Georgios Theodorou

Acute mastitis induces upregulation of expression of plasminogen activator-related genes by blood monocytes and neutrophils in dairy ewes.

The main objective of the present study was to examine whether genes implicated in the plasminogen activating cascade: urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR) and plasminogen activator inhibitors type 1 (PAI-1) and type 2 (PAI-2) are expressed in a differential manner in ovine blood monocytes and neutrophils obtained from healthy and mastitic dairy ewes. A total of 48 blood samples were collected from 8 healthy and 8 mastitic dairy ewes over a period of 3 weeks. Streptococcus agalactiae was detected in milk samples isolated from mastitic animals. Results indicated that expression of all four genes was very low in monocytes and neutrophils isolated from healthy animals. In contrast, there was a 2- to 5-fold increase (P<0.05) in expression of all four genes in monocytes and an 18- to 38-fold increase (P<0.01) in neutrophils isolated from mastitic animals. In conclusion, upregulation of expression of u-PA and u-PAR by monocytes and neutrophils is probably related to the rapidity of migration of these cells towards the mammary gland, while the upregulation of PAI-1 and PAI-2 is a rather enigmatic observation and it is probably related to the successful localization of the infection.

Effect of growth factors and lactogenic hormones on expression of plasminogen activator-related genes and cell proliferation in a bovine mammary epithelial cell line.

There is conflicting evidence in the literature as to whether up-regulation of urokinase plasminogen activator (u-PA) expression is related to bovine mammary epithelial cell growth. The role of u-PA receptor (u-PAR) and that of the plasminogen activator inhibitors type 1 and type 2 (PAI-1 and PAI-2) in bovine mammary epithelial cell proliferation is not known. The effect of growth factors and various hormones known to affect mammary function on expression of u-PA, u-PAR, PAI-1, PAI-2 and cell proliferation using the BME-UV1 bovine mammary epithelial cell line was examined. Cell proliferation was measured using the MTT assay and direct cell enumeration. Results showed that both IGF-1 and EGF increased cell proliferation but EGF was a more potent mitogen than IGF-1. Furthermore, IGF-1 increased by 2-fold expression of both u-PA and u-PAR while EGF increased by 3·8-fold the expression of only u-PAR. Both growth factors had no effect on expression of PAI-1 and PAI-2. In a manner consistent with changes in gene expression, EGF and to a lesser extent IGF-1 up-regulated total cell associated, membrane-bound and secreted u-PA activity. Thus, a strong correlation exists between u-PAR gene expression along with the activity of u-PA present on cell membranes and cell proliferation. Dexamethasone, prolactin and surprisingly insulin had no effect on cell proliferation. Dexamethasone alone and when combined with insulin or prolactin up-regulated gene expression of both PAI- and PAI-2 but not that of u-PA and u-PAR. Decreased total cell-associated, membrane-bound and secreted u-PA activity was detected in cells cultured in the presence of dexamethasone when combined with insulin or prolactin. However no such effect was observed in the presence of dexamethasone alone. Thus, dexamethasone acting synergistically with prolactin or insulin inhibits the activation of the plasmin-plasminogen system but this inhibition is not correlated with any changes in cell proliferation.

Effects of soyabean meal- or whey-based diets on lipid metabolism in weaned piglets.

