Análisis molecular de los repertorios de receptores de antígeno en las respuestas alorreactivas contra HLA-B27 y su contribución al estudio comparativo de la estructura antigénica de B*2705 y B*2703

Tesis de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Microbiología II, leída el 16-02-1999Depto. de Microbiología y ParasitologíaFac. de FarmaciaTRUEpu

Vav1 Phosphorylation Is Induced by β2 Integrin Engagement on Natural Killer Cells Upstream of Actin Cytoskeleton and Lipid Raft Reorganization

The guanine nucleotide exchange factor Vav1 regulates actin polymerization and contributes to cytotoxicity by natural killer (NK) cells. An open question is how Vav1 becomes activated and what receptor can signal upstream of actin cytoskeleton rearrangement upon NK cell contact with target cells. Using transfected insect cells that express ligands of human NK cell receptors, we show that engagement of the β2 integrin LFA-1 on NK cells by intercellular adhesion molecule (ICAM)-1 led to a tyrosine phosphorylation of Vav1 that was not sensitive to cholesterol depletion and to inhibition of actin polymerization. Vav1 phosphorylation was blocked by an inhibitor of Src-family kinases, and correlated with activation of its downstream effector PAK. Binding of activation receptor 2B4 to its ligand CD48 was not sufficient for Vav1 phosphorylation. However, coengagement of 2B4 with LFA-1 resulted in an enhancement of Vav1 phosphorylation that was sensitive to cholesterol depletion and to inhibition of actin polymerization. Vav1 was recruited to a detergent-resistant membrane (DRM) fraction only when 2B4 and LFA-1 were coengaged, but not after LFA-1 engagement. Therefore, binding of LFA-1 to ICAM-1 on target cells may initiate an early signaling cascade in NK cells through activation of Vav1, leading to cytoskeleton reorganization and amplification of signals from other activation receptors

Polymeric Nanoparticles that Combine Dexamethasone and Naproxen for the Synergistic Inhibition of Il12b Transcription in Macrophages

Recent studies have demonstrated in vivo synergistic immunosuppressive and anti-inflammatory capacity of dexamethasone (Dx) and naproxen (NAP) in collagen-induced arthritis (CIA) rats. However, the molecular basis of this synergistic effect is barely understood. The low solubility of these drugs and their adverse effects hamper their efficacy on the treatment of inflammatory processes making nanoparticulated systems promising candidates to overcome these drawbacks. The aim of this work is the preparation of polymeric nanoparticles (NPs) that combine NAP and Dx in different concentrations, and the evaluation of the expression of key genes related to autoimmune diseases like CIA. To do so, self-assembled polymeric NPs that incorporate covalently-linked NAP and physically entrapped Dx are designed to have hydrodynamic properties that, according to bibliography, may improve retention and colocalization of both drugs at inflammation sites. The rapid uptake of NPs by macrophages is demonstrated using coumarine-6-loaded NPs. Dx is efficiently encapsulated and in vitro biological studies demonstrate that the Dx-loaded NAP-bearing NPs are noncytotoxic and reduce lipopolysaccharide- induced NO released levels at any of the tested concentrations. Moreover, at the molecular level, a significant synergistic reduction of Il12b transcript gene expression when combining Dx and NAP is demonstrated.Authors would like to thank the Spanish Ministry of Science, Innovation and Universities (MAT2017-84277-R and SAF2017-82223-R) and CIBER-BBN for the financial support of this project. E.E.-C. and Y.P. would like to thank the training program for Academic Staff (FPU15/06109 and FPU15/06170, respectively) of the Spanish Ministry of Education Culture and Sport. The kind support by Alvaro González- Gómez, Rosana Ramírez, and David Gómez, in the synthesis, cell culture and SEM experiments is greatly appreciatedPeer reviewe

Component-resolved diagnosis of pollen allergy based on skin testing with profilin, polcalcin and lipid transfer protein pan-allergens

