The role of the intracellular second messenger cAMP in the induction of long-term morphological changes in sensory neurons of {\it Aplysia\/}

The present work examines the role of cAMP in the induction of the type of long-term morphological changes that have been shown to be correlated with long-term sensitization in Aplysia.^ To examine this issue, cAMP was injected into individual tail sensory neurons in the pleural ganglion to mimic, at the single cell level, the effects of behavioral training. After a 22 hr incubation period, the same cells were filled with horseradish peroxidase and 2 hours later the tissue was fixed and processed. Morphological analysis revealed that cAMP induced an increase in two morphological features of the neurons, varicosities and branch points. These structural alterations, which are similar to those seen in siphon sensory neurons of the abdominal ganglion following long-term sensitization training of the siphon-gill withdrawal reflex, could subserve the altered behavioral response of the animal. These results expose another role played by cAMP in the induction of learning, the initiation of a structural substrate, which, in concert with other correlates, underlies learning.^ cAMP was injected into sensory neurons in the presence of the reversible protein synthesis inhibitor, anisomycin. The presence of anisomycin during and immediately following the nucleotide injection completely blocked the structural remodeling. These results indicate that the induction of morphological changes by cAMP is a process dependent on protein synthesis.^ To further examine the temporal requirement for protein synthesis in the induction of these changes, the time of anisomycin exposure was varied. The results indicate that the cellular processes triggered by cAMP are sensitive to the inhibition of protein synthesis for at least 7 hours after the nucleotide injection. This is a longer period of sensitivity than that for the induction of another correlate of long-term sensitization, facilitation of the sensory to motor neuron synaptic connection. Thus, these findings demonstrate that the period of sensitivity to protein synthesis inhibition is not identical for all correlates of learning. In addition, since the induction of the morphological changes can be blocked by anisomycin pulses administered at different times during and following the cAMP injection, this suggests that cAMP is triggering a cascade of protein synthesis, with successive rounds of synthesis being dependent on successful completion of preceding rounds. Inhibition at any time during this cascade can block the entire process and so prevent the development of the structural changes.^ The extent to which cAMP can mimic the structural remodeling induced by long-term training was also examined. Animals were subjected to unilateral sensitization training and the morphology of the sensory neurons was examined twenty-four hours later. Both cAMP injection and long-term training produced a twofold increase in varicosities and approximately a fifty percent increase in the number of branch points in the sensory neuron arborization within the pleural ganglion. (Abstract shortened by UMI.) ^

Mechanisms of adenovirus 5 E1A -mediated tumor suppression and E1A-mediated apoptosis

The adenovirus type 5 E1A gene products have numerous functions in cells, which serve as useful tools in studying the mechanisms of either oncogenesis or tumor suppression. To understand the mechanisms of E1A-mediated tumor suppression, we introduced an Ad5 E1A gene into murine melanoma cells, and characterized E1A-mediated biological functions both in vitro and in vivo. The results of the study indicated that: (i) Ad5 E1A mediated tumor suppression in rodent tumor cells; (ii) E1A-mediated tumor suppression is associated with E1A-mediated apoptosis in vivo.^ To determine which functional region(s) of E1A is(are) required for E1A-mediated apoptosis and whether E1A-mediated apoptosis is required for E1A-mediated tumor suppression, we established stable transfectants of E1A mutants, which have deletion mutation at either the N-terminal (p300-binding) or the CR2 (pRb-binding) domain or both, and then characterized biological functions both in vitro and in vivo. The results of the study indicate that the CR2 domain of E1A is required for E1A-mediated apoptosis, while the N-terminal domain of E1A is dispensable. Interestingly, either of the two domains is able to mediate tumor suppression, since mutant E1A with a single deletion at either domain still suppressed tumor growth. Importantly, deletion mutations at both the N-terminal and the CR2 domains of E1A abrogated E1A-mediated tumor suppression, suggesting both regions are required for E1A-mediated tumor suppression. The results demonstrate that E1A-mediated apoptosis is not the only mechanism for E1A-mediated tumor suppression. Thus, the N-terminal and CR2 domains of E1A mediated two independent mechanisms of tumor suppression.^ To understand the mechanism of E1A-mediated apoptosis, we examined the temporal relationship of molecular events during the apoptotic cascades after UV radiation and serum depletion in both the E1A-expressing cells and parental cells. Kinetic analysis of JNK activity indicates that the JNK pathway is greatly increased in response to UV light in E1A transfectants, suggesting that extracellular stress stimuli have been converted into intracellular stress signals with greater magnitude in E1A transfectants than those in parental cells. Thus, E1A-mediated sensitization precedes these events. As ceramide has been proposed as second messenger and upstream activator of JNK pathway for stress-induced apoptosis, we also examined the roles of ceramide in apoptosis and the relationship with JNK pathway. The results indicate that E1A transfectants do not have increased sensitivity to ceramide. Therefore, E1A-mediated sensitization to UV radiation cannot be attributed to an increased sensitivity to ceramide. Furthermore, UV-induced JNK activation correlates with UV-induced apoptosis, while lethal dose of ceramide does not activate JNK. Thus, activation of JNK pathway is independent of the ceramide pathway. In addition, E1A transfectants also have increased activation of NF-kB in response to UV. These results suggest that E1A-mediated sensitization is an early event which associates with conversion of extracellular stress stimuli into amplified intracellular signals. The mechanism of E1A-mediated sensitization and its relationship with other pathways are discussed. ^

