Miguel Angel de la Fuente

Mechanism of recruitment of WASP to the immunological synapse and of its activation following TCR ligation

F-actin polymerization following engagement of the T cell receptor (TCR) is dependent on WASP and is critical for T cell activation. The link between TCR and WASP is not fully understood. In resting cells, WASP exists in a complex with WIP, which inhibits its activation by Cdc42. We show that the adaptor protein CrkL binds directly to WIP. Further, TCR ligation results in the formation of a ZAP-70-CrkL-WIP-WASP complex, which is recruited to lipid rafts and the immunological synapse. TCR engagement also causes PKCtheta-dependent phosphorylation of WIP, causing the disengagement of WASP from the WIP-WASP complex, thereby releasing it from WIP inhibition. These results suggest that the ZAP-70-CrkL-WIP pathway and PKCtheta link TCR to WASP activation.

WIP Deficiency Reveals a Differential Role for WIP and the Actin Cytoskeleton in T and B Cell Activation

WIP stabilizes actin filaments and is important for filopodium formation. To define the role of WIP in immunity, we generated WIP-deficient mice. WIP(minus sign/minus sign) mice have normal lymphocyte development, but their T cells fail to proliferate, secrete IL-2, increase their F-actin content, polarize and extend protrusions following T cell receptor ligation, and are deficient in conjugate formation with superantigen-presenting B cells and anti-CD3 bilayers. In contrast, WIP-deficient B lymphocytes have enhanced proliferation and CD69 expression following B cell receptor ligation and mount normal antibody responses to T-independent antigens. Both WIP-deficient T and B cells show a profound defect in their subcortical actin filament networks. These results suggest that WIP is important for immunologic synapse formation and T cell activation.

WIP is a chaperone for Wiskott–Aldrich syndrome protein (WASP)

Wiskott–Aldrich syndrome protein (WASP) is in a complex with WASP-interacting protein (WIP). WASP levels, but not mRNA levels, were severely diminished in T cells from WIP−/− mice and were increased by introduction of WIP in these cells. The WASP binding domain of WIP was shown to protect WASP from degradation by calpain in vitro. Treatment with the proteasome inhibitors MG132 and bortezomib increased WASP levels in T cells from WIP−/− mice and in T and B lymphocytes from two WAS patients with missense mutations (R86H and T45M) that disrupt WIP binding. The calpain inhibitor calpeptin increased WASP levels in activated T and B cells from the WASP patients, but not in primary T cells from the patients or from WIP−/− mice. Despite its ability to increase WASP levels proteasome inhibition did not correct the impaired IL-2 gene expression and low F-actin content in T cells from the R86H WAS patient. These results demonstrate that WIP stabilizes WASP and suggest that it may also be important for its function.

CD84 functions as a homophilic adhesion molecule and enhances IFN-gamma secretion: adhesion is mediated by Ig-like domain 1.

CD84 is a member of the CD2 subset of the Ig superfamily of cell surface molecules. Its cytoplasmic tail binds to Src homology 2 domain-containing protein 1A (signaling lymphocytic activation molecule-associated protein), a protein encoded by the X-linked lymphoproliferative disease gene. It is preferentially expressed on B lymphocytes, monocytes, and platelets. We show that it is also expressed on thymocytes and T cells. CD84 was positive on CD4−CD8− thymocytes, and its expression decreased with cell maturation. It is expressed on mature T cells preferentially on CD45RO+. To identify the CD84 ligand, we generated a soluble Ig fusion protein containing the human CD84 extracellular domains (CD84-Ig). Because receptor-ligand interactions occur between several members of this subfamily, we assayed CD84-Ig binding with all members of the CD2 family. CD84-Ig bound to CD84-transfected cells, whereas no binding was detected with cells expressing other CD2 subfamily receptors, showing that CD84 binds to itself. Anti-CD84 mAbs recognizing epitopes wholly within domain 1 of CD84 blocked the binding of the CD84-Ig fusion protein to CD84-transfected cells and platelets. Data from CD84 domain human/mouse chimeras further revealed that only the first extracellular domain of the molecule is involved in the ligand receptor recognition. The CD84-CD84 interaction was independent of its cytoplasmic tail. Finally, concurrent ligation of human CD84 with mAbs or CD84-Ig and CD3 enhanced IFN-γ secretion in human lymphocytes. Thus, CD84 is its own ligand and acts as a costimulatory molecule.

