[ES] El objetivo de la presente tesis doctoral es el estudio, desarrollo y validación de diferentes métodos inmunoquímicos que permitan determinar contaminantes químicos en alimentos de origen vegetal y en agua, de manera que contribuyan a mejorar su calidad y por ende la seguridad del consumidor. Spirotetramat es un plaguicida de nueva generación altamente eficiente, comercializado mundialmente para su uso como insecticida en multitud de cultivos agrícolas. Tiene propiedades sistémicas, ya que después de la absorción se transloca tanto a través del xilema como del floema, gracias a que es transformado por la planta en spirotetramat-enol, mucho más polar. En consecuencia, la definición de residuo de este insecticida en alimentos de origen vegetal para fines analíticos incluye también dicho metabolito. Por otro lado, anatoxina-a es un alcaloide secundario con neurotoxicidad aguda que se pueden encontrar en agua dulce. Esta toxina es producida por siete géneros diferentes de cianobacterias, y se ha detectado en lagos y otras fuentes de agua de todos los continentes. El análisis de sustancias como spirotetramat y anatoxina-a se lleva a cabo actualmente mediante métodos cromatográficos como HPLC-MS. Estas técnicas presentan una elevada sensibilidad y fiabilidad; sin embargo, requieren personal altamente cualificado y un equipamiento caro y no portable. Una opción complementaria son los métodos inmunoquímicos, como el ELISA (Enzyme-Linked ImmunoSorbent Assay) o el inmunoensayo de flujo lateral (LFIA, Lateral Flow ImmunoAssay), ya que son métodos de análisis rápidos y económicos, y además son muy versátiles permitiendo adaptarlos a necesidades analíticas particulares, como los ensayos de cribado de numerosas muestras o los ensayos portátiles con lectura visual de los resultados. A partir de una colección de bioconjugados y de anticuerpos de spirotetramat y de anatoxina-a se caracterizó la afinidad y especificidad de los inmunorreactivos con el fin de seleccionar parejas conjugado/anticuerpo aptas para el desarrollo de inmunoensayos tipo ELISA y LFIA competitivos. Se optimizaron las condiciones de ensayo, y se llevó a cabo un estudio de la influencia de diferentes factores fisicoquímicos sobre los parámetros analíticos de los ensayos seleccionados. Posteriormente se evaluó la influencia de la matriz alimentaria, particularmente uva, zumo de uva y vino, así como de aguas de diferente procedencia, sobre la señal y la sensibilidad de los inmunoensayos. La diferente afinidad de los anticuerpos hacia spirotetramat y spirotetramat-enol nos llevó a optimizar el tratamiento de muestra, incluyendo una etapa de hidrólisis para transformar spirotetramat en spirotetramat-enol de manera controlada, rápida y cuantitativa. De este modo se hizo posible aportar resultados en forma de suma de la concentración de ambos compuestos en la muestra, tal y como exige la legislación vigente. Además, para la extracción de residuos de este insecticida a partir de muestras de uva se puso a punto un procedimiento empleando la tecnología QuEChERS, y para la reducción de interferencias de vinos y zumos se utilizó polivinilpolipirrolidona. En el caso de las aguas, se aplicó una simple filtración para eliminar partículas en suspensión. Los inmunoensayos enzimáticos en microplaca optimizados para determinar de manera competitiva residuos de spirotetramat presentaron valores de IC50 para spirotetramat-enol en torno a 0.1 ng/mL, y límites de detección alrededor de 0.02 ng/mL. El estudio de la precisión y exactitud del método empleando muestras de alimentos dopados reflejó límites de cuantificación de 2.5 ng/mL para uva, zumos de uva y vinos, tanto blancos como tintos, muy por debajo de los límites máximos de residuos autorizados en la Unión Europea para este insecticida en dichos alimentos. / [EN] The aim of this doctoral thesis is the study, development and validation of different immunochemical methods for determining chemical contaminants in produce and water, in a way that they may contribute to improving food quality and therefore to assure consumer safety. Spirotetramat is a highly efficient new-generation pesticide, marketed worldwide for use as insecticide in many agricultural crops. It has systemic properties, since short after absorption it translocates through both the xylem and the phloem, thanks to the fact that it is transformed by the plant into the much more polar spirotetramat-enol. Consequently, the residue definition for this insecticide in foods of plant origin with analytical purposes also includes said metabolite. On the other hand, anatoxin-a is a secondary alkaloid with acute neurotoxicity that can be found in fresh water. This toxin is produced by seven different genera of cyanobacteria, and has been detected in lakes and other water resources on all continents. The analysis of substances like spirotetramat and anatoxin-a is currently carried out by chromatographic methods like HPLC-MS. These techniques are highly sensitive and reliable; however, they require highly qualified personnel and expensive, non-portable equipment. Nowadays, the immunochemical methods, such as the ELISA (Enzyme-Linked