Leonardo Sáenz

The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts

Porcine circovirus type 2 (PCV2)-associated diseases are considered to be the biggest problem for the worldwide swine industry. The PCV2 capsid protein (Cap) is an important antigen for development of vaccines. At present, most anti-PCV2 vaccines are produced as injectable formulations. Although effective, these vaccines have certain drawbacks, including stress with concomitant immunosuppresion, and involve laborious and time-consuming procedures. In this study, Saccharomyces cerevisiae was used as a vehicle to deliver PCV2 antigen in a preliminary attempt to develop an oral vaccine, and its immunogenic potential in mice was tested after oral gavage-mediated delivery. The cap gene with a yeast-optimized codon usage sequence (opt-cap) was chemically synthesized and cloned into Escherichia coli/Saccharomyces cerevisiae shuttle vector, pYES2, under the control of the Gal1 promoter. Intracellular expression of the Cap protein was confirmed by Western blot analysis and its antigenic properties were compared with those of baculovirus/insect cell-produced Cap protein derived from the native PCV2 cap gene. It was further demonstrated by electron micrography that the yeast-derived PCV2 Cap protein self-assembles into virus-like particles (VLPs) that are morphologically and antigenically similar to insect cell-derived VLPs. Feeding raw yeast extract containing Cap protein to mice elicited both serum- and fecal-specific antibodies against the antigen. These results show that it is feasible to use S. cerevisiae as a safe and simple system to produce PCV2 virus-like particles, and that oral yeast-mediated antigen delivery is an alternative strategy to efficiently induce anti-PCV2 antibodies in a mouse model, which is worthy of further investigation in swine.

Effect of Leuprolide and Cetrorelix on Cell Growth, Apoptosis, and GnRH Receptor Expression in Primary Cell Cultures from Human Prostate Carcinoma

Contradictory data have been reported regarding the effect of GnRH agonists and antagonists on cell growth and survival, using prostate cancer-derived cell lines expressing either endogenous or exogenous GnRH receptors. We addressed the issue studying the effect of leuprolide (agonist) and cetrorelix (antagonist) on cell growth, apoptosis and GnRH receptor expression using a primary cell coculture system. Also, binding characteristics of prostate GnRH receptor in this culture system are described. Epithelial and stromal cells were obtained from prostate adenocarcinoma samples and cocultured in a bicameral system. Expression of GnRH receptors was evaluated by semiquantitative RT-PCR (transcript level) and Western blot (protein level). Cell growth was estimated by MTT method and apoptosis by DNA fragmentation using COMET assay. Saturation and competition binding studies were carried out using 125I-GnRH as radioligand. GnRH receptors from cell cultures of prostate cancer exhibited a single class of binding sites with a Kd of 1.11 ± 0.28 nM and a Bmax of 2.81 ± 0.37 pmol/mg of membrane protein for GnRH. Leuprolide and cetrorelix showed no effect on GnRH receptor expression. Both analogues showed a significant reduction in cell growth rate and an increase in DNA-fragmented cell number. These effects were dependent on the analogue concentrations (from 5–20 ng/mL). Considering that the culture system used in this work represents more closely the in vivo conditions of tumor cells than metastatic derived cell lines, we conclude that GnRH analogues have a significant inhibitory effect on cell viability of cells expressing GnRH receptors. In addition, GnRH receptors expressed in tumor prostatic cells seem not discriminate between agonist and antagonist, both analogues activating these receptors. Also, leuprolide and cetrorelix treatments did not influence GnRH receptor expression in our culture system. These differences with pituitary receptors may be explained by differences in affinity, transduction mechanism and molecular context in prostatic tissue.

Chitosan formulations improve the immunogenicity of a GnRH-I peptide-based vaccine.

Peptide vaccines using specific antigens with poor immunogenicity like GnRH-I are unable to develop an effective adaptive immune response and require the presence of adjuvants, essential to lymphocytic activation. Three chitosan formulations were evaluated for their ability as adjuvant of a poor immunogenic peptide vaccine against GnRH-I. Male Sprague-Dawley rats were immunized subcutaneously with recombinant His-GnRH-tandem-repeat peptide in high, low and phosphorylated high molecular weight chitosan solution at 0.5% (w/v). Freunds complete adjuvant was used as a positive control of immune response. Our results suggest that different chitosan formulations as adjuvant, with high or low viscosity degree allow inducing a high and persistent immune response against a poor immunogenic recombinant peptide. We found that the immune response was mediated by a increasing of IgG isotype 1, which were significantly greater than levels presented by the animals immunized with Freunds complete adjuvant. Nevertheless, chitosan with low molecular weight and highest acetylation degree was able to induce an immune response mediated by IgG isotype 2a. Additionally, high molecular weight phosphorylated chitosan, in which the phosphate groups were linked to N-acetyl-d-glucosamine unit, the immune response was reduced. All the immune responses obtained with chitosan as adjuvant were able to neutralize effectively the GnRH hormone proves by reducing of animal steroidogenesis and spermatogenesis demonstrating its capacity to improve immunogenicity in peptide vaccine.