BACKGROUND Allergy diagnosis needs to be improved in patients suffering from pollen polysensitization due to the existence of possible confounding factors in this type of patients. OBJECTIVE To evaluate new diagnostic strategies by comparing skin responses to pan-allergens and conventional allergenic extracts with specific IgE (sIgE) to purified allergen molecules. METHODS One thousand three hundred and twenty-nine pollen-allergic patients were diagnosed by a combination of an in vitro method with a panel of 13 purified allergens, including major allergens and pan-allergens, using a high-capacity screening technology (ADVIA-Centaur®) and skin prick test (SPT) to pan-allergens and conventional extracts. RESULTS There was a high concordance (κ index) between in vitro (sIgE to major allergens) and in vivo (SPT to conventional extracts) methods in patients who were not sensitized to pan-allergens, but SPT with conventional extracts failed to diagnose patients with sensitization to pan-allergens. In patients who were simultaneously sensitized to polcalcins and profilins, there was a duplication both in the number of sensitizations to major allergens and in the years of disease evolution. There was a statistical association between sensitization to profilins and/or lipid transfer proteins and food allergy (P<0.0001). CONCLUSION The novel diagnostic strategy has proven to be a valuable tool in daily clinical practice. Introduction of routine SPT to pan-allergens is a simple and feasible way of improving diagnostic efficacy. Patients sensitized to pan-allergens should be tested by an adequate panel of allergenic molecules in order to identify the allergens that are responsible for the allergic disease

T cells loaded with magnetic nanoparticles are retained in peripheral lymph nodes by the application of a magnetic field

T lymphocytes are highly dynamic elements of the immune system with a tightly regulated migration. T cell-based transfer therapies are promising therapeutic approaches which in vivo efficacy is often limited by the small proportion of administered cells that reaches the region of interest. Manipulating T cell localisation to improve specific targeting will increase the effectiveness of these therapies. Nanotechnology has been successfully used for localized release of drugs and biomolecules. In particular, magnetic nanoparticles (MNPs) loaded with biomolecules can be specifically targeted to a location by an external magnetic field (EMF). The present work studies whether MNP-loaded T cells could be targeted and retained in vitro and in vivo at a site of interest with an EMF.Results: T cells were unable to internalize the different MNPs used in this study, which remained in close association with the cell membrane. T cells loaded with an appropriate MNP concentration were attracted to an EMF and retained in an in vitro capillary flow-system. MNP-loaded T cells were also magnetically retained in the lymph nodes after adoptive transfer in in vivo models. This enhanced in vivo retention was in part due to the EMF application and to a reduced circulating cell speed within the organ. This combined use of MNPs and EMFs did not alter T cell viability or function.Conclusions: These studies reveal a promising approach to favour cell retention that could be implemented to improve cell- based therapy

Phosphoinositide 3–kinase γ participates in T cell receptor–induced T cell activation

Class I phosphoinositide 3–kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase–associated receptors or G protein–coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase–associated receptor, p110γ deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110γ, as well as the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gαq/11, lymphocyte-specific Tyr kinase, and ζ-associated protein. TCR stimulation activates p110γ, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110γ controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110γ in TCR-induced T cell activation

Cytolytic granule polarization and degranulation controlled by different receptors in resting NK cells

The relative contribution to cytotoxicity of each of the multiple NK cell activation receptors has been difficult to assess. Using Drosophila insect cells, which express ligands of human NK cell receptors, we show that target cell lysis by resting NK cells is controlled by different receptor signals for cytolytic granule polarization and degranulation. Intercellular adhesion molecule (ICAM)-1 on insect cells was sufficient to induce polarization of granules, but not degranulation, in resting NK cells. Conversely, engagement of the Fc receptor CD16 by rabbit IgG on insect cells induced degranulation without specific polarization. Lysis by resting NK cells occurred when polarization and degranulation were induced by the combined presence of ICAM-1 and IgG on insect cells. Engagement of receptor 2B4 by CD48 on insect cells induced weak polarization and no degranulation. However, coengagement of 2B4 and CD16 by their respective ligands resulted in granule polarization and cytotoxicity in the absence of leukocyte functional antigen-1–mediated adhesion to target cells. These data show that cytotoxicity by resting NK cells is controlled tightly by separate or cooperative signals from different receptors for granule polarization and degranulation

PI3K p110δ is expressed by gp38(-)CD31(+) and gp38(+)CD31(+) spleen stromal cells and regulates their CCL19, CCL21, and LTβR mRNA levels.

