IMMUNOLOGICAL BIOCHEMISTRY

Author: JIMÉNEZ CALIANI ANTONIO JAVIER.
Year: 2004.
University: SEVILLA [www.us.es].
Place of defense: FACULTAD MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: It has recently been discovered and opened a new field of study of metatonina and its action on the system inmune.Ampliando this study, we decided to investigate the role it would play melatonin on two diseases autounmunes, rheumatoid arthritis and systemic lupus erythematosus. The administration of melatonin in rats with induced arthritis turned pro collagen have adverse effects by raising the levels of anti-collagen antibodies, elevated levels of cytokines inflematorias 11-1 and 11-6 contributing to the degradation and erotión cartilage, not reduced the levels of oxidative stress and UO LPO and motrífica hormone levels: Increasing cortisol and modifying the production of mormonas sex. The administration of malatona in mice MRL-lpr turned out to have the opposite effect in females as sex, the administration of melatonin has beneficial effects because it reduces levels of autoantiguerpos and causes a change in the response of the type TH2 antinflamatiria males poseee harmful effects for having a response proinflamatoría with elevated levels of autoantibodies melatonin acts on the LUPUS so directly at the cellular level and demodo indirectly on the production of mormonas sex, surely through the GNRH.

Author: ESTÉVEZ ALBEROLA M. CARMEN.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE QUÍMICAS.
Place of preparation: FACULTAD DE QUÍMICAS.

Summary: The thesis has been submitted to the preparation of polyclonal antibodies and MIPs (Footprint Molecular Polymer) for the development of analytical methodologies for the detection of environmental contaminants. There have been antibodies for Nonylphenol, the main degradation product of non-ionic surfactants poliretoxilados, which has developed an ELISA with a detectability of 2.3 ug / l. The nature of the analyte and the haptenos used as competitors has proved key to the development of testing with high reproducibility (especially due to its lipophilicity), so it has been necessary to produce antigens upholstery using a macromolécular hidrofílica as aminodextrano. On the other hand, there have been antibodies for sulfofenilcarboxilatos (SPCs), degradation products of LAS, ANOTHER family of surfactants. This was considered a heterologous immunization strategy, based on the mixture of compounds of inoculation. It was designed with two haptenos different that occurred 3 families antisera. With each family developed a ELISa, by in each case has studied the effect of hapteno immunization used on the specificity and sensitivity of the same. Moreover, it has opened a line of work led to the preparation and evaluation of MIPs. Whereas as analyte the Irgarol 1051, it has perfected a method of preparing the receiver as well as assessment and characterization of it as selective molecular receptor (by determining the affinity and capacity) and selective extraction stage.

Author: CHACÓN FERNÁNDEZ PEDRO JOSÉ.
Year: 2004.
University: SEVILLA [www.us.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: Although allergy is a disease in man described at the beginning of the twentieth century, its causes and mechanisms are still being studied. For years known to allergic reactions or atopic originate in individuals who suffer as a result of an immune reaction "wrong" against foreign substances or allergens which are mediated through immunoglobulin E-type synthesized against these allergens ( IgE). This IgE performs its function on the surface of immune effector cells (lymphocytes, macrophages, eosinophils, basophils and neutrophils). They joined through specific receptors (Fc epsilon RI, Fcepsilon RII and galectina-3), resulting in the activation of the same, resulting in the release of inflammatory mediators. The protaglandinas are soluble mediators synthesized by oxygenation of arachidonic acid, by action of the enzyme cyclooxygenase (COXs). There are two types of COXs: COX-1 is constitutively expressed in most tissues and is responsible for the generation of prostaglandins that regulate normal physiological conditions (gastric secretion, for example), whereas COX-2 is not appearing expressed in normal tissues in, and can be induced in inflammatory conditions, stress, etc.. There is increasing data suggest that the induction of COX-2 and prostaglandin synthesis by the same, can be a key element in the pathophysiologic state of a large number of disorders, including allergy. IL-8 is a cytokine capable proinflamatoria, due to its main function: it is chemotactic (attractive in terms of chemical gradient) to immune cells. Also features angiogénicas and mitogénicas. For this reason has been associated c on a large number of diseases that circulate with inflammation, such as allergic reactions. In this study we demonstrated that lymphocytes (T, B and NK) of subjects allergic express COX-2 when grown with specific allergens to which they are aware, without altering the expression of COX-1, the process being mediated by IgE and going through the receiver Fcepsilon RII. Induction of COX-2 is expressed at the level of mRNA, protein and enzyme activity is mediated by the routes of transmission of signals involving protein kinases activated by mitogenic (MAPKs) and the transcription factor NF-kappa B is modulated by glucocorticoiedes. Besides the enzymatic inhibition of the enzyme alters the expression of inflammatory cytokines by lymphocytes. Moreover we show that the cultivation of neutrophils subjects allergic to the allergen responsible for the clinical picture induces the production of IL-8 in culture medium, which was parallel to the expression of its mRNA. The process was dependent on IGE, and occurred mostly through the receiver Fcepsilon RI, through signaling pathways involving the calcium, reactive oxygen species and the phosphatase calcineurin. This is the first evidence of the activation of both proteins (COX-2 and IL-8) in immune cells (lymphocytes and neutrophils) that are actively involved in the development of allergic reactions. Our data opens up new prospects for knowing the functional role of both cell types in diseases associated with IgE.

