Immunogenicity, safety and dual DIVA-like character of a recombinant candidate vaccine against neosporosis in cattle.

Neosporosis is the major infectious cause of abortion and reproductive losses in cattle worldwide; however, there are no available vaccines or drugs to control this disease. Recently, a dual (positive and negative) DIVA-like (Differentiation of Infected from Vaccinated Animals) vaccine was evaluated in a pregnant mouse model of neosporosis, showing promising immunogenic and protective results. The current report aimed to study the safety, the dose-dependent immunogenicity and the dual DIVA-like character of a recombinant subunit vaccine composed of the major surface antigen from Neospora caninum (rNcSAG1) and the carrier/adjuvant Heat shock protein 81.2 from Arabidopsis thaliana (rAtHsp81.2) in cattle. Healthy heifers were separated and assigned to experimental groups A-F and subcutaneously immunized with 2 doses of vaccine formulations 30 days apart as follows: A (n = 4): 50 µg rNcSAG1 + 150 µg rAtHsp81.2; B (n = 4): 200 µg rNcSAG1 + 600 µg rAtHsp81.2; C (n = 4): 500 µg rNcSAG1 + 1,500 µg rAtHsp81.2; D (n = 3): 150 µg rAtHsp81.2; E (n = 3):1,500 µg rAtHsp81.2, and F (n = 3) 2 ml of sterile PBS. The immunization of heifers with the different vaccine or adjuvant doses (groups A-E) was demonstrated to be safe and did not modify the mean value of the evaluated serum biomarkers of metabolic function (GOT/ASP, GPT/ALT, UREA, Glucose and total proteins). The kinetics and magnitude of the immune responses were dose-dependent. The higher dose of the vaccine formulation (group C) stimulated a broad and potent humoral and cellular immune response, characterized by an IgG1/IgG2 isotype profile and IFN-γ secretion. In addition, this was the first time that dual DIVA-like character of a vaccine against neosporosis was demonstrated, allowing us to differentiate vaccinated from infected heifers by two different DIVA compliant test approaches. These results encourage us to evaluate its protective efficacy in infected pregnant cattle in the future.

Regulation of the ovarian oxidative status by leptin during the ovulatory process in rats.

Leptin exerts both stimulatory and inhibitory effects on the ovulatory process. In this study, we investigated whether these opposite effects involve changes in the oxidative status in response to different levels of leptin. To this end, we performed both in vivo and in vitro assays using ovaries of immature rats primed with gonadotropins to induce ovulation. Superoxide dismutase (SOD) and catalase (CAT) activity, lipid peroxidation, glutathione (GSH) content, and reactive oxygen species (ROS) were studied as oxidative damage-related parameters. The expression of BCL2, BAX, and caspase 3 were measured by western blot as apoptosis-related biomarkers. The acute treatment with leptin, which inhibits ovulation, decreased SOD activity and increased active caspase 3 expression. No differences were found in CAT activity, lipid peroxidation, or total GSH. In contrast, the daily administration of leptin, which induces ovulation, decreased GSH content, ROS levels, and Bax and active caspase 3 expression, but caused no changes in other parameters. In addition, the daily administration of leptin induced follicular growth, measured by the number of antral follicles in ovarian sections. Using ovarian explant cultures, we found increased BCL2 expression and decreased SOD activity at low and high concentrations of leptin respectively. Thus, leptin can modulate the oxidative status of the ovarian tissue, during the ovulatory process, by acting on different targets according to its circulating levels. At low concentration, leptin seems to play a protective role against the oxidative stress, whereas at high concentrations, this protein seems to be involved in cell death.

Neuropeptide Y regulates the leptin receptors in rat hypothalamic and pituitary explant cultures.