The present study aimed to test the hypothesis that dietary protein source influences lipid metabolism-related parameters weaned piglets. The effects of soyabean meal (SB) and whey proteins (WP) on gene expression of several genes involved in the lipogenic process in liver, visceral (VAT) and subcutaneous (SAT) adipose tissues, plasma insulin concentration and fatty acid (FA) profile were investigated in 18 weaned piglets. Weaned piglets were fed one of two diets containing either SB or WP as the main protein source. Following a 10-h fasting period, plasma insulin concentration and FA profile were assessed at 56 and 72 days of age, whereas gene expression in liver, VAT and SAT was assessed at 72 days of age. Plasma insulin concentration was not affected by diet, although it was 40% lower in SB fed pigs. The SB pigs had lower 14:0 (p < 0.01) and higher 18:3n-3 (p < 0.001) levels in plasma in comparison with WP pigs. However, these changes were attributed to background differences in the dietary FA profile and not to a direct protein source effect. Gene expression of sterol regulatory element-binding protein 1 (SREBP-1) in liver and VAT were lower (p < 0.01 and p < 0.05, respectively) in SB compared to WP fed piglets, but no differences occurred in SAT. No changes were observed in sterol regulatory element-binding protein 2, liver X receptor, peroxisome proliferator-activated receptors α and γ and plasminogen activator inhibitor 1 mRNA levels, either in liver or in adipose tissues. In conclusion, dietary protein source, accompanied likely by side alterations in the dietary composition, affects lipid metabolism in pigs through the downregulation of SREBP-1, which is a crucial determinant of lipogenic process.

Dietary inclusion level effects of a phytogenic characterised by menthol and anethole on broiler growth performance, biochemical parameters including total antioxidant capacity and gene expression of immune-related biomarkers

The supplementation of a phytogenic feed additive (PFA) characterised by menthol and anethole was evaluated at three levels on broiler growth performance, nutrient digestibility, biochemical parameters, total antioxidant capacity of plasma, breast and thigh meat as well as on the relative gene expression of immune-related biomarkers. A total of 225 1-day-old male Cobb-500 were assigned into three treatments with five replicates of 15 chicks each. Wheat-soybean meal basal diets were formulated according to a three-phase (i.e. starter, grower and finisher) feeding program. Dietary treatments were: no PFA, PFA at 100 mg/kg diet and PFA at 150 mg/kg diet. Feed and water were available ad libitum. Performance parameters were monitored weekly and all other biological responses were determined at 42 days of broiler age. Increasing PFA level increased (P = 0.044) bodyweight gain at finisher period, decreased quadratically (P = 0.035) overall feed intake, and quadratically improved (P = 0.024) overall feed conversion ratio. Moreover, increasing PFA level increased plasma total antioxidant capacity linearly (P = 0.001) whereas linearly decreased (P = 0.005) triglyceride concentration. Thigh meat cholesterol decreased linearly (P = 0.016) with increasing PFA level. Expression of pro-inflammatory cytokine IL-2 in caecal tonsils increased quadratically (P = 0.046) with increasing PFA level. In conclusion, PFA inclusion at 100 mg/kg diet affected positively performance whereas a stronger improvement mainly in plasma total antioxidant capacity and triglyceride as well as in meat cholesterol was noted for the 150 mg/kg diet level. Inclusion of PFA resulted in increasing pro-inflammatory biomarker IL-2 at local caecal level.

Leucocyte expression of genes implicated in the plasminogen activation cascade is modulated by yoghurt peptides.

The urokinase-plasminogen activator (u-PA), its receptor (u-PAR) and the inhibitors of u-PA (PAI-1 and PAI-2) provide a multi-molecular system in leucocytes that exerts pleiotropic functions influencing the development of inflammatory and immune responses. The objective of the present study was to examine the ability of water soluble extracts (WSE) obtained from traditional Greek yoghurt made from bovine or ovine milk to modulate the expression of u-PA, u-PAR, PAI-1 and PAI-2 in ovine monocytes and neutrophils. WSE were obtained from 8 commercial traditional type Greek yoghurts made from ovine or bovine milk. WSE upregulated the expression of all 4 u-PA related genes in monocytes but the upregulation was much higher in the PAI-1 (10-fold) than in u-PA and u-PAR (3-4 fold) thus, shifting the system towards inhibition. In line with this observation, WSE reduced total and membrane-bound u-PA activity in monocytes. In neutrophils, WSE caused small (50-60%) but significant (P < 0·05) reductions in expression of u-PAR and PAI-2 but had no effect on expression of u-PA, PAI-1 and on total cell-associated and membrane-bound u-PA activity. WSE from yoghurts made from bovine or ovine milk were essentially equally effective in affecting the u-PA system except for the u-PAR gene in ovine neutrophils that was affected (reduced) by the ovine and not the bovine WSE. In conclusion, peptides present in WSE modulated the expression of u-PA related genes but the effect was much more prominent in monocytes than in neutrophils.

