Optimal combinations of acute phase proteins for detecting infectious disease in pigs

Peer reviewedPublisher PD

Desarrollo de metodologías analíticas y estudios sobre la aplicabilidad de las proteínas de fase aguda como biomarcadores de salud y bienestar animal en el perro. Caracterización de ITIH4 como nueva proteína de fase aguda canina

La concentración de proteínas de fase aguda (PFA) se eleva en el perro en diferentes situaciones patológicas. Aunque no es un parámetro que permita determinar la causa de la elevación y no pueden utilizarse como un test primario para la detección de una determinada enfermedad, estas proteínas presentan una elevada sensibilidad para la detección de situaciones que alteran la salud del animal y pueden proporcionar evidencia de que el animal presenta inflamación o infecciones subclínicas. Se han publicado numerosos estudios que describen las PFA como biomarcadores en diferentes tipos de enfermedades que afectan a la especie canina y el interés de su uso como biomarcadores de salud y bienestar animal ha ido incrementando en las últimas décadas. Sin embargo la falta de una metodología específica de especie adecuadamente validada se ha considerado como una de las principales limitaciones para la incorporación de estos parámetros en la clínica. El origen de esta tesis se encuentra en trabajos pioneros llevados a cabo en el departamento de Bioquímica y Biología Molecular y Celular por el grupo del profesor Lampreave, que llevaron a cabo la caracterización de la respuesta de fase aguda en la especie porcina y el descubrimiento de la Pig-MAP, nueva proteína sérica y principal PFA en el cerdo, también conocida como ITIH4. Posteriormente se estudió la nueva proteína ITIH4 en otras especies como la humana, bovina y ovina donde también se comporta como una PFA positiva.En esta tesis se continúan estos estudios centrados en la especie canina. El objetivo general de este trabajo ha sido caracterizar la respuesta de la ITIH4, junto con otras PFA caninas ya conocidas (CRP, Hp y GPA) en diferentes procesos inflamatorios o enfermedades caninas. En este trabajo se ha aislado y caracterizado por primera vez la ITIH4 en la especie canina, que al igual que en el resto de las especies en las que se ha estudiado, consta de una única cadena polipeptídica de 120 KDa. Además se han purificado CRP, Hp y GPA con un alto grado de pureza. Las proteínas purificadas (ITIH4, CRP, Hp y GPA) se utilizaron para inmunizar conejos y obtener antisueros específicos que reconocían la proteína de interés. Los antisueros específicos se utilizaron para desarrollar y validar métodos analíticos robustos, que han permitido cuantificar cada una de estas proteínas en muestras de suero de perro. Se ha desarrollado un método de inmunodifusión radial (IR) y un ELISA tipo sándwich, que han permitido cuantificar por primera vez la ITIH4 canina en muestras de suero. A su vez, también se han desarrollado tres métodos de IR para la cuantificación de CRP, Hp y GPA caninas y un ELISA tipo sándwich para la Hp. La determinación de la concentración de ITIH4 canina, junto con el resto de PFA purificadas (CRP, Hp y GPA) en muestras de suero de animales sometidos a estímulos inflamatorios de diferente origen (cirugía, piometra, infección experimental con Leishmania infantum) ha permitido caracterizar a esta proteína como proteína de fase aguda moderada en la especie canina, tanto por el incremento que experimenta en su concentración tras los diferentes estímulos inflamatorios, como por la cinética mostrada tras la infección experimental con L. infantum. La toma de muestra en el tiempo inmediato tras la infección experimental con L. infantum ha permitido observar por primera vez una respuesta inflamatoria fuerte y temprana, caracterizada por un aumento de CRP, ITIH4 y Hp con valores máximos a las 24 -48 h, dependiendo de la PFA. Los valores de PFA volvieron a aumentar de forma mas moderada entre los 8 – 10 meses post-infección, coincidiendo en términos generales con la aparición de los síntomas clínicos. CRP e ITIH4 mostraron una correlación positiva y moderada con el título de anticuerpos anti Leishmania que presentaba el animal y también con la sintomatología clínica. La cuantificación de CRP, ITIH4, Hp y GPA en sueros de perros clínicamente sanos, ha permitido establecer un intervalo de referencia, para cada una de estas proteínas, para los métodos analíticos desarrollados. Adicionalmente se ha realizado un estudio en colaboración con la Asociación Española de Veterinarios Municipales (AVEM), que ha permitido evaluar el uso de estas PFA como marcadores de bienestar animal en perros alojados en centros de protección. En este estudio se ha encontrado una asociación de los niveles séricos de CRP y Hp presentes en animales alojados en centros de protección y las características de los diferentes centros estudiados, pudiendo asociarse niveles mas elevados de estas proteínas a características de los centros que pueden producir un menor estado de bienestar en el animal.<br /

