Evaluation of the reactivity of commercially available monoclonal antibodies with equine cytokines.

Research on equine cytokines is often performed by analyses of mRNA. For many equine cytokines an analysis on the actual protein level is limited by the availability of antibodies against the targeted cytokines. Generation of new antibodies is ongoing but time consuming. Thus, testing the reactivity of commercially available antibodies for cross-reactivity with equine cytokines is of particular interest. Fifteen monoclonal antibodies against IL-1ß, IL-6, IL-8, IL-12, IL-18 and Granulocyte Macrophage Colony stimulating factor (GM-CSF) of different species were evaluated for reactivity with their corresponding equine cytokines. Dot Blot (DB) and Western Blot (WB) analyses were performed using recombinant equine cytokines as positive controls. Immunohistochemistry (IHC) was carried out on equine tissue and flow cytometry on equine PBMC as positive controls. As expected, three equine IL-1ß antibodies detected equine IL-1ß in DB, WB and IHC. For these, reactivity in IHC has not been described before. One of them was also found to be suitable for intracellular staining of equine PBMC and flow cytometric analysis. Two antibodies raised against ovine GM-CSF cross-reacted with equine GM-CSF in DB, WB and IHC. For these anti-GM-CSF mAbs this is the first experimental description of cross-reactivity with equine GM-CSF (one mAb was predicted to be cross-reactive in WB in the respective data sheet). The other clone additionally proved to be appropriate in flow cytometric analysis. Two mAbs targeting porcine IL-18 cross-reacted in IHC, but did not show specificity in the other applications. No reactivity was shown for the remaining five antibodies in DB, although cross-reactivity of two of the antibodies was described previously. The results obtained in this study can provide beneficial information for choosing of antibodies for immunological tests on equine cytokines.

Enhanced protocol for CD14+ cell enrichment from equine peripheral blood via anti-human CD14 mAb and automated magnetic activated cell sorting.

REASONS FOR PERFORMING STUDY: CD14 positive (CD14+) cells are the precursor cells of monocyte-derived dendritic cells (DCs). In horses their potent antigen-presenting capacity and ability to induce an effective immune response classify these cells suitable for several therapeutic approaches such as for equine sarcoid. However, in horses, the generation efficiency of DCs from adherent peripheral blood mononuclear cells (PBMCs) is currently still poor. OBJECTIVES: Establishment of a simple short protocol to enhance DC generation in horses by using a human CD14 monoclonal antibody (mAb) and an automated magnetic activated cell sorting (MACS) system. METHODS: Peripheral blood mononuclear cells were isolated from fresh heparinised blood samples of 3 horses and primarily stained for flow cytometric analysis (FACS) with a mAb against human CD14 as well as a secondary phycoerythrin (PE) conjugated antibody to determine the initial percentage of CD14 cells in the sample. Peripheral blood mononuclear cells were used for automated MACS using the same primary and secondary antibodies and analysed by FACS. CD14+ selected cells were cultured for 4 days adding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to the culture media. Dendritic cell generation was assessed analysing cell morphology and surface marker expression (hCD83, hCD86, eqMHCII). RESULTS: Prior to selection, the mean percentage of CD14+ cells in the total cell population was 5.5%, further gaiting of this cell population resulted in 78.46% CD14+ monocytes. After our positive selection the mean percentage of CD14+ cells in the population was 98% without affecting viability. After culture, DC yield was 2-fold higher than in previous published outcomes. CONCLUSIONS: The additional CD14 cell separation step after PBMC isolation significantly amplified the number of CD14+ cells, increasing the number of generated DCs. POTENTIAL RELEVANCE: The number of DCs available is critical for further use of these cells and the herein described protocol will therefore help to improved DC generation for therapeutic approaches in horses.

hCG-induced ovulation in thoroughbred mares does not affect corpus luteum development and function during early pregnancy.

UNLABELLED: Our aim was to compare Corpus luteum (CL) development and blood plasma concentration of progesterone ([P4]) in thoroughbred mares after spontaneous ( CONTROL: C) or human chorionic gonadotrophin (hCG)-induced ovulation. Lactating mares (C=12; hCG=21) were daily teased and mated during second oestrus post-partum. Treated mares received 2500 IU hCG i.v. at first day of behavioural oestrus when dominant follicular size was >35, or=45 mm. Dominant follicle before ovulation, CL and gestational sac were measured by ultrasound and [P4] by radioimmunoassay (RIA). Blood sampling and ultrasound CL exams were done at days 1, 2, 3, 4, 8, 12, 16, 20, 25, 30, 35, 40, 45, 60 and 90 after ovulation and gestational sac from day 12 after ovulation in pregnant (P) mares; non-pregnant (NP) were followed until oestrus returned. Data analyses considered four subgroups: hCG-P, hCG-NP, C-P and C-NP. Preovulatory follicular size was smaller in hCG mares than in C: 39.2+/-2.7 mm vs 51.0+/-1.8 mm (p<0.0001). All hCG mares ovulated 24-48 h after treatment and presented similar oestrus duration as controls. C. luteum size in P mares showed the same pattern of development through days 4-35, presenting erratic differences during initial establishment. Thus, on days 1 and 3, CL was smaller in hCG-P (p<0.05); while in hCG-NP, CL size was greater than in C-NP on day three (p=0.03). Corpus luteum size remained stable until day 90 in hCG-P mares, while in C-P a transient and apparently not functional increase was detected on days 40 and 45 (p<0.05) and the decrease from day 60 onwards, made this difference to disappear. No differences were observed in [P4] pattern between P, or between NP subgroups, respectively. So, hCG-induced ovulation does not affect CL development, neither [P4] during early pregnancy. One cycle pregnancy rate tended to be lower in hCG mares while season pregnancy rates were similar to controls.

