L. Starvaggi Cucuzza

Gene expression profiling of thymus in beef cattle treated with prednisolone

Glucocorticoids (GCs) are extensively used in livestock production, not only for their anti-inflammatory properties but also to improve the quality and quantity of meat in veal and beef production. In Italy, an increase in GC-positive cases has been observed in cattle since 2008, particularly prednisolone (PDN). Recent studies clearly demonstrate that both histopathological analysis and high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) were unable to detect PDN treatments. The aim of this study was to identify transcriptomic signatures of PDN administration in the thymus of experimentally treated animals by comparison with untreated controls, in order to identify gene expression changes or pathways alteration induced by the corticosteroid treatment. Microarray data analysis showed substantial modifications in thymus gene expression profiles after PDN treatment. Several of the 388 differentially expressed genes encoded pro-inflammatory and anti-inflammatory mediators or immune regulators which showed that PDN might have a role in the regulation of immunologic homeostasis, act on both innate and acquired components of the immunity and mainly induce the activation of immune tolerance and anti-inflammatory pathways. Thus, this study allowed to deepen the effects of PDN on the immune system and showed the potentiality of gene expression profiling by DNA-microarray as a powerful tool to complement the existing methods against the illegal use of growth promoting hormones, especially when working on samples collected after slaughtering.

Epidermal growth factor and hepatocyte growth factor receptors collaborate to induce multiple biological responses in bovine mammary epithelial cells

The aim of this work was to explore whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) could increase the biological responses of a mammary epithelial cell line of bovine origin when added simultaneously. We also investigated a possible molecular mechanism underlying this cooperation. The development of mammary gland requires several circulating and locally produced hormones. Hepatocyte growth factor and its tyrosine kinase receptor, mesenchymal-epithelial transition factor (MET), are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor and its ligands have also been implicated in the growth and morphogenesis of the mammary epithelium. Both EGF and HGF seem to exert a morphogenic program in this tissue; therefore, we hypothesized that these cytokines could act cooperatively in bovine mammary epithelial cells. We have already shown that the bovine BME-UV cell line, a nontumorigenic mammary epithelial line, expresses both MET and EGF receptor. Simultaneous treatment with HGF and EGF elicited an increase in proliferation, dispersion, degradation of extracellular matrix, and motility. Following EGF treatment, BME-UV mammary cells exhibited an increase in MET expression at both the mRNA and protein levels. Long-term treatment of BME-UV cells with HGF and EGF together increased the level of activation of the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathways when compared with HGF or EGF alone. These data outline a possible cooperative role of the EGF and HGF pathways and indicate that cross-talk between their respective receptors may modulate mammary gland development in the cow.

Oxytocin precursor gene expression in bovine skeletal muscle is regulated by 17β-oestradiol and dexamethasone.

Growth promoter administration, in livestock, potentially poses a major threat to public health, due to the potential endocrine and carcinogenic activity of residues, accumulating in edible tissues, such as skeletal muscle. Therefore, development of new screening tests and methods for the detection of illicit treatments of food animals would be useful. In this study the serum concentrations of oxytocin peptide were measured in beef cattle receiving 17β oestradiol, dexamethasone or placebo over a period of 40 days. Changes in gene expression of oxytocin precursor in skeletal muscle were also examined in these animals. Serum analysis using an oxytocin EIA kit indicated a significant up-regulation of the biosynthesis of this nonapeptide only in cattle after 17β oestradiol, but not after dexamethasone or placebo treatment. Quantitative PCR (qPCR) analysis showed a significant overexpression of the oxytocin precursor gene by 33.5 and 13.3-fold in cattle treated with 17β oestradiol and dexamethasone, respectively, in comparison to placebo treated animals. Regulation of gene expression by some myogenic regulatory factors in skeletal muscle was also evaluated in these animal groups, confirming the activity of both growth promoters on this gene. To investigate the use of the oxytocin precursor gene as biomarker for 17β oestradiol and dexamethasone treatment in beef cattle, an absolute quantification of this gene by qPCR was developed. A standard curve was generated and developed with TaqMan® technology and optimal criterion value, sensitivity and specificity of this screening method were established through ROC analysis. This analysis suggested that the up-regulation of oxytocin precursor gene expression in skeletal muscle tissue is a valid marker for detection of illicit 17β oestradiol and/or dexamethasone use in beef cattle. This method may serve as a novel diagnostic tool in the screening phase, and, if introduced in routine testing, may significantly improve overall efficacy and success of the food screening process ordered by state authorities.

Positive effect of silymarin on cell growth and differentiation in bovine and murine mammary cells.