Acute-phase inter-alpha-trypsin inhibitor heavy chain 4 levels in serum and milk of cows with subclinical mastitis caused by Streptococcus species and coagulase-negative Staphylococcus species

The aim of the study was to investigate the concentrations of acute-phase inter-a-trypsin inhibitor heavy chain 4 (ITIH4) in serum and milk of cows with subclinical mastitis caused by Streptococcus sp. (STR) and coagulase-negative Staphylococcus sp. (CNS) and healthy cows. The blood and milk samples were obtained from 60 mid-lactation, multiparous Holstein-Friesian cows from 7 herds in the Lublin region of Poland. In the milk samples from 40 cows with subclinical mastitis, Streptococcus sp. and CNS were isolated. The ITIH4 was significantly higher in serum of cows with subclinical mastitis caused both by STR and CNS compared with healthy cows. One hundred percent of animals infected with Streptococcus sp. and 89% of animals infected with Staphylococcus sp. showed ITIH4 concentration in sera higher than 0.5 mg/mL. The concentration of ITIH4 in milk also was significantly higher in cows with subclinical mastitis caused by Streptococcus sp. and Staphylococcus sp. compared with the control group. Seventy percent of cows infected by STR and CNS showed ITIH4 concentration in milk higher than 2.5 µg/mL. Milk ITIH4 concentration higher than 5 µg/mL was found in 55% of animals infected with Streptococcus sp. and in 40% of animals infected with Staphylococcus sp. No statistically significant differences were observed in ITIH4 concentrations both in serum and in milk between the studied unhealthy animal groups. These results suggest that ITIH4 may be used in the future as a novel diagnostic marker in serum and in milk of subclinical mastitis in cows

The representation of the inmigrant characters in the programs of fiction

[EN] This article presents the results of a research study into the image of immigration as presented in television fiction after realizing an analysis of content to more than 2.000 characters and more than 100 programs of prime-time fictional programming broadcast in 2011 on six television channel. The results have allowed to conclude that there exists an infrarepresentación of the immigrant characters, as well as a construction based on the use of stereotypes.[ES] En la presente investigación se ha analizado la imagen de la inmigración en la ficción televisiva, tomando como referencia la Teoría del Cultivo y la investigación previa sobre el análisis de la representación de las minorías étnicas e inmigrantes en la ficción televisiva (Greenberg, Mastro y Brand, 2002). El presente artículo presenta los resultados a los que se ha llegado tras realizar un análisis de contenido a más de 2.000 personajes y más de 100 programas emitidos en horario de prime time en seis cadenas televisivas. Los resultados han permitido concluir que existe una infrarepresentación de los personajes inmigrantes, así como una construcción basada en el uso de estereotipos

A new wine Torulaspora delbrueckii killer strain with broad antifungal activity and its toxin-encoding double-stranded RNA virus

Wine Torulaspora delbrueckii strains producing a new killer toxin (Kbarr-1) were isolated and selected for wine making. They killed all the previously known Saccharomyces cerevisiae killer strains, in addition to other non-Saccharomyces yeasts. The Kbarr-1 phenotype is encoded by a medium-size 1.7 kb dsRNA, TdV-Mbarr-1, which seems to depend on a large-size 4.6 kb dsRNA virus (TdV-LAbarr) for stable maintenance and replication. The TdV-Mbarr-1 dsRNA was sequenced by new generation sequencing techniques. Its genome structure is similar to those of S. cerevisiae killer M dsRNAs, with a 5′-end coding region followed by an internal A-rich sequence and a 3′-end non-coding region. Mbarr-1 RNA positive strand carries cis acting signals at its 5′ and 3′ termini for transcription and replication respectively, similar to those RNAs of yeast killer viruses. The ORF at the 5′ region codes for a putative preprotoxin with an N-terminal secretion signal, potential Kex2p/Kexlp processing sites, and N-glycosylation sites. No relevant sequence identity was found either between the full sequence of Mbarr-1 dsRNA and other yeast M dsRNAs, or between their respective toxin-encoded proteins. However, a relevant identity of TdV-Mbarr-1 RNA regions to the putative replication and packaging signals of most of the M-virus RNAs suggests that they are all evolutionarily related.This work was funded by grants GR10088 from the Extremadura Regional Government and AGL2011-25711 from the Spanish Ministry of Education and Science. RV acknowledges support by a fellowship from the Extremadura Regional Government.Peer Reviewe

Haptoglobin and pig-major acute protein are increased in pigs with postweaning multisystemic wasting syndrome (PMWS)