ImmunoSorbent Assay) or the LFIA (Lateral Flow ImmunoAssay), constitute excellent complementary analytical options to instrumental strategies, since they are fast and inexpensive analytical methods, and are also very versatile so they can be adapted to particular analytical needs, such as screening assays for large numbers of samples or portable tests with visual reading of the results. The antibody affinity and specificity from a collection of spirotetramat and anatoxin-a immunoreagents was characterized in order to select conjugate/antibody pairs suitable for the development of competitive ELISA and LFIA tests. The assay conditions were optimized, and a study of the influence of different physicochemical factors over the analytical parameters of the selected immunoassays was carried out. Subsequently, the influence of the food matrix, particularly grape, grape juice and wine, as well as water from different sources, over the assay signal and sensitivity was evaluated. The different affinity of the available antibodies towards spirotetramat and spirotetramat-enol led us to optimize the sample treatment procedure, so a hydrolysis step to transform spirotetramat into spirotetramat-enol in a controlled, rapid and quantitative way, was included. Thus, it was possible to provide results in the form of the sum of the concentration of both compounds in the sample, as required by current legislation. In addition, a procedure using QuEChERS technology was developed to extract residues of this insecticide from grape samples, and polyvinylpolypyrrolidone was used to reduce interferences from wines and juices. In the case of waters, a simple filtration was applied to remove suspended particles. Microplate enzyme immunoassays that were optimized to competitively determine spirotetramat residues showed IC50 values for spirotetramat-enol around 0.1 ng/mL, and limits of detection around 0.02 ng/mL. Precision and accuracy studies with these immunoassays using fortified food samples reflected limits of quantification of 2.5 ng/mL for grapes, grape juices and wines, both white and red, well below the maximum residue limits authorized by the European Union for this insecticide in such foodstuffs. Finally, a comparative study with HPLC-MS/MS validated the studied immunoassay for analyzing spirotetramat residues in grape samples within a wide range of concentrations. / [CA] L’objectiu de la present tesi doctoral és l’estudi, desenvolupament i validació de diferents mètodes immunoquímics que permeten determinar contaminants químics en aliments d’origen vegetal i en aigua, de manera que contribuïsquen a millorar la seua qualitat i per tant la seguretat dels consumidors. Spirotetramat és un plaguicida de nova generació altament eficient, comercialitzat mundialment per a l’ús com insecticida en multitud de cultius agrícoles. Té propietats sistèmiques, ja que en ser absorbit es transloca tant a través del xilema com del floema, gràcies a que és transformat per la planta en spirotetramat-enol, molt més polar. Conseqüentment, la definició de residu d’aquest insecticida en aliments d’origen vegetal amb finalitats analítiques inclou també l’esmentat metabòlit. D’una altra banda, anatoxina-a és un alcaloide secundari amb neurotoxicitat aguda que es pot trobar en aigua dolça. Aquesta toxina és produïda per set gèneres de cianobacteris diferents, i s’ha detectat en llacs i altres fonts d’aigua de tots els continents. L’anàlisi de substàncies com spirotetramat i anatoxina-a es duu a terme actualment mitjançant mètodes cromatogràfics, com HPLC-MS. Aquestes tècniques presenten una elevada sensibilitat i fiabilitat; tanmateix, requereixen personal altament qualificat i un equipament car i no portable. Una opció complementària són els mètodes immunoquímics, com l’ELISA (Enzyme-Linked ImmunoSorbent Assay) o l’immunoassaig de flux lateral (LFIA, Lateral Flow Immunoassay), ja que són mètodes d’anàlisi ràpids i econòmics, i a més són molt versàtils, la qual cosa permet adaptar-los a necessitats analítiques particulars, com són els assaigs per destriar nombroses mostres o els assaigs portàtils amb lectura visual dels resultats. A partir d’una col·lecció de bioconjugats i d’anticossos de spirotetramat i d’anatoxina-a es va caracteritzar l’afinitat i especificitat dels immunorreactius amb la finalitat de seleccionar parelles conjugat/anticòs aptes per desenvolupar immunoassaigs tipus ELISA i LFIA competitius. S’optimitzaren les condicions d’assaig, i es va dur a terme un estudi de la influència de diferents factors fisicoquímics sobre els paràmetres analítics dels assaigs seleccionats. Posteriorment, es va avaluar la influència de la matriu alimentària, particularment raïm, suc de raïm i vi, així com d’aigües de diferent procedència, sobre el senyal i la sensibilitat dels assaigs. La diferent afinitat dels anticossos cap a spirotetramat i spirotetramat-enol ens va dur a optimitzar el tractament de mostra mitjançant la inclusió d’una etapa d’hidròlisi per transformar spirotetramat en spirotetramat-enol de manera ràpida, controlada i quantitativa. D’aquesta manera es va fer possible aportar resultats en forma de suma de la