P Morales

Hospital Universitario 12 de Octubre, Madrid, Madrid, Spain

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Publications (59)176.58 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Mutations in hemochromatosis gene cause an inappropriately high absorption of iron that induces iron overload and deposition in several tissues, such as liver, pancreas, and heart. Iron overload in the liver has been associated with a high risk of hepatocarcinoma and susceptibility to viral and bacterial infections. The aim of this study was to describe the frequencies of HFE mutations among a kidney transplant population with versus without hepatitis C virus (HCV) infection, and its influence on liver and kidney status parameters. We selected 3 populations: 2 groups of kidney transplant recipients-59 with and 60 without HCV infection-and a third control group of 50 healthy subjects. We collected clinical data concerning liver and kidney status, such as iron, ferritin, albumin, creatinine, gamma GGT, GOT, proteinuria, %prothrombin, and Bilirubin. HFE mutations among patients and controls were determined by polymerase chain reaction-restriction fragment length polymorphism using DNA from the peripheral blood. We observed no significant difference with respect to the frequencies of HFE mutations between controls and patients with versus without HCV infection. Finally comparison of HFE positive versus negative mutation carriers in both groups suggest that any clinical parameter is associated with HFE mutations.
    Transplantation Proceedings 07/2009; 41(6):2422-4. · 0.95 Impact Factor
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    ABSTRACT: The limited availability of organs for liver transplantation has focused interest on the use of cell transplants to restore hepatic function. Advances have been made in rodent models, but efficacy is limited in humans due to low engraftment efficiency. In rodents, pretransplantation treatment of the liver with engraftment enhancers (EE) shows that repopulation is feasible, although the toxicity of the substances impedes their application in humans. Evaluation of low-toxicity engraftment enhancers for human use requires testing in animal models, a time-consuming, expensive process that also raises ethical issues. To reduce animal use in the preliminary evaluation of a new EE, we designed an easily quantitated in vitro method that mimics an intraportal cell transplant. It is based on EE-mediated disruption of intercellular adhesion in confluent endothelial cell cultures.
    Transplantation Proceedings 07/2009; 41(6):2487-90. · 0.95 Impact Factor
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    ABSTRACT: Human leucocyte antigen-G (HLA-G) is a natural immunosuppressant produced in human placentas that binds differently to the inhibitory leucocyte immunoglobulin-like receptors LILRB1 (ILT2) and LILRB2 (ILT4) according to its biochemical structure. To predict the binding functions of the HLA-G5 soluble isoform synthesized in placental villous cytotrophoblast (vCTB) cells, we investigated structural features of this protein. Biochemical and immunological studies showed that vCTB cell HLA-G5 heavy (H)-chain proteins are disulphide-bonded homodimers unassociated with beta(2)-microglobulin (beta2m) light-chain proteins. Although comparatively low levels of beta2m messenger RNA (mRNA) were identified by real-time reverse transcription-polymerase chain reaction, immunoprecipitation studies failed to detect beta2m protein even when specific mRNA was doubled by transduction of a lentivirus-beta2m complementary DNA into vCTB cells. No abnormalities were identified in the translational start codon of vCTB cell beta2m mRNA and differentiation into syncytium did not promote beta2m synthesis. The failure of vCTB cells to exhibit beta2m in vitro was paralleled by a lack of detectable beta2m in vCTB cells in vivo. Lack of the beta2m protein could be the result of low levels of beta2m transcripts or of as yet unidentified translational defects. Experiments with recombinant ectodomains of LILRB indicate that beta2m-free HLA-G binds strongly to LILRB2, a receptor that is expressed by macrophages. This potentially immunosuppressive cell type is abundant in the pregnant uterus. Thus, our findings are consistent with the postulate that the natural beta2m-free homodimeric form of HLA-G5 synthesized in primary vCTB cells could comprise a particularly effective tolerogenic molecule at the maternal-fetal interface.
