Lucio Gómez

Identification of amino acid residues crucial for chemokine receptor dimerization.

Chemokines coordinate leukocyte trafficking by promoting oligomerization and signaling by G protein–coupled receptors; however, it is not known which amino acid residues of the receptors participate in this process. Bioinformatic analysis predicted that Ile52 in transmembrane region-1 (TM1) and Val150 in TM4 of the chemokine receptor CCR5 are key residues in the interaction surface between CCR5 molecules. Mutation of these residues generated nonfunctional receptors that could not dimerize or trigger signaling. In vitro and in vivo studies in human cell lines and primary T cells showed that synthetic peptides containing these residues blocked responses induced by the CCR5 ligand CCL5. Fluorescence resonance energy transfer showed the presence of preformed, ligand-stabilized chemokine receptor oligomers. This is the first description of the residues involved in chemokine receptor dimerization, and indicates a potential target for the modification of chemokine responses.

Inhibition of programmed cell death impairs in vitro vascular-like structure formation and reduces in vivo angiogenesis

Tissue remodeling during embryonic development and in the adult organism relies on a subtle balance between cell growth and apoptosis. As angiogenesis involves restructuring of preexisting endothelium, we examined the role of apoptosis in new vessel formation. We show that apoptosis occurs before capillary formation but not after vessels have assembled. Using the human umbilical vein endothelial cell (HUVEC) in vitro Matrigel angiogenesis model, we show that vascular‐like structure formation requires apoptotic cell death through activation of a caspasedependent mechanism and mitochondrial cytochrome c release. Vascular‐like structure formation was further blocked by caspase inhibitors such as z‐VAD or AcD‐EVD‐CHO, using HUVEC and human lung microvascular endothelial cells. Overexpression of anti‐apoptotic human Bcl‐2 or baculovirus p35 genes in HUVEC altered endothelial cell rearrangement during in vitro angiogenesis, causing impaired vessel‐like structure formation. Caspase inhibitors blocked VEGF‐ or bFGF‐induced HUVEC angiogenesis on 2‐ or 3‐D collagen gels, respectively, confirming that apoptosis was not the result of nonspecific cell death after seeding on the matrix. In an in vivo angiogenesis assay, caspase inhibitors blocked VEGF‐dependent vascular formation at the alignment step, as demonstrated histologically. This evidence indicates that endothelial cell apoptosis may be relevant for precise vascular tissue rearrangement in in vitro and in vivo angiogenesis.—Segura, I., Serrano, A., González de Buitrago, G., González, M. A., Abad, J. L., Clavería, C., Gómez, L., Bernad, A., Martínez‐A, C., Riese, H. H. Inhibition of programmed cell death impairs in vitro vascular‐like structure formation and reduces in vivo angiogenesis. FASEB J. 16, 833–841 (2002)

Leukocyte attraction through the CCR5 receptor controls progress from insulitis to diabetes in non-obese diabetic mice

Lymphocyte infiltration to pancreatic islets is associated to chemoattraction, as are other inflammatory autoimmune processes. We examined whether development of insulitis and diabetes dependson chemoattraction of lymphocytes via the CCR5 chemokine receptor. In non‐obese diabetic (NOD) mice, a substantial fraction of peripheral T cells and virtually all B cells expressed high CCR5 levels. CCR5 expression characterized the effector T cell phenotype, suggesting their potential involvement in disease development. In view of these findings and the CCL5 (RANTES, the CCR5 ligand) expression by pancreatic islets, we treated NOD mice with a neutralizing anti‐CCR5 antibody. This did not influence peri‐insulitis advancement, but inhibited β‐cell destruction and diabetes. These data demonstrate a role of CCR5‐dependent chemoattraction in insulitis progression to islet destruction, suggesting the potential value of therapeutic intervention by CCR5 targeting.

STAG2 and Rad21 mammalian mitotic cohesins are implicated in meiosis

STAG/SA proteins are specific cohesin complex subunits that maintain sister chromatid cohesion in mitosis and meiosis. Two members of this family, STAG1/SA1 and STAG2/SA2, ‡ are classified as mitotic cohesins, as they are found in human somatic cells and in Xenopus laevis as components of the cohesinSA1 and cohesinSA2 complexes, in which the shared subunits are Rad21/SCC1, SMC1 and SMC3 proteins. A recently reported third family member, STAG3, is germinal cell‐specific and is a subunit of the meiotic cohesin complex. To date, the meiosis‐specific cohesin complex has been considered to be responsible for sister chromatid cohesion during meiosis. We studied replacement of the mitotic by the meiotic cohesin complex during mouse germinal cell maturation, and we show that mammalian STAG2 and Rad21 are also involved in several meiosis stages. Immunofluorescence results suggest that a cohesin complex containing Rad21 and STAG2 cooperates with a STAG3‐specific complex to maintain sister chromatid cohesion during the diplotene stage of meiosis.

