Estudio químico biodirigido y caracterización farmacológica del aceite de oliva virgen extra en el lupus eritematoso sistémico experimental

El lupus eritematoso sistémico (LES) es una enfermedad autoinmune inflamatoria crónica que puede afectar a múltiples órganos, provocando un deterioro progresivo que afecta a la calidad de vida de los pacientes. En la actualidad no se dispone de una terapia efectiva y eficaz para el LES, y tanto la propia enfermedad como sus tratamientos contribuyen a incrementar la mortalidad y morbilidad de la misma. La terapia nutricional puede ser un prometedor enfoque en el abordaje del LES, ya que más allá de su soporte dietético, presenta potenciales efectos profilácticos sin los efectos adversos asociados a la farmacoterapia clásica, contribuyendo a reducir comorbilidades y mejorar la calidad de vida de los pacientes con LES. En este sentido, recientes estudios han confirmado que el consumo habitual de aceite de oliva virgen extra (AOVE), alimento funcional de primera magnitud dentro del contexto de la dieta mediterránea, es eficaz en la prevención y tratamiento de ciertas patologías relacionadas con la inflamación crónica y el sistema inmune. A pesar del esfuerzo investigador llevado a cabo en los últimos años, aún se desconoce el potencial antiinflamatorio e inmunomodulador del AOVE y de sus componentes bioactivos en el LES. Previamente, diversos estudios preclínicos y clínicos han puesto de manifiesto que el consumo regular de AOVE dentro de la dieta mediterránea posee diversas propiedades beneficiosas que podrían ser de interés en el manejo del LES. No obstante, no existían hasta la fecha estudios experimentales concluyentes que validasen los posibles beneficios concretos del AOVE o de sus componentes bioactivos en el desarrollo y progresión de esta enfermedad autoinmune, así como de los mecanismos bioquímicos y moleculares involucrados. En vista a estos antecedentes, el objetivo general de la presente tesis doctoral ha sido desarrollar un estudio experimental multidisciplinar y transversal para investigar el funcionalismo del AOVE y de sus fracciones bioactivas en el LES. Los resultados de la presente tesis doctoral ponen de manifiesto que el AOVE presenta efectos beneficiosos en la aparición y desarrollo del LES experimental, destacando el papel de los compuestos fenólicos del AOVE. A la vista de los resultados obtenidos, el AOVE, junto con sus compuestos fenólicos, podría constituir la base para el desarrollo de nuevas estrategias nutricionales para el tratamiento de esta enfermedad inmunoinflamatori

Extra virgin olive oil polyphenolic extracts downregulate inflammatory responses in LPS-activated murine peritoneal macrophages suppressing NFκB and MAPK signalling pathways

Extra virgin olive oil (EVOO) is obtained from the fruit of the olive tree Olea europaea L. Phenolic compounds present in EVOO have recognized anti-oxidant and anti-inflammatory properties. However, the activity of the total phenolic fraction extracted from EVOO and the action mechanisms involved are not well defined. The present study was designed to evaluate the potential anti-inflammatory mechanisms of the polyphenolic extract (PE) from EVOO on LPS-stimulated peritoneal murine macrophages. Nitric oxide (NO) production was analyzed by the Griess method and intracellular reactive oxygen species (ROS) by fluorescence analysis. Moreover, changes in the protein expression of the pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1), as well as the role of nuclear transcription factor kappa B (NFκB) and mitogen-activated protein kinase (MAPK) signalling pathways, were analyzed by Western blot. PE from EVOO reduced LPS-induced oxidative stress and inflammatory responses through decreasing NO and ROS generation. In addition, PE induced a significant down-regulation of iNOS, COX-2 and mPGES-1 protein expressions, reduced MAPK phosphorylation and prevented the nuclear NFκB translocation. This study establishes that PE from EVOO possesses anti-inflammatory activities on LPS-stimulated murine macrophages.Ministerio de Ciencia e Innovación AGL 2008-02475Ministerio de Ciencia e Innovación AGL 2011-26949Junta de Andalucía P-10AGR-6609Ministerio de Economía y Competitividad INNCORPORA-PT

