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No / The effects of a conjugated linoleic acid (CLA) mixture of single isomers (50:50, w/w, cis9,trans11:trans10,cis12) and the individual isomers on (a) the production of resting and calcium ionophore stimulated 14C-eicosanoids and (b) the incorporation of 14C-arachidonic acid (AA) into membrane phospholipids of human saphenous vein endothelial cells were investigated. The CLA mixture and the individual isomers were found to inhibit resting production of 14C-prostaglandin F2a by 50, 43 and 40%, respectively. A dose dependent inhibition of stimulated 14C-prostaglandins was observed with the CLA mixture (IC50 100 ¿M). The cis9,trans11 and trans10,cis12 (50 ¿M) isomers individually inhibited the overall production of stimulated 14C-prostaglandins (between 35 and 55% and 23 and 42%, respectively). When tested at a high concentration (100 ¿M), cis9,trans11 was found to inhibit eicosanoid production in contrast to trans10,cis12 that caused stimulation. The overall degree of 14C-AA incorporation into membrane phospholipids of the CLA (mixture and individual isomers) treated cells was found to be lower than that of control cells and the cis9,trans11 isomer was found to increase the incorporation of 14C-AA into phosphatidylcholine. Docosahexaenoic acid, eicosapentaenoic acid and linoleic acid did not alter the overall degree of incorporation of 14C-AA. The results of this study suggest that both isomers inhibit eicosanoid production, and although trans10,cis12 exhibits pro-inflammatory activity at high concentrations, the CLA mixture maintains its beneficial anti-inflammatory action that contributes to its anti-carcinogenic and anti-atherogenic properties.

No / Chronic wounds often result from prolonged inflammation involving excessive polymorphonuclear leukocyte activity. Studies show that the omega-3 polyunsaturated fatty acids eicosapentaenoic and docosahexaenoic acids found in fish oils generate bioactive lipid mediators that reduce inflammation and polymorphonuclear leukocyte recruitment in numerous inflammatory disease models. The purpose of this study was to test the hypotheses that boosting plasma levels of eicosapentaenoic and docosahexaenoic acids with oral supplementation would alter lipid mediator levels in acute wound microenvironments and reduce polymorphonuclear leukocyte levels. Eighteen individuals were randomized to 28 days of either eicosapentaenoic + docosahexaenoic acid supplementation (Active Group) or placebo. After 28 days the Active Group had significantly higher plasma levels of eicosapentaenoic (p<0.001) and docosahexaenoic acid (p<0.001) than the Placebo Group and significantly lower wound fluid levels of two 15-lipoxygenase products of omega-6 polyunsaturated fatty acids, [9- hydroxyoctadecadienoic (HODE) acid (p = 0.033) and15-hydroxyeicosatrienoic acid (HETrE) (p = 0.006)], at 24 hours post wounding. The Active Group also had lower mean levels of myeloperoxidase, a leukocyte marker, at 12 hours and significantly more re-epithelialization on Day 5 post wounding. We suggest that lipid mediator profiles can be manipulated by altering polyunsaturated fatty acid intake to create a wound microenvironment more conducive to healing.

Yes / Background: Oral administration of purified omega-3 (ω-3) PUFAs is associated with changes to the fecal microbiome. However, it is not known whether this effect is associated with increased PUFA concentrations in the gut. Objectives: We investigated the luminal bioavailability of oral ω-3 PUFAs (daily dose 1 g EPA and 1g DHA free fatty acid equivalents as triglycerides in soft-gel capsules, twice daily) and changes to the gut microbiome, in the ileum. Methods: Ileostomy fluid (IF) and blood were obtained at baseline, after first capsule dosing (median 2 h), and at a similar time after final dosing on day 28, in 11 individuals (median age 63 y) with a temporary ileostomy. Fatty acids were measured by LC–tandem MS. The ileal microbiome was characterized by 16S rRNA PCR and Illumina sequencing. Results: There was a mean 6.0 ± 9.8-fold and 6.6 ± 9.6-fold increase in ileal EPA and DHA concentrations (primary outcome), respectively, at 28 d, which was associated with increased RBC ω-3 PUFA content (P ≤ 0.05). The first oral dose did not increase the ileal ω-3 PUFA concentration except in 4 individuals, who displayed high luminal EPA and DHA concentrations, which reduced to concentrations similar to the overall study population at day 28, suggesting physiological adaptation. Bacteroides, Clostridium, and Streptococcus were abundant bacterial genera in the ileum. Ileal microbiome variability over time and between individuals was large, with no consistent change associated with acute ω-3 PUFA dosing. However, high concentrations of EPA and DHA in IF on day 28 were associated with higher abundance of Bacteroides (r2 > 0.86, P < 0.05) and reduced abundance of other genera, including Actinomyces (r2 > 0.94, P < 0.05). Conclusions: Oral administration of ω-3 PUFAs leads to increased luminal ω-3 PUFA concentrations and changes to the microbiome, in the ileum of individuals with a temporary ileostomy.

