Leptin and its Receptor Expression in Swine Pituitaries Cultured under Deprived Conditions

Veterinary Research Communications -     

Peripheral T lymphocyte changes in neonatal piglets: Relationship with growth hormone (GH), prolactin (PRL) and cortisol changes

Taking into account the role played by the neuroendocrine network in affecting the early development of the immune response, the present study aims to assess neonatal immunity in piglets by testing peripheral lymphocyte age-related changes in relationship to plasma levels of some relevant immunoregulatory hormones, such as growth hormone (GH), prolactin (PRL) and cortisol. For this purpose, we studied the peripheral lymphocyte age-related changes in relationship to plasma levels of GH, PRL and cortisol in conventional piglets from birth (day 0) to 41 days of age. A significant decrease was observed in the total number of lymphocytes at day 0, with a subsequent constant increment up to 41 days of age. Concomitantly, the number of T cell subsets (mainly CD8(+) cells and double positive CD4(+)CD8(+)) was low at birth, with strong increments between the 19th and 41st days of life. The CD4(+) T cell number subset was less diminished at birth than that of CD8(+), albeit with significant increments in the post-weaning period. Of interest, gammadelta T cells, which are more involved in innate immune efficiency, displayed the same trend as CD8(+) T cells from birth to the 41st day of life. From day 0 up to the 19th day, significant inverse correlations were found between T cell subsets and GH or PRL or cortisol, albeit with more significant inverse correlations with cortisol. The high levels of GH and PRL in the pre-weaning period may be due to the fact that they have to counteract the cortisol-mediated negative effect on lymphocyte production and development. These findings suggest that stress condition occurs at birth with decreases in the immune parameters, in the same way as in human newborns, with a subsequent gradual normalisation and immune development, as shown by decreased cortisol, GH and PRL normalisation and concomitant increments in T cell subsets.     

Infection, immunity and the neuroendocrine response

The Central Nervous (CNS) and Immune Systems (IS) are the two major adaptive systems which respond rapidly to numerous challenges that are able to compromise health. The defensive response strictly linking innate to acquired immunity, works continuously to limit pathogen invasion and damage. The efficiency of the innate response is crucial for survival and for an optimum priming of acquired immunity. During infection, the immune response is modulated by an integrated neuro-immune network which potentiates innate immunity, controls potential harmful effects and also addresses metabolic and nutritional modifications supporting immune function. In the last decade much knowledge has been gained on the molecular signals that orchestrate this integrated adaptive response, with focus on the systemic mediators which have a crucial role in driving and controlling an efficient protective response. These mediators are also able to signal alterations and control pathway dysfunctions which may be involved in the persistence and/or overexpression of inflammation that may lead to tissue damage and to a negative metabolic impact, causing retarded growth.This review aims to describe some important signalling pathways which drive bidirectional communication between the Immune and Nervous Systems during infection. Particular emphasis is placed on pro-inflammatory cytokines, immunomodulator hormones such as Glucocorticoids (GCs), Growth hormone (GH), Insulin-like Growth Factor-1 (IGF-1), and Leptin, as well as nutritional factors such as Zinc (Zn).Finally, the review includes up-to-date information on this neuroimmune cross-talk in domestic animals. Data in domestic animal species are still limited, but there are several exciting areas of research, like the potential interaction pathways between mediators (i.e. cytokine-HPA regulation, IL-6-GCS-Zn, cytokines-GH/IGF-1, IL-6-GH-Leptin and thymus activity) that are or could be promising topics of future research in veterinary medicine.     

Lymphocyte activation as cytokine gene expression and secretion is related to the porcine reproductive and respiratory syndrome virus (PRRSV) isolate after in vitro homologous and heterologous recall of peripheral blood mononuclear cells (PBMC) from pigs vaccinated and exposed to natural infection