Olive‐Oil‐Derived Polyphenols Effectively Attenuate Inflammatory Responses of Human Keratinocytes by Interfering with the NF‐κB Pathway

Scope: Extra virgin olive oil (EVOO) is rich in phenolic compounds, including hydroxytyrosol (HTy) and hydroxytyrosyl acetate (HTy-Ac), which have presented multiple beneficial properties. Their impact on inflammatory responses in human keratinocytes and modes of action have not been addressed yet. Methods and results: Primary human keratinocytes are pretreated with HTy-Ac or HTy for 30 min and stimulated with IL-1β or Toll-like receptor 3 ligand (TLR3-l). Thymic stromal lymphopoietin (TSLP), measured by ELISA, is attenuated by both polyphenols in a dose-dependent manner. The expression of several inflammation-related genes, including distinct TSLP isoforms and IL-8, are assessed by quantitative RT-PCR and likewise inhibited by HTy-Ac/HTy. Mechanistically, EVOO phenols counteracts I κB degradation and translocation of NF-κB to the nucleus, a transcription factor of essential significance to TSLP and IL-8 transcriptional activity; this is evidenced by immunoblotting. Accordingly, NF-κB recruitment to critical binding sites in the TSLP and IL-8 promoter is impeded in the presence of HTy-Ac/HTy, as demonstrated by chromatin immunoprecipitation. Promoter reporter assays finally reveal that the neutralizing effect on NF-κB induction has functional consequences, resulting in reduced NF-κB-directed transcription. Conclusion EVOO phenols afford protection from inflammation in human keratinocytes by interference with the NF-κB pathway

Frequencies and TCR Repertoires of Human 2,4,6-Trinitrobenzenesulfonic Acid-specific T Cells

Allergic contact dermatitis is a widespread T cell-mediated inflammatory skin disease, but in vitro monitoring of chemical-specific T cells remains challenging. We here introduce short-term CD154/CD137 upregulation to monitor human T cell responses to the experimental sensitizer 2,4,6-trinitrobenzenesulfonic acid (TNBS). Peripheral blood mononuclear cells (PBMC) from healthy donor buffy coats were TNBS-modified and incubated with unmodified PBMC. After 5 and 16 h, we detected TNBS-specific activated CD154+CD4+ and CD137+CD8+ T cells by multi-parameter flow cytometry, respectively. Activated cells were sorted for restimulation and bulk T cell receptor (TCR) high-throughput sequencing (HTS). Stimulation with TNBS-modified cells (3 mM) induced CD154 expression on 0.04% of CD4+ and CD137 expression on 0.60% of CD8+ memory T cells, respectively (means, n = 11–17 donors). CD69 co-expression argued for TCR-mediated activation, which was further supported by TNBS-specific restimulation of 10/13 CD154+CD4+ and 11/15 CD137+CD8+ T cell clones and lines. Major histocompatibility complex (MHC) blocking antibodies prevented activation, illustrating MHC restriction. The high frequencies of TNBS-specific T cells were associated with distinct common changes in the TCR β-chain repertoire. We observed an overrepresentation of tryptophan and lysine in the complementarity determining regions 3 (CDR3) (n = 3–5 donors), indicating a preferential interaction of these amino acids with the TNBS-induced epitopes. In summary, the detection of TNBS-specific T cells by CD154/CD137 upregulation is a fast, comprehensive and quantitative method. Combined with TCR HTS, the mechanisms of chemical allergen recognition that underlie unusually frequent T cell activation can be assessed. In the future, this approach may be adapted to detect T cells activated by additional chemical sensitizers