concentració d’ambdós composts en la mostra, tal i com exigeix la legislació vigent. A més a més, per extraure residus d’aquest insecticida a partir de mostres de raïm es va posar a punt un procediment emprant la tecnologia QuEChERS, i per reduir interferències de vins i sucs es va utilitzar polivinilpolipirrolidona. En el cas de les aigües, es va aplicar una simple filtració per eliminar partícules en suspensió. Els immunoassaigs enzimàtics en microplaca optimitzats per determinar de manera competitiva residus de spirotetramat presentaren valors d’IC50 per spirotetramat-enol al voltant de 0.1 ng/mL, i límits de detecció propers a 0.02 ng/mL. L’estudi de la precisió i exactitud del mètode emprant mostres d’aliments dopats va reflectir límits de quantificació de 2.5 ng/mL per raïm, sucs de raïm i vins, tant blancs com negres, molt per sota dels límits màxims de residus autoritzats per la Unió Europea per a aquest insecticida en els esmentats aliments. Finalment, un estudi comparatiu amb HPLC-MS/MS va validar l’immunoassaig estudiat per analitzar residus de spirotetramat en mostres de raïm en un ampli rang de concentracions. En el cas d’anatoxina-a, es van optimitzar dos immunoassaigs tipus ELISA competitiu, els valors d’IC50 dels quals van estar entre 0.5 i 1.0 ng/mL, amb límits de detecció per davall de 0.1 ng/mL. L’anàlisi de diferents tipus d’aigües fortificades amb anatoxina-a ens va revelar que els immunoassaigs desenvolupats permeten quantificar aquesta cianotoxina entre 0.5 i 500 ng/mL. Addicionalment es van optimitzar i caracteritzar assaigs immunocromatogràfics, tipus tires reactives, tant per spirotetramat com per anatoxina-a, vàlids com a tècniques portables i ràpides per determinar semi-quantitativament aquestes substàncies tòxiques en vi i aigües, respectivament. Seguint la normativa europea per a mètodes ràpids front a petites molècules orgàniques, es va determinar el senyal indicatiu del llindar per distingir les mostres positives, que superen la concentració de destriament establerta, de les negatives. En el cas de spirotetramat, el mètode desenvolupat permet el triatge, tant instrumental com visual, amb un interval de confiança del 99%, de mostres de vi amb una concentració de residu de 1000 ng/mL, equivalent al límit màxim de residus, expressada como la suma de spirotetramat més spirotetramat-enol. Per anatoxina-a, les tires immunocromatográfiques desenvolupades van poder detectar mostres d’aigua amb 2 ng/mL de l’esmentada cianotoxina amb una fiabilitat del 99%, i mostres amb 1 ng/mL amb una probabilitat del 40%, mentre que el límit de detecció visual va ser de 3 ng/mL. / A la Secretaría Nacional de Educación Superior, Ciencia, Tecnología e Innovación (SENESCYT), del gobierno de la República de Ecuador que al adjudicarme la beca bajo el “PROGRAMA DE BECAS PARA DOCTORADO (PHD) PARA DOCENTES DE UNIVERSIDADES Y DE ESCUELAS POLITÉCNICAS 2015”, permitió formarme como persona y profesional en mis estudios de doctorado. / Cevallos Cedeño, RE. (2020). Desarrollo de métodos inmunoquímicos para la determinación de sustancias tóxicas en alimentos y aguas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/149570

Background: In recent years, several new ELISAs for the detection of antibodies against the porcine reproductive and respiratory disease virus (PRRSV) in pig serum have been developed. To interpret the results, specificity and sensitivity data as well as agreement to a reference ELISA must be available. In this study, three commercial ELISAs (INgezim PRRS 2.0 - ELISA II, Priocheck® PRRSV Ab porcine – ELISA III and CIVTEST suis PRRS E/S PLUS - ELISA IV, detecting PRRSV type 1 antibodies) were compared to a standard ELISA (IDEXX PRRS X3 Ab Test - ELISA I). The serum of three pigs vaccinated with an attenuated PRRSV live vaccine (genotype 2) was tested prior to and several times after the vaccination. Furthermore, serum samples of 245 pigs of PRRSV positive herds, 309 pigs of monitored PRRSV negative herds, 256 fatteners of assumed PRRSV negative herds with unknown herd history and 92 wild boars were tested with all four ELISAs. Results: ELISAs II and III were able to detect seroconversion of vaccinated pigs with a similar reliability. According to kappa coefficient, the results showed an almost perfect agreement between ELISA I as reference and ELISA II and III (kappa > 0.8), and substantial agreement between ELISA I and ELISA IV (kappa = 0.71). Sensitivity of ELISA II, III and IV was 96.0%, 100% and 91.5%, respectively. The specificity of the ELISAs determined in samples of monitored PRRSV negative herds was 99.0%, 95.1% and 96.4%, respectively. In assumed negative farms that were not continually monitored, more positive samples were found with ELISA II to IV. The reference ELISA I had a specificity of 100% in this study. Conclusions: All tested ELISAs were able to detect a PRRSV positive herd. The specificity and sensitivity of the tested commercial ELISAs, however, differed. ELISA II had the highest specificity an ELISA III had the highest sensitivity in comparison to the reference ELISA. ELISA IV had a lower sensitivity and specificity than the other ELISAs.

