I. Politis

Establishment and characterization of a bovine mammary epithelial cell line with unique properties.

SummaryClonal cell lines (BME-UV) were established from primary epithelial cells by stable transfection with a plasmid, carrying the sequence of the simian virus 40 early region mutant tsA58, encoding the thermolabile large T antigen. The BME-UV cells have undergone more than 300 population doublings and produce intranuclear large T antigen. At low confluency, growing islands of cells are apparent exhibiting the characteristic cobblestone morphology of epithelial cells. The BME-UV cells expressed functional markers such as microvilli and desmosomes and biochemical markers of mammary epithelial cells such as a repertoire of cytokeratins. The BME-UV cells are capable of synthesizing low levels of α-lactalbumin and α8l (50 ng/ml of medium/24 h). One of the cell lines, BME-UV1 showed enhanced proliferation in the presence of epidermal growth factor (EGF) and insulinlike growth factor I (IGF-I). The BME-UV1 cell line is the only known bovine mammary epithelial cell line responsive to EGF. The BME-UV cells grown on collagen at low confluency are capable of developing very long projections that most likely allow for communication between cells at a distance from each other. The BME-UV cells may become a valid model system to examine bovine mammary epithelial proliferation and differentiation and cell-to-cell communication.

Evaluation of the protective effects of α-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.

Effect of mastitis on plasminogen activator activity of milk somatic cells

This study was conducted to examine the effects of mastitis and stage of lactation on plasminogen activator (PA) activity in milk somatic cells. An assay system, which measures the plasmin-mediated hydrolysis of the chromogenic substrate D-valyl-leucyl-lysine p-nitroanilide, was used to assess PA activity present within milk somatic cells. Milk cell associated PA activity was increased (P < 0.05) by 50% in the presence of fibrin fragments. This suggests that milk somatic cells contain tissue PA which, unlike urokinase PA, is preferentially activated in the presence of fibrin fragments. An increase of the milk somatic cell count from < 5 x 10(4) to > 10(6) cells/ml resulted in an 8-fold increase in PA activity per cell. Elevated levels of PA activity were associated with milk somatic cells isolated from mastitic quarters obtained from cows in early (< 4 months in lactation) or late lactation (> 8 months in lactation). We conclude that PA activity is increased during severe mastitic inflammation. Although the physiological function of this enzyme is as yet unclear, we propose that it may be involved in the conversion of plasminogen to plasmin, contributing to the higher levels of plasmin occurring in milk isolated from mastitic quarters.

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 μg/kg), OTA plus TRM at three inclusion rates (104 CFU/g of feed, 105 CFU/g, 106 CFU/g) and TRM alone at 105 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 μM): 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 30 d before the immunosuppressive effect became apparent. The dietary inclusion of TRM completely blocked the detrimental effects of OTA on several immune properties in broilers.

Changes in plasmin-plasminogen-plasminogen activator system in milk from Italian Friesian herds

Changes in plasmin, plasminogen and plasminogen activator (PA) throughout the lactation were investigated in individual milk samples obtained from 32 Friesian cows from four commercial herds located in Northern Italy. Herds were chosen to represent four different, yet typical for Italy, diets. Increased levels of plasmin and PA (P < 0.05) were observed with advancing lactation. Plasminogen peaked during the fifth month of lactation. The increased levels of plasmin during the fifth month of lactation are partly due to increased plasminogen, which reflects increased permeability of mammary epithelium. However, the ratio of plasminogen to plasmin decreased with advancing lactation, suggesting accelerated conversion of plasminogen to plasmin. Major differences were observed between herds with respect to plasmin levels. These differences probably reflect differences in diets and management practices. This could be very important for Northern Italy where most of the milk produced is used for cheese manufacture. Plasmin, PA and somatic cell counts (SCC) were negatively correlated with casein/protein with coefficients of −0.38, −0.43 and −0.40, respectively. A significant correlation existed between PA and SCC (r = 0.50). PA was positively correlated with plasmin (r = 0.49).

Effect of vitamin E supplementation on neutrophil function, milk composition and plasmin activity in dairy cows in a commercial herd