    Immunology 11/2007; 122(2):179-88. · 3.71 Impact Factor
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    ABSTRACT: The human leukocyte antigen-G (HLA-G) gene encodes a protein that is highly expressed at the human maternal-fetal interface during pregnancy and may be critical to the survival of the semiallogenic fetus. A unique feature of this gene is a 13-bp deletion in the proximal promoter that renders it unresponsive to transactivation by the nuclear factor-kappaB (NF-kappaB). We previously showed that the proximal promoter of Paan-AG, the functional homologue of HLA-G in the olive baboon (Papio anubis), is intact. We cloned the promoters of two putative Paan-AG alleles (AG1 and AG2) and identified a number of regulatory elements including two kappaB sites. In the current study, binding and activity of the two kappaB elements in each putative allele were assessed by electrophoretic mobility shift and supershift assays. Functional activity was determined using luciferase reporter assays. The kappaB1 and kappaB2 elements in AG1 bound NF-kappaB with similar affinity. In contrast, the kappaB1 element of AG2 bound NF-kappaB with a much higher affinity than AG-1 kappaB1 (a 30-fold increase), whereas kappaB2 did not bind. Mutagenesis analysis showed that the difference in binding intensities was due to two nucleotides in the 3' end of kappaB1. Similarly, failure of AG2 kappaB2 binding was a result of the last nucleotide in the 3' end that differed from the consensus; mutating this nucleotide to match the consensus reestablished binding. Functional activity of the two putative alleles also differed; AG1 luciferase activity was consistently lower than that of AG2. Mutating the last two nucleotides in the 3' end of AG1 kappaB1 resulted in increased luciferase activity to levels comparable to that of AG2. Overall, these results show that in vitro variations in the promoter region may influence transcription of Paan-AG.
    Immunogenetics 06/2007; 59(5):359-66. · 2.89 Impact Factor
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    ABSTRACT: HLA-G is an HLA class Ib gene that is highly expressed in human trophoblast cells. The single HLA-G mRNA is alternatively spliced to generate at least seven transcripts, three of which encode soluble isoforms. Many studies have shown that high levels of soluble antigens are associated with successful implantation and graft acceptance. To study expression, regulation and functions of two of the soluble isoforms, HLA-G5 and HLA-G6, we generated recombinant proteins in eukaryotic cells and developed monoclonal antibodies specific for each of the two proteins. In addition, we investigated the olive baboon Paan-AG gene as a potential functional correlate of HLA-G. Here, we present summaries of the studies that have been conducted in our laboratory using these tools and discuss the results within the context of the research on this topic that is ongoing in ours and other laboratories worldwide. Collectively, the data indicate that soluble HLA-G is a critical contributor to immune privilege in pregnancy and imply that this placenta-derived substance may impact other pathways leading to successful reproduction.
    Placenta 05/2007; 28 Suppl A:S57-63. · 3.12 Impact Factor
  • Blood 11/2006; 109(10):2623. · 9.78 Impact Factor
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    ABSTRACT: Pregnancy in mammals featuring hemochorial placentation introduces a major conflict with the mother's immune system, which is dedicated to repelling invaders bearing foreign DNA and RNA. Numerous and highly sophisticated strategies for preventing mothers from rejecting their genetically different fetus(es) have now been identified. These involve production of novel soluble and membrane-bound molecules by uterine and placental cells. In humans, the placenta-derived molecules include glycoproteins derived from the HLA class Ib gene, HLA-G. Isoforms of HLA-G saturate the maternal-fetal interface and circulate in mothers throughout pregnancy. Uteroplacental immune privilege for the fetus and its associated tissues is believed to result when immune cells encounter HLA-G. Unequivocally demonstration of this concept requires experiments in animal models. Both the monkey and the baboon express molecules that are similar but not identical to HLA-G, and may comprise suitable animal models for establishing a central role for these proteins in pregnancy.