Effects of peptides derived from traditional Greek yoghurt on expression of pro- and anti-inflammatory genes by ovine monocytes and neutrophils

ABSTRACT The objective of the present study was to examine the effect of yoghurt peptides on expression of various pro- and anti-inflammatory genes by ovine monocytes and neutrophils. A total of eight commercial traditional-type Greek yoghurts made with ovine or bovine milk were used. Water-soluble extracts (WSEs) were obtained from all samples. Peptides present in WSE induced cyclooxygenase-2 gene expression and down-regulated inducible NO synthase, Interleukin-1 Receptor Antagonist and Ιnterleukin-12β gene expression in both cell types. Treatment of neutrophils with WSE resulted in a decrease in Interleukin-1β gene expression relative to control. There was a different response from monocytes and neutrophils regarding Transforming Growth Factor-β1 gene expression with an increase by monocytes and a decrease by neutrophils relative to control. Expression of Ιnterferon-γ and Ιnterleukin-10 remained unaffected. In conclusion, peptides derived from Greek yoghurt exhibited a mixed effect (pro- and anti-inflammatory) on two immunocompetent cell populations, although the anti-inflammatory effect was more pronounced.

Effects of dietary acidifier supplementation on broiler growth performance, digestive and immune function indices

The effect of dietary acidifier (AC) supplementation in combination or not with avilamycin (AV) was evaluated on broiler performance, nutrient digestibility, gizzard pH, activity of digestive enzymes and relative expression of cytokines. A 2 × 2 factorial experimental design with inclusion of AC (0 and 1 g/kg of diet) and AV (0 and 2.5 mg/kg of diet) as the main factors was implemented. Subsequently, 544-day-old male Cobb broilers were allocated in four treatments (i.e. C – no additions, AC; AV and ACAV), each having eight replicates of 17 broilers, for 6 weeks. Overall bodyweight gain (BWG) was improved (PAC = 0.035) by AC addition. BWG and feed conversion ratio were improved (P ≤ 0.05) by AV addition in all growth periods and overall. Dry matter, organic matter digestibility and energy retention were improved by AC (PAC = 0.050, PAC = 0.045 and PAC = 0.020, respectively), or AV (PAV = 0.018, PAV = 0.013 and PAV = 0.012, respectively). In addition AV supplementation improved digestibility of ether extracts (PAV = 0.022) and crude protein (PAV = 0.042). Pancreatic trypsin activity was lower in AC (PAC = 0.027) or AV (PAV ≤ 0.001) supplemented diets. An AC × AV effect on gizzard pH (PAC × AV = 0.034) was noted. Expression of interferon-γ (PAV = 0.010), interleukin (IL) 18 (PAV = 0.048) and inducible nitric oxide synthase 2 (PAV = 0.035) in spleen were downregulated by AV. An upregulation of IL10 (PAC × AV = 0.018), IL18 (PAC × AV = 0.002) and IL22 (PAC × AV = 0.030) in the caecal tonsils was shown for the combined ACAV supplementation. In conclusion, dietary AC supplementation improved performance, nutrient digestibility and energy retention, irrespective of AV inclusion. This study has provided evidence for potential links between digestive and immune function indices with growth performance improvements.