Chemokine Receptor 2 Blockade Prevents Asthma in a Cynomolgus Monkey Model

The pathophysiology of asthma is characterized by accumulation and activation of several cell types in the lung, which correlates with coordinated production of specific cytokines and chemokines. To study the effect of selective CCR2 chemokine receptor blockade on leukocyte recruitment to the lung and on bronchial function, we used a nonhuman primate model of allergic airway disease that closely resembles human asthma. Allergic cynomolgus monkeys were treated with the antagonist anti-CCR2 (CCR2-05) monoclonal antibody and then challenged with Ascaris suum antigen; the effect of antibody treatment on macrophage and eosinophil infiltration was determined. Pulmonary function was calculated by measurement of lung resistance and dynamic compliance. Local inflammatory responses were analyzed after intradermal challenge with A. suum antigen. CCL2 up-regulation in bronchoalveolar lavage (BAL) was analyzed by enzyme-linked immunosorbent assay, and in vitro CCR2-05 antagonistic activity was tested in monkey peripheral blood mononuclear cells using chemotaxis and calcium mobilization assays. The results show that neutralization of CCR2 reduces antigen-induced bronchial hyper-responsiveness and attenuates macrophage and eosinophil accumulation in the BAL of asthmatic monkeys. The results confirm that selective blockade of a single chemokine receptor involved in early stages of asthma can condition later disease stages and suggest the utility of anti-CCR2-neutralizing monoclonal antibodies in the treatment of asthma in man.

Influence of surfactants on atomic diffusion

Abstract We have used Monte Carlo simulations with realistic interatomic potentials, combined with experimental results obtained by He diffraction (thermal energy atom scattering) and STM to investigate the effect of a surfactant agent such as Pb on the mechanisms of atomic diffusion involved in epitaxial metal growth. We find that the main role of the surfactant is to hinder fast diffusion by hopping over the surface, which is the dominant mechanism on a compact face such as Cu(111), and to promote exchange. As a side effect, this facilitates interlayer diffusion and hence layer-by-layer growth, because islands are smaller and have rougher borders; adatoms reaching an edge have more opportunities to cross them by exchange with a step atom.

Antitumor effects of a monoclonal antibody to human CCR9 in leukemia cell xenografts

Tumor expression of certain chemokine receptors is associated with resistance to apoptosis, migration, invasiveness and metastasis. Because CCR9 chemokine receptor expression is very restricted in healthy tissue, whereas it is present in tumors of distinct origins including leukemias, melanomas, prostate and ovary carcinomas, it can be considered a suitable candidate for target-directed therapy. Here, we report the generation and characterization of 91R, a mouse anti-human CCR9 IgG2b monoclonal antibody that recognizes an epitope within the CCR9 N-terminal domain. This antibody inhibits the growth of subcutaneous xenografts from human acute T lymphoblastic leukemia MOLT-4 cells in immunodeficient Rag2−/− mice. Tumor size in 91R-treated mice was reduced by 85% compared with isotype-matched antibody-treated controls. Tumor reduction in 91R-treated mice was concomitant with an increase in the apoptotic cell fraction and tumor necrotic areas, as well as a decrease in the fraction of proliferating cells and in tumor vascularization. In the presence of complement or murine natural killer cells, 91R promoted in vitro lysis of MOLT-4 leukemia cells, indicating that this antibody might eliminate tumor cells via complement- and cell-dependent cytotoxicity. The results show the potential of the 91R monoclonal antibody as a therapeutic agent for treatment of CCR9-expressing tumors.

Nonstochastic Behavior of Atomic Surface Diffusion on Cu(111) down to Low Temperatures

Atomic diffusion is usually understood as a succession of random, independent displacements of an adatom over the surfaces potential energy landscape. Nevertheless, an analysis of molecular dynamics simulations of self-diffusion on Cu(111) demonstrates the existence of different types of correlations in the atomic jumps at all temperatures. Thus, the atomic displacements cannot be correctly described in terms of a random walk model. This fact has a profound impact on the determination and interpretation of diffusion coefficients and activation barriers.

ZAP-70 upregulation in transformed B cells after early pre-BI cell transplant into NOD/SCID mice

Understanding of the signal transduction pathways that lead to B cell development is of extreme interest to learn how alterations in these pathways might initiate malignant transformation. Long-term cultured early pre-BI cells can differentiate into IgM+ B cells after transplant into NOD/SCID mice, offering the possibility to explore checkpoints in B cell development. Using DNA microarray and Western blot analysis of IgM+ B cells vs parental early pre-BI cells, we demonstrated that zeta-associated protein 70 (ZAP-70) is upregulated in our B cell differentiation model. Unlike parental ZAP-70− early pre-BI cells, ZAP-70+ IgM+ B cells exhibited a transformed phenotype, as indicated by BCL-6 expression, a high Ki-67 proliferation index, resistance to IL-7 deprivation-induced apoptosis, and an increased repopulation rate in NOD/SCID mice. These data show the characterization and generation of a novel murine leukemia model with many similarities to human ZAP-70+ B cell chronic lymphocytic leukemia.

Atomic View of Surfactant Action in Epitaxial Growth: From STM to Computer Simulation

We have combined experiments and computer simulations to visualize the atomic mechanisms behind the surfactant role of Pb, Co, or Cu atoms deposited on Cu(111) covered with a Pb monolayer quickly getting buried beneath the latter. The presence of the surfactant induces a strong buckling in several substrate layers, which, in turn, modifies many of the system’s properties. Atomic diffusion takes place below the surfactant by atomic exchange, while on the clean surface hopping dominates. The activation energy for surface diffusion or the adsorption energies are also significantly altered. These findings have important implications for the synthesis of novel heterostructures such as magnetic superlattices.