The objective of this study was to determine the serum concentration levels of selected acute phase proteins (APP), haptoglobin (HPT) and pig-major acute phase protein (pig-MAP), in postweaning multisystemic wasting syndrome (PMWS) affected pigs and PCV2-subclinically infected pigs. In a first study, a group of 15 eight-week-old conventional pigs from a PMWS affected farm were bled and a complete necropsy, histopathology and in situ hybridisation to detect PCV2 were performed. Based on the results, pigs were classified as suffering from PMWS (n = 10) or healthy animals (n = 5). In a second study, a group of 45 pigs from another PMWS affected farm were selected and bled at 3, 7, 12 and 28 weeks of age. The assessment of PCV2 infection status in these pigs was retrospectively done by PCV2 PCR in serum samples. Selected APP were measured in the serum of all studied pigs by means of radial immunodiffusion. Mean HPT and pig-MAP levels were significantly increased (p = 0.004 and p = 0.0006 respectively) in PMWS-affected pigs when compared to levels found in healthy pigs (2.52 ± 0.88 mg/mL vs. 1.06 ± 0.73 mg/mL for HPT and 3.81 ± 1.53 mg/mL vs. 0.76 ± 0.34 mg/mL for pig-MAP). In the second study, no significant difference in mean HPT and pig-MAP values were observed between PCV2 PCR positive and negative pigs of any age. However, both APP increased significantly with age in PCV2 PCR negative pigs. Altogether, the present results suggest that APP levels are significantly increased in pigs that develop PMWS, but not in animals with a PCV2 subclinical infection

Translation regulation after taxol treatment in NIH3T3 cells involves the elongation factor (eEF)2

Changes to the translational machinery that occur during apoptosis have been described in the last few years. The two principal ways in which translational factors are modified during apoptosis are: (i) changes in protein phosphorylation and (ii) specific proteolytic cleavages. Taxol, a member of a new class of anti-tubulin drugs, is currently used in chemotherapeutic treatments of different types of cancers. We have previously demonstrated that taxol induces calpain-mediated apoptosis in NIH3T3 cells [Piñeiro et al., Exp. Cell Res., 2007, 313:369–379]. In this study we found that translation was significantly inhibited during taxol-induced apoptosis in these cells. We have studied the phosphorylation status and expression levels of eIF2a, eIF4E, eIF4G and the regulatory protein 4E-BP1, all of which are implicated in translation regulation. We found that taxol treatment did not induce changes in eIF2α phosphorylation, but strongly decreased eIF4G, eIF4E and 4E-BP1 expression levels. MDL28170, a specific inhibitor of calpain, prevented reduction of eIF4G, but not of eIF4E or 4E-BP1 levels. Moreover, the calpain inhibitor did not block taxol-induced translation inhibition. All together hese findings demonstrated that none of these factors are responsible for the taxol-induced protein synthesis inhibition. On the contrary, taxol treatment increased elongation factor eEF2 phosphorylation in a calpain-independent manner, supporting a role for eEF2 in taxol-induced translation inhibition.Depto. de Farmacología y ToxicologíaFac. de MedicinaTRUEpu

Increase in the number of diagnosed cases of whooping cough; Do we think more in it?

Debido al aumento de casos diagnosticados de tosferina en nuestra área, en los últimos años, decidimos realizar una revisión con el objetivo de conocer las características clínico-epidemiológicas de esta enfermedad en nuestro medio.Debido ao aumento de casos diagnósticos de tosferina na nosa área, nos últimos anos, decidimos realizar unha revisión co obxectivo de coñecer as características clínico-epidemiolóxicas desta enfermidade no noso medioDue to the increase of diagnostic cases of pertussis in our area, in recent years, we decided to carry out a review with the objective of knowing the characteristics clinical-epidemiological aspects of this disease in our medium

Calpain inhibition stimulates caspase-dependent apoptosis induced by taxol in NIH3T3 cells

Taxol is an anticancer drug that triggers apoptosis in a wide spectrum of cancers such as ovarian, breast, lung, head and neck, and bladder carcinoma by both caspase-dependent and -independent apoptosis mechanisms. However, the exact signaling pathways involved in taxol-induced apoptosis strongly depend on the cellular background and they are not completely established yet. In this study we demonstrate that taxol induces caspase-3- independent apoptosis in NIH3T3 cells by a calpain-mediated mechanism. Taxol treatment produced changes in the mitochondrial membrane potential (ΔΨm) which could be responsible of Ca2+ release from the mitochondria and the consequent calpain activation. Interestingly, we show that calpain produced proteolysis of caspase-3 and demonstrate that, accordingly, calpain inhibition increased taxol-induced apoptosis. In addition, we reveal that poly (ADP-ribose) polymerase (PARP) was processed by calpain in taxol-treated cells and by caspase-3 after calpain inhibition. In conclusion, these results demonstrate for the first time that calpain could play an important role modulating taxol-induced apoptosis. Further studies are needed to address the potentiality of inducing apoptosis by a combined use of taxol and calpain inhibitors in cells with increased calpain activity.Depto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasTRUEpu