Retinoids,retinoid analogs,and lactoferrin interact and differentially affect cell viability of 2 bovine mammary cell types in vitro

Two bovine mammary cell types (BME-UV1 and MeBo cells) were used to evaluate the effect of natural retinoids, retinoid analogs, and bovine lactoferrin (bLf) on cell viability in vitro. Experiments with Alamar Blue showed a linear relationship between fluorescence and cell viability index. The BME-UV1 cells exhibited twice the metabolic activity but required half the doubling time of the MeBo cells. The BME-UV1 cells were very sensitive to all-trans retinoic acid (atRA) inhibition of cell viability (P < 0.05) and exhibited a dose-dependent inhibition with 9-cisRA (9cRA; P < 0.05). The MeBo cells exhibited some inhibition with these natural ligands (P < 0.05), but they were not as sensitive. The addition of bLf had similar inhibitory effects (P < 0.05) on cell viability of the 2 mammary cell types. Applications of RA receptor (RAR) agonist indicated that the stimulation of the RAR in both mammary cell types was highly effective in inhibition of cell viability (P < 0.05), whereas the application of an RAR antagonist stimulated MeBo cell viability (P < 0.05) and inhibited BME-UV1 cell viability (P < 0.05). Finally, the use of the RAR antagonist in conjunction with bLf indicated a rescue of the bLf effect in the MeBo cells, suggesting that bLf is acting through the RAR receptor. Conversely, bLf reverted inhibition of cell viability by 9cRA in the BME-UV1 cell type (P < 0.05). We conclude that RAR interaction in bovine mammary cell types regulates cell viability in vitro; we hypothesize that the natural ligands mediate regulation of bovine mammary cell viability in vivo and that bLf can either enhance or reverse the retinoid-induced inhibition of cell viability, depending on the type of bovine mammary cell studied.     

Evaluation of the protective effect of bovine lactoferrin against lipopolysaccharides in a bovine mammary epithelial cell line

Lactoferrin (Lf) is a non-haem iron-binding glycoprotein with a molecular weight of about 80 kDa, synthesized by glandular epithelial cells and stored in the secondary granules of neutrophils. The physiological significance of Lf is related to non-specific immune defence against pathogens, immunomodulatory activity, iron homeostasis, antioxidant properties and regulation of cell growth. Lf is a bioactive component of the mammary secretions and its modulatory and defensive functions do affect the newborn and the mammary gland as well. In this work a bovine mammary epithelial cell line (BME-UV1) was used as an in vitro model of the bovine mammary epithelium to examine the protective role of exogenous bovine Lf (bLf) against the cytotoxic damage induced by bacterial lipopolysaccharides (LPS) and the endogenous bLf mRNA expression after LPS exposure. In the in vitro model used, exogenous bLf exerts a protective effect against endotoxin cytotoxicity, which could be mediated by the LPS-neutralizing capability of bLf. In addition, in BME-UV1 cells the response to LPS exposure does not involve bLf mRNA expression, suggesting that this cell line lack of functional LPS-responsive elements.     

Colostral and milk insulin-like growth factors and related substances: mammary gland and neonatal (intestinal and systemic) targets

The identification of hormones and regulatory factors in colostrum and milk has led to intensive investigations on their roles in the development and maintenance of the mammary and neonatal tissues. Insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in transgenic mice influence mammary biology gland towards the end of lactation. In the bovine, IGFBP-3 is the major IGFBP in mammary secretions. In addition to binding IGFs, IGFBP-3 also binds to lactoferrin (Lf). Secreted IGFBP-3 re-enters mammary epithelial cells and with the presence of a nuclear localization sequence, IGFBP-3 and Lf enter the nucleus. Nuclear IGFBP-3 affects apoptotic signaling through the retinoic-x-receptors, while Lf affects apoptotic events through unknown mechanisms. Such interactions likely influence mammary development and involution. Furthermore, ingested colostral bioactive factors can exert regulatory functions in neonates. Intestinal receptors for IGFs and insulin are modified by age and/or diet. Feeding IGF-I had no effect, but colostrum extracts had small intestinal effects (stimulation of proliferation and villus size), suggesting that several factors, rather than one single bioactive factor were responsible. Systemic changes of metabolic and endocrine profiles in neonates depend on composition, amounts, time and duration of feeding colostrum. Early postnatal colostrum intake is not only important for the provision and absorption of immunoglobulins. Thus, in neonatal calves the lack of colostrum intake during the first 24h after birth results in a low immunoglobulin G, beta-carotene and Vitamin A status that persists for weeks and plasma patterns of fatty acids, essential amino acids and the glutamine/glutamate ratios are affected. In calves oral administration of IGF-I had no and feeding of colostrum whey extracts had only minor effects on metabolic and endocrine traits. Thus, mammary secretions influence regulatory functions of mammary and neonatal tissues.     