The role of p110δ PI3K in lymphoid cells has been studied extensively, showing its importance in immune cell differentiation, activation and development. Altered T cell localization in p110δ-deficient mouse spleen suggested a role for p110δ in non-hematopoietic stromal cells, which maintain hematopoietic cell segregation. We tested this hypothesis using p110δ(WT/WT) mouse bone marrow to reconstitute lethally irradiated p110δ(WT/WT) or p110δ(D910A/D910A) (which express catalytically inactive p110δ) recipients, and studied localization, number and percentage of hematopoietic cell subsets in spleen and lymph nodes, in homeostatic conditions and after antigen stimulation. These analyses showed diffuse T cell areas in p110δ(D910A/D910A) and in reconstituted p110δ(D910A/D910A) mice in homeostatic conditions. In these mice, spleen CD4(+) and CD8(+) T cell numbers did not increase in response to antigen, suggesting that a p110δ(D910A/D910A) stroma defect impedes correct T cell response. FACS analysis of spleen stromal cell populations showed a decrease in the percentage of gp38(-)CD31(+) cells in p110δ(D910A/D910A) mice. qRT-PCR studies detected p110δ mRNA expression in p110δ(WT/WT) spleen gp38(-)CD31(+) and gp38(+)CD31(+) subsets, which was reduced in p110δ(D910A/D910A) spleen. Lack of p110δ activity in these cell populations correlated with lower LTβR, CCL19 and CCL21 mRNA levels; these molecules participate in T cell localization to specific spleen areas. Our results could explain the lower T cell numbers and more diffuse T cell areas found in p110δ(D910A/D910A) mouse spleen, as well as the lower T cell expansion after antigen stimulation in p110δ(D910A/D910A) compared with p110δ(WT/WT) mice

Long term biotransformation and toxicity of dimercaptosuccinic acid-coated magnetic nanoparticles support their use in biomedical applications

Although iron oxide magnetic nanoparticles (MNP) have been proposed for numerous biomedical applications, little is known about their biotransformation and long-term toxicity in the body. Dimercaptosuccinic acid (DMSA)-coated magnetic nanoparticles have been proven efficient for in vivo drug delivery, but these results must nonetheless be sustained by comprehensive studies of long-term distribution, degradation and toxicity. We studied DMSA-coated magnetic nanoparticle effects in vitro on NCTC 1469 non-parenchymal hepatocytes, and analyzed their biodistribution and biotransformation in vivo in C57BL/6 mice. Our results indicate that DMSA-coated magnetic nanoparticles have little effect on cell viability, oxidative stress, cell cycle or apoptosis on NCTC 1469 cells in vitro. In vivo distribution and transformation were studied by alternating current magnetic susceptibility measurements, a technique that permits distinction of MNP from other iron species. Our results show that DMSA-coated MNP accumulate in spleen, liver and lung tissues for extended periods of time, in which nanoparticles undergo a process of conversion from superparamagnetic iron oxide nanoparticles to other non-superparamagnetic iron forms, with no significant signs of toxicity. This work provides the first evidence of DMSA-coated magnetite nanoparticle biotransformation in vivo.RM holds a post-doctoral contract supported by EU-FP7 MULTIFUN project (no. 262943), LG holds a Sara Borrell post-doctoral contract (CD09/00030) from the Carlos III Health Institute, Spanish Ministry for Health, Social Services and Equality (MSSSI), and TMZ received a FPU pre-doctoral fellowship from the Spanish Ministry of Economy and Competitiveness (MINECO). This work was partially supported by grants from the MINECO (SAF-2011-23639 to DFB and MAT2011-23641 and CSD2007-00010 to MPM), the Research Network in Inflammation and Rheumatic Diseases (RIER) of the ISCIII-MSSSI Cooperative Research Thematic Network program (RD08/0075/0015 to DFB), the Madrid regional government (S009/MAT-1726 to MPM), and EU-FP7 MULTIFUN project (no. 262943 to DFB and MPM).S2009/MAT-1726/NanobiomagnetPeer Reviewe

Dynamic redistribution of raft domains as an organizing platform for signaling during cell chemotaxis

Spatially restricted activation of signaling molecules governs critical aspects of cell migration; the mechanism by which this is achieved nonetheless remains unknown. Using time-lapse confocal microscopy, we analyzed dynamic redistribution of lipid rafts in chemoattractant-stimulated leukocytes expressing glycosyl phosphatidylinositol–anchored green fluorescent protein (GFP-GPI). Chemoattractants induced persistent GFP-GPI redistribution to the leading edge raft (L raft) and uropod rafts of Jurkat, HL60, and dimethyl sulfoxide–differentiated HL60 cells in a pertussis toxin–sensitive, actin-dependent manner. A transmembrane, nonraft GFP protein was distributed homogeneously in moving cells. A GFP-CCR5 chimera, which partitions in L rafts, accumulated at the leading edge, and CCR5 redistribution coincided with recruitment and activation of phosphatidylinositol-3 kinase γ in L rafts in polarized, moving cells. Membrane cholesterol depletion impeded raft redistribution and asymmetric recruitment of PI3K to the cell side facing the chemoattractant source. This is the first direct evidence that lipid rafts order spatial signaling in moving mammalian cells, by concentrating the gradient sensing machinery at the leading edge