The aim of this work was to investigate whether the expression of leptin receptors (OBR) in the hypothalamic-pituitary (HP) axis is regulated by the orexigenic neuropeptide Y (NPY) during ovulation. To this end, we performed in vitro assays, using cultures of both hypothalamic and anterior pituitary explants from immature rats primed with gonadotropins to induce ovulation. In hypothalamic explants, protein expression of both the long and short OBR isoforms was increased by the presence of NPY at 100-500 ng/ml and at 300-500 ng/ml, respectively. Similarly, in pituitary explants, protein expression of the long isoform was increased between 30 and 300 ng/ml while that of the short isoform was increased only at 300 ng/ml. When both tissues were incubated with NPY and BIBP3226, a specific antagonist of the NPY Y1 receptor subtype, the NPY-induced protein expression was totally reversed by the antagonist at almost every concentration assayed. However, this antagonist was not always capable of blocking the increase caused by the presence of NPY at transcript level. In conclusion, our results indicate that NPY is able to regulate the expression of both the long and the short isoforms of OBR in the HP axis, at least in part, through the NPY Y1 receptor. These results reinforce the fact that NPY and its NPY Y1 receptor play a critical role in reproduction by modulating leptin sensitivity.

Ovarian signalling pathways regulated by leptin during the ovulatory process.

Leptin, a protein secreted by different tissues, is able to exert both stimulatory and inhibitory effects on the ovulatory process. Thus, we investigated whether these opposite effects involve changes in the ovarian signalling pathways in response to different levels of leptin. To this end, we performed both in vivo and in vitro assays using immature rats primed with gonadotrophins to induce ovulation. The acute treatment with leptin, which inhibits the ovulatory process, caused a significant decrease in the phosphorylation of both STAT3 and ERK1/2 and a simultaneous increase in suppressors of cytokine signalling 3 (SOCS3) protein. However, daily administration of a low dose of leptin, which induces the ovulatory process, showed increased phosphorylation of both STAT3 and ERK1/2 and a decreased expression of SOCS3 protein. Using ovarian explant cultures, we also found that leptin was able to activate both STAT3 and ERK1/2 at 10 ng/ml but only STAT3 at 300-500 ng/ml. In addition, at 100-300 ng/ml, leptin increased protein but not mRNA expression of SOCS3. The addition of specific inhibitors of JAK/STAT and MAPK signalling pathways suppressed both the increase and the decrease in leptin-induced progesterone secretion. These results indicate that i) different levels of leptin are able to regulate STAT3, ERK1/2 and SOCS3 at both intra- and extra-ovarian level and that ii) the dual action of leptin on steroidogenesis seems to occur, at least in part, through both the ERK and STAT cascades.

Different levels of leptin regulate different target enzymes involved in progesterone synthesis.

OBJECTIVE: To study the effects of different doses of leptin on the expression of proteins involved in P synthesis, such as steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ßHSD). DESIGN: Experimental studies. SETTING: Research laboratory. ANIMAL(S): Immature rats primed with gonadotropins to induce ovulation. INTERVENTION(S): In vivo studies: rats received either an acute or daily treatment with leptin. In vitro studies: ovarian explants were cultured in the absence or presence of leptin (0.3-500 ng/mL). MAIN OUTCOME MEASURE(S): The expression of both messenger RNA and protein of StAR, P450scc, and 3ßHSD were measured by reverse transcription-polymerase chain reaction (PCR) and Western blot, respectively. RESULT(S): The acute treatment with leptin, which inhibits the ovulatory process, caused a significant reduction in the ovarian expression of P450scc without changes in StAR or 3ßHSD. In contrast, the daily treatment, which induces the ovulatory process, showed an increased expression of the ovarian 3ßHSD protein, without differences in the other proteins measured. We also found that leptin increased the protein of both P450scc and 3ßHSD at physiological levels and inhibited both messenger RNA and protein of 3ßHSD at higher concentrations. CONCLUSION(S): The results indicate that 1) leptin is able to regulate the expression of the 3ßHSD protein in a dose-dependent manner; and 2) leptin seems to exert its dual effects on P synthesis on different targets in a dose-dependent manner.