Expresión diferencial de proteínas en el corazón de ratas espontáneamente hipertensas con hipertrofia cardíaca / Differential protein expression in hearts of spontaneously hypertensive rats with cardiac hypertrophy

Introducción. La hipertensión arterial afecta a alrededor del 20% de la población del mundo occidental. A pesar de la disponibilidad de excelentes fármacos antihipertensivos, la repercusión orgánica sobre los órganos diana (corazón, cerebro y riñón) sigue siendo elevada. La hipertrofia ventricular izquierda es un hallazgo común en pacientes hipertensos. Aunque se han asociado diversos patrones transcripcionales celulares y genéticos con la hipertrofia cardíaca, sus mecanismos siguen siendo desconocidos. Los objetivos de este trabajo son: a) estudiar el perfil proteico de ratas hipertensas con hipertrofia cardíaca en comparación con animales normotensos, y b) identificar las proteínas de interés. Materiales y métodos. Los estudios se realizaron en ratas macho espontáneamente hipertensas (SHR) y se utilizaron como controles ratas normotensas Wistar Kyoto (WKY). Después de 48 semanas de vida se sacrificó a los animales y se les extrajo el corazón. Para detectar cambios proteicos en la hipertensión grave, analizamos el patrón de expresión de las proteínas cardíacas por electroforesis bidimensional. Resultados. De los más de 1.000 spots resueltos en el rango de pH 4 a 7 del ventrículo izquierdo, nos centramos en 432 bien definidos. En la comparación con los corazones normotensos, de los 432 spots analizados 360 permanecieron invariables y 72 se encontraron alterados en el corazón de las ratas SHR. La identificación de los spots alterados se está realizando mediante espectrometría de masas (MALDI-TOF). Por el momento se han identificado algunos spots tales como la tropomiosina 1-α y el polipéptido Va de la citocromo C oxidasa. Conclusiones. A través de un enfoque proteómico hemos analizado las diferencias entre los proteomas del corazón hipertrófico de animales hipertensos y de controles sanos. En el corazón hipertrófico se observó un cierto número de proteínas sobre o infraexpresadas. Estos datos ilustran un uso inexplorado de la proteómica como herramienta para el descubrimiento de nuevas dianas terapéuticas (AU)

Introduction. Arterial hypertension affects approximately 20% of the Western world. Despite the availability of excellent antihypertensive drugs, damage to target organs (heart, brain and kidney) remains significant. Left ventricular hypertrophy is a common finding in hypertensive patients. Although different cellular and gene transcription patterns have been associated with cardiac hypertrophy, the molecular mechanisms of this process remain mostly unknown. The aims of the present work were: a) to analyze the differential cardiac protein expression in spontaneously hypertensive rats (SHR) with cardiac hypertrophy compared with normotensive rats, and b) identify proteins of interest. Materials and methods. Studies were conducted in male spontaneously hypertensive rats (SHR), with normotensive Wistar-Kyoto rats (WKY) used as controls. At 48 weeks of age, rats were euthanized and hearts removed on block. Analysis of protein expression patterns by two-dimensional polyacrylamide gel electrophoresis (2-DE) was performed to detect changes in heart proteins associated to severe hypertension. Results. From the more than 1000 spots resolved in the 4-7 pH range by 2-DE of myocardial tissue from WKY and SHR rats, we focused on 432 well-defined spots. In comparison with those obtained in normotensive rat hearts, 360 spots remained invariable, 43 increased and 29 decreased or were absent. The identification by mass spectrometry (MALDI-TOF) of altered spots is under study. To date, few spots such as alphatropomyosin and the Va polypeptide of cytochrome c-oxidase have been identified. Conclusions. Via a proteomic approach, we analyzed the difference between proteomes of hypertrophic hearts of hypertensive and normotensive rats and observed a number of over or under expressed proteins in damaged hearts. These data illustrate a hitherto unexplored use of proteomics to discover new therapeutic targets for antihypertensive drugs (AU)

Hemorrhagic gastroesophageal ulceration by pulmonary infection in extrahepatic cholestatic pigs.

We developed a biliary and pulmonary microbiologic study in 22 Large-White pigs that underwent bile-duct ligation in order to demonstrate that sepsis has a biliary and pulmonary origin which may be involved in the gatroesophageal pathology. All the pigs died at 18.2 +/- 8.9 days of the post-operative period. The cause of death was hemorrhagic ulceration of the gastroesophageal region in 36.3% (n = 8) of the animals that also presented multiple bilateral miliary lung abscesses. High infestation rates with intestinal germs were found in the bile and lung. In conclusion, the experimental model of extrahepatic cholestasis in the Large-White pig could be useful for the study of etiopathogenic mechanisms by which the pulmonary infection produces a hemorrhagic gastroesophageal ulceration considered as stress ulcer.

Biochemical changes during the preservation stage of ripe olive processing.

The influences of initial sodium chloride (6% and 0% w/v in tap water) and acetic acid concentrations (0.3%, and 0.6% v/v), use of starter culture, and aerobic versus anaerobic conditions on the biochemical changes that take place throughout the preservation stage of ripe olive processing were investigated. Glucose, fructose and sucrose were completely consumed during preservation. Mannitol and malic acid were metabolized only in the presence of lactic acid bacteria or oxidative yeast (aerobic treatment). The main metabolites produced were lactic and acetic acid in aerobic or anaerobic treatments inoculated with Lactobacillus plantarum. Methanol and ethanol were present in all the brines although in a lower concentration when conditions were aerobic. Thus, induced lactic fermentation led to the most efficient utilization of carbohydrates and yielded the most suitable physicochemical characteristics for ripe olive preservation.