    Reproductive Biology and Endocrinology 02/2006; 4 Suppl 1:S10. · 2.41 Impact Factor
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    ABSTRACT: The human major histocompatibility complex (MHC) contains genes encoding the Human Leukocyte Antigens (HLA). Of these antigens, placental immunologists need study only the HLA class I molecules, because HLA class II expression is repressed in the fetal placental cells that are in direct contact with maternal blood and tissues containing maternal immune cells. The class I antigens are subdivided into two general categories. The class Ia antigens are highly polymorphic and are typified by HLA-A, -B, and -C; these are expressed by nearly all somatic cells and stimulate graft rejection when foreign to the host. By contrast, the HLA class Ib antigens, HLA-E, -F, and -G, have restricted expression, few variants, and appear rarely to be immunostimulatory. One class Ia antigen, HLA-C, and the three class Ib antigens are differentially expressed by trophoblast cell subpopulations. In order to understand immune privilege in the pregnant uterus and placenta, it is essential to study the unique structural and functional features of these four genes and their glycoprotein products. In this chapter, we focus on the first class Ib gene identified in human placentas, HLA-G, with emphasis on its two soluble isoforms, HLA-G5 and HLA-G6. We describe methods developed in our laboratory to distinguish mRNAs encoding HLA-G5 and HLA-G6, and antibody-based protocols for identification of the soluble isoforms.
    Methods in molecular medicine 02/2006; 122:181-203.
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    ABSTRACT: The human major histocompatibility complex (MHC) contains genes encoding the Human Leukocyte Antigens (HLA). Of these antigens, placental immunologists need study only the HLA class I molecules, because HLA class II expression is repressed in the fetal placental cells that are in direct contact with maternal blood and tissues containing maternal immune cells. The class I antigens are subdivided into two general categories. The class Ia antigens are highly polymorphic and are typified by HLA-A, -B, and -C; these are expressed by nearly all somatic cells and stimulate graft rejection when foreign to the host. By contrast, the HLA class Ib antigens, HLA-E, -F, and -G, have restricted expression, few variants, and appear rarely to be immunostimulatory. One class Ia antigen, HLA-C, and the three class Ib antigens are differentially expressed by trophoblast cell subpopulations. In order to understand immune privilege in the pregnant uterus and placenta, it is essential to study the unique structural and functional features of these four genes and their glycoprotein products. In this chapter, we focus on the first class Ib gene identified in human placentas, HLA-G, with emphasis on its two soluble isoforms, HLA-G5 and HLA-G6. We describe methods developed in our laboratory to distinguish mRNAs encoding HLA-G5 and HLA-G6, and antibody-based protocols for identification of the soluble isoforms. Key WordsEnzyme-linked immunosorbent assay (ELISA)–flow cytometry–HLA-G–human–immunoblotting–immunohistochemistry–placenta–reverse transcriptase polymerase chain reaction (RT-PCR)–recombinant HLA-G–trophoblast cells
    12/2005: pages 181-203;
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    ABSTRACT: The major histocompatibility complex (MHC)-F class Ib locus shows a limited polymorphism, and the function of its mainly intracellular protein is not clear. We have identified human leucocyte antigen (HLA)-F orthologous DNA sequences in Pongidae in order to study the MHC-F gene evolution and its products' function. HLA-F orthologous cDNA transcripts are found in chimpanzee and in the new primate species studied (bonobo, gorilla and orangutan). Analyses of the predicted amino acid sequences and their comparison with other primate MHC-F proteins show that MHC-F may be a protein with a typical class I structure and that the key residues of the peptide-binding region (PBR) are highly conserved in MHC-F in all studied primates species. Thus, MHC-F conservation along the primate evolution suggests an important role in cellular physiology. It is possible that the MHC-F protein could be involved, together with MHC-G and MHC-E, in the natural killer (NK) cell activity regulation, although rhesus macaque does not express MHC-G and MHC-E orthologues. The evolutionary pathway of the six-base-pair deletion at exon 2 existing in some primates is put forward.
