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In the current study, we investigated the effect of lead chloride (PbCl2) administration (50 and 100 ppm) on organ and body weight as well as its bioaccumulation during pregnancy and the postnatal period in mice. We showed that lead has no effect on the body weight of mice. However, spleen weight is affected by the two doses of PbCl2 while liver and kidney weights are altered only by the 100-ppm dose. Inductively coupled plasma atomic emission spectrometry (ICP-AES) analysis showed that lead accumulates in the blood, spleen, and thymus. Both doses of PbCl2 significantly reduced splenocyte and thymocyte cell counts after stimulation with lipopolysaccharide (LPS) and phytohemagglutinin A (PHA), respectively. On the other hand, we showed that the levels of Th1 cytokines (interleukin-2 (IL-2), interferon gamma (IFN-γ)), and tumor necrosis factor alpha (TNF-α) were reduced in the serum of mice treated with PbCl2 in a dose-dependent manner, as measured by ELISA. The levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) were very low in untreated mice and were also reduced by treatment with PbCl2. The levels of IL-2, IFN-γ, IL-4, IL-10, and TNF-α secretion differentially decreased in LPS-stimulated splenocytes in lead-treated mice. Using PHA-stimulated thymocytes, we observed a reduction in the levels of IL-2, IL-4, IL-10, and TNF-α in the PbCl2-treated groups. However, IFN-γ concentration in the supernatant of these cells was not decreased when mice were treated with 50 ppm of lead.

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Periostin is an extracellular matrix protein that actively contributes to tumor progression and metastasis. Here, we hypothesized that it could be a marker of bone metastasis formation. To address this question, we used two polyclonal antibodies directed against the whole molecule or its C-terminal domain to explore the expression of intact and truncated forms of periostin in the serum and tissues (lung, heart, bone) of wild-type and periostin-deficient mice. In normal bones, periostin was expressed in the periosteum and specific periostin proteolytic fragments were found in bones, but not in soft tissues. In animals bearing osteolytic lesions caused by 4T1 cells, C-terminal intact periostin (iPTN) expression disappeared at the invasive front of skeletal tumors where bone-resorbing osteoclasts were present. In vitro, we found that periostin was a substrate for osteoclast-derived cathepsin K, generating proteolytic fragments that were not recognized by anti-periostin antibodies directed against iPTN. In vivo, using an in-house sandwich immunoassay aimed at detecting iPTN only, we observed a noticeable reduction of serum periostin levels (- 26%; P < 0.002) in animals bearing osteolytic lesions caused by 4T1 cells. On the contrary, this decrease was not observed in women with breast cancer and bone metastases when periostin was measured with a human assay detecting total periostin. Collectively, these data showed that mouse periostin was degraded at the bone metastatic sites, potentially by cathepsin K, and that the specific measurement of iPTN in serum should assist in detecting bone metastasis formation in breast cancer.

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Analytical techniques are essential in the process of standardizing and validating vaccines. In this study we described a methodology to establish an ELISA sandwich for the quantification of a new vaccine against avian influenza virus H5N1 based on the main antigenic determinant of the virus, the extracellular domain of the glycoprotein hemagglutinin (HA), fused to the extracellular domain of the chicken CD154 glycoprotein (HACD). The chimerical proteins HA and HACD were produced in SiHa cells and the experiments were performed by using three monoclonal antibodies (MAb-HA1, MAb-HA2 and MAb-HA3), alone or conjugated to horseradish peroxidase (HRP-HA1, HRP-HA2 and HRP-HA3). The hemagglutination inhibition assay was carried out with a negative and a positive H5N2 reference serum, together with the antigen H5N1 A/Mallard/Italy/3401/05, all purchased from the "Istituto Zooprofilattico delle Venezie", Italy. After demonstrating the similar recognition pattern between the HA and the HACD proteins, the MAb-HA2 at a concentration of 2,5 µg/mL was selected as the capture antibody and the HRP-HA3 at a dilution of 1/20000 was selected as the detection antibody due to their optimal values of optical density at these conditions. The best dynamic range of the standard curve using the protein HACD was achieved at concentrations from 100 to 1,56 ng/mL. There were no significant differences when five batches of HACD were quantified by the ELISA sandwich and the bicinchoninic acid method linked to densitometry. In conclusion, the final parameters for the quantification of the chimeric protein HACD using an ELISA sandwich were described, which could contribute to develop a large-scale process for the final vaccine production.

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La fasciolosis es una parasitosis cosmopolita de importancia médico-veterinaria ocasionada por Fasciola hepatica, que afecta al ganado ovino, caprino y vacuno; y accidentalmente al hombre ocasionando una infección endemo-epidémica de difícil diagnóstico. El objetivo fue desarrollar un ELISA sándwich indirecto, empleando 3 anticuerpos, para identificar antígenos de secreción-excreción de Fasciola hepatica (ESFh). Para el ELISA se emplearon anticuerpos policlonales de ratón anti ESFh como anticuerpos de captura, anticuerpos policlonales de conejo anti ESFh como anticuerpos de detección, a las concentraciones de 10 y 5 μg/mL, respectivamente. Como conjugado se emplearon anticuerpos monoclonales de ratón anti-inmunoglobulinas totales de conejo ligado a peroxidasa 1/1000). Se analizaron 31 muestras de heces de ganado ovino y los resultados se compararon con los obtenidos por el examen coproparasitológico directo (CD) y contrainmunoelectroforesis (CIEF). El límite de detección obtenido para ELISA sándwich indirecto fue 100 ng/mL. La prueba presentó una sensibilidad de 100%, especificidad de 96.6% y valores predictivos positivos y negativos de 50% y 96.6% respectivamente; con relación al examen CD. Al comparar ELISA tipo sándwich indirecto con CIEF se obtuvo una especificidad de 93.5% y un valor predictivo negativo del 100%. Se concluye que la prueba de ELISA sándwich indirecto diseñada es capaz de detectar antígenos metabólicos en muestras de heces de ovino y se puede utilizar para el diagnóstico Fasciola hepatica.

Fasciolosis is a cosmopolitan parasitosis medical-veterinary importance caused by Fasciola hepatica, which affects sheep, goats and cattle; and it affects man accidentally causing an epidemic-endemic infection difficult to diagnose. The aim was to develop an indirect sandwich ELISA with 3 antibodies for detecting excretory-secretory antigens of Fasciola hepatica (ESFh). For the development of indirect sandwich ELISA were used, as capture antibody, mouse polyclonal antibodies anti ESFh and polyclonal antibodies rabbit anti-ESFh as detection antibody, at the concentrations of 10 and 5 μg/mL respectively. The conjugate used was mouse monoclonal anti- total immunoglobulins rabbit linked to peroxidase (1/1000). Were analized 31 sheep fecal samples, and the results were compared with those obtained by direct coproparasitological examination (DC) and counterimmunoelectrophoresis (CIEP). The detection limit obtained for indirect sandwich ELISA was 100 ng/mL. The test had a 100% sensitivity, 96.6% specificity, positive and negative predictive values of 50% and 96.6% respectively, in relation to DC test. Comparing with CIEP the specificity obtained for indirect sandwich ELISA was 93.5% and a negative predictive value of 100%. We concluded that indirect sandwich ELISA designed is able to detect metabolic antigens in ovine feces samples and can be used for Fasciola hepatica diagnosis.