oral administration of carvedilol.

In vivo measurements of human dorsal hand vein diameters have become a valuable tool in investigating vasoactive agents. This study in 13 healthy volunteers was performed to establish the influence of locally infused clonidine, angiotensin II, norepinephrine, and prostaglandin F2 alpha (PGF2 alpha) on hand vein diameter in a nonsystemically active dose range. The study was intended to select agents suitable for venous preconstriction before and after oral administration of 50 mg carvedilol to obtain information on the mechanisms of vasodilation of this combined alpha- and beta-blocker. Infusions of norepinephrine (ED50, 20-40 ng/min) and PGF2 alpha (ED50, 480-1,170 ng/min) into the hand vein under investigation led to nearly complete constriction of the vessel. Infusions with clonidine and angiotensin II led to approximately 35% diameter reduction under the same conditions. A wide interindividual variety of hand vein dose response was observed for all vasoconstrictors. Oral administration of 50 mg carvedilol led to a rightward shift in the dose-response curves of angiotensin II, norepinephrine, and PGF2 alpha. On a 5% significance level, the venoconstrictive effects of norepinephrine and PGF2 alpha were attenuated by carvedilol compared with placebo. We conclude that a mechanism of vasodilation by carvedilol in addition to alpha-adrenoceptor antagonism could be responsible for the attenuation of venoconstriction induced by PGF2 alpha.

The effect of oral cilazapril and prazosin on the constrictor effects of locally infused angiotensin I and noradrenaline in human dorsal hand veins.

The effects of the ACE inhibitor cilazapril (5 mg p.o.) and the alpha 1-adrenoceptor blocker prazosin (2 mg p.o.) were investigated on the dose-response curves to angiotensin I and to noradrenaline, administered locally in the hand veins in six healthy male volunteers in doses not producing systemic effects. Both angiotensin I and noradrenaline produced a dose-dependent constriction of the congested veins. The angiotensin I effects were completely abolished after the administration of cilazapril but not significantly altered after the administration of prazosin. The noradrenaline dose-response curves were shifted to the right (dose ratio about 10) by prazosin, but not by cilazapril. The data suggest that angiotensin I, after having been converted to angiotensin II exerts direct venoconstrictor effects which under resting conditions are not mediated by noradrenaline release.