Ever since the emergence of the hypothesis that linked the aetiology of schizophrenia with abnormal membrane phospholipids composition, an increasing number of evidences have suggested reduced membrane polyunsaturated fatty acids in patients with schizophrenia. This has led to a conduct of several studies to evaluate the efficacy of omega-3 fatty acid supplement in the modification of membrane phospholipids and treatment of schizophrenia. The two main omega-3 fatty acid classes, EPA and DHA, play a vital role in membranes. This project work reviews omega-3 fatty acid studies and summarizes their outcomes. Eight original articles (nine studies) were reviewed. Six out of nine studies measured RBC membrane fatty acids levels and all six studies reported a significant increase in EPA after EPA supplement. Two studies reported increased DHA post omega-3 fatty acid and DHA supplement, respectively. One study observed a dose-dependent increment in DHA after EPA supplement. Improved symptoms were observed in seven studies, while one study found a worsening of symptoms in patients with low baseline PUFA. Moreover, out of the six studies that evaluated the correlation between symptom change and membrane fatty acids change, three studies observed a correlation between increased EPA and symptom improvement. One study reported an increased AA associated with improved symptoms, in contrast to another study, which found a correlation between increased AA and worsened symptoms. The conclusion from this project work is that EPA supplement can increase the EPA levels in membranes; however, its therapeutic effect in schizophrenia requires further investigation using larger studies. / Ända sedan tillkomsten av hypotesen som länkade etiologin av schizofreni med onormala sammansättningar av membranfosfolipider, har bevis för nedsatt membranfettsyror hos patienter med schizofreni ökat. Detta har lett till genomförandet av flera studier för att utvärdera effekten av omega-3 supplement i modifieringen av membranfosfolipider och i behandling av schizofreni. De två viktigaste omega-3 klasserna, EPA och DHA, spelar en viktig roll i membran. Detta projektarbete granskar de omega-3 studierna och sammanfattar deras resultat. Åtta originalartiklar (nio studier) granskades. Sex av nio studier mätte nivåer av RBC membranfettsyror och alla sex studierna rapporterade en signifikant ökning av EPA efter EPA behandling. Två studier rapporterade ökad DHA efter omega-3 och DHA behandling, respektive. En studie observerade en dosberoende ökning i DHA efter EPA behandling. Förbättrade symtom observerades i sju studier, medan en studie fann en försämring av symtom hos patienter med låg baseline PUFA. Av de sex studier som utvärderade sambandet mellan symtomförändring och förändring i membranfettsyror, hittade två studier samband mellan ökad EPA och symtomförbättring. En studie rapporterade en ökad AA i samband med förbättrade symtom, i motsats till en annan studie, som fann ett samband mellan ökad AA och försämrade symtom. Slutsatsen från detta projektarbete är att EPA tillägg ökar nivåer av EPA i membranfosfolipider; men dess terapeutiska effekt vid schizofreni kräver ytterligare utredning med hjälp av större studier.

La maladie d’Alzheimer (MA) est une démence progressive caractérisée dans ses stades précoces par une perte synaptique et une dégénérescence neuronale. Aucun traitement efficace n’est disponible à ce jour et les mécanismes moléculaires impliqués sont encore mal connus. Des études récentes, menées notamment par notre équipe, indiquent que les oligomères de peptide amyloïde-ß (Aß) interagissent avec la membrane plasmique et y induisent un stress cellulaire impliquant des cascades apoptotiques. Ce travail a été consacré à l’étude de l’influence du statut lipidique sur la sensibilité des neurones au peptide Aß soluble et sur la signalisation pro-apoptotique. Nous avons montré que ce peptide induit un remodelage membranaire conduisant à la mort neuronale en activant des voies pro-apoptotiques et en inhibant des voies de survie. Par contre, la supplémentation du milieu en acide docosahexaénoïque (DHA), acide gras polyinsaturé de type ?3, protège les neurones de la mort induite par le peptide Aß. Cet effet préventif semble dépendre d’une incorporation du DHA pouvant localement induire un remodelage membranaire discret capable de préserver des voies de croissance et de survie cellulaire. Le DHA n’agit toutefois pas en inhibant l’activation de la phospholipase A2 cytosolique (cPLA2) et le stress oxydant induits par le peptide Aß, mécanismes que nous avons observés être impliqués dans la voie pro-apoptotique menant à la production de céramides suite à l’activation des sphingomyélinases. Ces enzymes occupent une position essentielle dans la toxicité du peptide Aß, puisqu’un prétraitement par le DHA ou par la sphingosine-1-phosphate, connue pour son potentiel antiapoptotique, protège les neurones en inhibant la sphingomyélinase acide. Les travaux de cette thèse ont donc montré que les lipides, molécules structurales mais aussi médiateurs de signalisation, sont des acteurs essentiels à considérer pour le développement de stratégies préventives ou thérapeutiques contre la MA / Alzheimer’s disease (AD) is a progressive dementia characterised in the early stages by synaptic loss and neurodegeneration. Currently, no effective treatments are available and the molecular mechanisms implicated are still unknown. Recent studies, including ours, indicate that the direct interaction between soluble amyloid-ß (Aß) oligomers and the plasma membrane initiates a cellular stress involving apoptotic cascades. The present work focuses on the impact of lipid status on neuronal susceptibility to the toxicity of soluble Aß peptide and on the pro-apoptotic signalling pathways. We showed that this peptide induces membrane remodelling, thereby activating pro-apoptotic pathways and inhibiting survival signalling, which leads to neuronal cell death. This was however prevented in neurons cultured in a medium supplemented with docosahexaenoic acid (DHA), an ?3 polyunsaturated fatty acid. This protective effect seems to rely on subtle local membrane remodelling due to DHA incorporation, which maintains active survival and growth pathways. DHA did not prevent either cytosolic phospholipase A2 (cPLA2) or oxidative stress upon soluble Aß peptide exposure. These two events have already been reported by our team to be implicated in a pro-apoptotic cascade leading to ceramide production due to the activation of sphingomyelinases. These enzymes very likely play a central role in Aß peptide toxicity, as pretreatments with DHA or sphingosine-1-phosphate, a well-known anti-apoptotic compound, lead to neuronal protection through the inhibition of acid sphingomyelinase. This PhD thesis demonstrates that lipids, as structural molecules as well as signalling mediators, are essential players that could be used to develop strategies for the prevention or treatment of AD