Inflammatory effect of cleaved bovine lactoferrin by elastase on staphylococcal mastitis

Elastase activity and concanavalin A (Con A) low affinity bovine lactoferrin (bLf) molecule were detected in mammary gland secretions (MGSs) from mammary glands (MGs) with clinical staphylococcal mastitis. Changes in clinical symptoms correlated with increases in both elastase activity and the concentration of Con A low-affinity Lf in MGSs from mastitic MGs. Bovine Lf treated with elastase (elastase-Lf) showed various small bLf molecules and the same image on Con A two-dimensional immunoelectrophoresis as low Con A affinity bLf in MGSs. We confirmed the presence of four common bLf peptides for the elastase-bLf and low Con A affinity bLf molecules in mastitic MGSs, and synthesized four peptides. Strong mRNA expression of interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) was induced in bovine mammary epithelial cells on stimulation with low Con A affinity bLf, elastase-bLf, and GQRDLLFKDSAL, a synthesis bLf peptide based on nuclear factor kappa B (NFkappaB) activation. These results suggest that bLf was cleaved by elastase, and that this cleavage changed the physical function of Lf. Our results indicate that elastase induced production of low Con A affinity bLf, including the bLf peptide GQRDLLFKDSAL, and had an inflammatory effect on staphylococcal mastitis.     

Small molecule lactoferrin with an inflammatory effect but no apparent antibacterial activity in mastitic mammary gland secretion

We have identified various lactoferrin (Lf) molecules in mastitic mammary gland secretions (MGSs), and these Lf molecules were examined for their physiological function in MG. These Lf molecules were isolated by Con A affinity chromatography, and then analyzed by various electrophoresis methods and N-terminal amino acid sequencing. The low Con A affinity Lf was found to have low molecular peptides as compared with the 86 kDa of the high Con A affinity Lf, which is usually detected in healthy MGSs. The N-terminal amino acid sequence of each of the small molecular Lfs were confirmed as fragments of 86 kDa Lf. This low Con A affinity Lf stimulated spleen adherent cells to produce more O(2)(-) than 86 kDa Lf. Furthermore, the low Con A affinity Lf showed low antibacterial activity against E. coli and S. aureus, and had decreased iron-binding capacity in comparison with 86 kDa Lf. Moreover, the 86 kDa Lf could stimulate bovine T cells or macrophages to produce IFN-gamma, IL-4, IL-6, and IL-1alpha. However low Con A affinity Lf induced the production of TNFalpha, but not physiological T cell or macrophage cytokines. It was also found that when the healthy MGs of dry cows were injected with the low Con A affinity Lf, there was an increase in polymorphonuclear cells together with TNFalpha, MCP-1, and IL-8 production. These results suggested that low Con A affinity Lf in mastitic MGSs differed from 86 kDa Lf in physiological characteristics, and, that it induced an inflammatory reaction in MGs.     

A new diagnostic indicator using concanavalin a low-affinity lactoferrin levels in mammary gland secretion in mastitic drying cows

We examined the effective diagnostic indicator using the concanavalin A (Con A) low-affinity lactoferrin (Lf) to mastitic drying cows. The concentrations of both Lf and Con A low-affinity Lf in mammary gland secretions (MGSs) were lower than normal MGSs at the early and middle dry periods and colostrums. On the other hand, the levels of Con A low-affinity Lf in MGSs increased following the appearance of mastitis symptoms, and decreased when the mastitic symptoms were cured. Moreover, IgG1 concentrations of colostrums decrease on the quarters where a high level of Con A low-affinity Lf was determined after the onset of dry period. These results suggest that this method could be used as a useful indicator to mastitic drying cows.     

Effect of combination therapy with lactoferrin and antibiotics against staphylococcal mastitis on drying cows

We examined combination therapy with both lactoferrin (Lf) and antibiotics on clinical mastitis due to Staphylococcus aureus (S.aureus) on drying cows. The clinical symptoms of mastitic quarters were cured 81% of combination therapeutic quarters at 7 days post injection (dpi). Moreover, most of mammary gland secretions (MGSs) in combination therapeutic quarters were normal at 7 days after parturition. In the quarters with combination therapy, S.aureus counts, Lf concentrations and content rate of concanavalin A (Con A) low-affinity Lf decreased and were lower than in the quarters treated with Lf or antibiotics alone. The mRNA expression of tumor necrosis factor alpha (TNFalpha) of the quarters with combination therapy also decreased and was lower than that of the Lf or antibiotics treated. The mRNA expression of pro-inflammatory cytokines and chemokines in bovine mammary gland epithelial lined cells (BMEC) stimulated with Lf were lower than those of Con A low-affinity Lf stimulated BMEC. Moreover, Lf showed an inhibitory effect to the induction of pro-inflammatory cytokine mRNA expression when co-stimulated with Lf and Con A low-affinity Lf. Nuclear factor kappa B (NFkappaB) activation was also induced with Con A low-affinity Lf, and the inhibitory effects of Lf were also confirmed on BMEC co-stimulated with Lf and Con A low-affinity Lf. These results indicated that the efficacy of combination therapy with antibiotics and Lf caused antibacterial effect of antibiotics and inhibition of pro-inflammatory cytokine and chemokine production with Lf via the inhibition of NFkappaB activation.     

Effects of bovine lactoferrin by the intramammary infusion in cows with staphylococcal mastitis during the early non-lactating period

To evaluate the clinical effects of bovine lactoferrin on staphylococcal mastitis in Holstein cows during the early non-lactating period, 41 mammary quarters were selected randomly from 36 cows on 3 dairy farms. Twelve quarters were infused intramammarily with bovine lactoferrin. Twenty-nine quarters were infused with antibiotic as a control. In the bovine lactoferrin-infused group, 91.7% of mastitic quarters were cured at 7 days after calving, compared with 48.3% in the control group. Furthermore, the changes in mammary secretion induced by the infusion of bovine lactoferrin were investigated. Mean numbers of staphylococci in mammary gland secretions were significantly decreased in both 5 bovine lactoferrin-infused quarters and 5 antibiotic-infused control quarters (p<0.05). Unlike in the control quarters, the mean total cell concentration in the mammary gland secretions increased in bovine lactoferrin-infused quarters. Similar results were obtained in 6 healthy quarters which were infused with bovine lactoferrin. In these quarters, the cell population contained mainly phagocytes such as polymorphonuclear leukocytes and cells positive for CD11b which is known as a complement receptor. The mean concentration of C3 in mammary gland secretions was significantly increased in 5 mastitic quarters infused with bovine lactoferrin (p<0.05), but showed no significant change in 5 mastitic control quarters. These results suggested that bovine lactoferrin treatment for staphylococcal mastitis in the early non-lactating period might increase the rate of cure through the induction of innate immunity in the host.     

The antiproliferative effect of bovine lactoferrin on canine mammary gland tumor cells

Lactoferrin has several biological activities, including antitumor activities in some human and animal tumor cells. Clinical trials have been carried out in human medicine based on these effects. However, the antitumor effects of lactoferrin in veterinary medicine remain unknown. In this in vitro study, we demonstrated that co-incubation of canine mammary gland tumor cells (CIPp and CHMp) and bovine lactoferrin induced growth arrest of tumor cells. This growth arrest was associated with induction of G1 arrest. Furthermore, this effect was stronger in tumor cells than in normal cells. These findings demonstrate that bovine lactoferrin has anti-tumor activity in canine mammary tumors and has the potential for use in tumor-bearing dogs.     