*Ras proteins and human tumor cell radiosensitivity

Ras proteins (H-, N-, K4A-, and K4B) are associated with cellular resistance to ionizing radiation (IR) and, consequently, may provide a potential target for radiosensitization strategies in cancer treatment. Several approaches have been used to compromise Ras activity and enhance IR-induced cell killing; however, these techniques either target proteins in addition to Ras or only target one member of the Ras family. In this study, I have used an adenovirus (AV1Y28) that expresses a single-chain antibody fragment directed against Ras proteins to investigate the mechanism(s) responsible for Ras-mediated radiation resistance. AV1Y28 enhanced the radiosensitivity of a number of human tumor cell lines without affecting the radiosensitivity of normal human fibroblasts. Whereas AV1Y28-mediated sensitization was independent of ras gene mutational status, it was dependent on active Ras proteins suggesting that AV1Y28 may be useful against a broad range of tumors. AV1Y28-mediated cell killing was not the result of redistributing cells into a more radiosensitive phase of the cell cycle and did not enhance IR-induced apoptosis. Given that Ras proteins transduce environmental signals to the nucleus, the effect of AV1Y28 on the IR-inducible transcription factor NF-κB were determined. Although AV1Y28 inhibited IR-induced NF-κB through the suppression of IKK, additional work established that NF-κB did not play a role in AV1Y28-mediated radiosensitization. However, a novel component of the signaling pathway responsible for IR-induced NF-κB was identified. Previous studies had suggested a relationship between mutant ras genes and IR-induced G2 delay; therefore the effects of AV1Y28 on the progression of cells from G2 to M after IR were determined. Pretreatment of cells with AV1Y28 prevented the IR-induced G2 arrest. AV1Y28-mediated abrogation of IR-induced G2 arrest correlated with those cell line lines that were sensitized by AV1Y28. Moreover, a significant increase in cells undergoing mitotic catastrophe was found after IR in AV1Y28 treated cells. The abrogation of G2 arrest by AV1Y28 was the result of maintaining the active form of cdc2, an inducer of mitosis, after exposure to IR. This study identified the mechanism of AV1Y28-mediated radiosensitization and has provided insight into the signal transduction pathways responsible for Ras-mediated radiation resistance. ^

Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs) using microarray in a multicenter study.

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.

The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray.

Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited mayor que50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.

Component-resolved diagnosis of wheat flour allergy in baker's asthma.

Baker's asthma is one of the most common types of occupational asthma and its prevalence is increasing in the last years. Diagnosis of occupational asthma is complex. The poor specificity of current diagnostic approaches may be associated with insufficient purity of wheat extracts or lack of inclusion of major allergens in them. In this work, we use microarray technology to characterize the allergenic profiles of baker's asthma patients from three regions in Spain and to analyze the influence of other environmental allergens on the sensitization pattern.

"Deporte inclusivo en la escuela": diseño y análisis de un programa de intervencion para promover la inclusión del alumnado con discapacidad en Educación Física