    Tissue Antigens 11/2005; 66(4):277-83. · 2.93 Impact Factor
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    ABSTRACT: The baboon major histocompatibility complex (MHC) class Ib gene, Paan-AG, is structurally similar to the human MHC class Ia gene, HLA-A, but exhibits characteristics similar to those of the class Ib gene HLA-G. These include limited polymorphism, alternative splicing of a single message, and restricted tissue distribution, with high expression in the placenta. In order to determine whether regulatory elements controlling expression of Paan-AG resemble those of HLA-A or HLA-G, we cloned the 5' and 3' untranslated regions of Paan-AG. Unexpectedly, sequence comparisons showed that potential regulatory elements in Paan-AG strikingly resembled those in HLA-A and differed in major respects from those in HLA-G. Unlike HLA-G, Paan-AG contained an intact interferon-gamma stimulated response element (ISRE) in the promoter. Studies using luciferase reporter assays showed that the Paan-AG ISRE was functional. The basal activity of the Paan-AG ISRE and its response to interferon-gamma was similar to that of class Ia MHC genes. Further, we identified an ISRE in the 3' untranslated region of Paan-AG that is known to be functional in HLA-A2 but is deleted in HLA-G. These experiments predict that functional studies may demonstrate differences in regulation of expression of Paan-AG and HLA-G genes, which could restrict the use of the baboon as a primate model for studying HLA-G expression and function.
    Immunogenetics 01/2005; 56(9):657-66. · 2.89 Impact Factor
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    ABSTRACT: Throughout human pregnancy, activated maternal macrophages producing anti-inflammatory cytokines comprise a stable cell population in the uterus. This organ is also massively infiltrated with semiallogeneic, placenta-derived, invasive cytotrophoblast cells, which produce membrane and soluble isoforms of human leukocyte antigen (HLA)-G. Here, we investigated the possibility that two soluble isoforms of HLA-G, HLA-G5 and -G6, program macrophage production of cytokines. The model system consisted of human U937 myelomonocytic cells treated with phorbol 12-myristate 13-acetate (PMA) and interferon-gamma (IFN-gamma), which induced differentiation and activation but did not affect their viability or decrease their expression of the two inhibitory immunoglobulin-like transcript (ILT) receptors for HLA-G, ILT2 and ILT4. Exposure of the PMA/IFN-gamma-treated U937 cells to increasing concentrations of recombinant HLA-G5 or -G6 (rG5 and rG6) stimulated effects common to the two isoforms. High doses of both significantly decreased interleukin (IL)-10 and dramatically increased transforming growth factor-beta1. Differential effectiveness between the isoforms was demonstrated in dose-response studies, as was differential binding to ILT2 and ILT4 in receptor-blocking studies. No effects on production of IL-4, IL-1 receptor antagonist, IL-15, tumor necrosis factor alpha, IL-1beta, or IL-6 were observed. Collectively, the results are consistent with the postulate that environmental programming of decidual macrophages may be dictated in part by their proximity to soluble HLA-G-producing fetal cytotrophoblast cells.
    Journal of Leukocyte Biology 01/2005; 76(6):1220-8. · 4.57 Impact Factor
  • Human Immunology 09/2004; 65(9):972-973. · 2.30 Impact Factor
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    ABSTRACT: The human class Ib major histocompatibility complex (MHC) molecule, HLA-G, is unique in its limited polymorphism, high expression in the placenta and generation of multiple transcripts by alternative splicing. The proteins encoded by these transcripts are believed to modulate maternal-fetal immunological relationships during pregnancy. The baboon placenta expresses Paan-AG, a novel MHC molecule that is evolutionarily related to the MHC-A locus but shares unique characteristics with HLA-G. In this brief review, we present evidence suggesting that Paan-AG may be the functional homologue of HLA-G, and propose that the baboon would compromise an excellent animal model for functional studies of HLA-G proteins in human pregnancy.
    Gynecologic and Obstetric Investigation 02/2004; 57(1):33-6. · 1.10 Impact Factor
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    ABSTRACT: The HLA-G message is alternatively spliced into multiple transcripts, two of which encode soluble isoforms. To initiate studies on the specific functions of the soluble isoforms, we produced soluble rHLA-G1 (rsG1) and rsG2 in human embryonic kidney 293 cells and characterized the proteins. Both isoforms were glycosylated and formed disulfide-bonded oligomers. Recombinant sG1 associated with beta(2)-microglobulin, whereas rsG2 did not. Mouse mAb generated to rsG1 (1-2C3), which identified exclusively sG1, and mAb generated to rsG2 (26-2H11), which identified both soluble and membrane G2 (m/sG2), were used for immunohistochemical isoform mapping studies on placental tissue sections. Soluble G1 protein was abundant in many subpopulations of trophoblast cells, whereas m/sG2 protein was present exclusively in extravillous cytotrophoblast cells. Although both isolated placental villous cytotrophoblast cells and chorion membrane extravillous cytotrophoblast cells contained mRNAs encoding sG1 and sG2, protein expression was as predicted from the immunostains with m/sG2 present only in the invasive trophoblast subpopulation. Analysis of function by Northern and Western blotting demonstrated that both rsG1 and rsG2 inhibit CD8alpha expression on PBMC without changing CD3delta expression or causing apoptotic cell death. Collectively, the studies indicate that: 1) both sG1 and m/sG2 are produced in placentas; 2) transcription and translation are linked for sG1, but not G2; 3) expression of G2 is exclusively associated with the invasive phenotype; and 4) the two isoforms of sG may promote semiallogeneic pregnancy by reducing expression of CD8, a molecule required for functional activation of CTL.
    The Journal of Immunology 01/2004; 171(11):6215-24. · 5.52 Impact Factor
  • Human Immunology - HUM IMMUNOL. 01/2004; 65(9):972-972.
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    J S Hunt, J L Pace, P J Morales, C Ober
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    ABSTRACT: Soluble class Ib HLA-G glycoproteins synthesized in the placenta are abundant in the pregnant uterus and circulate in maternal blood throughout pregnancy. To establish immunogenicity of these proteins, we tested sera from 64 women with at least one successful pregnancy (multigravid), 21 women who had never been pregnant, and 54 males for antibodies to epitopes present on recombinant sHLA-G isoforms (sHLA-G1, sHLA-G2) derived from HLA 6.0 cDNA (HLA-G*0101 allele). By indirect enzyme-linked immunosorbent assay, antibodies to sHLA-G isoforms were identified in six sera, all from multigravid women; all other sera were negative (P = 0.0083). Immunoblots showed that two of the positive sera reacted exclusively with sHLA-G1 and -G2 whereas four reacted to both sHLA-G and pooled HLA class I antigens. To establish potential relationships between anti-sHLA-G and exposure to foreign paternal alleles (*0101, *0103, *0104, *0106), all multigravid women and their partners were genotyped. No relationship between allelic disparity and antibody production was identified. Taken together, these results indicate that (i) tolerance to HLA-G is the usual condition as antibodies to HLA-G were not detected in 91% (58/64) multigravid women, and (ii) pregnancy stimulates loss of tolerance in 9% (6/64) of multigravid women. All six women delivered healthy babies, demonstrating that maternal antibodies to epitopes on sHLA-G do not abrogate pregnancy.
    Molecular Human Reproduction 12/2003; 9(11):729-35. · 4.54 Impact Factor
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    ABSTRACT: Macrophages - together with natural killer (NK) cells - constitute the majority of bone marrow derived infiltrating cells in the decidua. As interferon-gamma (IFN-gamma), a cytokine produced by NK cells, has been reported to induce expression of human leukocyte antigen (HLA-G) in monocytic cells, suggesting expression of HLA-G on decidua macrophages potentially stimulated by IFN-gamma, the question arises whether decidua macrophages in normal pregnancy express HLA-G. The study was based on immunohistochemistry and flow cytometry. In order to exclude that potentially elusive soluble HLA-G was not detected by immunohistochemistry, we performed in addition RT-PCR of flow-sorted decidua macrophages. Our findings indicate that HLA-G is not present on macrophages of first trimester or term decidua in either membrane-bound or soluble form. Transcripts for soluble HLA-G1 and -G2 were not detected. We exclude a role of HLA-G on the surface of decidua macrophages or of soluble HLA-G1 or -G2 as a secretory product of decidua macrophages with regard to interaction with HLA-G receptors present in or outside the decidua.
