Short communication: Associations between blood fatty acids, ß-hydroxybutyrate, and α-tocopherol in the periparturient period in dairy cows: An observational study.

The objective of the present study was to examine the relationships between blood concentrations of fatty acids, ß-hydroxybutyrate (BHB), and α-tocopherol during the periparturient period in dairy cows. Blood samples were collected from 131 cows belonging to 4 different commercial dairy farms in southeastern Europe (Greece and Italy). We determined blood concentrations of fatty acids, BHB, and α-tocopherol at dry-off, at calving, and 30d postpartum. Results indicated that fatty acid concentrations were low at dry-off, reached maximum value at calving, and then declined at 30d postpartum. In fact, fatty acid concentrations at 30d postpartum were 50% lower than at calving. In contrast, BHB concentrations were low at dry-off, increased by 27% at calving, and continued to increase by another 20% at 30d postpartum. Overall, we found a weak correlation between fatty acids and BHB throughout the periparturient period. Concentrations of α-tocopherol were lowest at calving, and we detected no differences in α-tocopherol concentrations at dry-off or 30d postpartum. Negative correlations between fatty acids and α-tocopherol were highly significant at 30d postpartum and approached the level of significance at dry-off. However, both correlations became nonsignificant following the adjustment of α-tocopherol with cholesterol, indicating that the correlations were a reflection of changes in lipid transport. We found significant negative correlations (strong at dry-off and weak at 30d postpartum) between BHB and α-tocopherol after adjustment with cholesterol. The physiological basis for the negative correlations between BHB and α-tocopherol, especially that at dry-off, is not known and should not be taken to imply a cause-effect relationship. However, it opens the door to investigating the effects of vitamin E on liver function in dairy cows.

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.

Short communication: Oxidative status and incidence of mastitis relative to blood α-tocopherol concentrations in the postpartum period in dairy cows.

Vitamin E supplementation, when combined with high blood α-tocopherol (>6.25 µg/mL) at dry off, has been reported to unexpectedly increased the risk for clinical mastitis in dairy cows. Furthermore, higher levels of oxidative stress in the postpartum period were related to higher risk of mastitis. The objective of the present study was to determine the relationship between various serum biomarkers of oxidative status, incidence of mastitis, and blood α-tocopherol concentrations at dry off and at calving. A total of 146 dairy cows from a commercial farm were used in an observational field study. All cows were supplemented with 3,000 and 50 IU/cow per day of all-rac-α-tocopherol during the dry period and lactation, respectively. Blood samples were collected at dry off and at calving. Serum was analyzed for α-tocopherol, levels of reactive oxygen metabolites (ROM), thiol groups (SH), and ferric-reducing ability. Three α-tocopherol groups at calving were created: high (>3 µg/mL), medium (2-3 µg/mL), and low (<2 µg/mL). Three α-tocopherol groups at dry off were created: high (>6.25 µg/mL), medium (4.25-6.25 µg/mL), and low (<4.25 µg/mL). All cases of clinical mastitis that occurred during the dry period and the entire subsequent lactation were verified by a veterinarian. No differences were observed in the incidence of mastitis between the 3 α-tocopherol groups based on the serum levels at dry off. Incidence of mastitis was 4 times lower in the high and medium groups when compared with the corresponding value for the low-α-tocopherol group based on the serum levels at calving. Lower levels of ROM and SH at dry off and at calving were found in the group of cows with the highest α-tocopherol values at dry off when compared with the corresponding values in the low-α-tocopherol group. The ROM values at dry off but not at calving were lower in the group of cows with the highest α-tocopherol values at calving when compared with the corresponding values in the low-α-tocopherol group. No differences were observed in ferric-reducing ability values between the 3 α-tocopherol groups at dry off or calving. No differences were observed in all biomarkers of oxidative status between healthy cows and those with mastitis. Thus, blood α-tocopherol is inversely related to certain biomarkers of oxidative stress in the postpartum period and incidence of mastitis. However, reduction in the incidence of mastitis is not mediated through a reduction in the levels of various biomarkers of oxidative stress.

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.

Serum adiponectin acutely after an ischemic stroke: implications for a long-lasting, suppressed anti-inflammatory role.

