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.

Reevaluation of vitamin E supplementation of dairy cows: bioavailability, animal health and milk quality.

Although vitamin E has been known as an essential nutrient for almost 80 years, we are far from a complete understanding of all the aspects related to bioavailability and its effects on health and milk quality in dairy cows. Vitamin E is a generic descriptor for two families of lipid-soluble compounds, the tocopherols and the tocotrienols, of which α-tocopherol has the highest biological activity. Commercially available α-tocopherol supplements for dairy cows contain either the natural RRR form or the synthetic (all-rac) form, which contains all the eight possible stereoisomers (four possessing the 2R and four possessing the 2S configuration) in equimolar amounts. Recent data clearly suggest that an almost complete discrimination against the 2S isomers occurs in dairy cows. Thus, 1 g of the all-rac form is essentially equivalent to 0.5 g of the RRR form. With respect to the effect of vitamin E supplementation of dairy cows on health and milk quality, the majority of published studies suggests that vitamin E supplementation at the level 1000 to 4000 IU/cow per day during the dry period reduces both the frequency of intramammary infection and that of clinical mastitis and improves milk quality, as shown by a reduction in the levels of somatic cell count (SCC)/ml in milk, decreased plasmin activity and increased oxidative stability of milk. However, a recent study from the Netherlands suggested that vitamin E supplementation at the 3000 IU/cow per day level during the dry period when combined with high levels of plasma vitamin E at dry-off (>14.5 µmol/l) increases the incidence of mastitis. Data from previously unpublished survey studies and those from published vitamin E feeding trials, in which high levels of blood vitamin E were observed, were reanalyzed. All farms selected for the analysis implemented oral administration of vitamin E at the 3000 IU/cow per day level throughout or during the late dry period (4 weeks before the expected day of parturition). Dairy cows were divided into three groups, depending on blood α-tocopherol levels at dry-off: high (>6.25 µg/ml), medium (between 6.25 and 4.25 µg/ml) and low (<4.25 µg/ml). Data indicate that there were no differences in the incidence of mastitis and in the level of SCC/ml of milk between the three groups. Thus, supplementation of 3000 IU vitamin E/cow per day in the late dry period remains recommended because it is generally associated with decreased risk of mastitis. Conditional or opposite effects have not been repeated and require further research before changing recommendations for vitamin E supplementation.

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.

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.

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.

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.

The effect of two bovine beta-casein peptides on various functional properties of porcine macrophages and neutrophils: differential roles of protein kinase A and exchange protein directly activated by cyclic AMP-1.

The effects of two bovine beta-casein peptides on the urokinase plasminogen activator (u-PA) system and superoxide anion (SA) production by porcine macrophages and neutrophils activated by phorbol myristate acetate (PMA) were investigated. Macrophages and neutrophils were obtained from fourteen weaned piglets and were cultured in vitro for 24 h with or without one of two chemically synthesised peptides: tripeptide leucine-leucine-tyrosine (residues 191-193 of beta-casein) (LLY) and hexapeptide proline-glycine-proline-isoleucine-proline-asparagine (residues 63-68 of beta-casein). Following incubation, cells were stimulated with 80 microM-PMA. Total cell-associated u-PA, membrane-bound u-PA, free u-PA binding sites along with SA production were determined after stimulation with PMA. Both peptides suppressed the u-PA system and SA production of PMA-stimulated macrophages isolated from piglets during weeks 1-2 after weaning. Only the tripeptide LLY suppressed the u-PA system and SA production of PMA-stimulated neutrophils during the same time period. None of the peptides tested had any effect (P>0.05) on the u-PA system and SA production of PMA-stimulated macrophages and neutrophils isolated from the same piglets during weeks 5-6 after weaning. Thus, peptides are effective only in the early post-weaning period. Using cyclic AMP analogues that are highly specific activators of protein kinase A (PKA) or exchange protein directly activated by cyclic AMP-1 (Epac-1), we found that activation of PKA, but not Epac-1, was responsible for the downregulation of the u-PA system, whereas activation of PKA and/or Epac-1 was responsible for the downregulation of SA system in both macrophages and neutrophils.

Effect of vitamin E supplementation on lymphocyte distribution in gut-associated lymphoid tissues obtained from weaned piglets.

Fifteen piglets were used to determine the effect of vitamin E supplementation on the number of CD4-immunoreactive (CD4+) T-lymphocytes, CD8-immunoreactive (CD8+) T-lymphocytes and IgA-immunoreactive (IgA+) B-lymphocytes per follicle in the Peyer's patch of distal ileum and the mesenteric lymph nodes of weaned piglets. Piglets, following a 3-day adaptation period after weaning, were assigned to one of three experimental groups: control (no vitamin E supplementation), vitamin E supplementation of 100 mg/kg of diet and vitamin E supplementation of 300 mg/kg of diet. Supplementation of vitamin E lasted for a period of 36 days. The basal diet contained 80 mg alpha-tocopherol/kg of diet. All piglets were killed at day 39 after weaning and samples of the distal ileum and adjacent mesenteric lymph nodes were collected. The number of cells for each lymphocyte subset was counted in the Peyer's patch and the mesenteric lymph nodes follicles, in cryostat sections processed for immunohistochemistry. Results showed that vitamin E supplementation (300 mg/kg diet) of piglets caused an increase (P < 0.05) in the number of IgA+ B-lymphocytes in the Peyer's patch, but not in the mesenteric lymph nodes, compared with the corresponding values in control animals. Vitamin E supplementation had no effect (P > 0.05) on the number of CD4+ and CD8+ T-lymphocytes in the follicles of the Peyer's patch and the adjacent mesenteric lymph nodes. Thus, vitamin E had relatively minor effects on distribution of the major immunocompetent cells in the gut. The numbers of CD4+ and CD8+ T-lymphocytes as well as IgA+ B-lymphocytes per follicle were higher by 26-77% (P < 0.05) in the mesenteric lymph nodes than the corresponding values in the Peyer's patch.

Use of Trichosporon mycotoxinivorans to suppress the effects of ochratoxicosis on the immune system of broiler chicks.

(1) The objective of this study was to determine whether the dietary inclusion of Trichosporon mycotoxinivorans (TRM) could suppress the detrimental effects of ochratoxin A (OTA) on the immune system of broiler chicks. (2) Six experimental treatments were tested in 300 1-d-old broiler chicks. Treatments included addition to a standard broiler ration of neither OTA nor TRM (Diet 1), OTA alone (500 microg/kg), OTA plus TRM at three inclusion rates (10(4) CFU/g of feed, 10(5) CFU/g, 10(6) CFU/g) and TRM alone at 10(5) CFU/g of feed. The ration was fed to chicks for 42 d. (3) Blood samples were collected at d 10, 20, 30 and 40 and macrophages and heterophils were isolated. The following variables were determined in macrophages and heterophils activated by phorbol myristate acetate (65 microM): cell viability, total cell-associated urokinase-plasminogen activator (u-PA), membrane-bound u-PA, free u-PA binding sites and superoxide production. (4) There was a decrease in the viability of macrophages and heterophils from chicks receiving OTA-contaminated feed compared to the viability of cells from control birds at d 40. Dietary TRM completely blocked the effect of OTA on cell viability; all three inclusion rates were equally effective. There was a decrease in total cell-associated and membrane-bound u-PA in macrophages and heterophils of chicks receiving OTA-contaminated feed compared to the corresponding values in control birds for heterophils at d 30 and 40 and for the macrophages at d 40. (5) Similarly, dietary TRM abolished the effect of OTA on total cell-associated and membrane-bound u-PA activity. All three inclusion rates of yeast were equally effective. Heterophils, but not macrophages, isolated from chicks receiving OTA-contaminated diet produced less superoxide anion compared to all other diet groups at d 30 and 40. (6) The immune system is a primary target of OTA toxicity in broilers: several functional properties of macrophages and heterophils were depressed in chicks fed OTA-contaminated feed. There was a delay of 30d before the immunosuppressive effect became apparent. The dietary inclusion of TRM completely blocked the detrimental effects of OTA on several immune properties in broilers.

Effect of vitamin E supplementation on various functional properties of macrophages and neutrophils obtained from weaned piglets.

Sixteen piglets were used to determine the effect of vitamin E supplementation on several functional properties of macrophages and neutrophils obtained from weaned piglets. Piglets, immediately following weaning, were assigned to one of three experimental groups: control (no vitamin E supplementation), low level of vitamin E supplementation (100 mg DL-alpha-tocopheryl acetate/kg diet) and high level of vitamin E supplementation (300 mg DL-alpha-tocopheryl acetate/kg diet). Supplementation of vitamin E lasted for a period of 36 days, following a 3-day adaptation period after weaning. Blood samples were collected on days 0, 12, 24 and 36 of the experimental period, monocytes/macrophages and neutrophils were isolated and the following parameters were determined in macrophages and neutrophils activated by phorbol myristate acetate: total cell-associated and membrane-bound urokinase plasminogen activator (u-PA) activity and superoxide anion production. Results showed that macrophages and neutrophils isolated from piglets that received supplemental vitamin E had higher (P < 0.05) total and membrane-bound u-PA activities as well as higher (P < 0.05) superoxide anion production compared with the values of the corresponding cells obtained from control piglets on day 12 of the experimental period. Both levels of vitamin E supplementation (low and high) were equally effective. In contrast, vitamin E supplementation had no effect (P > 0.05) on total and membrane-bound u-PA activities and superoxide anion production by porcine macrophages and neutrophils on days 24 and 36 of the experimental period. In conclusion, the low level of vitamin E supplementation is recommended for piglets for the first 2 weeks after weaning.

Evaluation of the protective effects of alpha-tocopherol and retinol against ochratoxin A cytotoxicity.

Ochratoxin A (OTA), a mycotoxin frequently present in food and feedstuffs, produces a wide range of toxic effects, including cell death via lipid peroxidation. In one human and four animal cell lines we determined the half lethal concentration (LC50) of OTA, its effect on reactive oxygen species (ROS) production, and its ability to induce cytochrome p450 activity. We also examined the protective effect of alpha-tocopherol and all-trans-retinol in the most sensitive cell lines (i.e. bovine mammary epithelia, for which LC50 was 0.8 microg/ml (24 h), and Madin Darby canine kidney, for which LC50 was 4.3 microg/ml (48 h)). Pre-incubation for 3 h with either antioxidant significantly (P<0.05) ameliorated the OTA-induced reduction in cell viability and significantly decreased (P<0.05) ROS production. These findings indicate that oxidative stress is an important factor in OTA cytotoxicity. Supplementation with antioxidant molecules may counteract the short-term toxicity of this mycotoxin.

Effects of dietary conjugated linoleic acid isomers on several functional properties of macrophages and heterophils in laying hens.

1. Effects of dietary conjugated linoleic acid (CLA) on various functional properties of macrophages and heterophils in the laying hen were determined. 2. Seventy two 28-week-old-ISA brown hens were randomly assigned to one of two experimental groups and were fed diets containing 0 or 1% CLA for a total period of 18 weeks. Blood samples were collected from 6 hens per diet group every 3 weeks and macrophages and heterophils were isolated. The following variables were determined in resting and phorbol myristate acetate-activated (65 microM) macrophages and heterophils: total cell-associated urokinase plasminogen activator (u-PA), membrane-bound u-PA, free u-PA binding sites and superoxide production. 3. There were no significant differences between diet groups throughout the whole experimental period in total cell-associated u-PA, membrane-bound u-PA, free u-PA binding sites and superoxide production by resting macrophages and heterophils. 4. Activated heterophils and macrophages isolated from hens fed the CLA-supplemented diet had higher membrane-bound u-PA activity compared with the corresponding values for activated heterophils and macrophages obtained from hens fed the basal diet. These differences were significant for heterophils during weeks 6, 12, 15 and 18 and for macrophages during weeks 6, 9 and 12 of the experiment. 5. Macrophages and heterophils from hens fed the CLA diet had lower superoxide production compared with the corresponding cells from the control hens on weeks 12, 15 and 18 of the experiment. 6. Dietary CLA modulated certain aspects of the immune system in the laying hen. The increased quantity of u-PA on the membrane of macrophages and heterophils isolated from hens fed the CLA diet may facilitate the ability of these cells to reach the point of a potential inflammation (pro-inflammatory effect). 7. In contrast to the effect on the u-PA system, dietary CLA reduced superoxide production by activated macrophages and heterophils during the second half (last 9 weeks) of the experimental period suggesting that CLA may exert an anti-inflammatory effect in the laying hen.

Rumen-protected choline administration to transition cows: effects on milk production and vitamin E status.

We investigated the influence of rumen-protected choline (RPC) supplementation on milk production, lipid metabolism and vitamin E status in dairy cows receiving a silage-based diet. Twenty-six Italian Holstein multiparous cows were assigned by weight and average production in the previous lactation, to one of two groups: control (no RPC supplementation) and RPC (supplemented with 20 g/day rumen-protected choline chloride). Treatment began 14 days before expected calving and continued for 30 days after parturition. Choline administration significantly increased milk production during the first month of lactation and also the concentration (and total secretion) of choline in milk, but did not affect fat or protein concentrations in milk, or plasma levels of glucose, beta-hydroxybutyrate, cholesterol and non-esterified fatty acids (NEFA). However, around parturition, NEFA concentrations in plasma were lower in treated animals than in controls, suggesting improved lipid metabolism as a result of choline supplementation. Choline supplementation also increased alpha-tocopherol plasma concentrations, suggesting a novel aspect in dairy cows.

The effect of various vitamin E derivatives on the urokinase-plasminogen activator system of ovine macrophages and neutrophils.

The effect of vitamin E derivatives on the urokinase-plasminogen activator (u-PA) system of resting and phorbol myristate acetate (PMA)-activated ovine macrophages and neutrophils were investigated. Blood monocyte-macrophages and neutrophils were isolated from twenty-four animals. Macrophages or neutrophils were cultured in vitro for 3 or 24 h with or without various vitamin E derivatives: free alpha-tocopherol (alpha-T), alpha-tocopheryl acetate (alpha-TA), or alpha-tocopheryl succinate (alpha-TS). Following incubation, cells were stimulated with 80 microm-PMA. Total cell-associated u-PA, membrane-bound u-PA and free u-PA binding sites were determined before and after stimulation with PMA. Results showed that none of the vitamin E derivatives had any effect (P>0.05) on the u-PA system of resting monocyte-macrophages or neutrophils. In contrast, alpha-TS, but not alpha-TA or alpha-T, increased (P<0.01) total cell-associated u-PA and membrane-bound u-PA of PMA-stimulated macrophages and neutrophils. alpha-TS had no effect (P>0.05) on total u-PA and membrane-bound u-PA activities of macrophages and neutrophils cultured in the presence of 4-phorbol 12,13 didecanoate, a phorbol ester that does not activate protein kinase (PK) C. Addition of H7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), which is a potent inhibitor of both PK A and C, completely abolished the effect of alpha-TS on total cell-associated u-PA and membrane-bound u-PA of PMA-activated macrophages and neutrophils. Addition of HA1004 (N-(2-quanidinoethyl)-5-isoquinoline sulfonamide hydrochloride), which is a potent PK A but a weak PK C inhibitor, had no effect (P>0.05) on total cell-associated u-PA and membrane-bound u-PA of PMA-activated macrophages and neutrophils cultured in the presence of alpha-TS. Thus, PK C modulates the effect of alpha-TS on the u-PA system of ovine macrophages and neutrophils.

Effects of vitamin E on urokinase-plasminogen activator receptor expression by bovine neutrophils.

OBJECTIVE: To determine the effect of vitamin E supplementation on urokinase-plasminogen activator (u-PA) receptor (u-PAR) expression by neutrophils of dairy cows. ANIMALS: 16 healthy Holstein dairy cows. PROCEDURE: 16 cows were assigned to 1 of 2 experimental groups: control (no vitamin E supplementation) and vitamin E supplementation. Supplementation of vitamin E started 4 weeks prior to and continued up to 4 weeks after parturition and included oral administration of vitamin E at 3,000 U/cow per day; these cows also received 1 injection of vitamin E (5,000 units), 1 week prior to the expected date of parturition. Blood samples were collected, and neutrophils were isolated weekly throughout the experimental period. The following variables were measured: u-PA (mRNA), total cell-associated u-PA activity, membrane-bound u-PA activity, and free unoccupied u-PA binding sites on the cell membrane of neutrophils. RESULTS: Stimulated neutrophils isolated from cows that received vitamin E supplementation had significantly higher u-PA mRNA and total cell-associated and membrane-bound u-PA activity at postpartum week 1, compared with those of stimulated neutrophils isolated from control cows. There were no differences between groups throughout the whole experimental period in u-PA binding sites of neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: The increased total cell-associated and membrane-bound u-PA activity in neutrophils isolated from cows that received vitamin E may facilitate the ability of neutrophils to extravasate and reach the mammary gland at postpartum week 1. Rapid recruitment of neutrophils is critical for proper defense of the gland.