Científicas españolas: un mundo por descubrir

Objetivos: La Unión Europea sugiere, entre otros puntos, que la formación universitaria sea complementada con los avances que se vayan produciendo en la investigación científica y, además, pone un especial énfasis en la digitalización de contenidos y en la difusión a través de internet en la Educación Superior. Por ello, el objetivo del proyecto llevado a cabo fue realizar una actividad en la que los alumnos entrevistaran a una mujer científica española que trabajara en alguno de los temas estudiados en clases e ir creando un blog, vinculado a Facebook, con las entrevistas realizadas. De esta forma, además de dar visibilidad a las científicas de nuestro país, ya que en muchos casos la mujer está infravalorada por la sociedad en el ámbito científico, se pretendía conseguir la participación activa del alumnado y favorecer y fomentar su aprendizaje activo, desarrollar la capacidad del alumno de profundizar en un tema de investigación y de plantearse y de que tomaran conciencia del relevante nivel científico que existe en nuestro país y, en concreto, de la importancia que tienen las mujeres. Metodología: Se propuso la actividad a alumnos de 5 Grados diferentes (Farmacia, Doble Grado de Nutrición y Dietética, Ciencia y Tecnología de los Alimentos, Terapia Ocupacional y Óptica y Optometría). Los alumnos interesados en participar formaron grupos de trabajo y eligieron el tema sobre el que querían trabajar del listado propuesto por los profesores. Buscaron una mujer científica española de reconocido prestigio que trabajara en el tema elegido y, después de comunicárselo al profesor, se pusieron en contacto con ella para solicitarles la realización de la entrevista. Una vez que esta accedía a su realización, los alumnos prepararon un dossier de preguntas basándose en los trabajos publicados de la misma, y, tras ser supervisado y corregido por el profesor, eran formuladas a la científica elegida. Una vez realizada la entrevista, los alumnos redactaron un artículo y tras su revisión por el profesor, se hacía público en el blog. Para dar mayor visibilidad al blog, se creó una cuenta abierta de Facebook en la que se iba vinculando el mismo. Al final de todo el proceso se realizó una valoración de la actividad global por parte de los alumnos mediante una encuesta de opinión tipo Likert. Resultados: Se ofertó la actividad a 581 alumnos y participaron 195 (33,6%). Con la actividad propuesta se ha conseguido la participación activa de un porcentaje considerable del alumnado, destacando en los Grados de CYTA (78,4%), Doble Grado de Farmacia y Nutrición (72,3%) y Farmacia (24,1%). Dentro de los que participaron en la actividad, el porcentaje de aprobados fue mayor que el de suspensos (78 vs. 22%; p<0,05, respectivamente). Por otra parte, la valoración otorgada a la actividad fue bastante buena (3,8 sobre 5 puntos), aunque muchos estudiantes manifestaron que les llevó bastante tiempo su realización. Por último, según los alumnos, la actividad les hizo tomar conciencia del alto nivel científico de muchas científicas españolas (4,5 puntos sobre 5)

Squalene targets pro- and anti-inflammatory mediators and pathways to modulate over-activation of neutrophils, monocytes and macrophages

Squalene is a natural triterpene consumed as an integral part of the human diet. Increasing evidence demonstrates that squalene has antioxidant, cardioprotective and anti-carcinogenic activities. Nevertheless, its anti-inflammatory properties remain unclear. The effects of squalene on lipopolysaccharide (LPS)-mediated inflammatory response in murine macrophages and human monocytes and neutrophils were investigated. Squalene reduced intracellular levels of ROS, nitrites, cytokines (TNF-α, IL-1β, IL-6 and IFN-γ) and pro-inflammatory enzymes (iNOS, COX-2 and MPO), including a decreased expression of TLR4 and key proteins for signalling pathways mediated by NF-κB (IκBα), MAPKs (JNK) and MMPs (1, 3 and 9). In addition, squalene enhanced expression levels of anti-inflammatory enzymes (HO-1) and transcription factors (Nrf2 and PPARγ). This study establishes that squalene has significant potential for management of inflammatory conditions characterized by an over-activation of neutrophils/monocytes/macrophages and thereby for the efficient termination of the inflammatory response.This work was supported by funds from the Spanish Ministerio de Ciencia e Innovación (MICINN) (AGL2008-02475, AGL2011-26949 and AGL2011-29008) and Junta de Andalucía (P-10AGR-6609 and P09-CVI-5007). The authors gratefully acknowledge the assistance of Centre for Technology and Innovation Research, University of Seville (CITIUS). SM has the benefit of a FPI fellowship (BES-2012-056104) of MICINN. This work was supported by the University of Seville, “V Own Research Plan” contract to BB.Peer Reviewe