OBJECTIVE: Past ischemic stroke (IS) patients display suppressed adiponectin (ADPN) levels a few months after disease onset. It is still unclear whether hypoadiponectinemia is already present by the early stages of stroke or occurs as a delayed effect of the acute ischemic reaction. In the present study we investigated ADPN levels acutely after an IS. MATERIALS AND METHODS: Serum ADPN was measured in 82 consecutive acute IS patients, and 30 stroke-free subjects of similar age and sex distributions. RESULTS: Patients had significantly lower ADPN levels than controls. Higher ADPN was significantly associated with reduced odds for IS accounting for age, sex and high-density lipoproteins. This association was strengthened after further adjustments for potential confounders. ADPN levels remained suppressed even 6 months after stroke. CONCLUSIONS: ADPN is significantly suppressed already by the early phases of stroke, and remains unchanged 6 months later. We propose a stable-over-time anti-inflammatory role of ADPN in IS, unrelated to the acute ischemic reaction.

Prognostic value of phagocytic activity of neutrophils and monocytes in sepsis. Correlation to CD64 and CD14 antigen expression.

The role of the phagocytic function of monocytes and neutrophils in sepsis has been poorly investigated. The present study evaluated the impact of the phagocytic activity of neutrophils and monocytes on the outcome of patients with severe sepsis. Thirty-one patients and 30 healthy individuals were enrolled in the study. The phagocytic activity of monocytes and neutrophils was evaluated during 24 h after admission and the results were correlated to the expression of CD64 on neutrophils and monocytes, CD14 antigen on monocytes, the Simplified Acute Physiology Score II and the patients' survival. A reduced phagocytic activity of neutrophils during the first 24 h after admission was a negative predictor for survival. Increased expression of CD64 antigen on polymorphonuclear cells (PMNs) and monocytes was favourably correlated to the patients' survival. In multivariate analysis the phagocytic activity of PMNs was the only independent predictor factor for survival. Patients with PMN phagocytic activity <37% had lower expression of CD64 on monocytes and PMNs and worse outcome, while those with phagocytic activity >37% had higher expression of CD64 on monocytes and PMNs and better outcome. Reduced phagocytic activity of neutrophils may represent a state of neutrophil inactivation similar to that previously described for monocytes during the compensatory anti-inflammatory response.

T helper 1 (Th1)/Th2 cytokine expression shift of peripheral blood CD4+ and CD8+ T cells in patients at the post-acute phase of stroke.

Local humoral and cellular immune responses modulate the inflammatory processes involved in the development of atherosclerotic lesions, as well as in the evolution of brain infarcts in stroke patients. The role of systemic adaptive immunity on the progression of such disease manifestations is less clear. In the current study, we evaluated the percentages of T helper 1 (Th1) [interleukin (IL)-2, interferon (IFN)-gamma] and Th2 (IL-4, IL-10) cytokine-producing peripheral blood CD4+ and CD8+ T cells in 23 patients with a history of ischaemic stroke (IS) at the chronic stable phase of the disease (median post-stroke time 34.5 months). Seven stroke-free individuals matched for age and vascular risk factors (matched controls, MC) were collected for comparison. To measure cytokine values at baseline and after stimulation, we used a flow cytometry method of intracellular cytokine staining. Intrinsic Th1 and Th2 cytokine production in unstimulated T cells was negligible in all study participants. Following mitogenic stimulation with phorbol 12-myristate13-acetate/ionomycin, both the IS and the MC groups exhibited a similarly strong Th1 response; IL-2 production predominated in the CD4+ T cells and IFN-gamma in the CD8+ T cells. However, when measuring the Th2 cytokine-production capacity post-stimulation, a significant increase in the percentage of IL-4-producing T cells was observed in the IS groups, compared with the MC group, resulting in a significantly lower ratio of IFN-gamma-/IL-4-producing T cells. No such Th2 enhancement could be confirmed for the case of IL-10. We propose that in IS patients there is a systemic shift of the immune system towards Th2 responses at the late post-acute phase of stroke.

Procleix Ultrio transcription-mediated amplification vs. serological blood screening in south-western Greece.

We present here our overall experience after 27 months of performance of the Procleix Ultrio [HIV-1, hepatitis C virus (HCV), hepatitis B virus (HBV)] transcription-mediated amplification (TMA) assay. The aim of this report is to assess the impact of nucleic acid testing (NAT) implementation in blood screening of south-western Greek blood donors. We processed 38,264 units of blood as neat samples with the Procleix Ultrio TMA assay (Chiron/GenProbe, Emeryville/San Diego, CA, USA) between 1 January 2005 and 31 March 2007. NAT results were compared to those obtained from routine serology tests and quantitative polymerase chain reaction (PCR) assays. Overall, 52 units of blood tested positive for HBV (1.4 per thousand), 8 for HCV (0.2 per thousand) and none for HIV or multiple infections. The yield of TMA was 0.183 per thousand for HBV (7/38 264) and 0 for HCV. The TMA HBV-positive donations were tested for HBV DNA by a quantitative PCR assay and were found negative (below the detection limit of the method, 200 copies/mL). Follow-up testing showed that the TMA HBV-positive donations were positive for anti-hepatitis B core antigen immunoglobulin G antibodies. Implementation of the TMA assay in the individual donation configuration increased HBV detection compared to serological screening or a commonly used quantitative PCR assay. Follow-up studies will determine the impact of NAT implementation in HBV transmission in countries with an intermediate HBV incidence.