Expression of plasminogen activator-related genes in the adipose tissue of lactating dairy sheep in the early post-weaning period

There is growing evidence that plasminogen activator inhibitor type 1 (PAI-1) is expressed in adipose tissue and its expression is implicated in inflammation that accompanies obesity-associated diseases. The physiological role of other genes implicated in the plasminogen-activating cascade such as urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR) and plasminogen activator inhibitor type 2 (PAI-2) in ovine adipose tissue remains unknown. The objective of this study was to examine the changes in the expression of four plasminogen activator (PA)-related genes during the early post-weaning period in dairy ewes. A total of 21 subcutaneous adipose tissue samples were obtained from seven lactating dairy ewes of the Chios breed at weeks 1, 2 and 4 after weaning. Results indicated that expression of all PA-related genes was detected in most of the samples examined. Greatest expression of u-PAR corresponded to highest (week 1), while greatest expression of PAI-2 corresponded to lowest (week 4) rate of lipolysis, as indicated by the expression of hormone-sensitive lipase, in the ovine adipose tissue. There were no significant differences in the expression of the other two PA-related genes (u-PA, PAI-1) throughout the experimental period. Plasminogen activator-related genes are not expressed in a coordinated manner in the adipose tissue of lactating dairy sheep in the early post-weaning period. In conclusion, adipose tissue mobilization is correlated with highest expression of u-PAR and lowest expression of PAI-2.

Effect of dietary protein source on piglet meat quality characteristics

An experiment was conducted to examine the effects of different dietary protein sources (soybean meal vs whey protein) on piglet meat quality characteristics. Eighteen castrated male Large White × Duroc × Landrace piglets were randomly assigned to 2 groups. Piglets were kept in individual metabolic cages and fed ad libitum over a period of 38 days the following 2 diets: diet SB, which was formulated to meet the nutrient requirements of piglets using soybean meal as the main crude protein source and diet WP, where SB was totally replaced by a mixture of whey proteins on equal digestible energy and crude protein basis. At the end of the experiment, piglets were weighed and slaughtered. After overnight chilling, samples of Longissimus dorsi muscle were taken and were used for meat quality measurements. No significant differences were observed in the values of pH, color, water holding capacity, shear force and intramuscular fat content of L. dorsi muscle between the dietary treatments. Measurement of lipid oxidation values showed that dietary supplementation with different protein sources did not influence meat antioxidant properties during refrigerated storage. The SB piglets had lower C14:0 (P<0.01) and higher C18:3n-3 (P<0.001) levels in intramuscular fat in comparison with WP piglets. However, these changes were attributed to background differences in the dietary FA profile and not to a direct protein source effect. The results of this preliminary study indicate that the examined dietary protein sources do not have a significant effect on meat quality characteristics of piglets.

Allelic polymorphism of Ovar-DRB1 exon2 gene and parasite resistance in two dairy sheep breeds

The Ovar-DRB1 gene locus is one of the most polymorphic genes of the Major Histocompatibility Complex (Ovar-MHC) and holds a functional role to antigen presentation. The aim of this study was: a) to describe the Ovar-DRB1 locus variability in two dairy Greek sheep breeds and b) to investigate associations between this variability with resistance to gastrointestinal parasitosis. Blood and faecal samples were collected from 231 and 201 animals of Arta and Kalarrytiko breeds, respectively. The identification of alleles was performed using the sequence–base method. Faecal egg counting (FEC) of the gastrointestinal parasites and measures of blood plasma pepsinogen levels were performed in order to evaluate parasitological parameters. From this study in the overall examined animals, thirty-nine Ovar-DRB1 alleles were identified, among them, ten new alleles, reported for the first time in the literature. In Arta breed a total of twenty-four alleles were found. Among the detected alleles, ten were breed specific and five were new. Regarding the Kalarrytiko breed, twenty-nine alleles were found, fifteen of them were unique and nine were new. The studied breeds differed in their allelic profile, with only 12 common from the total of 134 different recorded genotypes. A higher number of animals with high parasitic load and high plasma pepsinogen values were found in Kalarrytiko. Associations between Ovar-DRB1 alleles with FEC values were found with certain heterozygous genotypes to present significantly reduced FEC values. The large number of detected alleles with low frequencies and the fact that the majority of animals were heterozygous, make hard to find strong associations