Fifty-six Holstein dairy cows from a commercial dairy herd in the Northern part of Greece were used to determine the effect of vitamin E supplementation on immune parameters, milk composition and milk quality. Cows were assigned to one of two experimental groups: control (no vitamin E supplementation) and vitamin E supplementation. Supplementation of vitamin E started 4 weeks prior to and continued up to 12 weeks after parturition. Supplementation included daily oral administration of vitamin E at 3000 i.u./cow prepartum and was reduced to 1000 i.u./cow post partum. Blood samples were collected weekly for 8 weeks starting 4 weeks before parturition, neutrophils were isolated and the following parameters were determined in neutrophils activated by phorbol myristate acetate: total cell-associated and membrane-bound urokinase plasminogen activator (u-PA) activity and superoxide production. Milk samples were collected weekly and fat, protein, lactose, somatic cell count (SCC), plasmin and plasminogen-derived activity were determined. Activated neutrophils isolated from cows that received supplemental vitamin E had higher (P<0.01) total and membrane-bound u-PA activities during the first 3 weeks after parturition and higher (P<0.01) superoxide production during week 1 prepartum and week 1 post partum compared with the corresponding values of activated neutrophils isolated from control cows. Vitamin E supplementation had no effect (P=0.28) on plasminogen-derived activity in milk. Milk obtained from cows that received supplemental vitamin E had SCC lower by 25% (P<0.05) and plasmin lower by 30% (P<0.01) than corresponding values in milk obtained from control cows. The reduction in plasmin as a result of vitamin E supplementation is very beneficial to the dairy industry because plasmin reduces the cheese-yielding capacity of milk, affects the coagulating properties of milk and its overall ability to withstand processing during cheesemaking. In conclusion, vitamin E supplementation had positive effects on the function of bovine neutrophils and milk quality in a commercial dairy herd.

Distribution of plasminogen activator in different fractions of bovine milk

The type and relative amounts of plasminogen activator (PA) in different fractions of bovine milk obtained from 15 Holstein cows were examined. Raw milk was centrifuged to separate skim milk and a somatic cell pellet. PA was mainly localized within the casein fraction, being 42 times that in the serum, and in association with somatic cells. The predominant form of PA in milk casein was isolated from SDS-PAGE gel extracts and had a molecular mass of approximately 75 kDa. Its activity was increased 4.1-fold (P < 0.01) in the presence of fibrin but was unaffected by the presence of amiloride, indicating that it was due to tissue-PA. The predominant forms of PA associated with milk somatic cells were isolated from SDS-PAGE gel extracts and had molecular masses of approximately 30 and approximately 50 kDa. The activity of both proteins was unaffected by the presence of fibrin but was dramatically reduced by the presence of amiloride, indicating that they represented urokinase-PA.

Function of bovine mammary macrophages as antigen-presenting cells

The ability of mammary macrophages treated with Staphylococcus aureus to induce antigen-specific T-cell proliferation was compared to that of the autologous blood monocytes. Induction of T-cell proliferation has been correlated with changes in major histocompatibility complex (MHC) class II antigen expression and interleukin 1 (IL-1) production by mammary macrophages and blood monocytes. The present study showed that both monocytes and mammary macrophages treated with S. aureus induced T-cell proliferation. However, there was a 3-fold decrease (P less than 0.05) in T-cell proliferation in macrophage cultures compared to those of blood monocytes, when these cells were treated with S. aureus. Mammary macrophages, the cells less effective in stimulating T-cell proliferation, expressed lower levels (2-fold) of MHC class II molecules and produced less IL-1 (3-fold) than blood monocytes. These data suggest that S. aureus may affect macrophage-T cell interaction by modulating the expression of MHC class II molecules and the synthesis of IL-1 by macrophages.

Milk Peptides and Immune Response in the Neonate

Bioactive peptides encrypted within the native milk proteins can be released by enzymatic proteolysis, food processing, or gastrointestinal digestion. These peptides possess a wide range of properties, including immunomodulatory properties. The first months of life represent a critical period for the maturation of the immune system because a tolerance for nutrient molecules should be developed while that for pathogen-derived antigens is avoided. Evidence has accumulated to suggest that milk peptides may regulate gastrointestinal immunity, guiding the local immune system until it develops its full functionality. Our data using the weaning piglet as the model suggest that several milk peptides can downregulate various immune properties at a time (one to two weeks after weaning) that coincides with immaturity of the immune system. The protein kinase A system and/or the exchange protein directly activated by cyclic AMP (Epac-1) are implicated in the mechanism through which milk peptides can affect immune function in the early postweaning period. Despite the fact that the research in this field is in its infancy, the evidence available suggests that milk protein peptides may promote development of neonatal immune competence. Milk contains a variety of components that provide immunological protection and facilitate the development of neonatal immune competence. Two main categories of milk compounds are thought to be associated with immunological activity. The first category includes cytokines, which neonates do not produce efficiently. Cytokines present in milk are thought to be protected against intestinal proteolysis and could alleviate immunological deficits, aiding immune system maturation (Kelleher & Lonnerdal, 2001; Bryan et al., 2006). The second category of milk compounds includes milk protein peptides. Milk peptides may affect mucosal immunity possibly by guiding local immunity until it develops its full functionality (Baldi et al., 2005). This chapter focuses on the effects of milk peptides on immune function and attempts to provide an overview of the knowledge available in this field.

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.