The present study evaluated the lymphocyte activation in PRRSV-vaccinated pigs subsequently exposed to natural infection by in vitro stimulation of peripheral blood mononuclear cells (PBMC) with homologous vaccine and two heterologous PRRSV isolates. The responsiveness was assessed by determining IFN-γ secreting cells by ELISpot assay, lymphocyte CD8 phenotype by intracellular staining/flow cytometry, cytokine gene expression by real-time quantitative PCR and cytokine secretion by ELISA. Conventional pigs were weaned at 28 days of age and inoculated intramuscularly (IM) or needle-less intradermally (ID) with a modified-live PRRSV vaccine suspended in adjuvant, while control pigs were injected with adjuvant alone (ADJ). Blood samples were collected at vaccination, 35 days post-vaccination and after 35 days post-exposure to natural infection by a heterologous field strain. Thirty-five days post-vaccination, PRRSV vaccine induced a low but significant virus-specific IFN-γ secreting cell response upon stimulation with both the vaccine strain and the two isolates in vaccinated pigs. Conversely, after 35 days post-exposure, only the vaccine strain and the BS/114/S isolate triggered this response. Intracellular staining showed that PRRSV-specific immune cells reacting upon vaccine strain and BS/114/S stimulation were mostly CD8⁺ IFN-γ producing cells whereas the stimulation with BS/55 isolate induced an IFN-γ production associated to the CD8^?IFN-γ⁺ phenotype. At 35 days post-vaccination, PBMC from vaccinated pigs showed lower IL-10 expression and release, and higher TNF-α gene expression upon stimulation with both the vaccine and viral isolates. After infection, both cytokines were not differently modulated in different groups. Immune parameters give evidence that IFN-γ secreting cells in the peripheral blood can be elicited upon PRRSV infection although vaccination itself does not stimulate high levels of these reactive cells. Moreover, the cross-reactivity against divergent PRRS viruses can show a different intensity and be differently associated with cytotoxic CD8⁺IFN-γ⁺ as well as CD8^?IFN-γ⁺ cells. Overall, the obtained data confirmed that the immune activation against PRRSV is not dependent on the genetic divergence of the virus. Especially after infection, a different immune reactivity was evident upon stimulation with the different isolates in terms of frequency and CD8 phenotype of PRRSV-specific IFN-γ producing cells. The modulation of cytokines in vaccinated pigs appeared to be more dependent on vaccination or infection conditions than on stimulation by different isolates, and the changes of IL-10 more relevant than those of TNF-α at gene and protein levels. Moreover, under the conditions of this study, the PRRSV vaccine administered via the intradermal route by a needle-less device was confirmed to induce an immune response comparable or in some cases higher than the intramuscular route.     

Systemic and local immune response in pigs intradermally and intramuscularly injected with inactivated Mycoplasma hyopneumoniae vaccines

The systemic and respiratory local immune response induced by the intradermal administration of a commercial inactivated Mycoplasma hyopneumoniae whole-cell vaccine (Porcilis^® MHYO ID ONCE – MSD AH) in comparison with two commercial vaccines administered via the intramuscular route and a negative control (adjuvant only) was investigated. Forty conventional M. hyopneumoniae-free pigs were randomly assigned to four groups (ten animals each): Group A = intradermal administration of the test vaccine by using the needle-less IDAL^® vaccinator at a dose of 0.2 ml; Group B = intramuscular administration of a commercially available vaccine (vaccine B); Group C = intramuscular administration of the adjuvant only (2 ml of X-solve adjuvant); Group D = intramuscular administration of a commercially available vaccine (vaccine D). Pigs were vaccinated at 28 days of age. Blood and bronchoalveolar lavage (BAL) fluid samples were collected at vaccination (blood only), 4 and 8 weeks post-vaccination. Serum and BAL fluid were tested for the presence of antibodies by ELISA test. Peripheral blood monomorphonuclear cells (PBMC) were isolated to quantify the number of IFN-γ secreting cells by ELISpot. Moreover, cytokine gene expression from the BAL fluid was performed. Total antibodies against M. hyopneumoniae and specific IgG were detected in serum of intradermally and intramuscularly (vaccine B only) vaccinated pigs at 4 and 8 weeks post-vaccination. M. hyopneumoniae specific IgA were detected in BAL fluid from vaccinated animals (Groups A and B) but not from controls and animals vaccinated with the bacterin D (p < 0.05). Significantly higher gene expression of IL-10 was observed in the BAL fluid at week 8 post-vaccination in the intradermally vaccinated pigs (p < 0.05). The results support that the intradermal administration of an adjuvanted bacterin induces both systemic and mucosal immune responses. Moreover, the intramuscularly administered commercial vaccines each had a different ability to stimulate the immune response both systemically and locally.     