Author: VEGA RIOJA ANTONIO.
Year: 2004.
University: SEVILLA [www.us.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: The research presented can be summarized as follows: * In this research paper discusses the regulatory role of endogenous hydrogen peroxide on the expression of the gene for the inducible nitric oxide (NOS2) induced by LPS / IFN-gamma in macrophages peritoneal rat. Initially it was found that the hydrogen peroxide added exogenously inhibited the expression of the NOS2, which took us to analyze the effects of the activity of monoamina oxidasa-A / B (MAO / B) and the amine oxidase sensitive semicarbazida ( SSA), as enzymes made of hydrogen peroxide on the expression of NOS2. In the presence of their substrates, tyramine for MAO-A/By benzilamina for SSAO, is the intracellular synthesis of hydrogen peroxide and inhibiting the expression of the NOS2, as we found by Western-blotting, flow cytometry and microscopy fluorescence. The pargilina and semicarbazida, specific inhibitors of the MAO-A/By of SSAO, respectively, offset these negative effects on the expression of NOS2. In the presence of cations Fe2 + and Cu2 + is a greater inhibition of the expression of the NOS2, suggesting that reactive oxygen species derived from the reaction of Fenton involved in this process. In addition it was found that the effect of hydrogen peroxide generated by monoaminas oxidasas occurs at the level of mRNA. These results provide a new checkpoint in the expression of NOS2 through hydrogen peroxide or other reactive oxygen species. A possible scenario might be that the inhibition of expression of the NOS2 by hydrogen peroxide is a cellular protection mechanism against the cytotoxic effects produced as a result of the activation of enzymes generate reactive oxygen species, finding, therefore, a new paper and unique to the family of protein from the monoaminas oxidasas. * The cyclooxygenase (COX) is a key enzyme in prostaglandin synthesis. The regulation of the expression of its isoform COX-2 is responsible for the increase of prostaglandins that takes place during inflammatory conditions, and is also involved in allergic diseases. In this work we show that the expression of COX-2 is induced strongly nuetrófilos of allergic patients when they are exposed to anti-IgE antibodies or allergens to which the patient is sensitive. This induction was detected at the level of mRNA and protein was accompanied by the release of prostaglandin E2. We also show evidence that specific inhibitors of the NADPH oxidase, as are the HMAP and AEBSF, completely inhibited the expression of COX-2 dependent IgE, suggesting that this process is mediated by reactive oxygen species derived from the activity of the NADPH oxidase . Besides the MAP kinases (MAPKs), p38 and ERK and the transcription factor NF-kappaB, are also involved in regulating the expression of COX-2, since specific inhibitors of these two kinases, such as SB03580 and PD098059 , and the route of NF-kappaB, as MG132, inhibiting the expression of COX-2 dependent IgE. It also presents evidence that radical. OH generated via Fenton reactions from hydrogen peroxide, may constitute a potential candidate able to regulate the expression of COX-2 dependent IgE through MAPKs and NF- KappaB. The results presented show a new role for reactive oxygen species as second messengers in regulating the expression of COX-2 in human neutrophils during allergic reactions. * "Nuclear factor of activated T cells" (NFATs) is a family of transcription factors that play a key role in the transcription of cytokine genes keys in a wide variety of cells. The presence of isoforms of NFAT has 8 been 5f7 scrita virtually all cell types of the immune system, with the exception of neutrophils. In the paper presented, we have described for the first time the expression of human neutrophils in the messenger RNA encoding isoforms coding isoforms NFAT1, NFAT2, NFAt4 and NFTA5 but not dela isoform NFAT3. However, only protein NFAT2 was expressed by these cells. We have also described that allergens to which a patient has been sensitized, are capable of promoting translocation of NFAT2 the core of neutrophils in these patients, an effect that was mimetizado for treatment of neutrophils with anti-IgE antibodies. These antibodies also increased the activity of calcineurin and the DNA binding activity of NFAT2. Finally we found that the calcineurin was able to interact physically with NFAT2. Our findings provide clear evidence that FAT2 is constitutively expressed in human neutrophils and is activated under the action of allergens and anti-IgE antibodies in a calcineurin-dependent mechanism.