    American journal of reproductive immunology (New York, N.Y.: 1989) 09/2002; 48(2):96-102. · 3.32 Impact Factor
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    ABSTRACT: About 15–20% of bone marrow derived cells in the endometrium and the decidua are CD14-positive macrophages coexpressing the scavenger receptor CD163. The costimulatory molecule CD86 (B7-2) but not CD80 (B7-1) is found expressed on first trimester and term decidua macrophages (D-Mph). The number of D-Mph increases at the implantation site in the basal decidua, where they are in contact with the HLA-G-positive invading extravillous cytotrophoblast. As D-Mph express the inhibitory receptors CD85d (ILT4, LIR-2) and CD85e (ILT2, LIR-1), they are potentially susceptible to inhibition by class Ia and class Ib molecules. D-Mph may have specific functions relating to successful pregnancy, and phenotypic and functional properties may differ between early and term pregnancy. A number of data make their molecular repertoire comparable to those of alternatively activated macrophages which are immunosuppressive. For instance, first trimester decidua macrophages express IL-10 and the MS-1 high molecular weight antigen on the cell surface fibronectin and F XIIIa are detectable. Functional data are required to complement these studies.
    American Journal Of Reproductive Immunology 08/2002; 48(3):142 - 142. · 3.32 Impact Factor
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    ABSTRACT: The human class Ib major histocompatibility complex (MHC) molecule, HLA-G, is unique in its limited polymorphism, high expression in the placenta and generation of multiple transcripts by alternative splicing. The proteins encoded by these transcripts are believed to modulate maternal-fetal immunological relationships during pregnancy. The baboon placenta contains messages encoded by a novel MHC gene, Paan-AG, which is evolutionarily related to the HLA-A locus, but shares unique characteristics with HLA-G. In this study, we show that the Paan-AG message is alternatively spliced to generate at least seven transcripts. One of these transcripts retains intron 4 and encodes a soluble glycoprotein with three external domains and a unique 21-amino-acid sequence at the carboxyl terminus, similar to soluble HLA-G1. This glycoprotein was detected in first trimester placental villous cytotrophoblast and syncytiotrophoblast, and in extravillous cytotrophoblast cells in the basal plate in term placenta. Four of the transcripts ( Paan-AG1, Paan-AG2, Paan-AG3, Paan-AG4) encode membrane-bound class Ib MHC glycoprotein isoforms. Paan-AG1 protein expression was similar to that of sPaan-AG, while Paan-AG2 protein was not detected in these tissues. The other two transcripts ( Paan-AGx and Paan-AGxi) contain a truncated exon 3 and multiple stop codons. Paan-AG1 and Paan-AGx transcripts were detected in a number of non-placental tissues, but these transcripts contained multiple stop codons. Because of the structural similarities and common features of organ-specific expression and splicing of the message, studies on Paan-AG may be of value in dissecting the functions of the class Ib proteins in human pregnancy.
    Immunogenetics 07/2002; 54(3):164-73. · 2.89 Impact Factor

Publication Stats

923 Citations
176.58 Total Impact Points

Institutions

  • 1993–2009
    • Hospital Universitario 12 de Octubre
      Madrid, Madrid, Spain
  • 2002–2007
    • University of Kansas
      • Department of Anatomy and Cell Biology
      Kansas City, KS, United States
    • Kansas City VA Medical Center
      Kansas City, Missouri, United States
  • 1993–2005
    • Complutense University of Madrid
      • Departamento de Bioquímica y Biología Molecular II
      Madrid, Madrid, Spain
  • 1989–2000
    • Hospital 12 de Octubre
      Madrid, Madrid, Spain
  • 1992
    • Comunidad de Madrid
      Madrid, Madrid, Spain
  • 1991
    • University of Alcalá
      Cómpluto, Madrid, Spain