La Educación Física se muestra, a priori, como un área idónea para trabajar intervenciones educativas para facilitar la inclusión de alumnos con discapacidad. Sin embargo, poco se conoce sobre el potencial que presentan los deportes adaptados y paralímpicos como contenido para fomentar la sensibilización y concienciación del alumnado sin discapacidad, especialmente en situaciones inclusivas de práctica. Por otro lado, la actitud del docente y su formación se presentan como claves en este contexto, ya que es quien en última instancia selecciona los contenidos a trabajar. Entendemos que en EF disponemos de una ocasión única en el curriculum para fomentar la participación activa y efectiva del alumno con discapacidad en clase (especialmente a nivel de desarrollo de la competencia motriz, entre otras), si bien esto depende de factores relacionados con los dos agentes anteriores. Es por todo ello que la Educación Física, como protagonista y como contenido, se muestra como un contexto adecuado para la investigación de los procesos de inclusión de alumnos con discapacidad en el ámbito educativo. Este trabajo de investigación pretende arrojar luz a los interrogantes que condicionan y limitan este contexto, desde una perspectiva multidisciplinar, con distintas metodologías, sobre los tres agentes indicados. La falta de consenso en la literatura en cuanto a las características y tipo de intervenciones eficaces para facilitar esta sensibilización del alumnado, unido a que es un ámbito relativamente reciente como tema de investigación, nos ha impulsado a trabajar en esta línea. El primer objetivo de este trabajo de investigación fue diseñar e implementar un programa de sensibilización y concienciación hacia la discapacidad basado en los deportes adaptados en el área de Educación Física para alumnos de secundaria y bachillerato. Inicialmente, se realizó una búsqueda bibliográfica tanto a nivel nacional como internacional, con el fin de definir las características principales que aporta la literatura científica en este aspecto. Apoyándonos por un lado, en el programa educativo Paralympic School Day (CPI, 2004) y por otro, en la citada revisión, desarrollamos un planteamiento inicial de estructura y fases. Dicho proyecto, fue presentado al Comité Paralímpico Español y a las federaciones deportivas españolas para personas con discapacidad, con la finalidad de recabar su apoyo institucional en forma de aval y recursos no solo a nivel económico sino también como apoyo logístico y de difusión. Tras su aprobación y gracias también al apoyo de la UPM, la Fundación Sanitas y Liberty Seguros, se procedió a diseñar el programa. Para el desarrollo de los materiales didácticos se contactó con expertos en la materia de EF y Actividad Física Adaptada tanto del ámbito educativo (profesores de educación secundaria y profesorado universitario) como del deportivo a nivel nacional. A su vez, se comenzó a difundir entre el profesorado de los centros con el fin de detectar su interés en participar durante el curso académico (2012-2013) en el programa “Deporte Inclusivo en la Escuela”. Con la finalización del desarrollo de los materiales didácticos, se visitó a los centros educativos para presentar el dossier informativo donde se explicaba el programa, así como las características y fases para su implementación. El programa está fundamentado en la Teoría del Contacto (Allport, 1954) y basado en los deportes adaptados y paralímpicos, planteado con una metodología inclusiva, seleccionando la información, la simulación y el contacto directo como estrategias para el fomento de la sensibilización y concienciación hacia la inclusión. En la reunión celebrada en la Facultad de Ciencias de la Actividad Física y del Deporte (INEF-UPM) en febrero de 2013, se coordinó junto con el profesorado la implementación del programa en cada uno de los 13 centros educativos, con acciones concretas como la adecuación de la propuesta didáctica en la planificación anual del profesor, el préstamo de material o la ponencia del deportista paralímpico entre otras cuestiones. Para la consecución del objetivo 2 de este trabajo, analizar el efecto del programa en los distintos agentes implicados en el mismo, alumnos sin discapacidad, profesorado de EF y alumnos con discapacidad, se calendarizó la toma de datos y la administración de las diferentes herramientas metodológicas para antes (pretest) como después de la intervención (posttets). En el caso de los alumnos sin discapacidad (N= 1068), se analizó el efecto de la intervención sobre la actitud hacia la inclusión, utilizando el cuestionario “Children Attitude Integrated Physical Education-Revised” (CAIPE-R; Block, 1995) de carácter cuantitativo tras su validación y adaptación al contexto español. Los resultados mostraron cambios significativos positivos en la actitud el grupo que mantuvo un contacto no estructurado con alumnos con discapacidad. En esta muestra también se midió la actitud hacia el juego cooperativo con compañeros con discapacidad en clases de EF usando el cuestionario “Children's Beliefs Toward Cooperative Playing With Peers With Disabilities in Physical Education” (CBIPPD-MPE; Obrusnikova, Block, y Dillon, 2010). El desarrollo de un sistema de categorías fundamentado en la Teoría del Comportamiento Planificado (Azjen, 1991) sirvió como base para el análisis de las creencias del alumnado sin discapacidad. Tras la intervención, las creencias conductuales emergentes se mantuvieron, excepto en el caso de los factores identificados como obstáculos de la inclusión. En el caso de las creencias normativas también se mantuvieron tras la intervención y respecto a las creencias de control, los alumnos identificaron al profesor como principal agente facilitador de la inclusión. En el caso de los profesores de EF participantes en el programa (N=18), se analizó el efecto del programa en su actitud hacia la inclusión de alumnos con discapacidad en EF con el cuestionario “Attitud toward inclusion of individual with physical disabilities in Physical Education” (ATISDPE-R; Kudláèek, Válková, Sherrill, Myers, y French, 2002). Los resultados mostraron que no se produjeron diferencias significativas tras la intervención en la actitud general, encontrando algunas diferencias en determinados ítems relacionados con los beneficios de la inclusión en los alumnos sin discapacidad relacionados con los docentes con experiencia previa con discapacidad y en EF antes de la intervención. La otra dimensión analizada fue el efecto de la intervención en la autoeficacia del profesor en la enseñanza de la EF en condiciones inclusivas, habiendo utilizado el cuestionario "Self-efficacy in teaching PE under inclusive conditions" (SEIPE; Hutzler, Zach, y Gafni, 2005). Los resultados en este caso muestran diferencias significativas positivas en cuestiones