Author: CAPARRÓS CAYUELA ESTHER.
Year: 2005.
University: MURCIA [www.um.es].
Place of defense: UNIVERSIDAD DE MURCIA.
Place of preparation: UNIVERSIDAD MIGUEL HERNÁNDEZ.

Summary: In this thesis has studied the mechanism for regulating populations T CD4 + T CD8 + by different receivers, both as cytotoxic T lymphocytes in human dendritic cells. On the one hand, the study of coagregación in membrane of heterodímero activator CD94/NKG2H and molecule MHC-I in cytotoxic T lymphocytes indicated that there is a blockade in different ways activation, as well as the mobilization of the organizer of a microtubule the area of contact between the T cell efectora and potential target cell. Biologically, recognition receptors involved in inhibition of cytotoxic activity in CD8 + T cells by ligands on antigen presenting cells would block the lysis of these cells by cytotoxic cells. Moreover, we study the mechanisms of transmission of signals that are derived from laligación in membrane of the molecule CD70,. Aggregation of this type II transmembrane protein induces activation of Erk MAPK, as well as Akt and the transcription factor NFkB, besides activating cytotoxicity in T lymphocytes CD8 and CD4 +. The analysis of these mutants of the protein generated in the laboratory revealed the importance of Cys 17 and Ser 6 and 9 of the molecule in the induction of the transcription factor NF-kB and survival. Finally, we analyze the effects of aggregation in the membrane molecule DC-SIGN in dendritic cells derived from monocytes. DC-SIGN is a type C lectina own immature dendritic cells. It is linked to the interaction with different pathogens that induce chronic infections such as cytomegalovirus, Aspergillus fumigatus, HIV or Ebola virus. Activating DC-SIGN by their ligands induces activation of MAPK and Akt and Erk mobilization of calcium intracelula L even this molecule is located in lipid rafts and interacts with other proteins involved in signal transmission. In addition, DC-SIGN modulates the signals from receptors as TLRs, favoring differentiation of a population Th2 efectora or regulator, which contributes to blocking the response Th1, inflammatory response, leading to the elimination of the pathogen. In this way, pathogens used as target DC-SIGN for its entry to dendritic cells, not only what used to enter the cell, but also induce a change in the polarization of the immune response.

Author: HERRANZ FALCON PATRICIA.
Year: 2005.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The quimioquinas are a family of low molecular weight proteins that regulate the recruitment of leukocytes to the site of inflammation and regulate cellular traffic through the body, by binding to G protein coupled receptor (GPCR), which are expressed in the membrane of these cell. It has been described that when a quimioquinas joins the receiver and after the stabilization of the receptor conformation dimérica, there is the association of proteins Janus kinases (JAK) to the receiver. These kinases will activate by transfosforilación and fosforilan receptor tyrosine residue, after which is the association of G protein receptor. Following the activation of these proteins will trigger a series of signaling cascades, and is the result of activation of all those molecules which will lead to the final cellular response. Mutation of these residues generated non-functional receptors not dimerizaban and also were not able to signal. Tests in vitro and in vivo in human cell lines and primary T cells showed that synthetic peptides containing debris were able to block the response induced by the ligand of CCR5, CCL5. Using techniques of energy transfer between fluorochromes (FRET) demonstrate the existence of preformed dimers in the cell membrane, so that the role of quimioquina seems to be to stabilize the active conformation of the receptor. This is the first description of the waste involved in dimerización of quimioquinas receivers, and are potential targets for changing the responses of the quimioquinas. Besides using the receptor CCR2 human model, we identified the domains of the receptor through which the kinase JAK2 interacts. We have seen that the waste Leu140 and Ala141 in the second loop intracellular receptor are essential for the association of JAK. At mutate debris obutvimos receivers mutants CCR2 that are normally expressed in the cell membrane and were capable of uniting ligand but that they were not capable of uniting JAK2. These mutants were unable to signal, which indicates that the association of the JAK quimioquinas receptors is a key step in the functionality of the quimioquinas. Through bioinformatics analysis, technical transfer enrgía between fluorochromes, tests in vitro and in vivo, we have identified waste which may constitute potential targets for designing drugs that affect the functionality of the quimioquinas for specifics.

