Detecting Candida albicans in Human Milk

Procedures for diagnosis of mammary candidosis, including laboratory confirmation, are not well defined. Lactoferrin present in human milk can inhibit growth of Candida albicans, thereby limiting the ability to detect yeast infections. The inhibitory effect of various lactoferrin concentrations on the growth of C. albicans in whole human milk was studied. The addition of iron to the milk led to a two- to threefold increase in cell counts when milk contained 3.0 mg of lactoferrin/ml and markedly reduced the likelihood of false-negative culture results. This method may provide the necessary objective support needed for diagnosis of mammary candidosis

Benefits of Lactoferrin, Osteopontin and Milk Fat Globule Membranes for Infants

The provision of essential and non-essential amino acids for breast-fed infants is the major function of milk proteins. In addition, breast-fed infants might benefit from bioactivities of milk proteins, which are exhibited in the intestine during the digestive phase and by absorption of intact proteins or derived peptides. For lactoferrin, osteopontin and milk fat globule membrane proteins/lipids, which have not until recently been included in substantial amounts in infant formulas, in vitro experiments and animal models provide a convincing base of evidence for bioactivities, which contribute to the protection of the infant from pathogens, improve nutrient absorption, support the development of the immune system and provide components for optimal neurodevelopment. Technologies have become available to obtain these compounds from cow's milk and the bovine compounds also exhibit bioactivities in humans. Randomized clinical trials with experimental infant formulas incorporating lactoferrin, osteopontin, or milk fat globule membranes have already provided some evidence for clinical benefits. This review aims to compare findings from laboratory and animal experiments with outcomes of clinical studies. There is good justification from basic science and there are promising results from clinical studies for beneficial effects of lactoferrin, osteopontin and the milk fat globule membrane complex of proteins and lipids. Further studies should ideally be adequately powered to investigate effects on clinically relevant endpoints in healthy term infants

Cattle Mammary Bioreactor Generated by a Novel Procedure of Transgenic Cloning for Large-Scale Production of Functional Human Lactoferrin

Large-scale production of biopharmaceuticals by current bioreactor techniques is limited by low transgenic efficiency and low expression of foreign proteins. In general, a bacterial artificial chromosome (BAC) harboring most regulatory elements is capable of overcoming the limitations, but transferring BAC into donor cells is difficult. We describe here the use of cattle mammary bioreactor to produce functional recombinant human lactoferrin (rhLF) by a novel procedure of transgenic cloning, which employs microinjection to generate transgenic somatic cells as donor cells. Bovine fibroblast cells were co-microinjected for the first time with a 150-kb BAC carrying the human lactoferrin gene and a marker gene. The resulting transfection efficiency of up to 15.79×10−2 percent was notably higher than that of electroporation and lipofection. Following somatic cell nuclear transfer, we obtained two transgenic cows that secreted rhLF at high levels, 2.5 g/l and 3.4 g/l, respectively. The rhLF had a similar pattern of glycosylation and proteolytic susceptibility as the natural human counterpart. Biochemical analysis revealed that the iron-binding and releasing properties of rhLF were identical to that of native hLF. Importantly, an antibacterial experiment further demonstrated that rhLF was functional. Our results indicate that co-microinjection with a BAC and a marker gene into donor cells for somatic cell cloning indeed improves transgenic efficiency. Moreover, the cattle mammary bioreactors generated with this novel procedure produce functional rhLF on an industrial scale

Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

BACKGROUND: There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. METHODOLOGY/PRINCIPAL FINDINGS: We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. CONCLUSIONS/SIGNIFICANCE: Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale

MiR-584 mediates post-transcriptional expression of lactoferrin receptor in Caco-2 cells and in mouse small intestine during the perinatal period

MicroRNAs function as gene expression modulators that are critical for mammalian development. Lactoferrin receptor on the apical membrane of enterocytes has been suggested to play key roles in the absorption of lactoferrin-bound iron from breast milk. The objective of this study was to identify mechanisms of microRNA mediated post-transcriptional regulation of the lactoferrin receptor. Sequence analyses revealed that the miR-584 sequence is identical in human, mouse and rat, and there is a conserved region complementary to the seed region (5' nucleotides 2-8) of miR-584 within the lactoferrin receptor mRNA-3'-untranslated region. miR-584 was further found to co-localize with lactoferrin receptor mRNA in mouse small intestine. The 3'-untranslated region of human lactoferrin receptor mRNA was cloned into pGL3-control luciferase reporter vector. By luciferase reporter assays in HEK293 cells, miR-584 mimic specifically repressed the reporter activity in a dose-dependent manner. miR-584 mimic reduced endogenous lactoferrin receptor protein expression in Caco-2 cells, without significantly affecting the mRNA level. We also determined that miR-584 expression is inversely correlated with lactoferrin receptor mRNA and protein expression. Taken together, we propose that miR-584 contributes to the post-transcriptional expression of lactoferrin receptor during the perinatal period. These findings demonstrate a novel example of how microRNAs may be involved in regulation of nutrient metabolism in the newborn