Bovine intelectins: cDNA sequencing and expression in the bovine intestine

Intelectins (Itlns) are lectins with potential roles in innate immunity, capable of binding bacteria via galactofuranose residues. Itlns also function as intestinal receptors for the antimicrobial glycoprotein lactoferrin (Lf). Since Lf binds strongly to enterohemorrhagic Escherichia coli O157:H7 (EHEC), we aimed to determine the expression of Lf receptor in terminal rectum, the site of predilection of EHEC in cattle. We sequenced two bovine intelectins (Itln1 and Itln2) and showed that both were expressed in abomasum and rectum, but expression appeared minimal in the jejunum. There was significantly higher expression of Itln2 in terminal rather than proximal rectum. Lactoferrin was expressed in all samples examined. Thus, we have demonstrated two novel bovine Itlns and shown that they are expressed along with Lf in the gastrointestinal tract, where they may interact with microbial pathogens.     

Detection of bovine lactoferrin binding protein on Jurkat human lymphoblastic T cell line

Lactoferrin (Lf), a member of the transferrin family protein, is an iron-binding protein that is known to interact with mammalian cells through a specific receptor. We examined binding of Lf to Jurkat human lymphoblastic T cell line (Jurkat cells) by far Western blotting, and found that bovine Lf and human Lf bound to the same protein components of Jurkat cells, and that pepsin digestion of Lf disrupts the sites responsible for binding to cellular proteins. We also found that the sugar chains of bovine Lf are not involved in binding between bovine Lf and Jurkat cells. Bovine Lf, bovine transferrin and ovotransferrin bound to the same proteins of Jurkat cells, which had molecular weights of about 35 kDa.     

Disposition kinetics of lactoferrin in milk after intramammary administration

Disposition kinetics of lactoferrin (Lf) purified from cheese whey was studied in the milk of Finnish Ayrshire cows after intramammary administration of 1 g of Lf into one udder quarter. Intramammary administration of 1 g of Lf increased Lf concentration in milk for several hours. Mean elimination half-life of Lf was 2.2 h and a mean maximum concentration of 6.3 g/L was reached between 1 and 4 h. After 8 h of administration, Lf concentrations in milk decreased to almost the same level as before the infusion. Forty-eight hours postinfusion, the mean Lf concentration was again higher than in the milk samples taken before the infusion of Lf, being on average 1.5 g/L. Lactoferrin caused some local tissue irritation in the udder quarter. Severity of the irritation reactions varied between cows. The udder quarters of primiparous cows reacted faster than those of multiparous cows, but irritation reactions decreased more rapidly in the older cows than in primiparous cows. The cows had no general signs such as fever or anorexia. The somatic cell count returned to baseline level 4 days after the administration.     

The in vitro effect of bovine lactoferrin on the activity of organ leukocytes in rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla) and wels catfish (Silurus glanis)

Lactoferrin (LF) is a glycoprotein found in milk, neutrophil granules, secretions and selected organs of mammals. Lactoferrin exhibits antibacterial, antiviral, fungicidal, immunoregulatory and other functions. Although fish are devoid of this protein and its cell receptors, LF effect on the immune mechanisms of fish has been demonstrated. The objective of this study was to investigate the effect of bovine lactoferrin, applied in vitro, on the activity of head kidney and spleen leukocytes in three freshwater fish species: rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla) and wels catfish (Silurus glanis). The obtained results validate LF beneficial effect on the respiratory burst of phagocytes in rainbow trout and wels catfish despite the fact that the potential killing activity against Aeromonas hydrophila was not stimulated in any of the studied species. Bovine lactoferrin enhanced the proliferation of T-lymphocytes in rainbow trout and European eel, as well as of B-lymphocytes in rainbow trout.     

