The modulatory effects of prostaglandin-E on cytokine production by human peripheral blood mononuclear cells are independent of the prostaglandin subtype

The production of inflammatory mediators, relevant to (auto)immune diseases and chronic inflammatory conditions, can be modulated by dietary intake of n-3 and n-6 long chain polyunsaturated fatty acids (PUFAs). It was suggested that these effects are related to the formation of different series of eicosanoids, in particular prostaglandin-E (PGE). In this study we investigated whether prostaglandin subtypes metabolized from arachidonic acid (PGE(2)), dihomo-γ-linolenic acid (PGE(1)) or eicosapentaenoic acid (PGE(3)) have different effects on T-cell proliferation and cytokine production in vitro. Freshly isolated human peripheral blood mononuclear cells (PBMC) were stimulated with concanavalin A (ConA) or lipopolysaccharide (LPS) in the presence or absence of exogenous PGE(1), PGE(2) or PGE(3). We found that tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and to a lesser extent interleukin (IL)-10 production was inhibited by all PGE-subtypes in ConA-stimulated PBMC concomitant with unaffected IL-2 levels. The modulated cytokine production of ConA stimulated cells was independent of T-cell proliferation. PGE(2) and PGE(1) moderately stimulated proliferation, while PGE(3) inhibited the proliferative response to some extent. In LPS-stimulated PBMC, TNF-α production was inhibited by all PGE-subtypes, whereas IL-6 remained unaffected and IL-10 production was increased. Time course experiments on the effects of PGE-subtypes on cytokine production after ConA or LPS stimulation showed these effects to be time dependent, but indifferent of the prostaglandin subtype added. Overall, the modulatory effects of PGE on cytokine production were irrespective of the subtype. This may implicate that the immunomodulatory effects of PUFAs, with respect to cytokine production, are not caused by a shift in the subtype of PGE

Polyunsaturated fatty acids support epithelial barrier integrity and reduce IL-4 mediated permeability in vitro.

Item does not contain fulltextBACKGROUND: The intestinal mucosa functions as a barrier against harmful dietary and microbial antigens. An intact gut barrier forms a prerequisite for protection against infection and allergy. Both allergic and inflammatory mediators (e.g. IL-4, IFN-gamma) are known to compromise the epithelial barrier integrity by enhancing permeability. Breast milk provides protection against infection and allergy and contains polyunsaturated fatty acids (PUFA). AIM OF THE STUDY: Although PUFA are commonly used in infant formulas their effect on intestinal barrier is still poorly understood. Therefore the effects of distinct PUFA (n-6: LA, GLA, DGLA, AA; n-3: ALA, EPA, DHA) and a fat blend with PUFA composition similar to that of the human breast milk fat fraction, on barrier integrity were investigated. METHODS: Human intestinal epithelial cells (T84) were pre-incubated with individual PUFA or a lipase treated fat blend, with or without subsequent IL-4 exposure. Barrier integrity was evaluated by measuring transepithelial resistance and permeability. Membrane phospholipid composition was determined by capillary gas chromatography. RESULTS: DGLA, AA, EPA, DHA and to a lesser extend GLA enhanced basal TER and strongly reduced IL-4 mediated permeability, while LA and ALA were ineffective. Furthermore, the lipase treated fat blend effectively supported barrier function. PUFA were incorporated in the membrane phospholipid fraction of T84 cells. CONCLUSIONS: Long chain PUFA DGLA, AA, EPA and DHA were particularly effective in supporting barrier integrity by improving resistance and reducing IL-4 mediated permeability. Fat blends that release specific PUFA upon digestion in the gastrointestinal tract may support natural resistance

In-Depth Glycoproteomic Characterization of γ-Conglutin by High-Resolution Accurate Mass Spectrometry

The molecular characterization of bioactive food components is necessary for understanding the mechanisms of their beneficial or detrimental effects on human health. This study focused on γ-conglutin, a well-known lupin seed N-glycoprotein with health-promoting properties and controversial allergenic potential. Given the importance of N-glycosylation for the functional and structural characteristics of proteins, we studied the purified protein by a mass spectrometry-based glycoproteomic approach able to identify the structure, micro-heterogeneity and attachment site of the bound N-glycan(s), and to provide extensive coverage of the protein sequence. The peptide/N-glycopeptide mixtures generated by enzymatic digestion (with or without N-deglycosylation) were analyzed by high-resolution accurate mass liquid chromatography–multi-stage mass spectrometry. The four main micro-heterogeneous variants of the single N-glycan bound to γ-conglutin were identified as Man(2)(Xyl) (Fuc) GlcNAc(2), Man(3)(Xyl) (Fuc) GlcNAc(2), GlcNAcMan(3)(Xyl) (Fuc) GlcNAc(2) and GlcNAc (2)Man(3)(Xyl) (Fuc) GlcNAc(2). These carry both core β1,2-xylose and core α1-3-fucose (well known Cross-Reactive Carbohydrate Determinants), but corresponding fucose-free variants were also identified as minor components. The N-glycan was proven to reside on Asn(131), one of the two potential N-glycosylation sites. The extensive coverage of the γ-conglutin amino acid sequence suggested three alternative N-termini of the small subunit, that were later confirmed by direct-infusion Orbitrap mass spectrometry analysis of the intact subunit