Kinetic behavior of three preparations of alpha-tocopherol after oral administration to postpubertal heifers.

OBJECTIVE: To assess the kinetic behavior of 3 preparations of alpha-tocopherol (vitamin E) after oral administration to heifers. ANIMALS: 8 postpubertal Friesian heifers. PROCEDURE: A single oral bolus of 5,000 U of alpha-tocopherol in oil or encapsulated in liposomes or cyclodextrin was administered to each cow, using a 4 X 4 design with 8 days between treatments. Blood samples for kinetic analyses were obtained at various times for 168 hours after treatment. RESULTS: Mean (+/- SEM) maximal plasma concentrations of alpha-tocopherol were 4.86 +/- 0.49 microg/ml, 5.03 +/- 0.39 microg/ml, and 5.08 +/- 0.56 microg/ml after administration of oil, liposomal, and cyclodextrin preparations, respectively. Plasma concentrations peaked 21 to 34 hours after administration. The disappearance rate constant (Kd) was less after administration of alpha-tocopherol encapsulated in liposomes, compared with the other 2 preparations. Area under the concentration versus time curve was greater after administration of either encapsulated form of alpha-tocopherol, compared with alpha-tocopherol in oil, but these differences were not significant. CONCLUSIONS AND CLINICAL RELEVANCE: The lower Kd determined for alpha-tocopherol encapsulated in liposomes suggests that this formulation may result in longer persistance of the vitamin in plasma than the other 2 preparations. Dietary supplementation with alpha-tocopherol encapsulated in liposomes may enhance plasma availability of this vitamin in cattle and could be useful during periods of increased vitamin E requirements, such as parturition and early stages of life.

Metallothionein expression prevents apoptosis. II: Evaluation of the role of metallothionein expression on the chemotherapy-induced apoptosis during the treatment of acute leukemia.

Metallothioneins (MT) are low molecular weight cysteine-rich proteins, present in a wide variety of eukaryotes. Although their physiological function is not entirely understood, recently it was found that in vitro human MTs (hMTs) expression prevents apoptosis. In the present study, the apoptosis preventing effect of hMTs is evaluated in vivo, in order to correlate the apoptotic effect of chemotherapy during the treatment of acute leukemia with the expression of hMTs. The expression of hMTs was studied immunocytochemically in bone marrow smears and peripheral blood cytocentrifugations of 47 children with acute leukemia at diagnosis and during treatment. Apoptosis was quantitatively studied in peripheral blood samples during the induction therapy. Eighteen cases were found to be positive for hMTs expression at diagnosis and the mean apoptosis curve of these cases showed maximal effect on the second day of treatment, the apoptotic action of chemotherapy being completed on the tenth day. The mean apoptosis curve of the hMTs negative cases (29 cases) showed maximal effect on the first day of treatment and the apoptotic action of chemotherapy was completed on the sixth day. When considering the day on which the maximal apoptotic effect appeared and the day on which the apoptotic action of treatment was completed, the results indicated retardation of the chemotherapy-induced apoptosis dependent on hMTs expression, as a result of resistance to treatment. Furthermore, the study of hMTs expression during treatment, showed that although the apoptotic action of chemotherapy eliminates blast cells, a cell population positive for hMTs survived and increased during treatment, since they were able to escape apoptotic cell death. These findings, indicated that in vivo, hMTs constitute a cellular protective mechanism preventing chemotherapy-induced apoptosis, thus regulating the response of patients to treatment.