Leptin in sow: Influence on the resumption of cycle activity after weaning and on the piglet gain

In sows, a strong relationship exists between body condition and reproductive efficiency and milk yield. Leptin may act as a metabolic gate which permits the activation of reproductive axis: in the sow, serum concentration of leptin was positively correlated with adiposity at farrowing. An interesting aspect useful to clarify the biology of leptin, was the discovery that the placenta expresses the ob gene, the ob receptor gene and it is a site of leptin production, suggesting a possible role of the hormone in fetal growth; after birth, the placenta functions were taken over from milk, especially to the delivery of maternal hormones and growth factors to the neonate. The exact role of maternal leptin in the physiology of neonatal piglets remains to be determined. Our aim was to evaluate if maternal leptin levels at the beginning of lactation and at weaning could predict the resumption of cycle activity and/or the piglet gain. Thirty-eight Large White × Landrace pregnant sows (16 nulliparous and 22 pluriparous) were used. Blood samples were taken from sows and piglets at d 5 and d 21 after farrowing; in the same days, milk samples were taken after oxytocin injection by means of complete manual milking of all mammary glands of one side. On the basis of the blood leptin at d 5, sows were divided into 3 groups (Low: < 2.3 ng/ml; Medium: 2.3 to 2.6 ng/ml; High: > 2.6 ng/ml). Our results show a correlation at d 5 between backfat thickness and blood leptin (r = 0.342; P < 0.05). The resumption of the cyclic activity was faster in sows with a leptin level at d 5 greater than 2.3 ng/ml (P < 0.01). Milk composition at d 5 and 21 was not affected by parity and leptin. Piglet ADG was significantly (P < 0.05) influenced by sow leptin groups (0.180 kg day^− 1 for piglets from Low group and 0.224 for High group). Piglets weaned by High group sows have shown a greater blood leptin content at weaning (P < 0.01) than other groups. In conclusion we have found a significant correlation between leptin and productive and reproductive performances of pigs. This paper underlines the pleiotropic actions exerted by leptin in the productive sow.     

Factors Modulating Apoptosis: an in-vitro Study in Swine Granulosa Cells

The aim of this study was to investigate the effects of some endocrine and intra‐ovarian factors on the activation/inhibition of apoptosis in swine granulosa cells. Upon incubation in a 10% FCS‐supplemented M199, granulosa cells from small (< 3 mm) follicles programmed their death after 24–48 h of culture; in the absence of FCS, apoptosis was reduced after 24 h of culture. Cells cultured in the presence of FCS were treated with db‐cAMP, LH, FSH, Insulin‐like Growth Factor‐I IGF‐I or PMSG to verify the role of these substances in apoptotic death: all these molecules inhibited apoptosis after 48 h of incubation. A further aim of the study was to investigate the possible involvement of nitric oxide (NO), an intra‐ovarian modulator, in the regulation of granulosa cell apoptosis and its possible role in the modulation of steroidogenesis. After a 48 h incubation with a substrate of NO synthesis ( l ‐arginine, 0.1 and 1 m m ), a NO donor [S‐nitroso‐N‐acetyl‐penicillamine (SNAP) , 0.2 and 1 m m ] or a NO synthase inhibitor [N^ω‐nitro‐ l ‐arginine‐methyl‐ester (NAME, 1 and 5 m m )], the onset of apoptotic death was evaluated: l . arginine and NAME did not induce any significant variation of apoptosis, whereas 1 m m SNAP exerted a protective action. A significant stimulatory effect of l ‐arginine on NO production, associated with a suppressive action on estradiol 17β concentrations was observed; NAME exerted an inhibitory effect on NO production, associated with an increase in estradiol secretion; estradiol 17β production was markedly inhibited by SNAP. In summary, the depletion of FCS could induce a cell cycle arrest in G₀ whereas apoptosis could be the consequence of cell cycle progression mediated by FCS; gonadotropins and IGF‐I could also act as survival factors. NO appeared to represent a ‘trophic’ signal for the follicle, whose involvement in the regulation of ovarian function is substantiated by its modulatory action on steroidogenesis.     

Effect of Weaning and Vaccinations on Immune and Hormonal Parameters in Neonatal Piglets

Veterinary Research Communications -