In Vitro Monitoring of Human T Cell Responses to Skin Sensitizing Chemicals&mdash;A Systematic Review

Background: Chemical allergies are T cell-mediated diseases that often manifest in the skin as allergic contact dermatitis (ACD). To prevent ACD on a public health scale and avoid elicitation reactions at the individual patient level, predictive and diagnostic tests, respectively, are indispensable. Currently, there is no validated in vitro T cell assay available. The main bottlenecks concern the inefficient generation of T cell epitopes and the detection of rare antigen-specific T cells. Methods: Here, we systematically review original experimental research papers describing T cell activation to chemical skin sensitizers. We focus our search on studies published in the PubMed and Scopus databases on non-metallic allergens in the last 20 years. Results: We identified 37 papers, among them 32 (86%) describing antigen-specific human T cell activation to 31 different chemical allergens. The remaining studies measured the general effects of chemical allergens on T cell function (five studies, 14%). Most antigen-specific studies used peripheral blood mononuclear cells (PBMC) as antigen-presenting cells (APC, 75%) and interrogated the blood T cell pool (91%). Depending on the individual chemical properties, T cell epitopes were generated either by direct administration into the culture medium (72%), separate modification of autologous APC (29%) or by use of hapten-modified model proteins (13%). Read-outs were mainly based on proliferation (91%), often combined with cytokine secretion (53%). The analysis of T cell clones offers additional opportunities to elucidate the mechanisms of epitope formation and cross-reactivity (13%). The best researched allergen was p-phenylenediamine (PPD, 12 studies, 38%). For this and some other allergens, stronger immune responses were observed in some allergic patients (15/31 chemicals, 48%), illustrating the in vivo relevance of the identified T cells while detection limits remain challenging in many cases. Interpretation: Our results illustrate current hardships and possible solutions to monitoring T cell responses to individual chemical skin sensitizers. The provided data can guide the further development of T cell assays to unfold their full predictive and diagnostic potential, including cross-reactivity assessments

Virgin olive oil and its phenol fraction modulate monocyte/macrophage functionality: a potential therapeutic strategy in the treatment of systemic lupus erythematosus