Bovine (Bos taurus) humoral immune response against Echinococcus granulosus and hydatid cyst infertility

Echinococcus granulosus, the agent of hydatid disease, presents an indirect life cycle, with canines (mainly dogs) as definitive hosts, and herbivores and human as intermediary ones. In intermediary hosts fertile and infertile cysts develop, but only the first ones develop protoscoleces, the parasite form infective to definitive hosts. We report the presence of bovine IgGs in the germinal layer from infertile cysts (GLIC), in an order of magnitude greater than in the germinal layer from fertile cysts (GLFC). When extracted with salt solutions, bovine IgGs from GLIC are associated with low or with high affinity (most likely corresponding to non specific and antigen specific antibodies, respectively). Specific IgGs penetrate both the cells of the germinal layer and HeLa cultured cells and recognize parasitic proteins. These results, taken together with previous ones from our laboratory, showing induction of apoptosis in the germinal layer of infertile hydatid cysts, provide the first coherent explanation of the infertility process. They also offer the possibility of identifying the parasite antigens recognized, as possible targets for immune modulation. J. Cell. Biochem. 112: 189–199, 2011.

Inhibition of Egr-1 by siRNA in prostate carcinoma cell lines is associated with decreased expression of AP-1 and NF-κB.

Previous studies suggest that the effects of Egr-1 on tumorigenesis are critically dependent on the levels of Egr-1 and the cellular context. For this reason, we examined the effects of blocking the Egr-1 activity by a short interfering RNA (siRNA) against Egr-1 on the expression of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) signaling in the PC-3 and LNCaP prostate carcinoma cell lines. We observed that the reduction in expression of Egr-1 in PC-3 and LNCaP cells by effects of the siRNA vector resulted in lower cell viability and increased apoptosis at 24 and 120 h after transfection. This reduced cell viability correlated well with a reduced activity of the NF-κB and AP-1 factors. We analyzed the expression and activity of these factors and found that p65 and IκB but not p50 were reduced in the siRNA-treated cells. Similarly, we found that c-Fos but not c-Jun was reduced in the siRNA treated cells. These effects were translated to reduced transcriptional activity of NF-κB over cellular inhibitor of apoptosis (cIAP) and p21 genes, as assayed by RT-PCR and of AP-1 over a luciferase reporter activated by AP-1 response elements. Egr-1 was also found to interact directly with p65 and IκB members of the NF-κB family, thereby was able to regulate directly their activity by post-transcriptional effects. These results suggest that Egr-1 may promote prostate cancer development by modulating the activity of factors NF-κB and AP-1, which are involved in cell proliferation and apoptosis.

Role of the phosphatase PP4 in the activation of JNK-1 in prostate carcinoma cell lines PC-3 and LNCaP resulting in increased AP-1 and EGR-1 activity

The specific signaling connections between the mitogen-activated protein kinases (MAPK) such as c-Jun N-terminal kinase (JNK-1) and phosphatases PP4 and M3/6, affecting the family of early nuclear factors, is complex and remains poorly understood. JNK-1 regulates cellular differentiation, apoptosis and stress responsiveness by up-regulating early nuclear factors such as c-Jun, a member of the activating protein (AP-1) family, and the Early Growth Factor (EGR-1). C-Jun, when phosphorylated by c-Jun N-terminal kinase (JNK-1) associates with c-Fos to form the AP-1 transcription factor that activates gene expression. We have investigated the regulation of the JNK-1 kinase by co-transfecting phosphatases PP4 and M3/6 in prostate cancer cell lines PC-3 and LNCaP, which have been previously stimulated with human EGF or cisplatin. Co-transfections of plasmids expressing the JNK-1 and the serine/threonine phosphatases PP4 resulted in a significant increase in JNK-1 activity in both PC3 and LNCaP cells. In contrast, co-transfection of JNK-1 with the dual specific phosphatase serine/threonine M3/6 showed only a marginal effect in JNK-1 activity. The phosphatase M3/6 also failed in blocking the induction of JNK-1 activity observed in presence of PP4. The higher activity of JNK-1 was associated with increased activities of the factors c-Jun/AP-1 and EGR-1. This suggests that JNK-1 activity in PC-3 and LNCaP cells requires not only active PP4 for stable maintenance but also suggests that the relative degree of phosphorylation of multiple cellular components is the determinant of JNK-1 stability.

Evaluation of glucose as a cryoprotectant for boar semen

Fertility parameters of boar spermatozoa were evaluated in vitro, after freeze-thawing the semen in three different extenders containing permeable and non-permeable cryoprotectants: A (111-OmM Tris, 31.4mM citric acid, 185.0mM glucose, 20 per cent egg yolk, 3 per cent glycerol and 100 iu/ml penicillin G); B (200mM Tris; 70.8mM citric acid, 55.5mM glucose, 20 per cent egg yolk, three per cent glycerol and 100 iu/ml penicillin G); C (200mM Tris, 70.8mM citric acid, 55.5mM fructose, 20 per cent egg yolk, 3 per cent glycerol and 100 iu/ml penicillin G). The freeze-thawing techniques were the same for each extender. Eight ejaculates from four boars were obtained; the sperm-rich fraction of each ejaculate was extended in each of the three media at a final concentration of 400 x 106 sperm/ml, loaded into 0.5 ml straws and frozen at a rate of 30°C/minute to - 196°C. The straws were thawed at 60°C for eight seconds. Sperm motility, acrosomal integrity and in vitro sperm penetration through the zona pellucida of gilt oocytes matured in vitro were evaluated. The motility of unfrozen spermatozoa was 93.1 per cent compared with 60.7 per cent, 48.2 per cent and 35 per cent for sperm frozen in extenders A, B and C respectively; these values were all significantly different (P<0.05). There was no significant decline in sperm motility after incubation for 30 minutes in extender A, but there were significant decreases in sperm motility after 30 minutes of incubation in B and C. The percentage acrosomal integrities were 97.2 per cent for the control and 45.5 per cent, 30.3 per cent and 16.8 per cent for the frozen-thawed spermatozoa in extenders A, B and C respectively. The results of the in vitro penetration assay were 80.7 per cent when using control spermatozoa, and 42.2 per cent, 18.4 per cent and 3.3 per cent when using frozen-thawed spermatozoa in extenders A, B and C respectively

Bovine IgG subclasses and fertility of Echinococcus granulosus hydatid cysts

Hydatidosis is an important zoonotic disease of worldwide distribution, causing important health problems to humans and major economical losses in infected livestock. Echinococcus granulosus, the etiological agent of hydatid disease, induces a humoral immune response in the intermediate host (human and herbivorous) against hydatid cyst antigens. Specifically, IgGs are found in the laminar and germinal layers and inside the lumen of fertile and infertile hydatid cysts. In the germinal layer of infertile cysts IgGs are found in an order of magnitude greater than in the germinal layer of fertile cysts; a fraction of those IgGs are associated with high affinity to germinal layer proteins, suggesting their binding to specific parasite antigens. We have previously shown that those immunoglobulins, bound with high affinity to the germinal layer of hydatid cysts, induce apoptosis leading to cyst infertility. In the present work the presence of IgG1 and IgG2 subclasses in the germinal layer of both fertile and infertile hydatid cysts is reported. IgG1 is the most relevant immunoglobulin subclass present in the germinal layer of infertile cysts and bound with high affinity to that parasite structure. Contrarily, though the IgG2 subclass was also found in the germinal and adventitial layers, those immunoglobulins show low affinity to parasite antigens. We propose that the binding of an IgG1 subclass to parasite antigens present in the germinal layer is involved in the mechanism of cyst infertility.

High prevalence of pathogenic Leptospira in alien American mink (Neovison vison) in Patagonia

BackgroundLeptospirosis is an important zoonosis with worldwide distribution caused by pathogenic bacteria of the genus Leptospira. The North American mink (Neovison vison) has an important role in the environmental contamination with Leptospira, as minks live in aquatic environments and are the predators of rodents.FindingsBlood and kidney samples were obtained from 57 minks in Southern Chile 39° S to 45° S. Pathogenic species of Leptospira were detected by PCR on 31/57 minks. To determine the species, we sequenced the 16S ribosomal RNA (rRNA) gene on nine of the positive samples. We predicted two pathogenic species: Leptospira interrogans (five samples) and Leptospira borgpetersenii (four samples).ConclusionsThis study showed that the American mink presents pathogenic species of Leptospira and confirm important environmental contamination of Patagonian rivers and lakes with pathogenic Leptospira.

The immune profile induced is crucial to determine the effects of immunocastration over gonadal function, fertility, and GnRH-I expression

Immunocastration or vaccination against the GnRH‐I hormone is a promising alternative to reproductive control in different animal species. Given the low immunogenicity of this hormone, the use of adjuvants becomes necessary.