relacionadas como sentirse capaces de mejorar las condiciones óptimas de enseñanza con alumnos con discapacidad física como movilidad reducida severa y amputación y discapacidad visual tanto en situaciones deportivas, como juegos o actividades fuera del centro educativo a favor de los docentes. En cuanto al género, los hombres obtuvieron valores superiores a las mujeres en relación a sentirse más capaces de incluir a alumnos con discapacidad física tanto en juegos durante el recreo como en la enseñanza de técnica deportiva. Los profesores con menos de 10 años de docencia mostraron valores más positivos en cuanto a sentirse capaces de incluir a un alumno con discapacidad fisica en deportes durante su tiempo libre. El análisis del diario del profesor muestra por un lado, las tendencias emergentes como principales elementos facilitadores u obstaculizadores de la inclusión en EF, identificando al propio alumno sin discapacidad, el propio profesor, los contenidos, los materiales y la organización. Por otro lado, el análisis de los contenidos propuestos en el programa. En el caso de los alumnos con discapacidad (N=22), se analizó el impacto del programa de intervención en el autoconcepto, con el cuestionario "Autoconcepto forma 5" (AF5; F. García y Musitu, 2001). Se encontraron diferencias significativas a favor de las mujeres antes de la intervención en la dimensión familiar, mientras que los hombres obtuvieron valores más altos en las dimensiones social y físico. En cuanto a la edad, se encontraron diferencias significativas antes de la intervención, con valores superiores en los alumnos más jóvenes (12-14 años) en la dimensión físico, mientras que los alumnos mayores (15-17 años) mostraron valores más altos en la dimensión social del cuestionario. Respeto al tipo de discapacidad, los alumnos con discapacidad motórica mostraron mejores valores que los que tienen discapacidad auditiva para la dimensión físico antes de la intervención. En cuanto al autoconcepto general, las diferencias significativas positivas se producen en la dimensión académica. En cuanto al efecto del programa en la autoestima de los alumnos con discapacidad, se utilizó la Escala de "Autoestima de Rosenberg" (Rosenberg, 1989), no obteniendo diferencias significativas en cuanto el género. Apareciendo diferencias significativas antes de la intervención en el caso de la variable edad en los alumnos más jóvenes, en cuanto a que desearían valorarse más, y en los alumnos con discapacidad auditiva en que no se sienten muy orgullosos de ellos mismos. Se produce una mejora en la autoestima general en cuanto a que se sienten menos inútiles tras la intervención. En relación al objetivo 3 de este trabajo, tras el análisis de los resultados y haber discutido los mismos con los autores de referencia, emergió la propuesta de orientaciones tanto para los programa de intervención en EF para la sensibilización y concienciación del alumnado hacia la inclusión como de cara a la formación específica del profesorado, como clave en este tipo de intervenciones. Creemos que el programa “Deporte Inclusivo Escuela” se convierte en un elemento transformador de la realidad, ya que responde a las necesidades detectadas a la luz de esta investigación y que vienen a dar respuesta a los distintos agentes implicados en su desarrollo. Por un lado, atiende la demanda del ámbito educativo en cuanto a las necesidades de formación del profesorado, sensibilización y concienciación del alumnado sin discapacidad, además de facilitar oportunidades de participación activa al alumno con discapacidad en las sesiones de EF. Por otro lado, satisface la demanda por parte de las instituciones deportivas del ámbito de la discapacidad en cuanto a la promoción y difusión de los deportes adaptados y paralímpicos. Por último, desde el ámbito universitario, se muestra como un recurso en la formación del alumnado del grado en Ciencias de la Actividad Física y del Deporte, por su participación directa con la discapacidad. Por estos motivos, este trabajo se muestra como un punto de partida adecuado para seguir avanzando en la investigación en esta área. ABSTRACT Physical Education (PE) seems a priori, as a suitable area to work educational interventions to facilitate the inclusion of students with disabilities. However, little is known about the potential that have adapted and Paralympic sports as content to raise awareness of students without disabilities, especially in inclusive practice context. On the other hand, teachers’ attitude and their training are presented as key in this context because it is who selects the content to work. We understand that PE have a unique opportunity in the curriculum to encourage active and effective participation of students with disabilities in class (especially at motor competence development, etc.), although this depends on factors related to the two agents. For these reasons that the PE, as actors and as content is displayed as a context for investigating the processes of inclusion of students with disabilities in education. This research aims to shed light on the questions that condition and limit this context, from a multidisciplinary perspective, with different methodologies on the three agents mentioned. The lack of accord in the literature regarding the characteristics and type of effective facilitators of awareness of students, and that is a new area as a research topic, has prompted us to work in this topic research. The first aim of this research was to design and implement a program of awareness towards disability based on adapted sports in the area of physical education for middle and high school students. Initially, a literature search was performed both nationally and internationally, in order to define the main features that brings the scientific literature in this area. On the one hand, we supported in the Paralympic School Day (IPC, 2004) educative program and on the other hand, in that review, we developed an initial approach to structure and phases. The project was presented to the Spanish Paralympic Committee and the Spanish Sports Federations for people with disabilities, in order to obtain institutional support in the form of guarantees and resources not only in economic terms but also as logistical support and dissemination. Thanks to the support of Fundación Sanitas, Liberty Seguros and Politechnical University of Madrid, we proceeded to design the program. For the development of teaching resources it was contacted experts in the field of Adapted Physical Activity and physical education and both the field of education (high school teachers and university professors) as the adapted sport national. In turn, it began to spread among the teachers of the schools in order to identify their interest in participating in the academic year (2012-2013) in