Author: Gómez Lozano Natalia.
Year: 2005.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: Biología Molecular. Facultad de CIencias. UAM.
Place of preparation: Biología Molecular. Facultad de CIencias. UAM.

Summary: KIR receptors (Killer-cell Immunoglobulin-like Receptors), after recognition of their ligands, the MHC class I molecules, governing the response of NK cells directed face potential target cells. KIR genes are located in the region chromosome 19q13.4, in a complex of up to 15 genes and 2 pseudogenes, all with a high degree of polymorphism and alternative forms of processing. KIR2DL5 is the family member described more recently, whose gene is present only in certain individuals. Our group has studied the inherent variability and its involvement in diversity haplotípica of KIR genes. This study has identified KIR2DL5 as a "hot spot" of the KIR diversity, in which there have been different recombination. We have demonstrated the existence of two loci coding for KIR2DL5 (KIR2DL5A and KIR2DL5B), which may occur separately or jointly in the same haplotype. We have also discovered three new alleles of KIR2DL5 (KIR2DL5A * 005, KIR2DL5B * 006 and * 007) and a sobrecruzamiento uneven between KIR2DL5 and KIR3DP1, which is presumably the source of two haplotypes unusual: those carrying a duplication of genes conserved KIR3DP1- KIR2DL4-KIR3DL/S1 or a deletion of the same genes. These haplotypes have enabled us to rule out an essential role for KIR2DL4, a receiver for HLA-G, reproduction, and to identify an allele chimeric of pseudogén KIR3DP1 that functional could encode a soluble receptor. The expression of KIR2DL5 is variable: it has alleles in lymphocyte transcribed NK yTy others who behave as pseudogenes not transcribed. This variation is correlated with polymorphisms in the promoter regions. We have analyzed the binding of transcription factors RUNX the sponsor of the alleles of KIR2DL5, activity in these regions in vitro and the effect of a drug desmetilate in expressing KIR2DL5. These tests show that the alleles of KIR2DL5 have not specified promoters potentially functional, but they are muted in vivo by epigenetic mechanisms, in which the factors RUNX could be implicated.

Author: Aceves Tejero Mónica.
Year: 2007.
University: VALLADOLID [www.uva.es].
Place of defense: Facultad de Medicina.
Place of preparation: Universidad de Valladolid - Facultad de Medicina.

Summary: The transcription factor NFAT is expressed in the immune response and inflammation. White is the immunosuppressive drug cyclosporin A and FK506 that revolutionized the field of organ transplantation in the mid-80. These drugs inhibit phosphatase calcineurin, which in turn inhibits desfosforilación of protein and transport it to the kernel. Furthermore, the activation of the role of NFAT is affected by the use of anti-inflammatory drugs such as salicylates and derivatives trifluorometilados. We noticed that salicylates inhibit the activation of transcription factor and inhibits the transport of the protein to the nucleus, however, salicylates and derivatives trifluorometilados inhibit the binding of NFAT DNA, and therefore inhibiting the expression of genes regulating . Since ámbos drugs inhibit the cascade of NFAT to trigger two different levels, we studied the combination of ámbos to try to reduce the unwanted effects of the use of CsA at high doses and high concentration. We managed to reduce the dose of current use of CsA combined along with a dose of triflusal used in terapética, in a way that inhibited the activity mediated NFATal Like when used in doses now considered as optimal. It remains to be resolved whether these effects could be used in the clinic as an alternative to current immunosuppressive therapy.