A Esclerose Lateral Amiotrófica (ELA) é uma doença progressiva e fatal causada pela degeneração seletiva dos neurônios motores do cérebro e medula. Dos casos familiares de ELA (fELA), 20% são causados por mutações pontuais no gene da sod1. O ácido docosahexaenoico (C22:6, n-3, DHA) é um ácido graxo altamente insaturado, sendo um dos principais ácidos graxos da massa cinzenta do cérebro. Estudos têm correlacionado mutações de SOD1 com a formação de agregados que poderiam ser induzidos por ácidos graxos insaturados. O objetivo deste estudo foi avaliar os efeitos e mecanismos do DHA e de seus hidroperóxidos (DHAOOH) na agregação de SOD1 in vitro. As análises de dicroísmo circular (CD) mostraram mudanças na estrutura secundária de ambas as proteínas apo-SOD1WT e G93A promovidas pelo DHA, resultando em aumento de superfície hidrofóbica e formação de estruturas do tipo beta-amilóide, como mostrado pelos ensaios do bis- ANS e Tioflavina, respectivamente. Estas mudanças resultam na formação de agregados amorfos como observado por microscopia eletrônica de varredura (MEV). Espécies de alto peso molecular foram observadas nas incubações do DHA com as formas apo da SOD1 por SDS-PAGE sob condições não redutoras e também por cromatografia de exclusão por tamanho. A formação dos agregados mostrou-se dependente de resíduos de Cys na sua forma desprotonada, visto que agregados não foram observados na presença de beta-mercaptoetanol e sua formação foi inibida na presença de bloqueador de tióis e em pH ácido. Além disso, análises por cromatografia de exclusão mostraram que a agregação é dependente da insaturação e conformação cis dos ácidos graxos. Comparativamente ao DHA, os hidroperóxidos do DHA tiveram um efeito menor na agregação de SOD1, porém revelaram a propriedade de induzir a dimerização covalente de SOD1. No geral, os dados mostram que o DHA induz a agregação de SOD1, através de um processo envolvendo a exposição de superfícies hidrofóbicas, formação de pontes dissulfeto e também de possíveis cross-links envolvendo reações do tipo \"ene-tiol\". / ALS is a progressive and fatal disease caused by selective degeneration of motor neurons in the brain and spinal cord. Twenty percent of familial ALS (fALS) cases are caused mainly by point mutations in the sod1 gene. Docosahexaenoic acid (C22:6, n-3, DHA) is a highly unsaturated fatty acid, wich is one of the main fatty acids in the cerebral gray matter. Studies have linked SOD1 mutations to the formation of aggregates that could be induced by unsaturated fatty acids. The aim of this study was to evaluate the effect of DHA on aggregation of SOD1 fALS mutants in vitro and its mechanisms. CD analysis shows changes in the secondary structure of both apo-SOD1WT and G93A promoted by DHA resulting in an increase in the surface hydrophobicity and formation of structures such as beta amyloid, which was also confirmed by bis-ANS assay and Thioflavin, respectively. These changes enhance the interaction of SOD1 and DHA, leading to amorphous aggregates as revealed by FESEM. Incubation of DHA with apo-SOD1 forms results in high-molecular weight species as detected by SDS-PAGE analyses under non-reducing conditions and also by size exclusion chromatography. This appears to require Cys residues in their thiolate forms because high aggregates are not observed under reducing conditions and also by size exclusion chromatography or at acidic pH. Also, size-exclusion chromatography indicates that the mutant apo-SOD1 aggregation is dependent on the unsaturation and cis-conformation of fatty acids. Compared to the DHA, DHAOOH had a minor effect on SOD1 aggregation, however revealed the ability to induce covalent dimerization of SOD1. Overall, the data suggest a mechanism of DHA aggregation, by a process involving exposure to hydrophobic surfaces, formation of disulfide bonds and also for possible cross-links involving reactions such \"thiol-ene\".