Silymarin, a naturally acknowledged hepatoprotector used in humans to treat liver diseases has been tested in murine (HC11) and bovine (BME-UV) mammary epithelial cell lines to evaluate a possible direct effect on cell growth and differentiation in mammary gland. Silymarin enhanced cell proliferation (p < 0.05) from 10 to 1000 ng/ml in association with growth factors, (up to 20%) or alone (up to 15%) versus controls. Furthermore, silymarin (100 ng/ml) was able to increase (p < 0.05) beta-casein gene expression alone or in association with prolactin (5 microg/ml). These effects may be related with protein kinase B (AKT) activation induced by silymarin treatment (p < 0.05) and/or by a dose-related inhibitory effect (p < 0.05) on caspase-3 activity related to a protective role in cell apoptosis. These data suggest that silymarin should be considered a candidate to support mammary gland activity during a lactogenetic state.

Effect of Echinacea augustifolia extract on cell viability and differentiation in mammary epithelial cells

Echinacea spp. are popularly used as an herbal medicine or food supplement for enhancing the immune system and activating biological property in different tissues. In this study we show the biological effect of Echinacea augustifolia extract on cell viability and cell differentiation in mammary epithelial cell lines. These effects have been observed in two different cell line derived from mouse (HC11) and bovine (BME-UV). Echinacea extract enhanced cell liability from 100 to 1000 ng/ml in association with growth factors, epidermal growth factor (EGF) or insulin, but also without EGF (p<0.05) up to 37% vs. control. This effect may be modulated by MAPK and Akt activation that Echinacea extract treatment increased and/or by a reduction of caspase 3 activity, showed a dose-response decrease after Echinacea treatment. Finally Echinacea extract was able to increase (p<0.05) at 100 ng/ml beta-casein expression in association with PRL (5 microg/ml). These data demonstrate that Echinacea angustifolia extract can stimulate mammary epithelial cell physiology and may be considered a candidate to support mammary gland activity during a mammogenetic and lactogenetic state.

Haematological and physiological responses of Piemontese beef cattle to different housing conditions

Public concern for animal welfare has progressively grown over the recent years. In this context, stress has a great economical impact on growth of animals and quality of animal products. The development and validation of methods to assess animal stress, particularly at the farm level, are desirable to evaluate animal production systems. Piemontese breed is traditionally tie-stall housed in the fattening period. Hence, the objective of this study was to characterise a profile of physiological and haematological changes of Piemontese beef cattle under different management conditions (tie-stall and loose housing). Our results suggest that the housing system is an important factor in animal welfare. Indeed, the values of the total protein, lysozyme, cortisol, serum and faecal corticosterone concentration and GR-α gene expression indicate that the tie-stall housing is more stressful than the loose system. All the alterations highlighted in this study considered together may be effective biomarkers of stress and disease susceptibility.

Role of FKBP51 in the modulation of the expression of the corticosteroid receptors in bovine thymus following glucocorticoid administration

The aim of this work was to study the transcriptional effects of glucocorticoids on corticosteroid hormone receptors, prereceptors (11β-hydroxysteroid dehydrogenase 1 and 2, 11β-HSD1 and 2), and chaperones molecules regulating intracellular trafficking of the receptors (FKBP51 and FKBP52) in thymus of veal calves. Moreover, the expression of FKBP51 and FKBP52 gene were investigated in beef cattle thymus. In the cervical thymus of veal calves, dexamethasone administration in combination with estradiol decreased FKBP51 expression (P < 0.01). The same treatment increased mineralocorticoid receptor (MR) (P < 0.01) and 11β-HSD1 expression (P < 0.05) compared to control group in the cervical thymus of veal calves. The thoracic thymus of veal calves treated with dexamethasone and estradiol showed a decrease of FKBP51 (P < 0.05), FKBP52 (P < 0.05), glucocorticoid receptor (P < 0.05), and MR expression (P < 0.05) compared to control group in the thoracic thymus of veal calves. The gene expression of FKBP51 decreased both in cervical (P < 0.01) and thoracic thymus (P < 0.01) of beef cattle treated with dexamethasone and estradiol. In addition, also prednisolone administration reduced FKBP51 expression in the cervical thymus (P < 0.01) and in the thoracic thymus of beef cattle (P < 0.01). The gene expression of FKBP52 increased only in the cervical thymus following dexamethasone administration (P < 0.01). The decrease of FKBP51 gene expression in thymus could be a possible biomarker of illicit dexamethasone administration in bovine husbandry. Moreover, so far, an effective biomarker of prednisolone administration is not identified. In this context, the decrease of FKBP51 gene expression in thymus of beef cattle following prednisolone administration could play an important role in the indirect identification of animals illegally treated with prednisolone.