Enhancement of amino acid catabolism in Cheddar cheese using α-ketoglutarate : amino acid degradation in relation to volatile compounds and aroma character

The effectiveness of the transaminase acceptor α-ketoglutarate in enhancing amino acid catabolism and manipulating the aroma profile of Cheddar cheese has been studied. Utilisation of α-ketoglutarate, catabolism of amino acids, volatiles production, and aroma profile of the cheese were monitored after 6, 12 and 24 weeks ripening. Glutamate and GABA were considerably enhanced on addition of the transaminase acceptor while levels of phenylalanine, leucine, isoleucine, alanine, valine, methionine and threonine were reduced. Addition of α-ketoglutarate increased volatile components originating from the catabolism of branched chain and aromatic amino acids. These compounds included acetic, propanoic, 2-methylpropanoic and 3-methylbutanoic acids, 3-methylbutanol, phenylacetaldehyde and benzaldehyde. Additionally enhanced production of 3-OH-2-butanone was evident. Addition of α-ketoglutarate increased aroma intensity, creamy and fruity aromas. Effects obtained must be verified by tasting cheeses made with food grade α-ketoglutarate, but results suggest potential benefits in accelerated maturation, low fat systems and manipulation of flavour profiles.

Authenticity Assessment of Dairy Products

The authenticity of dairy products has become a focal point, attracting the attention of scientists, producers, consumers, and policymakers. Among many others, some of the practices not allowed in milk and milk products are the substitution of part of the fat or proteins, admixtures of milk of different species, additions of low-cost dairy products (mainly whey derivatives), or mislabeling of products protected by denomination of origin. A range of analytical methods to detect frauds have been developed, modified, and continually reassessed to be one step ahead of manufacturers who pursue these illegal activities. Traditional procedures to assess the authenticity of dairy products include chromatographic, electrophoretic, and immunoenzymatic methods. New approaches such as capillary electrophoresis, polymerase chain reaction, and isotope ratio mass spectrometry have also emerged alongside the latest developments in the former procedures. This work intends to provide an updated and extensive overview since 1991 on the principal applications of all these techniques together with their advantages and disadvantages for detecting the authenticity of dairy products. The scope and limits of different tools are also discussed.

Changes in the mineral balance of milk submitted to technological treatments

Abstract The importance of the salt content and its distribution on the technological properties of milk is well known. In particular, the mineral balance between the colloidal and diffusible phases is decisive in the cheesemaking process. Although changes in the mineral balance produced by heating or cold storage have been widely studied, new developments have occurred in the past few years. In addition, new technological treatments have been developed, such as the preservation of milks at low temperature in a CO 2 atmosphere or the use of high pressure. All these treatments modify the distribution of salts and change the properties of the milk. This article reviews the effects of the different treatments on the mineral balance and discusses their technological implications.

Differential role of SLP-76 domains in T cell development and function

The adapter SLP-76 is essential for thymocyte development. SLP-76−/− mice were reconstituted with SLP-76 deletion mutant transgenes to examine the role of SLP-76 domains in T cell development and function. The N-terminal domain deletion mutant completely failed to restore thymocyte development. Mice reconstituted with Gads-binding site and SH2 domain deletion mutants had decreased thymic cellularity, impaired transition from double to single positive thymocytes, and decreased numbers of mature T cells in the spleen. Calcium mobilization and extracellular signal-regulated protein kinase activation were decreased in the Gads-binding site mutant but almost normal in the SH2 domain mutant. T cells from both mutants failed to proliferate following T cell antigen receptor ligation. Nevertheless, both mutants mounted partial cutaneous hypersensitivity responses and normal T cell dependent IgG1 antibody responses. These results indicate differential roles for SLP-76 domains in T cell development, proliferation and effector functions.