The detection by serology of nematode-transmitted polyhedral viruses (nepoviruses) in grapevines is often unreliable. Nepoviruses were detected by enzyme-linked immunosorbent assay (ELISA) in tissue-cultured and field-grown grapevines. Nepovirus detection in in vitro plants (plantlets) was affected by virus distribution and growth room temperature. The reliability of virus detection in field-grown grapevines was improved when modified grinding buffers were used. Arabis mosaic virus (AMV) was detected by ELISA, for the first time, in in vitro grapevines initiated from field-and screenhouse-grown plants throughout the summer. The virus was not reliably and repeatedly detected in in vitro plantlets grown at 25°C. AMV and grapevine fanleaf virus (GFLV) distribution was not uniform throughout the plantlets. This distribution was affected by the culture room temperature. The best plant parts to sample for virus detection came from the zones of rapidly proliferating shoots. The viruses were sometimes not detected in samples taken from other tissues. Growth room temperature had an important effect on virus detection in plantlets. The highest virus titres were found in plants growing at 15°C. Temperature increases in 5°C steps to 30°C reduced virus titre. AMV became undetectable in nearly all plantlets growing at 30°C for as little as 30 days. Growth at 30°C reduced ELISA absorbance values by 76% after 8 days and after 21 days the values were at 4% of pre-treatment levels. The virus titre dropped below detectable levels in most plantlets. AMV could not be detected in plantlets or rooted explants after being placed in a 30°C treatment for 2 months. Tomato ringspot virus was detected by ELISA, for the first time, in in vitro grapevine plants. The virus was repeatedly detected in in vitro plants growing at 20°C. Under the typical summer conditions experienced at Sidney, B.C., modifying the standard ELISA grinding buffer (0.01 M phosphate buffered saline, pH 7.4, 0.05% Tween-20, 0.2% ovalbumin, 2% polyvinylpyrrolidone) was essential for reliable detection of AMV or GFLV. AMV was reliably detected by ELISA in foliar samples from field or screenhouse plants throughout the summer when the grinding buffer was modified by increasing the pH to at least 8.2 and adding 1% nicotine or 0.15 M phosphate buffered saline. The most reliable results with GFLV were obtained with the nicotine enhanced buffers. In comparison, because of the increased workload associated with growing plants in vitro and the unreliable detection of viruses in these plants, it remained preferable to detect nepoviruses in field plants by ELISA. / Land and Food Systems, Faculty of / Graduate

Background: In recent years, several new ELISAs for the detection of antibodies against the porcine reproductive and respiratory disease virus (PRRSV) in pig serum have been developed. To interpret the results, specificity and sensitivity data as well as agreement to a reference ELISA must be available. In this study, three commercial ELISAs (INgezim PRRS 2.0 - ELISA II, Priocheck® PRRSV Ab porcine – ELISA III and CIVTEST suis PRRS E/S PLUS - ELISA IV, detecting PRRSV type 1 antibodies) were compared to a standard ELISA (IDEXX PRRS X3 Ab Test - ELISA I). The serum of three pigs vaccinated with an attenuated PRRSV live vaccine (genotype 2) was tested prior to and several times after the vaccination. Furthermore, serum samples of 245 pigs of PRRSV positive herds, 309 pigs of monitored PRRSV negative herds, 256 fatteners of assumed PRRSV negative herds with unknown herd history and 92 wild boars were tested with all four ELISAs. Results: ELISAs II and III were able to detect seroconversion of vaccinated pigs with a similar reliability. According to kappa coefficient, the results showed an almost perfect agreement between ELISA I as reference and ELISA II and III (kappa > 0.8), and substantial agreement between ELISA I and ELISA IV (kappa = 0.71). Sensitivity of ELISA II, III and IV was 96.0%, 100% and 91.5%, respectively. The specificity of the ELISAs determined in samples of monitored PRRSV negative herds was 99.0%, 95.1% and 96.4%, respectively. In assumed negative farms that were not continually monitored, more positive samples were found with ELISA II to IV. The reference ELISA I had a specificity of 100% in this study. Conclusions: All tested ELISAs were able to detect a PRRSV positive herd. The specificity and sensitivity of the tested commercial ELISAs, however, differed. ELISA II had the highest specificity an ELISA III had the highest sensitivity in comparison to the reference ELISA. ELISA IV had a lower sensitivity and specificity than the other ELISAs.

Master of Science / Department of Diagnostic Medicine/ Pathobiology / Lisa M. Pohlman / Background: Iron storage disease has proven to be a serious health concern for captive toucans. Physiologic mechanisms to efficiently extract iron from naturally iron-deficient diets appear the likely cause of iron overload when fed iron-sufficient diets in captivity. Iron overload can result in diabetes, heart failure, and even death. Serum ferritin concentrations are considered the most reliable screening tool to predict total body iron stores in many species, but an assay has not been available to measure serum ferritin in toucans. Objective: The purpose of this study was to develop an enzyme-linked immunosorbent assay (ELISA) to measure serum ferritin in toucans using a polyclonal antibody in a sandwich arrangement. Methods: Ferritin was isolated from toucan liver and used as a standard. A rabbit polyclonal anti-toucan antibody was used as the capture antibody and as a detection antibody conjugated to horseradish peroxidase. Linearity of toucan ferritin standards, effect of serum dilution, recovery of added ferritin standards, and intra- and inter-assay variability were determined. Results: Ferritin standards were linear from 0 to 50 ng/ml. The relationship between serum dilution and serum ferritin concentration was also linear. When 10, 20, 30, 40, or 50 ng/ml of purified toucan ferritin were added to diluted serum, the recoveries varied from 69% to 104%. The intra-assay variability for four test serum samples averaged 11% and the inter-assay variability for the same four samples averaged 11%. Conclusions: Although the results from the linearity and recovery studies are promising for assay development when viewed independently, preliminary ferritin concentrations from all toucans studied are much higher than expected. Upon further evaluation including Dot blot assays, Western blot assays, SDS-PAGE, and protein determination of the ferritin stock solution, it was determined that the ferritin stock solution did not contain a pure protein and therefore likely renders the assay invalid. Further testing is needed to confirm these findings.