the "Inclusive Sport in School" program. With the completion of the development of educational materials to schools he was visited to present the briefing where the program, as well as features and steps for its implementation are explained. The program is based on the Contact Theory (Allport, 1954) and based on adapted and Paralympic sports, raised with an inclusive approach, selecting strategies for promoting awareness and awareness to inclusion like information, contact and simulation of disability. At the meeting held at the Faculty of Sciences of Physical Activity and Sport (INEF-UPM) in February 2013, it was coordinated with the teachers implementing the program in each of the 13 schools with concrete actions such as adequacy of methodological approach in the annual planning of the teacher, the loan or the presentation of materials Paralympian among other issues. To achieve the objective 2 of this paper, to analyze the effect of the program on the various actions involved it, students without disabilities, PE teachers and students with disabilities, the date for management of the different methodological tools for before (pretest) and after the intervention (posttets). For students without disabilities (N= 1068), the effect of the intervention on the attitude towards inclusion was analyzed, using the quantitative questionnaire "Integrated Physical Education Attitude Children-Revised" (CAIPE-R; Block, 1995), after validation and adaptation to the Spanish context. The results showed significant positive changes in the attitude of the group with no structured contact with students with disabilities. This shows the beliefs towards the cooperative play was with peers with disabilities in PE classes also measured, using the questionnaire "Children's Beliefs Toward Cooperative Playing With Peers With Disabilities in Physical Education" (CBIPPD-MPE; Obrusnikova, Block, and Dillon, 2010). The development of a system of categories based on the Theory of Planned Behavior (Azjen, 1991) served as the basis for analysis of the beliefs of students without disabilities. After surgery, emerging behavioral beliefs remained, except in the case of the factors identified as barriers to inclusion. In the case of normative beliefs also they remained after surgery and regarding control beliefs, students identified the teacher as the main facilitator of inclusion. Regarding PE teachers participating in the program (N = 18), the effect of the program was analyzed their attitude toward inclusion of students with disability in PE with the questionnaire "Toward Attitude inclusion of Individual with in Physical Education "(ATISDPE-R; Kudláèek, Válková, Sherrill, Myers, and French, 2002). The results showed no significant difference occurred after surgery in the general attitude, finding some differences in certain related benefits of inclusion in students without disability relating to teachers with previous experience with disability in PE before intervention. The other dimension was analyzed the effect of the intervention on self-efficacy of teachers in the teaching of PE in inclusive terms, having used the questionnaire "Self-efficacy in PE teaching even under conditions" (SEIPE; Hutzler, Zach, and Gafni, 2005). The results showed significant differences positive in issues like being able to enhance the optimal conditions for teaching students with physical disabilities as amputation and severe visual impairment in both sports situations, such as games or activities outside the school to for teachers. Regard to gender, men earned higher values regarding women about feel more able to include students with physical disabilities in both games during recess and teaching sports technique. Teachers with less than 10 years of teaching showed more positive values as you feel able to include a student with physical disabilities in sports during their leisure time. The analysis of daily teacher shows on the one hand, emerging trends as key facilitators or barrier of the inclusion elements in PE, identifying the students without disabilities themselves, the professor, contents, materials and organization. Furthermore, the analysis of daily teacher about the contents proposed in the program. In the case of students with disabilities (N=22), the impact of the intervention program on self-concept was analyzed, with the questionnaire "Self-concept form 5" (AF5, F. Garcia and Musitu, 2001). The women showed significant differences before the intervention in family dimension, while men scored higher values in the social and physical dimensions were found. In terms of age, significant differences were found before the intervention, with higher values in younger students (12-14 years) in the physical dimension, while older children (15-17 years) showed higher values the social dimension of the questionnaire. Respect disabilities, students with motor disabilities showed better values than those with hearing impairment to the physical dimension before surgery. As for the general self-concept, positive significant differences occur in the academic dimension. As for the effect of the program on self-esteem of students with disabilities Scale "Rosenberg Self-Esteem" (Rosenberg, 1989) was used, not getting significant differences in gender. Only appear significant difference before the intervention in the case of younger students as they wish to be valued more, and students with hearing disabilities who do not feel very proud of themselves. Improved self-esteem generally occurs in that they feel less useless after surgery. With regard to the aim 3 of this research, after analyzing the results and have discussed them with the authors, it emerged the proposal of guidelines for both intervention program EF for sensitization and awareness of students towards inclusion as in the face of specific training for teachers, as key in such interventions. We believe that "Inclusive Sport Schools" program becomes a transforming element of reality, as it responds to the needs identified in the light of this research and come to respond to the various elements involved in its development. On the one hand, it meets the demand of the education sector in terms of the needs of teacher training, awareness of students without disability, and facilitates opportunities for students with disabilities for active participation in PE class. On the other hand, it meets the demand of sports institutions in the field of disability regarding the promotion and dissemination of adapted and Paralympic sports. Finally, it is shown as a resource from the university level for the training of degree in Physical Activity and Sport Science students, by its direct involvement with disability. For these reasons, this work is shown as a good starting point to further advance research in this area.