Growth inhibition of environmental mastitis pathogens during physiologic transitions of the bovine mammary gland

Ten dairy cows were infused intramammarily near drying off with concanavalin A (conA) or phytohemagglutinin (PHA). Mammary secretions were collected during physiologic transitions of the udder and were used in an in vitro microbiological assay to determine growth inhibition of mastitis pathogens. As mammary involution progressed, in vitro growth inhibition of Klebsiella pneumoniae, Escherichia coli, and Streptococcus uberis increased. Mammary secretions from conA- and PHA-treated glands had significantly increased bacterial growth inhibition. Secretions contained significantly increased concentrations of lactoferrin and a decreased citrate:lactoferrin molar ratio earlier in the dry period than did control mammary secretions. Greatest bacterial growth inhibition was observed in mammary secretions obtained 7 days before parturition. However, differences in secretion composition or bacterial growth inhibition were not found between conA- or PHA-treated and control udder halves during the prepartum period. Bacterial growth inhibition by mammary secretion decreased markedly during early lactation. A highly significant positive correlation was found between bacterial growth inhibition and concentrations of lactoferrin, serum albumin, and immunoglobulin G. A highly significant negative correlation was found in the citrate:lactoferrin molar ratio during early involution and the peripartum period.     

Expression of lactoferrin in the boar epididymis: effects of reduced estrogen

Lactoferrin is regulated by estrogen in the female reproductive tract and evidence in immature mice suggests that it may be estrogen regulated in males as well. The estrogen regulation of lactoferrin in the epididymis of the boar, a high estrogen-producing male, is unknown. This study was designed to test the hypothesis that lactoferrin expression in the boar epididymis is regulated by estrogen. Twenty-one littermate pairs of boars were treated with vehicle or Letrozole, an aromatase inhibitor, from 1 week of age until castration at 2 through 8 months. Epididymal tissue was collected at castration and fixed for immunolocalization of lactoferrin. Epididymal and testicular tissues were also collected from five mature boars (1-2.5 years) and fixed for immunocytochemistry (ICC). Lactoferrin was localized in the principal cell cytoplasm of the caput, corpus and cauda of developing boars but only in the corpus and cauda of mature boars. Basal cells were negative for lactoferrin. Sperm in the corpus and cauda was also positive for lactoferrin. The efferent ducts and testes were negative for lactoferrin. Intensity of lactoferrin immunostaining increased with age in the corpus and cauda regardless of treatment. Reduced endogenous estrogen in the epididymis during development did not affect the intensity of immunostaining between control and Letrozole-treated animals. Lactoferrin expression in the epididymis of the developing boar does not appear to be regulated by estrogen.     

乳铁蛋白生物学特性及其在断奶仔猪生产中的应用研究

乳铁蛋白是一种存在于哺乳动物外分泌液中的非血红素铁结合性糖蛋白,它具有多种生物活性功能.本文主要就乳铁蛋白的生物学功能及其在仔猪中生产中的应用前景做一综述.     

Growth inhibition of Escherichia coli and Klebsiella pneumoniae during involution of the bovine mammary gland: relation to secretion composition

Mammary secretions from 12 Holstein dairy cows were collected to evaluate growth inhibition of Escherichia coli and Klebsiella pneumoniae during involution and during physiologic transitions of the mammary gland. Mammary secretions obtained during late lactation poorly inhibited growth of E coli and K pneumoniae. However, as involution progressed, mammary secretions increasingly inhibited growth of both coliform mastitis pathogens. Greatest inhibition of E coli and K pneumoniae growth was observed when mammary glands were fully involuted. Growth inhibition remained high until 7 days before parturition, and then it decreased significantly (P less than 0.05) to that observed during late lactation. Inhibition of coliform mastitis pathogen growth was associated with high concentrations of lactoferrin and immunoglobulin G, decreased citrate concentration, and a low citrate to lactoferrin molar ratio. These data suggested that differences in susceptibility or resistance to new intramammary infection with coliform mastitis pathogens during the nonlactating period may be attributable, in part, to marked changes in mammary secretion composition that develop during physiologic transitions of the mammary gland. Resistance of the fully involuted mammary gland to coliform infection may be associated with high concentrations of natural protective factors.     