A quantitative competitive binding "triple sandwich" enzyme immunoassay was used to evaluate pathogen/class-specific antibody responses in Holstein-Friesian calves vaccinated against Clostridium perfringens B-toxin at various ages postpartum. Vaccination of dams at six weeks and again at two weeks prepartum increased pathogen-specific antibody levels in their colostrum and respective calf's serum. Calves initially vaccinated at three days produced both a primary and secondary pathogen-specific antibody response, whereas calves initially vaccinated at 12 and 21 days produced only secondary responses. Maternally-derived antibodies were found to suppress neonatal antibody production following primary immunization. They were also found to influence secondary humoral immune responses, although in a diminished capacity. Pathogen-specific IgG and IgM concentrations in dams' sera and colostra were found related to subsequent pathogen-specific IgG and IgM neonatal serum concentrations. Only pathogen-specific IgA in dams' colostra was correlated to neonatal levels, possibly owing to a different origin and role of this immunoglobulin class. All class-specific colostral immunoglobulin levels were related to subsequent neonatal concentrations. Based on results from this experiment, it is recommended that calves be vaccinated at three days postpartum with a booster administered at 63 days.

Die Organtransplantation stellt ein therapeutisches Verfahren für Patienten mit irreversibel geschädigten Organen dar. Doch weist dieses Behandlungskonzept weiterhin einen wesentlichen Nachteil auf: noch immer wird der langfristige Erfolg der Therapie zu oft durch die so genannte Transplantatabstoßung gefährdet. Hierbei handelt sich um eine vom Organtransplantat ausgelöste Immunantwort, die zu dessen Zerstörung führt. Die derzeit einzige Möglichkeit eine Abstoßung zu verhindern, ist die Unterdrückung des Immunsystems mit so genannten Immunsuppressiva. Auch wenn diese erstmals in den 60er Jahren des 20. Jahrhunderts erfolgreich eingesetzten Medikamente ständig verbessert werden, bleiben die von ihnen ausgelösten Nebenwirkungen weiterhin ein ernstzunehmendes Problem. Sie unterdrücken die gesamte körpereigene Abwehr, was zum Schutz der Organtransplantate vor Abstoßung gewünscht ist, doch fördern sie hierdurch die Entstehung von Tumoren und Infektionen. Bei der Transplantatabstoßung handelt es sich um eine von CD4+ T-Lymphozyten ausgelöste Immunantwort. Diese Lymphozyten werden von allogenen Peptiden, die von Spender-MHC-Molekülen stammen, über den indirekten Weg der Alloantigenerkennung aktiviert. An der Transplantatabstoßung ist zwar eine Vielzahl von Alloantigenen beteiligt, doch ist es möglich, Peptidantigene zu identifizieren, die einen nachweisbaren Effekt auf die Transplantatabstoßung ausüben. So wurde in der eigenen Arbeitsgruppe die für die Abstoßung allogener Organtransplantate beteiligten MHC (RT1u)-Peptidantigene charakterisiert. Insbesondere die Bedeutung des aus 19 Aminosäuren bestehenden allogenen Peptids P1 für die Alloimmunantwort wurde intensiv untersucht. So weisen P1-spezifische T-Lymphozyten ein ausgeprägtes Th1-Cytokin-Muster auf und beschleunigen die Abstoßung von Wistar-Furth-Organtransplantaten in Lewis-Ratten. Das Ziel dieser Arbeit war die Charakterisierung des T-Zellrezeptor Vb-Repertoires P1-spezifischer T-Lymphozyten mit der Methode des PCR-ELISA. In einem ersten Schritt wurden Thymozyten und T-Lymphozyten unterschiedlicher Lymphknotenstationen untersucht. Thymozyten exprimierten alle 22 TCR Vb-Elemente und einzig TCR Vb14 war überrepräsentiert. Die T-Lymphozyten der cervikalen, mesenterialen, iliakalen und poplitealen Lymphknoten zeigten ebenfalls eine charakteristische Überexpression bestimmter TCR Vb-Elemente. So exprimierten cervikale T-Lymphozyten bevorzugt die TCR Vb-Elemente 2, 6, 8.3 und 16, mesenteriale T-Lymphozyten die TCR Vb-Elemente 2, 4, und 8.1, illiakale T-Lymphozyten die TCR Vb-Elemente 2 und 6 und popliteale T-Lymphozyten die TCR Vb-Elemente 2, 4 und 9. Die Immunisierung mit dem nicht-immunogenen Kontrollpeptid (Autoantigen) Ac führte zu einer leichten Veränderung des T-Zellrezeptor-Repertoires, bei der die TCR Vb-Elemente 14 und 16 überexprimiert waren. Das Adjuvant TiterMax beeinflusste kaum das TCR Vb-Repertoire. Die Immunisierung mit dem allogenen Peptid P1 führte zu einer eindeutigen Beeinflussung des T-Zellrezeptor-Repertoires. Popliteale