5 FigurasMonocytes and macrophages are critical effectors and regulators of inflammation and innate immune response, which appear altered in different autoimmune diseases such as systemic lupus erythematosus (SLE). Recent studies suggested that virgin olive oil (VOO) and particularly its phenol compounds might possess preventive effects on different immune-inflammatory diseases, including SLE. Here, we evaluated the effects of VOO (and sunflower oil) on lipopolysaccharide (LPS)-activated peritoneal macrophages from a model of pristane-induced SLE in BALB/c mice, as well as those of the phenol fraction (PF) from VOO on the immune-inflammatory activity and plasticity in monocytes and monocyte-derived macrophages from healthy volunteers. The release of nitrite and inflammatory cytokines was lower in LPS-treated peritoneal macrophages from pristane-SLE mice fed the VOO diet when compared with the sunflower oil diet. PF from VOO similarly decreased the secretion of nitrite and inflammatory cytokines and expression of inducible nitric oxide, PPARγ and Toll-like receptor 4 in LPS-treated human monocytes. PF from VOO also prevented the deregulation of human monocyte subset distribution by LPS and blocked the genetic signature of M1 macrophages while favouring the phenotype of M2 macrophages upon canonical polarisation of naïve human macrophages. For the first time, our study provides several lines of in vivo and in vitro evidence that VOO and PF from VOO target and counteract inflammatory pathways in the monocyte–macrophage lineage of mice with pristane-induced SLE and of healthy subjects, which is a meaningful foundation for further development and application in preclinical and clinical use of PF from VOO in patients with SLE.M. A.-S. gratefully acknowledges support from a Postgraduate National Program of FPU fellowship and financial sponsorship from the Spanish Ministerio de Educación, Cultura y Deporte. S. M.-d. l. P. has the benefit of a FPI fellowship (BES-2012-056104) of MICINN. B. B. and S. M.-d. l. P. acknowledge support from ‘V Own Research Plan’ (University of Seville). The authors gratefully acknowledge the assistance of Centre for Technology and Innovation Research, University of Seville (CITIUS). The authors thank I+D+i of Oleoestepa SAC for kindly providing the VOO. This study was supported by research grants AGL2011-26949 and AGL2011-29008 (Spanish Ministry of Science and Innovation, MICINN) and P-10AGR-6609 (Junta de Andalucía). M. A.-S. and S. M.-d. l. P. performed cell cultures, cytokine measurements and RT-qPCR and western blot experiments. S. M.-d. l. P. and B. B. performed flow cytometry experiments and analysed the data. M. A.-S., S. M.-d. l. P. and F. J. G. M. wrote the main manuscript. C. A. d. l. L., M. S.-H. and F. J. G. M. designed and supervised the project and revised the paper. All authors discussed the results and implications and commented the manuscript at all stages. The authors declare that there are no conflicts of interest

Mast cells instruct keratinocytes to produce thymic stromal lymphopoietin: Relevance of the tryptase/protease-activated receptor 2 axis

Background: Thymic stromal lymphopoietin (TSLP) promotes TH2 inflammation and is deeply intertwined with inflammatory dermatoses like atopic dermatitis. The mechanisms regulating TSLP are poorly defined. Objective: We investigated whether and by what mechanisms mast cells (MCs) foster TSLP responses in the cutaneous environment. Methods: Ex vivo and in vivo skin MC degranulation was induced by compound 48/80 in wild-type protease-activated receptor 2 (PAR -2)-and MC-deficient mice in the presence or absence of neutralizing antibodies, antagonists, or exogenous mouse MC protease 6 (mMCP6). Primary human keratinocytes and murine skin explants were stimulated with lysates/supernatants of human skin MCs, purified tryptase, or MC lysate diminished of tryptase. Chymase and histamine were also used. TSLP was quantified by ELISA, real-time quantitative PCR, and immunofluorescence staining. Results: Mas-related G protein-coupled receptor X2 (Mrgprb2) activation elicited TSLP in intact skin, mainly in the epidermis. Responses were strictly MC dependent and relied on PAR-2. Complementarily, TSLP was elicited by tryptase in murine skin explants. Exogenous mMCP6 could fully restore responsiveness in MC-deficient murine skin explants. Conversely, PAR-2 knockout mice were unresponsive to mMCP6 while displaying increased responsiveness to other inflammatory pathways, such as IL-1a. Indeed, IL-1a acted in concert with tryptase. In primary human keratinocytes, MC-elicited TSLP generation was likewise abolished by tryptase inhibition or elimination. Chymase and histamine did not affect TSLP production, but histamine triggered IL-6, IL 8, and stem cell factor. Conclusion: MCs communicate with kerationocytes more broadly than hitherto suspected. The tryptase/PAR-2 axis is a crucial component of this cross talk, underlying MC-dependent stimulation of TSLP in neighboring kerationocytes. Interference specifically with MC tryptase may offer a treatment option for disorders initiated or perpetuated by aberrant TSLP, such as atopic dermatitis