Lactoferrin concentrations in milk from normal and subclinical mastitic cows

The concentrations of lactoferrin (Lf) in quarter milk from normal lactating cows and subclinical mastitic cows were measured to determine whether the Lf concentration in milk is influenced by the age of the cow, the stage of lactation, number of milk somatic cells and the presence of pathogens. Lf concentrations in 111 quarter milk samples from 28 normal lactating cows and 270 quarter milk samples from 198 subclinical mastitic cows were measured by means of a single radial immunodiffusion test. Lf concentrations (means +/- standard deviations; logarithmic form) in normal cows and subclinical mastitic cows were 2.23 +/- 0.39 and 2.70 +/- 0.39, respectively. The mean milk Lf concentration (log) in subclinical mastitic cows was significantly (p<0.01) higher than that in normal cows. The mean milk Lf concentration (log) in normal lactating cows aged 5 years was lower than those in normal lactating cows aged 2 years (p<0.01) and 3 years (p<0.05). The results showed that the milk Lf concentration (log) is associated with age of the dairy cow (one-way analysis of variance test, p<0.01). The mean milk Lf concentration (log) in the latter lactational period tended to be higher than those in the peak and middle periods. Milk Lf concentrations (log) tended to be proportional to the level of the somatic cell count (SCC) score. Mean milk Lf concentrations (log) in subclinical mastitic cows infected with Staphylococcus aureus and with other streptococci species were significantly (p<0.01) higher than those in cows infected with coagulase-negative staphylococci and with Corynebacterium bovis.     

Bovine lactoferrin binds to insulin-like growth factor-binding protein-3

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) has been shown to have IGF independent actions that appear to be mediated by specific IGFBP-3 binding proteins located on cell membranes. We show here using Western ligand blotting, a number of mammary membrane proteins that bind ¹²⁵I-labeled rhIGFBP-3. Immunoprecipitation studies demonstrated that the >70 kDa protein was identified from bovine mammary microsomes as bovine lactoferrin (bLf). In addition to being a secretory protein, Lf is tightly associated with cellular membranes. Labeled rhIGFBP-3 was shown to bind to commercially purchased and processed apo- or holo-human or bLf, but not bovine transferrin (bTf). Binding of [¹²⁵I]rhIGFBP-3 to other positively charged proteins was not detected nor was binding to rhIGFBP-5 or other mammary-secreted IGFBPs observed. Reciprocal specific binding of [¹²⁵I]bLf to rhIGFBP-3 was shown, but [¹²⁵I]bTf did not show binding to rhIGFBP-3. While [¹²⁵I]rhIGF-II does not bind to bLf, unlabeled rhIGF-II was shown to compete with [¹²⁵I]bLf for rhIGFBP-3 binding. More detailed analysis by dot blot showed that Lf competes (ED₅₀=3 μg/ml) or displaces (ED₅₀=1 mg/ml) bound [¹²⁵I]rhIGF-II from dot blotted rhIGFBP-3. In vitro studies with a bovine primary mammary epithelial cell culture showed that all-trans-retinoic acid stimulates the appearance of bovine IGFBP-3 and bLf in the conditioned media and that [¹²⁵I]rhIGFBP-3 could be utilized to detect conditioned media bLf. These findings reveal a novel role for bLf, binding to IGFBP-3 and perhaps disassociating IGFBP-3:IGF when in high concentration.     

Cryopreserved bovine mammary cells to model epithelial response to infection

Mammary gland epithelial cells are likely to be important effectors in defending against mastitis, yet little is known about their response mechanisms. Here, we describe a cryopreserved bovine mammary epithelial cell model to study the infection response. Primary cell cultures from four Holstein cows were prepared, and frozen after two passages. The cell cultures from each cow were then thawed and maintained separately, yet simultaneously, and exposed to treatments that included infection with Staphylococcus aureus or exposure to LPS from Escherichia coli. A clear inflammatory response was shown by a significant (P < 0.05), dose dependent, increase of lactoferrin and IL-8 secretion within 24h in response to S. aureus or LPS. Marked increases (P < 0.05) in lactoferrin, TNF-alpha and serum amyloid A (SAA) mRNA expression were also observed. The results indicate the usefulness of our model to study infection responses of mammary epithelial cells, where all cells are simultaneously exposed to the same infection pressure. These responses can be studied over time, and most importantly, biological replication is provided by the four different genotypes being investigated individually. Finally, the results indicate that mammary epithelial cells play an important role in inflammatory response, through the production of pro-inflammatory cytokines, an acute phase protein, and lactoferrin.