T-Lymphozyten, die 7 Tage nach Immunisierung analysiert wurden, zeigten ein Repertoire, bei dem die TCR Vb-Elemente 15, 16, 17 und 20 überexprimiert waren. Dieses Repertoire war am Tag 3 nach Immunisierung noch nicht so ausgebildet. Wurden die antigenspezifischen T-Lymphozyten nach ihrer Isolierung mit P1 in vitro restimuliert, so waren in diesem T-Zellrezeptor-Repertoire die TCR Vb-Elemente 8.3, 15, 16 und 20 überexprimiert. Zum Vergleich: in naiven T-Lymphozyten waren die Vb-Elemente 2, 4 und 9 überexprimiert. Damit war es zu einer deutlichen Verschiebung im T-Zellrezeptor-Repertoire antigenspezifischer T-Lymphozyten gekommen, die auf das Peptidantigen P1 zurückzuführen ist. Mit der Methode des PCR-ELISA wurde das T-Zellrezeptor-Repertoire antigenspezifischer T-Lymphozyten bestimmt. Hiermit sind wesentliche Voraussetzungen geschaffen worden, um T-Zellklone zu etablieren und ihre Bedeutung für die Transplantatabstoßung genauer zu untersuchen. / Organ transplantation is an important medical therapy for patients with irreversibly damaged organs. However, this therapeutic intervention has a great disadvantage because the long-term success of organ allografts is still too often endangered by the so called allograft rejection, an immune response directed to the allograft and leading to its destruction. The major approach for the prevention and management of allograft rejection is to suppress the immune system with immunosuppressive agents. These agents were introduced into transplantation medicine in the 1960s and since that time they have been continually improved. However, their side effects remain a seriousness problem because the suppression of the immune defence inhibits the allograft rejection on the one hand but causes infections and increases tumour incidence on the other hand. The allograft rejection is mediated by CD4+ T lymphocytes and the responsible antigens recognized by them are allogenic peptides processed from donor MHC molecules. Although a large number of allgenenic peptides are involved in allograft rejection, it is possible to identify certain peptide antigens involved in allograft rejection. In our group allogeneic peptides from MHC class I molecules from Wistar Furth rats were investigated. The significance of the allogeneic peptide P1, consisting of 19 amino acids, in inducing allograft rejection was analysed in detail. P1-specific T lymphocytes demonstrated a Th1-cytokine dominated profile and accelerated the rejection of allografts in Lewis rats donated by Wistar Furth rats. The aim of this study was to characterise the T cell receptor (TCR) Vb repertoire of P1-specific T lymphocytes with the PCR-ELISA technique. First, thymocytes and T lymphocytes from different lymph nodes were analysed. Thymocytes expressed all 22 TCR Vb elements and only TCR Vb14 was overrepresented. The T lymphocytes of cervical, mesenteric, iliacal und popliteal lymph nodes also demonstrated a certain repertoire of overrepresented TCR Vb elements. In cervical T lymphocytes the expression levels of the TCR Vb elements 2, 6, 8.3 and 16 were increased; in mesenteric T lymphocytes the TCR Vb elements 2, 4 and 8.1; in illiacal T lymphocytes the TCR Vb elements 2 and 6; and in popliteal T lymphocytes the TCR Vb elements 2, 4 and 9. The immunisation with the autoantigen Ac led to a small variation of the TCR Vb repertoire and the TCR Vb elements 14 and 16 were overrepresented. The adjuvant TiterMax did not influence the TCR Vb repertoire. In contrast, the immunisation with the allogeneic peptide P1 clearly influenced the T cell receptor repertoire. Popliteal T lymphocytes analysed 7 days after immunisation demonstrated a TCR Vb repertoire where the TCR Vb elements 15, 16, 17 und 20 were overrepresented. On day 3 after immunisation this repertoire was not yet clearly developed. In P1-specific T lymphocytes restimulated in vitro the expression levels of the TCR Vb elements 8.3, 15, 16 and 20 were increased. In comparison, in naïve T lymphocytes the TCR Vb elements 2, 4 and 9 were overrepresented. These results underline that the immunisation with peptide P1 induces a characteristic TCR Vb repertoire. The TCR Vb repertoire of P1-specific T lymphocytes was analysed with the PCR-ELISA technique. The determination of the T cell receptor repertoire is a prerequisite to establish T cell clones and to analyse their involvement in allograft rejection.