Effect of non-operative management (NOM) of splenic rupture versus splenectomy on the distribution of peripheral blood lymphocyte populations and cytokine production by T cells.

Post-traumatic splenectomy is associated with increased postoperative morbidity and mortality and long-term impairment of humoral and cellular immunity. Alternatives to surgery have been developed to minimize or avoid the immediate and/or long-term complications of splenectomy. Herein we investigated the long-term effect of non-operative management (NOM) of the traumatic rupture of the spleen on the distribution of peripheral blood (PB) lymphocyte populations and cytokine production by T cells. PB samples were drawn from six NOM patients, 13 age-matched adults who had undergone splenectomy after trauma (SP patients) and 31 age-matched controls. Cellular phenotypes and the intracellular production of interferon (IFN)-gamma, interleukin (IL)-2, IL-4 and IL-10 cytokines in T cells were determined in whole blood +/- mitogens by flow cytometry. NOM patients did not show any changes in the absolute numbers of lymphocytes or the distribution of their subsets, compared to the controls. In contrast, SP patients showed a sustained increase in the percentage and/or absolute numbers of lymphocytes, CD8 T cells, activated CD8 T cells, natural killer (NK) T cells, NK cells and gammadelta T cells, and a reduction in naive CD4 T cells. The constitutive or induced cytokine production by T cells of the NOM group was similar to the control group, whereas SP patients had increased percentages of constitutive IL-2- and IFN-gamma-producing CD8 T cells and IFN-gamma-producing CD4 T cells. Our findings indicate collectively that the healing process in NOM does not affect the architecture of the spleen to such an extent that it would lead to long-term alterations of the proportions of PB lymphocytes or the T cell cytokine profiles.

Factors affecting the plasmin-plasminogen system in milk obtained from three Greek dairy sheep breeds with major differences in milk production capacity.

The purpose of this study was to evaluate the effect of breed, stage of lactation, and health status of the udder on the plasmin-plasminogen system in ovine milk. A total of 38 ewes were used from 3 breeds [Boutsiko (n = 12), Chios (n = 12), and a synthetic breed (50% Boutsiko, 25% Arta, and 25% Chios, n = 14)] with major differences in their genetic potential with respect to milk yield. Milk samples were collected every 2 wk throughout the lactation period and were analyzed for fat, protein, lactose, and somatic cell count (SCC). In addition, milk plasmin (PL), plasminogen (PG), and plasminogen activator (PA) activities were determined. The Chios breed had the greatest average daily milk yield, the synthetic breed had an intermediate milk yield, and ewes of the Boutsiko breed had the lowest milk yield. Milk samples obtained from the Boutsiko breed had similar PL and PA activities, compared with those obtained from the other 2 breeds. The ratio of PG:PL was less in milk samples from the Boutsiko breed compared with the other 2 breeds, indicative of an increased rate of conversion of PG to PL for this breed. There was no correlation between PL activity and daily milk yield in ewes from all 3 breeds. Activities of PL, PG, and PA were greater in ovine milk with elevated SCC (>300,000/mL) compared with activities in milk with low SCC (<300,000/mL). The ratio of PG:PL was less in the high-SCC group compared with the low-SCC group, which indicates an increased rate of conversion of PG to PL for the high-SCC group. There was a decrease in PG and PA activities as well as in the PG:PL ratio in late lactation milk (mo 5 to 6) when compared with early or mid lactation milk (mo 1 to 4). Thus, the PL-PG system is affected by breed, stage of lactation, and the health status of the udder. No relationship was found between PL activity and daily milk yield in the 3 Greek dairy sheep breeds. Plasmin is not a marker for gradual involution in the Greek sheep breeds studied.

Short communication: study of immune parameters in three Greek dairy sheep breeds during the periparturient period.