B cell-intrinsic deficiency of the Wiskott-Aldrich syndrome protein (WASp) causes severe abnormalities of the peripheral B-cell compartment in mice

Wiskott Aldrich syndrome (WAS) is caused by mutations in the WAS gene that encodes for a protein (WASp) involved in cytoskeleton organization in hematopoietic cells. Several distinctive abnormalities of T, B, and natural killer lymphocytes; dendritic cells; and phagocytes have been found in WASp-deficient patients and mice; however, the in vivo consequence of WASp deficiency within individual blood cell lineages has not been definitively evaluated. By conditional gene deletion we have generated mice with selective deficiency of WASp in the B-cell lineage (B/WcKO mice). We show that this is sufficient to cause a severe reduction of marginal zone B cells and inability to respond to type II T-independent Ags, thereby recapitulating phenotypic features of complete WASp deficiency. In addition, B/WcKO mice showed prominent signs of B-cell dysregulation, as indicated by an increase in serum IgM levels, expansion of germinal center B cells and plasma cells, and elevated autoantibody production. These findings are accompanied by hyperproliferation of WASp-deficient follicular and germinal center B cells in heterozygous B/WcKO mice in vivo and excessive differentiation of WASp-deficient B cells into class-switched plasmablasts in vitro, suggesting that WASp-dependent B cell-intrinsic mechanisms critically contribute to WAS-associated autoimmunity.

Fatty acid composition of the triglyceride and free fatty acid fractions in different cows-, ewes- and goats-milk cheeses

ZusammenfassungEs wurde die Fettsäurezusammensetzung der Triglyceridfraktionen und der freien Fettsäuren in verschiedenen Käsesorten aus Kuh-, Schaf- und Ziegenmilch untersucht. Die Blauschimmelkäsesorten Cabrales, Roquefort und der Manchegokäse zeigten die höchsten freien Fettsäurewerte: 57.3; 26.0 und 32,4 g/kg, gefolgt von denen, die mit Hilfe von unreinem Lab hergestellt wurden (Majorero: 20.8 g/kg; Parmesan: 13.7 g/kg). Diese Proben zeigten ein pikantes Aroma. Die Schimmelkäsesorten wie Camembert und de la Vera hatten einen mittleren freien Fettsäuregehalt von 5.1 und 9.9 g/kg. Die anderen untersuchten Proben wiesen ebenfalls niedrige Lipolysewerte auf (Mahón: 8.7 g/kg; Roncal: 8.2 g/kg; Tetilla: 5.8 g/kg; Idiazabal: 5.6 g/kg. Im Vergleich der Fettsäureprofile der untersuchten Fraktionen wurde bei den freien Fettsäurefraktionen ein höherer Gehalt an kurzen Fettsäuren beobachtet, mit Ausnahme der Blauschimmelkäse und der Ziegenmilchkäsesorten.SummaryThe fatty acid composition of the triglyceride fraction and the individual and total free fatty acids in different cows-, ewes- and goats-milk cheeses were analysed. The highest total free fatty acid levels were recorded in blue cheeses (Roquefort: 26.0 g/kg; Cabrales: 57.3 g/kg) and in the aged Manchego cheese (32.4 g/kg), followed by those in hard cheeses manufactured using unpurified rennet pastes containing pregastric esterases (Parmesan: 13.7 g/kg; Majorero: 20.8 g/kg). These samples had sharp flavours. The soft cheeses with surface flora presented moderate levels of free fatty acids (Camembert: 5.1 g/kg; de la Vera: 9.9 g/kg). The remaining cheeses considered (Mahôn: 8.7 g/kg; Roncal: 8.2 g/kg; Tetilla: 5.8 g/kg; Idiazabal: 5.6 g/kg) also exhibited moderate levels of lipolysis. A comparison of the fattyacid profiles for the two fractions studied yielded higher contents for the short-chain fatty acids in the free fatty acid fraction, except in the blue and goats-milk cheeses.