Is Microarray Analysis Really Useful and Sufficient to Diagnose Nut Allergy in the Mediterranean Area?

Background: Component-based diagnosis on multiplex platforms is widely used in food allergy but its clinical performance has not been evaluated in nut allergy. Objective: To assess the diagnostic performance of a commercial protein microarray in the determination of specific IgE (sIgE) in peanut, hazelnut, and walnut allergy. Methods: sIgE was measured in 36 peanut-allergic, 36 hazelnut-allergic, and 44 walnut-allergic patients by ISAC 112, and subsequently, sIgE against available components was determined by ImmunoCAP in patients with negative ISAC results. ImmunoCAP was also used to measure sIgE to Ara h 9, Cor a 8, and Jug r 3 in a subgroup of lipid transfer protein (LTP)-sensitized nut-allergic patients (positive skin prick test to LTP-enriched extract). sIgE levels by ImmunoCAP were compared with ISAC ranges. Results: Most peanut-, hazelnut-, and walnut-allergic patients were sensitized to the corresponding nut LTP (Ara h 9, 66.7%; Cor a 8, 80.5%; Jug r 3, 84% respectively). However, ISAC did not detect sIgE in 33.3% of peanut-allergic patients, 13.9% of hazelnut-allergic patients, or 13.6% of walnut-allergic patients. sIgE determination by ImmunoCAP detected sensitization to Ara h 9, Cor a 8, and Jug r 3 in, respectively, 61.5% of peanut-allergic patients, 60% of hazelnut-allergic patients, and 88.3% of walnut-allergic patients with negative ISAC results. In the subgroup of peach LTP?sensitized patients, Ara h 9 sIgE was detected in more cases by ImmunoCAP than by ISAC (94.4% vs 72.2%, P<.05). Similar rates of Cor a 8 and Jug r 3 sensitization were detected by both techniques. Conclusions: The diagnostic performance of ISAC was adequate for hazelnut and walnut allergy but not for peanut allergy. sIgE sensitivity against Ara h 9 in ISAC needs to be improved.