Bovine lactoferrin and lactoferricin exert antitumor activities on human colorectal cancer cells (HT-29) by activating various signaling pathways

Lactoferrin (Lf) is an iron-binding glycoprotein and is present at high concentrations in milk. Bovine lactoferricin (LfcinB) is a peptide fragment generated by pepsin proteolysis of bovine lactoferrin (bLf). LfcinB consists of amino acid residues 17â 41 proximal to the N-terminal of bLf and a disulfide bond between residues 19 and 36 forming a cyclic loop. Both bLf and LfcinB have been demonstrated to have antitumor activities. Colorectal cancer is the 2nd most common cause of cancer deaths in developed countries. We hypothesized that bLf and LfcinB exert antitumor activities on colon cancer cells (HT-29) by triggering various signaling pathways. bLf and LfcinB significantly induced apoptosis in colon cancer cells but not in normal human intestinal epithelial cells (HIEC) revealed by the ApoTox-Glo triplex assay. The Live/dead cell viability assay showed that both bLf and LfcinB inhibited the viability of HT-29 cells. Transcriptome analysis indicated that bLf, cyclic LfcinB, and linear LfcinB exerted antitumor activities via differentially activating diverse signaling pathways, including p53, apoptosis, and angiopoietin signaling. Immunoblotting results validated that both bLf and LfcinBs increased expression of caspase-8, p53, and p21, critical proteins in tumor suppression. These results provide valuable information regarding bLf and LfcinB for potential clinical applications in colon cancer therapy.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Growth factor TGF- β induces intestinal epithelial cell (IEC-6) differentiation: MiR-146b as a regulatory component in the negative feedback loop

TGF-β is a potent pleiotropic factor that promotes small intestinal cell differentiation. The role of microRNAs in the TGF-β induction of intestinal epithelial phenotype is largely unknown. We hypothesized that microRNAs are functionally involved in TG

miR-214 regulates lactoferrin expression and pro-apoptotic function in mammary epithelial cells

Lactoferrin (Lf) is an abundantly expressed protein in human milk. Lactoferrin exhibits several important biological functions, and its expression is regulated by multiple environmental factors. Cellular endogenous factors, however, have not been extensively studied with regard to lactoferrin gene expression. In this study, we showed that lactoferrin gene expression and function are directly targeted by miR-214 in HC11 and MCF7 cells. In the lactoferrin mRNA 39 untranslated region (UTR) of human, mouse, rat, pig, bovine, camel, and goat species, there is a conserved region that perfectly matches the seed region of miR-214. Transfection of miR-214 mimic in HEK293 cells dose-dependently inhibited the activity of pGL3-control vector containing lactoferrin mRNA 3′UTR downstream of the luciferase gene. In HC11 cells, miR-214 overexpression inhibited the induction of lactoferrin expression by β-estradiol (E2) and dexamethasone-prolactin-insulin (DPI). Furthermore, in MCF7 cells, overexpression of miR-214 markedly decreased lactoferrin expression (P < 0.05), and inhibition of endogenous miR-214 expression increased lactoferrin expression and cellular apoptotic activities (P < 0.05). In summary, our data showed that miR-214 is directly involved in lactoferrin expression and lactoferrin mediated cancer susceptibility (proapoptotic activities) in mammary epithelial cells

Biochemical and molecular impacts of lactoferrin on small intestinal growth and development during early life

Postnatal modeling of the intestinal epithelium has long-term impacts on the healthy development of infants and relies largely on nutrient composition of the diet. Lactoferrin (Lf) is among the various human milk trophic factors that facilitate the infant intestinal adaptation. Hydrolysis of Lf is minimal at the prevailing postprandial pH of infants, and Lf may therefore have greater biological potential in infants than in adults. Lf bidirectionally stimulates concentration-dependent proliferation and differentiation of small intestinal epithelial cells, and therefore affects small intestinal mass, length, and epithelial digestive enzyme expression. A 105 kDa Lf receptor (LfR) specifically mediates the uptake of Lf into enterocytes and crypt cells. Mechanistically, the complex of Lf and LfR is internalized through clathrin-mediated endocytosis; both iron-free apo-Lf and iron-saturated holo-Lf activate the PI3K/Akt pathway, whereas only apo-Lf triggers ERK1/2 signaling. Lf enters the nucleus, where it can stimulate thymidine incorporation into crypt cells, regulating transcription of genes such as TGFb1. In the fetus, the plasma membrane LfR is at the highest abundance in the small intestine, and the receptor gene is tightly controlled at multiple levels. Aspecific microRNA, miR-584, is involved in the posttranscriptional regulation of LfR, and in the human LfR DNA promoter, 2 Sp1 binding sites have been characterized functionally. Finally, cell proliferation and global gene expression reveal that native bovine Lf can perform biological activities similar to those exerted by human Lf in postnatal small intestinal development