The objective of the present study was to evaluate whether immunosuppression occurs in 3 different Greek dairy sheep breeds during the periparturient period. A total of 33 ewes from 3 breeds [i.e., the low-producing Boutsiko breed (n = 11), which is highly adaptable to harsh environments; the high-producing but environmentally fragile Chios breed (n = 11); and an intermediate synthetic breed (50% Boutsiko, 25% Arta, and 25% Chios, n = 11)] were used. Blood samples were collected at 18 and 2 d before parturition and at 15 d after parturition. Total cell-associated and membrane-bound urokinase plasminogen activator (U-PA) activity, free U-PA binding sites on cellular membranes, and superoxide anion (SA) production by activated phagocytes were determined. Results indicated that all immune parameters measured remained constant during the periparturient period for the Boutsiko breed. In contrast, there were reductions in total cell-associated and membrane-bound U-PA activity by both monocytes-macrophages and neutrophils and in SA production by monocytes-macrophages at d 2 before parturition for the Chios breed. In the synthetic breed, there were reductions in total cell-associated and membrane-bound U-PA activity by monocytes-macrophages and in SA production by both monocytes-macrophages and neutrophils at d 15 after parturition. Thus, mild immunosuppression during the periparturient period was observed in the 2 breeds with the highest milk production.

Bioactivity characterization of 45S5 bioglass using TL, OSL and EPR: Comparison with the case of 58S sol-gel bioactive glass.

The current work exploits the effective application of thermoluminescence (TL), optically stimulated luminescence (OSL) and the possibility of applying Electron Paramagnetic Resonance (EPR) for the discrimination between different bioactive responses in the case of the 45S5 bioactive glass (SiO2 45, Na2O 24.5, CaO 24.5, P2O5 6 in wt%), which was synthesized through melting process. These techniques are suggested mainly due to their low spectroscopic detection thresholds. The original 45S5 in grain size range of 20-40µm was immersed in the Simulated Body Fluid (SBF) for various different immersion times ranging over one week. In this work the 110°C TL peak, a specific OSL component and the EPR signal at g=2.013 ascribed to oxygen hole center (OHC) are used due to their sensitivity to the different bioactive responses. For all luminescence and EPR components, the intensity plot versus immersion time yields sharp discontinuities, resulting in effective probes regarding the timescale for both the beginning as well as the end of the procedure of the crystalline HCAp formation respectively. On the contrary to the smooth decreasing pattern of both luminescence entities, the peak to peak amplitude of the EPR signal indicates an initial increase for the initial 16min of immersion, followed by a further decrease throughout the immersion time duration. The discontinuities monitored for both sensitivity of TL, OSL and EPR, in conjunction with the discontinuities monitored for the sensitization of TL and OSL, when plotted versus immersion time, provide an individual time scale for each one of the chemical reactions involved in the five steps of the aforementioned procedure. According to the authors' best knowledge, scarce characterization techniques could provide this time scale frame, while it is the first time that such an application of OSL and EPR is attempted. Finally, the bioactive response of the 45S5 bioglass was compared with that of the 58S sol-gel bioactive glass, in terms of the timescale of these five stages required for the final formation of the HCAp. The techniques of luminescence and EPR which take advantage of trapped charges are proposed as alternative cheap and prompt effective techniques towards discrimination between different bioactive responses in bioactive glasses.

Cloning of the 5' regulatory regions and functional characterization of the core promoters of ovine PLAU (u-PA) and SERPIN1 (PAI-1).

The activation of plasminogen plays a crucial role in various extracellular proteolytic events (fibrinolysis, cell migration, ovulation and involution of the mammary gland). In the present study we describe the isolation of the 5' proximal and distal promoter regions of ovine PLAU (urokinase plasminogen activator, u-PA) and SERPIN1 (plasminogen activator inhibitor 1, PAI-1) genes for the first time in ruminants. Analysis of the 5.645kb 5'-flanking region of u-PA revealed a putative TATA-less promoter. In contrast the isolated 2.787kb 5'-flanking region of PAI-1 included a TATA-box. It should be noted that both genes lack the initiator motif around the transcription start site. The two genes share a number of transcription factor binding sites, namely Nuclear Factor-kappa B, Stimulating Protein 1 and Activating protein 1, suggesting co-expression of the two genes. Moreover, additional, not shared, transcription factor binding sites were identified in u-PA and PAI-1. More important of these are the cis-regulatory elements for plasminogen activator inhibitor 2 located in the distal promoter region of u-PA, suggesting an involvement of the other specific inhibitor in the regulation of ovine u-PA gene expression, and the three stress response elements sites present in the proximal and distal promoter of PAI-1. Different genomic fragments of the two 5' flanking regions were directionally cloned into a suitable reporter vector upstream of a promoter-less luciferase gene. Transient transfection into bovine mammary epithelial (BME-UV) cells demonstrated that the regions of -384/+27 and -382/+22 for the u-PA and PAI-1genes, respectively, potentially function as core promoter regions.