Immune Polarization in Allergic Patients: Role of the Innate Immune System

Allergens come into contact with the immune system as components of a very diverse mixture. The most common sources are pollen grains, food, and waste. These sources contain a variety of immunomodulatory components that play a key role in the induction of allergic sensitization. The way allergen molecules bind to the cells of the immune system can determine the immune response. In order to better understand how allergic sensitization is triggered, we review the molecular mechanisms involved in the development of allergy and the role of immunomodulators in allergen recognition by innate cells.

Occupational allergic multiorgan disease induced by wheat flour

Bakers are repeatedly exposed to wheat flour (WF) and may develop sensitization and occupational rhinoconjunctivitis and/or asthma to WF allergens.1 Several wheat proteins have been identified as causative allergens of occupational respiratory allergy in bakery workers.1 Testing of IgE reactivity in patients with different clinical profiles of wheat allergy (food allergy, wheat-dependent exercise-induced anaphylaxis, and baker's asthma) to salt-soluble and salt-insoluble protein fractions from WF revealed a high degree of heterogeneity in the recognized allergens. However, mainly salt-soluble proteins (albumins, globulins) seem to be associated with baker's asthma, and prolamins (gliadins, glutenins) with wheat-dependent exercise-induced anaphylaxis, whereas both protein fractions reacted to IgE from food-allergic patients.1 Notwithstanding, gliadins have also been incriminated as causative allergens in baker's asthma.2 We report on a 31-year-old woman who had been exposed to WF practically since birth because her family owned a bakery housed in the same home where they lived. She moved from this house when she was 25 years, but she continued working every day in the family bakery. In the last 8 years she had suffered from work-related nasal and ocular symptoms such as itching, watery eyes, sneezing, nasal stuffiness, and rhinorrhea. These symptoms markedly improved when away from work and worsened at work. In the last 5 years, she had also experienced dysphagia with frequent choking, especially when ingesting meats or cephalopods, which had partially improved with omeprazole therapy. Two years before referral to our clinic, she began to have dry cough and breathlessness, which she also attributed to her work environment. Upper and lower respiratory tract symptoms increased when sifting the WF and making the dough. The patient did not experience gastrointestinal symptoms with ingestion of cereal products. Skin prick test results were positive to grass (mean wheal, 6 mm), cypress (5 mm) and Russian thistle pollen (4 mm), WF (4 mm), and peach lipid transfer protein (6 mm) and were negative to rice flour, corn flour, profilin, mites, molds, and animal dander. Skin prick test with a homemade WF extract (10% wt/vol) was strongly positive (15 mm). Serologic tests yielded the following results: eosinophil cationic protein, 47 ?g/L; total serum IgE, 74 kU/L; specific IgE (ImmunoCAP; ThermoFisher, Uppsala, Sweden) to WF, 7.4 kU/L; barley flour, 1.24 kU/L; and corn, gluten, alpha-amylase, peach, and apple, less than 0.35 kU/L. Specific IgE binding to microarrayed purified WF allergens (WDAI-0.19, WDAI-0.53, WTAI-CM1, WTAI-CM2, WTAI-CM3, WTAI-CM16, WTAI-CM17, Tri a 14, profilin, ?-5-gliadin, Tri a Bd 36 and Tri a TLP, and gliadin and glutamine fractions) was assessed as described elsewhere.3 The patient's serum specifically recognized ?-5-gliadin and the gliadin fraction, and no IgE reactivity was observed to other wheat allergens. Spirometry revealed a forced vital capacity of 3.88 L (88%), an FEV1 of 3.04 L (87%), and FEV1/forced vital capacity of 83%. A methacholine inhalation test was performed following an abbreviated protocol,4 and the results were expressed as PD20 in cumulative dose (mg) of methacholine. Methacholine inhalation challenge test result was positive (0.24 mg cumulative dose) when she was working, and after a 3-month period away from work and with no visits to the bakery house, it gave a negative result. A chest x-ray was normal. Specific inhalation challenge test was carried out in the hospital laboratory by tipping WF from one tray to another for 15 minutes. Spirometry was performed at baseline and at 2, 5, 10, 15, 20, 30, 45, and 60 minutes after the challenge with WF. Peak expiratory flow was measured at baseline and then hourly over 24 hours (respecting sleeping time). A 12% fall in FEV1 was observed at 20 minutes and a 26% drop in peak expiratory flow at 9 hours after exposure to WF,

Sensitive detection of major food allergens in breast milk: first gateway for allergenic contact during breastfeeding

Food allergy is recognized as a major public health issue, especially in early childhood. It has been hypothesized that early sensitization to food allergens maybe due to their ingestion as components dissolved in the milk during the breastfeeding, explaining reaction to a food, which has never been taken before. Thus, the aim of this work has been to detect the presence of the food allergens in breast milk by microarray technology. We produced a homemade microarray with antibodies produced against major food allergens. The antibody microarray was incubated with breast milk from 14 women collected from Fundación Jiménez Díaz Hospital. In this way, we demonstrated the presence of major foods allergens in breast milk. The analysis of allergens presented in breast milk could be a useful tool in allergy prevention and could provide us a key data on the role of this feeding in tolerance induction or sensitization in children.

A Recombinant Sal k 1 Isoform as an Alternative to the Polymorphic Allergen from Salsola kali Pollen for Allergy Diagnosis

