Preparation, characterisation and entrapment of a non-glycosidic threitol ceramide into liposomes for presentation to invariant natural killer T cells

Dendritic cells (DCs) are able to present glycolipids to invariant natural killer T (iNKT) cells in vivo. Very few compounds have been found to stimulate iNKT cells, and of these, the best characterised is the glycolipid a-galactosylceramide, which stimulates the production of large quantities of interferon-gamma (IFN-?) and interleukin-4 (IL-4). However, aGalCer leads to overstimulation of iNKT cells. It has been demonstrated that the aGalCer analogue, threitol ceramide (ThrCer 2), successfully activates iNKT cells and overcomes the problematic iNKT cell activation-induced anergy. In this study, ThrCer 2 has been inserted into the bilayers of liposomes composed of a neutral lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or dimethyldioctadecylammonium bromide (DDA), a cationic lipid. Incorporation efficiencies of ThrCer within the liposomes was 96% for DSPC liposomes and 80% for DDA liposomes, with the vesicle size (large multilamellar vs. small unilamellar vesicles) making no significant difference. Langmuir-Blodgett studies suggest that both DSPC and DDA stack within the monolayer co-operatively with the ThrCer molecules with no condensing effect. In terms of cellular responses, IFN-? secretion was higher for cells treated with small DDA liposomes compared with the other liposome formulations, suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs

La geografia a la Universitat Autònoma de Barcelona : un projecte d'Enric Lluch (II)

S'ha complert mig segle d'ensenyament de la geografia a la UAB (de 1969 a 2019). Al present article se n'hi expliquen els primers vint anys, fins a completar el desenvolupament de la Llei de reforma universitària (LRU) a la nostra universitat, i s'hi dona a conèixer quin era l'equip acadèmic que va acompanyar Enric Lluch (1928-2012) en aquesta experiència. També s'hi introdueix la geografia que recollia els enfocaments innovadors que arribaven principalment de França, dels països anglosaxons i d'Itàlia, i s'hi mostra la formació docent i investigadora del professorat, a més del desenvolupament de les eines científiques que han fet possible l'assentament de la geografia a la UAB. Bàsicament, es tracta d'explicar-hi la recerca, els contactes internacionals, els laboratoris i la revista Documents d'Anàlisi Geogràfica, entre d'altres qüestions. A les conclusions s'hi valora l'esforç col·lectiu que ha fet possible que aquest procés hagi continuat amb èxit, tant a Bellaterra com a Girona.Se ha cumplido medio siglo de enseñanza de la geografía en la UAB (de 1969 a 2019). En el presente artículo se explican sus primeros veinte años hasta completar el desarrollo de la Ley de reforma universitaria (LRU) en nuestra universidad y se da a conocer cuál era el equipo académico que acompañó a Enric Lluch (1928-2012) en esta experiencia. Se introduce la geografía que recogía los enfoques innovadores que llegaban principalmente de Francia, de los países anglosajones y de Italia, y se muestra la formación docente e investigadora del profesorado, así como el desarrollo de las herramientas científicas que han hecho posible el asentamiento de la geografía en la UAB. Básicamente, se trata de contar la investigación, los contactos internacionales, los laboratorios y la revista Documents d'Anàlisi Geogràfica, entre otros aspectos. En las conclusiones se valora el esfuerzo colectivo que ha hecho posible que este proceso haya continuado con éxito, tanto en Bellaterra como en Girona.Cinquante ans d'enseignement de la Géographie à l'UAB (1969 à 2019) viennent d'être célébrés. Les 20 premières années sont présentées, jusqu'à l'achèvement du développement de la loi sur la réforme de l'université (LRU) à l'UAB, ainsi que l'équipe académique qui a accompagné Enric Lluch (1928-2012) dans cette expérience. On a introduit la géographie qui a rassemblé les approches novatrices venues principalement de la France, des pays anglo-saxons et de l'Italie. Dans cette deuxième partie, nous expliquons la formation des enseignants en matière d'enseignement et de recherche, ainsi que le développement des outils scientifiques qui ont permis l'établissement de la géographie à l'UAB. Il s'agit essentiellement de recherche, de contacts internationaux, de laboratoires et du journal Documents d'Anàlisi Geogràfica, entre autres. Dans les conclusions, l'effort collectif qui a permis à ce processus de se poursuivre avec succès, à Bellaterra comme à Girona, est valorisé.Geography has been taught at the Autonomous University of Barcelona (UAB) for 50 years (1969-2019). This paper explores the first 20 years of geography until the entry into force of the University Reform Law (LRU) at the UAB and the academic team that accompanied Enric Lluch (1928-2012) in this experience. The introduction of geography based on innovative approaches coming mainly from France, English-speaking countries and Italy is discussed. In this second part we also describe the teaching and research training of teachers, and the development of scientific tools that have made the establishment of geography in the UAB possible: basically, research, international contacts, laboratories and the Documents d'Anàlisi Geogràfica journal, among others. To conclude, the collective effort that has contributed to the success of this process both in Bellaterra and in Girona is examined

Enzymatically oxidized phospholipids restore thrombin generation in coagulation factor deficiencies

Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell–derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid–phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed

Synthesis of Novel Glycolipid Agonists of the Protein CD1d

Invariant NKT (iNKT) cells are a subset of T lymphocytes that express an invariant $\alpha$ $\beta$ T cell receptor (TCR) as well as an NK1.1 marker. They play an important role in autoimmune diseases, such as type I diabetes and lupus. In contrast to conventional CD4+ and CD8+ T lymphocytes that recognise foreign peptides bound to the major histocompatibility complex (MHC) class I or MHC class II, iNKT cells recognise a range of foreign lipids and glycolipids bound to CD1d proteins. $\alpha$ $\beta$-Galactosyl Ceramide ($\alpha$ $\beta$GalCer), originally isolated from a marine sponge, is a powerful agonist of CD1d capable of triggering an immune response that results in the proliferation of a range of regulatory cytokines, including IFN-$_y$ (Th1), as well as IL-4 (Th2). This mixed cytokine response (i.e. Th1 and Th2), combined with the “unresponsive state” of iNKT cells after activation with $\alpha$GalCer, limits the therapeutic potential of this agonist. To address some of these issues, we have also synthesised an $\alpha$GalCer analogue, namely Threitol Ceramide (ThrCer), that exhibits attenuated activity relative to $\alpha$GalCer. ThrCer is a truncated analogue of $\alpha$GalCer that conserves the stereochemistry of the hydroxyl functions present in $\alpha$GalCer and that exhibit a much stronger ether bond under acidic hydrolysis linking the sugar moiety with ceramide than the glycosidic bond in $\alpha$GalCer. We have labelled ThrCer with a biotin residue and with a 14C radiolabel, and our collaborators have used these derivatives to show that ThrCer behaves similarly to $\alpha$GalCer in endogenous lipid trafficking and its tissue distribution in vivo. We have also made advances towards the stereoselective synthesis of recently discovered natural agonists of iNKT cells from pathogenic origin, namely $\alpha$-galactosyl diacylglycerol ($\alpha$GalDAG)

Steric analysis of epoxyalcohol and trihydroxy derivatives of 9-hydroperoxy-linoleic acid from hematin and enzymatic synthesis

We characterize the allylic epoxyalcohols and their trihydroxy hydrolysis products generated from 9R- and 9S-hydroperoxy-octadecenoic acid (HPODE) under non-enzymatic conditions, reaction with hematin and subsequent acid hydrolysis, and enzymatic conditions, incubation with Beta vulgaris containing a hydroperoxide isomerase and epoxide hydrolase. The products were resolved by HPLC and the regio and stereo-chemistry of the transformations were determined through a combination of 1H NMR and GC–MS analysis of dimethoxypropane derivatives. Four trihydroxy isomers were identified upon mild acid hydrolysis of 9S,10S-trans-epoxy-11E-13S-hydroxyoctadecenoate: 9S,10R,13S, 9S,12R,13S, 9S,10S,13S and 9S,12S,13S-trihydroxy-octadecenoic acids, in the ratio 40:26:22:12. We also identified a prominent δ-ketol rearrangement product from the hydrolysis as mainly the 9-hydroxy-10E-13-oxo isomer. Short incubation (5 min) of 9R- and 9S-HPODE with B. vulgaris extract yielded the 9R- and 9S-hydroxy-10E-12R,13S-cis-epoxy products respectively. Longer incubation (60 min) gave one specific hydrolysis product via epoxide hydrolase, the 9R/S,12S,13S-trihydroxyoctadecenoate. These studies provide a practical approach for the isolation and characterization of allylic epoxy alcohol and trihydroxy products using a combination of HPLC, GC–MS and 1H NMR

Design, Synthesis, and Functional Activity of Labeled CD1d Glycolipid Agonists

Invariant natural killer T cells (<i>i</i>NKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) <b>1</b>, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to <i>i</i>NKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR−α-GalCer–CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the <i>pro</i>-<i>S</i> hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR–glycolipid–CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the <i>pro</i>-<i>S</i>, and not the <i>pro</i>-<i>R</i>, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (<i>S</i>)-<b>10</b> displayed behavior comparable to that of ThrCer <b>5</b> itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 <b>4</b>, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (<b>11</b>) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for attaching a label to a range of CD1d agonists. The flexibility of the synthetic strategy, and late-stage incorporation of the label, opens up the possibility of using this labeling approach to study the in vivo behavior of a wide range of CD1d agonists

Design, Synthesis, and Functional Activity of Labeled CD1d Glycolipid Agonists

Invariant natural killer T cells (<i>i</i>NKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) <b>1</b>, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to <i>i</i>NKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR−α-GalCer–CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the <i>pro</i>-<i>S</i> hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR–glycolipid–CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the <i>pro</i>-<i>S</i>, and not the <i>pro</i>-<i>R</i>, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (<i>S</i>)-<b>10</b> displayed behavior comparable to that of ThrCer <b>5</b> itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 <b>4</b>, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (<b>11</b>) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for attaching a label to a range of CD1d agonists. The flexibility of the synthetic strategy, and late-stage incorporation of the label, opens up the possibility of using this labeling approach to study the in vivo behavior of a wide range of CD1d agonists

Quantitative assays for esterified oxylipins generated by immune cells

Phospholipid-esterified oxylipins include newly described families of bioactive lipids generated by lipoxygenases in immune cells. Until now, assays for their quantitation were not well developed or widely available. Here, we describe a mass spectrometric protocol that enables accurate measurement of several esterified oxylipins—in particular hydro(pero)xyeicosatetraenoic acids, hydroxyoctadecadienoic acids, hydroxydocosahexaenoic acids and keto-eicosatetraenoic acids—attached to either phosphatidylethanolamine or phosphatidylcholine. Lipids are isolated from cells or tissue using a liquid-phase organic extraction, then analyzed by HPLC–tandem mass spectrometry (LC/MS/MS) in multiple reaction–monitoring mode. The protocol can simultaneously monitor up to 23 species. Generation of standards takes ~2 d. Following this, extraction of 30 samples takes ~3 h, with LC/MS/MS run time of 50 min per sample