Os objetivos desse estudo foram: 1) Padronizar a pesquisa do anti-tTg, comparando-o com o anticorpo antiendomísio (AAE) e 2) Avaliar as interações da tTg com a fibronectina. 49 celíacos e 124 controles com AAE negativo foram avaliados. O AAE foi pesquisado por imunofluorescência indireta e a reatividade contra a tTg e a fibronectina por ELISA in house e com kits comerciais. O antitTg foi positivo em 46,9% e 100% dos celíacos com o ELISA in house e com kits comerciais, respectivamente. A adição de fibronectina não melhorou a sensibilidade do ELISA. Em conclusão: a detecção do antitTg por ELISA apresenta percentual elevado de falso-positivos, não podendo substituir a pesquisa do AAE / The aims of the current study were: to standardize the detection of anti-tTg antibodies, comparing them with antiendomysial antibodies (EMA) and to assess the interaction of tTg with fibronectin. 49 celiac patients and 124 controls were enrolled. EMA was detected by indirect immunofluorescence reaction and tTg and fibronectin reactivity by in house ELISA and with commercially available kits. Seropositivity to anti-tTG was found in 46.9% and 100% of patients by the in house technique and by commercial kits, respectively. Fibronectin addition did not improve the ELISA sensibility. In conclusion, ELISA for anti-tTG detection has a high rate of false positive results and does not replace EMA

No indivíduo adulto, a angiogênese ocorre particularmente em situações patológicas como nos tumores em crescimento, no desenvolvimento de metástases e no processo de cicatrização, sendo o fator de crescimento do endotélio vascular (VEGF) o principal fator envolvido na angiogênese tumoral. Por essa razão, o presente estudo teve como objetivo avaliar os níveis circulantes de VEGF no soro de cães com sarcoma de tecidos moles e sua relação com as células sanguineas, comparados com um grupo de animais sem câncer (controle), a expressão do VEGF e a densidade de microvasos nos espécimes tumorais dos cães com sarcoma de tecidos moles. O grupo controle foi composto de 30 cães machos e o grupo de animais com sarcoma de tecidos moles foi de 25 cães (18 machos e 7 fêmeas castradas) os quais foram avaliados prospectivamente. A coleta sangüínea foi realizada apenas uma vez no grupo controle e em três tempos nos animais com sarcoma (pré-operatório, duas semanas e três meses de pós-operatório) da mesma maneira. Após a colheita o sangue foi processado, para extração do soro e determinação dos níveis de VEGF a partir de um método quantitativo ELISA (enzime-linked immunosorbent assay). A expressão do VEGF e a densidade de microvasos foi investigada por meio da prova de imunoistoquiímica utilizando-se anticorpos anti-VEGF e anti-fator VIII respectivemente. Não houve diferença entre o nível sérico de VEGF dos animais controles e portadores de sarcoma de tecidos moles no tempo pré-operatório. O nível de VEGF sérico no pré-operatório mostrou-se discretamente aumentado em relação a duas semanas e três meses de pós-operatório. Houve correlação positiva entre VEGF sérico e contagem neutrófilos e correlação negativa entre o VEGF e quantidade de hemoglobina nos animais com sarcoma. Houve uma tendência dos animais com hemangiopericitoma apresentarem níveis maiores de VEGF sérico em relação aos portadores de tumor maligno da bainha neural periférica. Houve expressão do VEGF em 65% dos casos, sendo o hemangiopericitoma aquele que expressou maior quantidade de VEGF intratumoral. Não houve diferença na densidade de microvasos entre os tumores negativos e positivos ao VEGF. Os resultados encontrados sugerem contribuição das células do sangue circulante para os níveis de VEGF do soro de cães portadores de sarcomas de tecidos moles, células tumorais e outros tipos celulares parecem ser responsáveis pela angiogênese tumoral, mas não contribuem para elevação da concentração sérica desse fator. / In adult individual, angiogenesis occurs particularly in pathological situations as tumors growth, development of metastasis and in the wound healing process. The vascular endothelial growth factor (VEGF) is the main agent involved in tumor angiogenesis. Therefore, the aim of the present study was to evaluate the circulating levels of VEGF in the serum of dogs suffering from soft tissue sarcoma and its relation with the blood cells, and also the expression of VEGF and the micro vascular density in tumors specimens. A group of health animals was used as control. The control group and treatment group were composed of 30 male dogs 25 dogs (18 males and 7 castrated females) respectively. The blood collection was carried once in the control group and three times in the animals with sarcoma (timely, pre-surgery, two weeks and three months post-surgery) following the same protocol. The blood was processed and a quantitative method ELISA (enzime-linked immunosorbent assay) was used to determinate of the levels of VEGF. The expression of the VEGF and the microvascular density were investigated by means of the immunohistochemical test using anti-VEGF and anti-factor VIII antibodies respectively. There was no difference between serum level of VEGF of the controls animals and serum level of VEGF in the animal with soft tissue sarcoma in pre-surgical time. Serum level of VEGF in pre-surgical time revealed discrete increased in relation the two weeks and three months of post-surgery. There was a positive correlation between serum VEGF and neutrophils counting and negative correlation between the VEGF and amount of haemoglobin in the animals with sarcoma. The animals with hemangiopericytoma showed a trend to higher levels of VEGF in relation to the malignant peripheral nerve sheath tumor. The expression of the VEGF was detected in 65% of the cases; the hemangiopericytoma is the one that expressed greater intratumoral amount of VEGF. There was no difference in the microvascular density between the negative and positive tumors to the VEGF. The results suggest contribution of the circulating blood cells for the levels of serum VEGF of the of the dogs with of soft tissue sarcomas, tumors cells and other cells types seem to be responsible for tumor angiogenesis, but they don\'t contribute for rise serum level of VEGF.