The incidence of Amaranthaceae pollen allergy has increased due to the desertification occurring in many countries. In some regions of Spain, Salsola kali is the main cause of pollinosis, at almost the same level as olive and grass pollen. Sal k 1 - the sensitization marker of S. kali pollinosis - is used in clinical diagnosis, but is purified at a low yield from pollen. We aimed to produce a recombinant (r)Sal k 1 able to span the structural and immunological properties of the natural isoforms from pollen, and validate its potential use for diagnosis. METHODS: Specific cDNA was amplified by PCR, cloned into the pET41b vector and used to transform BL21 (DE3) Escherichia coli cells. Immunoblotting, ELISA, basophil activation and skin-prick tests were used to validate the recombinant protein against Sal k 1 isolated from pollen. Sera and blood cells from S. kali pollen-sensitized patients and specific monoclonal and polyclonal antisera were used. RESULTS: rSal k 1 was produced in bacteria with a yield of 7.5 mg/l of cell culture. The protein was purified to homogeneity and structural and immunologically validated against the natural form. rSal k 1 exhibited a higher IgE cross-reactivity with plant-derived food extracts such as peanut, almond or tomato than with pollen sources such as Platanus acerifolia and Oleaceae members. CONCLUSIONS: rSal k 1 expressed in bacteria retains intact structural and immunological properties in comparison to the pollen-derived allergen. It spans the immunological properties of most of the isoforms found in pollen, and it might substitute natural Sal k 1 in clinical diagnosis.

Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia

Immune mechanisms contribute to cerebral ischemic injury. Therapeutic immunosuppressive options are limited due to systemic side effects. We attempted to achieve immunosuppression in the brain through oral tolerance to myelin basic protein (MBP). Lewis rats were fed low-dose bovine MBP or ovalbumin (1 mg, five times) before 3 h of middle cerebral artery occlusion (MCAO). A third group of animals was sensitized to MBP but did not survive the post-stroke period. Infarct size at 24 and 96 h after ischemia was significantly less in tolerized animals. Tolerance to MBP was confirmed in vivo by a decrease in delayed-type hypersensitivity to MBP. Systemic immune responses, characterized in vitro by spleen cell proliferation to Con A, lipopolysaccharide, and MBP, again confirmed antigen-specific immunologic tolerance. Immunohistochemistry revealed transforming growth factor β1 production by T cells in the brains of tolerized but not control animals. Systemic transforming growth factor β1 levels were equivalent in both groups. Corticosterone levels 24 h after surgery were elevated in all sham-operated animals and ischemic control animals but not in ischemic tolerized animals. These results demonstrate that antigen-specific modulation of the immune response decreases infarct size after focal cerebral ischemia and that sensitization to the same antigen may actually worsen outcome.

Intrinsic responses to Borna disease virus infection of the central nervous system

Immune cells invading the central nervous system (CNS) in response to Borna disease virus (BDV) antigens are central to the pathogenesis of Borna disease (BD). We speculate that the response of the resident cells of the brain to infection may be involved in the sensitization and recruitment of these inflammatory cells. To separate the responses of resident cells from those of cells infiltrating from the periphery, we used dexamethasone to inhibit inflammatory reactions in BD. Treatment with dexamethasone prevented the development of clinical signs of BD, and the brains of treated animals showed no neuropathological lesions and a virtual absence of markers of inflammation, cell infiltration, or activation normally seen in the CNS of BDV-infected rats. In contrast, treatment with dexamethasone exacerbated the expression of BDV RNA, which was paralleled by a similarly elevated expression of mRNAs for egr-1, c-fos, and c-jun. Furthermore, dexamethasone failed to inhibit the increase in expression of mRNAs for tumor necrosis factor α, macrophage inflammatory protein 1β, interleukin 6, and mob-1, which occurs in the CNS of animals infected with BDV. Our findings suggest that these genes, encoding transcription factors, chemokines, and proinflammatory cytokines, might be directly activated in CNS resident cells by BDV. This result supports the hypothesis that the initial phase of the inflammatory response to BDV infection in the brain may be dependent upon virus-induced activation of CNS resident cells.

Thiazolidinediones and insulin resistance: Peroxisome proliferatoractivated receptor γ activation stimulates expression of the CAP gene

c-Cbl-associated protein (CAP) is a signaling protein that interacts with both c-Cbl and the insulin receptor that may be involved in the specific insulin-stimulated tyrosine phosphorylation of c-Cbl. The restricted expression of CAP in cells metabolically sensitive to insulin suggests an important potential role in insulin action. The expression of CAP mRNA and proteins are increased in 3T3-L1 adipocytes by the insulin sensitizing thiazolidinedione drugs, which are activators of the peroxisome proliferator-activated receptor γ (PPARγ). The stimulation of CAP expression by PPARγ activators results from increased transcription. This increased expression of CAP was accompanied by a potentiation of insulin-stimulated c-Cbl tyrosine phosphorylation. Administration of the thiazolidinedione troglitazone to Zucker (fa/fa) rats markedly increased the expression of the major CAP isoform in adipose tissue. This effect was sustained for up to 12 weeks of treatment and accompanied the ability of troglitazone to prevent the onset of diabetes and its complications. Thus, CAP is the first PPARγ-sensitive gene identified that participates in insulin signaling and may play a role in thiazolidinedione-induced insulin sensitization.