INTRODUÇÃO: Apesar de ser a droga de primeira escolha na prevenção da hemorragia pós-parto, o uso da ocitocina em cesarianas permanece empírico. O objetivo deste estudo foi dosar a ocitocina sérica após a administração profilática de diferentes regimes de ocitocina em pacientes submetidas à cesariana eletiva. MÉTODOS: 30 pacientes que se apresentaram para cesariana eletiva foram randomizadas para receber ocitocina intravenosa, após o clampeamento do cordão umbilical, nos seguintes grupos: G1 (n=9): 10 UI de ocitocina infundidas em 30 minutos (0,33 UI/min), G2 (n=11): 10 UI de ocitocina infundidas em 3 minutos e 45 segundos (2,67 UI/min) e G3 (n=10): 80 UI de ocitocina infundidas em 30 minutos (2,67 UI/min). Este estudo foi encoberto para as pacientes e para os cirurgiões. A avaliação do tono uterino foi realizada pela equipe cirúrgica e a dosagem da concentração sérica de ocitocina foi feita pela técnica de enzimoimunoensaio (ELISA), antes da anestesia (T0) e nos tempos 5 (T5), 30 (T30) e 60 (T60) minutos após o início da infusão da ocitocina. RESULTADOS: Os níveis de ocitocina sérica (média ± erro padrão, ng/mL) foram semelhantes entre os grupos em T0 (0,062 ± 0,021; 0,039 ± 0,019 e 0,067 ± 0,041; respectivamente, p = 0,76) e em T60 (0,648 ± 0,257; 0,356 ± 0,257 e 0,683 ± 0,257; respectivamente, p = 0,58). G3 apresentou níveis maiores de ocitocina que G1 em T5 (3,651 ± 0,741 versus 0,709 ± 0,268; p = 0,01). Em T30, G3 apresentou níveis de ocitocina sérica maiores que G1 (6,190 ± 1,195 versus 1,174 ± 0,375; p < 0,01) e, também, que G2 (6,190 ± 1,195 versus 0,411 ± 0,206; p < 0,01). Os parâmetros hemodinâmicos foram semelhantes entre os grupos. O tono uterino foi considerado satisfatório em todos os intervalos estudados e não houve a necessidade de utilização de uterotônico complementar. CONCLUSÃO: Foram demonstradas dosagens séricas de ocitocina por ELISA em gestantes submetidas à cesariana eletiva. A administração de 80 UI de ocitocina em 30 minutos resulta em níveis séricos de ocitocina maiores que os outros dois métodos de administração aos 5 e 30 minutos, porém, estas concentrações não diferem aos 60 minutos / BACKGROUND: The use of oxytocin to prevent postpartum hemorrhage after elective cesarean delivery still remains empirical. The purpose of this study was to determine oxytocin serum levels following differents regimens of prophylactic oxytocin administration in pregnant women undergoing elective cesarean delivery. METHODS: 30 healthy pregnant patients were randomized to receive intravenous oxytocin, after clamping of the umbilical cord, into the following groups: G1 (n=9), 10 IU of oxytocin infused over 30 minutes (0.33 IU/min); G2 (n=11), 10 IU of oxytocin infused over 3 minutes and 45 seconds (2.67 IU/min) and G3 (n=10), 80 IU of oxytocin infused over 30 minutes (2.67 IU/min). Both patient and surgeon were blinded to the study group allocation. Uterine tone was assessed by palpation by the surgeon. Serum oxytocin concentration was determined by enzyme immunoassay (EIA) before anesthesia (T0) and at 5 (T5), 30 (T30) and 60 (T60) minutes following the start of oxytocin infusion. RESULTS: Serum oxytocin levels (mean ± standard error, ng/mL) were similar in the groups at T0 (0.062 ± 0.021, 0.039 ± 0.019 and 0.067 ± 0.041, respectively, P = 0.76), and T60 (0.648 ± 0.257, 0.356 ± 0.257 and 0.683 ± 0.257, respectively, P = 0.58). G3 presented higher serum oxytocin than G1 at T5 (3.651 ± 0.741 versus 0.709 ± 0.268, P = 0.01). At T30, serum oxytocin levels of G3 were higher than G1 (6.190 ± 1.195 versus 1.174 ± 0.375, P < 0.01) and also than G2 (6.190 ± 1.195 versus 0.411 ± 0.206, P < 0.01). Hemodynamic data were similar in all groups. Uterine tone was considered satisfactory in all intervals studied and no additional uterotonic agent was needed. CONCLUSION: We demonstrate serum oxytocin determinations by EIA in healthy pregnant women presented for elective cesarean delivery. Administering 80 IU in 30 min results in higher serum oxytocin levels at 5 and 30 min than the other two methods of oxytocin administration, but the concentrations did not differ at 60 min